CN106267406B - AIDS blood purification - Google Patents

Abstract

A kind of AIDS blood purification for medical domain, it is characterized in that the separator of preparation energy washed corpuscles and blood plasma, former macrophage feature was not only retained with hybridoma technology preparation but also the hybridoma macrophage strain of energy indeterminate growth expands parallel, it prepares HIVgp120 and gp41 antibody and combines HIVgp120 the and gp41 antibody of goat-anti Ig, object made above is formulated in agar gel, clarifier is formed with high-biocompatibility material package, gel is set to form antibody titer from high to low from top to bottom and the layer distributed of agarose concentration from low to high, wherein in conjunction with goat-anti Ig and unbonded gp120 and gp41 antibody, hybridoma macrophage, which is fixed in agar gel, to be play a part of to adsorb HIV, made clarifier is applied in combination with separator and regulatory process , the blood in extracorporal circulatory system is divided into blood plasma and haemocyte by separator, and the purified device of blood plasma converges with haemocyte after filtering out HIV, then feeds back.

Description

AIDS blood purification

Technical field

The present invention relates to the preparations and application of AIDS blood purification in medical domain, are mainly used for AIDS patient's blood The removing of AIDS virus is starched, to achieve the purpose that treat AIDS.

Background technique

AIDS is passed caused by human immunodeficiency virus (Human Immunodeficiency Virus, HIV) It catches an illness, is widely current in the whole world, according to the related report of the World Health Organization (WHO) and The Joint Programme on AIDS, certainly Since U.S.'s discovery Patient With Aids cases in 1981, the whole world has 208 countries and regions to receive the serious of AIDS so far It threatens, there are about 40,000,000 people to have infected AIDS, and death toll is more than 20,000,000, and there are about 6000 people to become AIDS sense daily Dye person, while there are about more than 300 people to die of AIDS daily.It is in rapid growth period in HIV infected individuals in China, it is far super at present Cross 1,000,000.AIDS has become the great of after tumour, cardiovascular and cerebrovascular disease, tuberculosis, diabetes another facing mankind Infectious diseases, it has also become the serious public health of global concern and social concern.

Human immunodeficiency virus is a kind of slow virus (Lentivirus) for infecting human immune cells, belongs to reverse transcription disease One kind of poison, it is about 120 nanometers of diameter, substantially spherical in shape.Outer virionic membrane is lipoid coating (from host cell), and embedded with virus Albumen gp120 and gp41.Gp41 is transmembrane protein, and gp120 is located at surface, and with gp41 by noncovalent interaction in conjunction with.To It is inside the sphere matrix (Matrix) formed by albumen p17 and the half-cone capsid (Capsid) that albumen p24 is formed, capsid Under Electronic Speculum be in high electron density, include virulent rna gene group, enzyme (reverse transcriptase, integrase, protease) and other Ingredient from host cell.

It after HIV enters human body, is swallowed first by macrophage, but HIV changes certain positions in macrophage quickly Acidic environment creates the condition for being suitble to it to survive, is not killed not only and breeds in it instead.Because CD4 be HIV by Body, so the HIV bred in macrophage is through its envelope protein gp120 and under the auxiliary of gp41, (gp41 plays bridge Effect, utilizes itself hydrophobic effect mediate retroviral cyst membrane and cell membrane fusion) entering CD4+ cell, (cell, monokaryon macrophage are thin Born of the same parents, Dendritic Cells etc.), it is proliferated rapidly in the cell, generates 10 daily⁹~10¹⁰Virion, and it is normal constantly to enter other And regenerated CD4+ replicate into the cell, manufacture more virus infected cells, make peripheral blood CD4+T cell sustaining breakdown, subtract It is few.The T cell of infected Apoptosis or even infected by HIV can be directly activated to express in conjunction with gp120 and the CD4 receptor of HIV Envelope antigen can also start normal T-cell, cause a large amount of broken of CD4+ cell indirectly by cell surface CD4 molecule cross-link Bad, as a result cause the severe immune deficiency centered on CD4+T cell defect, patient mainly shows: periphery lymphocyte is reduced, T4/T8 proportional arrangement disappears to the reaction of phytohemagglutin phytolectin and certain antigens, delayed allergy decline, NK cell, macrophage Cell activity weakens, and the synthesis of the cell factors such as IL2, interferon is reduced.CD4+T cell is most important immunocyte, infection Person once loses a large amount of CD4+T cells, and entire immune system will all lose the infection of various diseases by deathblow Go resistance.HIV can also show as hiding without showing clinical symptoms, genome for a long time after entering host's CD4+ cell RNA reverse transcription enters in host cell nuclear at double-stranded DNA with viral integrase enzyme, under the action of integrase, double-stranded DNA integration Into host cell gene group, the viral DNA being integrated is known as provirus, and the several months that can hide does not replicate even for many years, causes The incubation period of AIDS several months to many years.In the incubation period of AIDS, HIV is mainly in the macrophage and Dendritic Cells of lymph node Breeding, these cells are intracorporal HIV depots, and releasably the CD4+T cell into peripheral blood or transfection peripheral blood, there is silk point Splitting original, antigen, TNF, IL-2 and lymph plain (LT) can excite HIV provirus gene multiple in the CD4+T Intracellular transcription of infection System.After being largely proliferated, inhibition of HIV particle constantly discharges from the infection cell being destroyed and is free on blood, then enters back into New CD4+T cell continues course of infection.And cell after HIV infection, be expressed in infection cell surface gp120 and Gp41 can mediated infection and merging between non-infected cells, such as infected by HIV the expression of CD4+T cell gp120 be uninfected by The CD4 of cell is combined, and is led to cell fusion and is formed multinucleate giant cell.

Body production envelope protein (Gp120, Gp41) antibody and core protein (P24) antibody can be stimulated after HIV infection.? Low-level antiviral neutralizing antibody is measured in HIV carriers, AIDS patients serum, wherein AIDS patients level is minimum, HIV carriers' highest illustrates that the antibody has protective effect in vivo.But antibody cannot be with the virus that retains in mononuclear macrophage Contact, and antigenic variation easily occurs for HIV envelope protein, original antibody is ineffective, keeps neutralizing antibody due from playing Effect.In the latent infection stage, HIV provirus is integrated into host cell gene group, therefore HIV will not be known by immune system Not, so relying solely on itself immune function can not be removed.Another critically important reason should be killed according to antibody It goes out, remove the mechanism speculate of antigen, after immune antibody and antigen binding, to generate immunological effect or by activating complement, Mediating A1) CC effect dissolves cellular antigen, but HIV is not cellular antigen；Attract phagocyte by chemotaxis Antigen is swallowed, but HIV is protected in phagocyte instead, is proliferated；Antibody and antigen binding play neutralization, are allowed to lose Appeal is gone, but HIV antigenic structure is changeable, is often difficult to antibody.

In terms of the treating AIDS method for having been used for clinic at present, effect is less ideal: (1) hiv reverse transcriptase inhibits Agent: being only capable of preventing the permissive cell of not yet infected by HIV from infecting, and does not have a therapeutic effect to the cell infected, and toxic side effect compared with It is more, including gland plastochondria toxicity, bone marrow suppression, globular anemia, granulocyte and decrease of platelet, pancreatitis, intersect in addition resistance to The generation of pharmacological property, this kind of medicine are not used alone, mainly do drug combination, and are also easy to produce drug resistance mutant, cause under clinical efficacy Drop or failure.(2) toxic side effects such as drug induced hepatic injury, disorders of lipid metabolism and drug resistance hiv protease inhibitor: are also easy to produce Property.(3) it hiv integrase inhibitor: by inhibiting inhibition of HIV DNA to play a role with host cell DNA integration, is reversed with HIV Transcriptase inhibitors and protease inhibitors are combined while use has synergistic effect to antiviral.(4) inhibition of HIV is inhibited to enter inhibition Agent: including block gp120 with CD4 ining conjunction with, blocking HIV in conjunction with accessory receptor, act on gp41 film subunit and act on T drench Bar cell surface CC-chemokine receptor 5 (CCR5) has side effect to liver and heart to block HIV to enter host cell.(5) Cytokine therapy: it is mainly used for regulatory T-cell dynamic equilibrium.(6) HIV vaccine is treated: such as intrinsic due to the particularity of HIV Immune to be not enough to resist HIV and its targeting destruction immune system, virus mutation is rapid, causes not yet to develop real safety so far Effective vaccine.(7) gene therapy: the research of HIV gene therapy is never interrupted, including antisense technology, RNA bait, RNA dry It disturbs, intrabody, dominant negative mutant, suicide gene etc., but enters the gene therapy in II clinical trial phase stage almost No.(8) monoclonal antibody passive immunization therapy: the neurological susceptibility of HIV is reduced by lowering CD4+T cell surface CCR5, is prolonged The progress of slow AIDS and the diffusion for reducing HIV, neutralizing monoclonal antibody 2G12,2F5 and 4E10, which are applied to HIV infection person, to be had Good tolerance and safety can delay but cannot prevent rebound (9) adoptive immunity cell therapy of virus: external a large amount of It will lead to viral massive amplification when the culture self CD4+T cell of HIV, increase the CD4+T cell quantity of virus infection, and feed back CD4+T cell may will increase the place of internal virus replication, lead to virus load bounce-back, and on the whole, adoptive immunity is thin Born of the same parents treat without apparent toxic side effect, also do not obtain satisfied therapeutic effect.

Ag-Ab precipitation reaction was initially applied to research Liesegang's phenomenon in 1905 in gel, applied within 1932 In identification bacterium bacterial strain, nineteen forty-six Oudin carries out immunodiffusion in test tube, for analyzing antigen mixture, 1948 Elek and Ouchterlony establishes the two-way double-diffusion process of agar respectively, for identifying simultaneously, more two or more antigens or anti- Body.The media gel agar or agarose of Ag-Ab precipitation reaction in gel are a kind of polysaccharide body containing sulfate, high temperature When can be dissolved in water, gel is congealed into after cold, inside forms a kind of porous reticular structure, and aperture is very big, allow macromolecular Substance (molecular weight is up to million or more) passes freely through.The size in aperture further depends on agar concentration, and agar concentration is big, aperture phase To smaller, agar concentration is small, and aperture is relatively large.The aperture of 1% agar gel is about 85nm, since agar or agarose have Good chemical stability, water content is big after gel, and transparency is good, and convenient sources are easy to handle, therefore is a kind of diffusion well Medium.The molecular weight of antigen and antibody in gel from high concentration region to low concentration region generally all 200,000 hereinafter, spread When suffered resistance very little, be substantially in free diffusing form.Due to the molecular weight of different antigen molecules, structure, shape and electricity Lotus amount is different, therefore its diffusion coefficient is different, and diffusion velocity is also just different in gel.When antigen and corresponding antibodies are after spreading It meets in gel, forms antigen antibody complex if the two is appropriate in place's ratio of meeting and form maximum compound.By In the molecular weight increase of compound, particle increases, thus does not continue to spread and generate to precipitate, and shows threadiness or band-like, this Kind precipitating is formed one " specific barrier ", and all antigen or antibody molecule same in immunology cannot pass through, And those of property difference molecule can continue to spread by this barrier, until forming the compound of themselves. In this way, synantigen is not formed by each have their own position of precipitating.Such reaction is known as agar gel diffusion or AGP test, or Immune proliferation, linear or band-like " specific barrier " formed are known as immuning lines or immunoprecipitation band, referred to as precipitate Line or sealed Belt.It is at present with the routine experiment checkup item of known antibodies detection unknown quantity corresponding antigens, and " middle traditional Chinese medicines Allusion quotation " standard method of the regulation for the detection of influenza virus vaccine hemagglutinin content in 2010 editions.Usually by a certain amount of goat-anti people Ig antiserum ingredient is mixed in agar gel, is made containing the specificity sero-fast agar plate of goat-anti people Ig, is beaten after to be solidified Hole, and human serum to be checked (IgG, IgA, IgM etc.) is added in corresponding aperture, spread serum to be checked around in agar plate, Properly locate to combine in antigen and antibody concentration ratio, forms macroscopic white precipitate ring and no longer spread.Thus may be used See, when a kind of solution passes through semi-solid gel, the gel pore detention that macromolecular solute therein is just acted on by molecular sieve is solidifying In glue, antibody that antigen especially therein can be fixed in advance in gel in conjunction with and be attracted in gel.So can root According to the principle that agar gel is spread, the clarifier of design treatment AIDS prepares the HIV antibody of different infection strains, HIV is resisted Body is fixed in gel in advance, and when patient flows through clarifier through the blood plasma that extracorporal circulatory system is isolated, the HIV in blood plasma is preparatory Be fixed on corresponding antibody in gel in conjunction with and detention in the gel in clarifier, while because of the aperture of 1% agar gel About 85nm can also be acted on by the HIV detention of 100~120nm in gel through the absorption of such antibody and gel molecular sieve, can HIV is removed outside prosthesis.

The phagocyte of the mankind has large and small two kinds, and microphage is the neutrophil leucocyte in peripheral blood, macrophagocyte It is the macrophage in the monocyte and a variety of organs, tissue in blood, the two constitutes mononuclear phagocyte system.Monocyte It is formed by the monocyte precursor Development And Differentiation in marrow, accounts for about 3% one the 8% of blood middle leukocytes sum, volume is relatively drenched Bar cell is bigger, and monocyte only stops 12-24 hours in blood, subsequently into connective tissue or organ, reach maturity for Macrophage, macrophage are highly differentiation, mature cell type in mononuclear phagocyte system, have stronger phagocytosis function Can, wandering macrophage is greater than monocyte several times, and it lasts a long time, can survive in the tissue some months, the macrophage of colonization There is different titles, be Kupffer Cell in liver, be in brain microglia, be osteoclast etc. in bone, expresses Fc Receptor, C3b receptor and CD14 play defense function in inherent immunity, and the professional antigen of participation adaptive immunity is offered Cell.The CD4 molecule of Expression of Macrophages, is the receptor of AIDS virus (HIV), thin by macrophage first after HIV enters human body The phagocytosis of born of the same parents, but HIV changes the acidic environment at certain positions in macrophage quickly, creates the condition for being suitble to it to survive, It is not killed mass propagation aggregation in it instead not only, then HIV is passed into CD4+T cell.So hybridoma can be used Technology prepares hybridoma macrophage strain, is used to prepare the clarifier for the treatment of AIDS, after massive amplification with macrophage Phagocytic function removes the HIV in blood plasma.

In short, various drugs and biological products can not effectively kill intracorporal AIDS virus, and price, side effect is big, So far the effective ways for the treatment of AIDS be there is no, it has also become attack the global problem being unable to long.

Summary of the invention

In order to solve to attack the global problem in the treating AIDS field being unable to long, present inventors have proposed the present invention.

The invention aims to provide AIDS blood purification；Another object is to provide for AIDS blood purification Preparation and application method.

The object of the present invention is achieved like this: the separator of preparation energy washed corpuscles and blood plasma prepares HIVgp120 With gp41 antibody, then by antigen of gp120 and gp41 antibody goat-anti gp120 and gp41 antibody is prepared, is prepared with hybridoma technology Not only former macrophage feature had been retained but also the hybridoma macrophage strain of energy indeterminate growth expands parallel, with high-biocompatibility material Package hybridoma macrophage strain and preparing can prevent cell and its fragment from filtering out and can provide the purification in place for removing HIV Gp120 and gp41 antibody and goat-anti gp120 and gp41 antibody are mixed, tie goat-anti gp120 and gp41 antibody completely by device It closes, but the surplus gp120 and gp41 antibody for having high titre, antibody is incorporated the heat preservation after 100 DEG C dissolve with reversed gradient titre and is existed The agarose of 39~42 DEG C of gradient concentration successively takes agarose that clarifier is added, is cooled to by antibody titer from down to height After semi-solid gel again plus next time, the gel in clarifier is made to form antibody titer from high to low from top to bottom and from as low as The layer distributed of high agar concentration is conducive to the effect of plasma perfusion and molecular sieve and immune clearance, wherein anti-with goat-anti HIV Body in conjunction with and unbonded gp120 and gp41 antibody, the strain of hybridoma macrophage be fixed on Ago-Gel and play absorption The effect of HIV, prepared clarifier are applied in combination with separator and regulatory process in turn, and the blood in extracorporal circulatory system is separated Device is divided into blood plasma and haemocyte, and the purified device of blood plasma converges with haemocyte after filtering out HIV, then feeds back.

Technological core of the invention is made of plasma separator and clarifier, and wherein plasma separator is used for washed corpuscles And blood plasma, cleanser in clarifier is by being fixed on HIV antibody, the HIV antibody in conjunction with goat-anti Ig, hybridoma of agar gel Macrophage strain is made, wherein the molecular weight of HIV antibody its conjugate in conjunction with goat-anti Ig is than unbonded HIV antibody molecule Amount is big, not easily pass through gel molecular sieve and contained goat-anti Ig easily with agar gel secure bond the characteristics of, HIV antibody also with Be easier to be fixed in agar gel, the HIV in blood plasma can be combined when meeting with the HIV antibody for being fixed in agar gel It reacts and forms antigen antibody complex, so that agar gel is fixed in by HIV antibody, because of the day of hybridoma macrophage So phagocytosis characteristic, can be fixed in agar gel, and agar gel is formed by filter by phagocytosis and therewith when HIV meets therewith Hole is reduced with increasing for agarose concentration, and clarifier entrance agarose concentration is low, and filter opening is just big, is conducive to plasma perfusion And the association reaction of high titre antibody or cell and HIV；And exit concentration is high, filter opening is just small, is easy to detention HIV or macromolecular Conjugate, clarifier are combined with a variety of special and non-specific HIV and remove composition, in order to avoid caused by extraordinary strain is because of immunity difference Futile treatment, thus in vitro circulation in, after blood isolates blood plasma and haemocyte by separator, when blood plasma flows through clarifier its In HIV be cleaned agent absorption and remove, purified blood plasma is fed back after converging with haemocyte, and the present invention is with external mechanical removal The method substitution of HIV can not effectively kill the conventional anti-reverse transcription drug treatment in vivo of HIV for a long time, realize artificial By HIV from the treatment new method of internal mechanical removal.

Specific embodiment

Fig. 1 is the application schematic diagram of the AIDS blood purification proposed according to the present invention.

Fig. 2 is the schematic diagram of internal structure of the separator proposed according to the present invention.

Fig. 3 is the schematic diagram of internal structure of the clarifier proposed according to the present invention.

In Fig. 1, one end of arterial blood line pipe (1) is connected with arteries, and the other end is through heparin pump (2) and blood pump (3) it is connected with plasma separator (4), plasma separator (4) purification in parallel with two through blood plasma pump (6) and circulation line (7) Device (8), clarifier (9) be connected, be then successively connected with circulation line (10), venous line (5), venous line (5) it is another End is connected with vein blood vessel.

In Fig. 2,1 is plasma separator, and 2 be plasma separator inner cavity, and 3 be the micropore on the tube wall of plasma separator inner cavity, 4 Being cannot be by the haemocyte of micropore (3), and 5 be the blood plasma and chemical analysis that can pass through micropore (3), and 6 be plasma separator exocoel, 7 be blood plasma outflux, and 8 be that there is the haemocyte of switchable valve to export.

In Fig. 3,1 is free HIV, and 2,4 be respectively the HIV antibody being fixed in agar gel (6), macrophage, 3,5 Respectively HIV in conjunction with HIV antibody, macrophage after be delayed at conjugate in agar gel (6), 7 is are coagulated by agar The large volume of HIV of glue (6) molecular sieve detention.

Below with reference to Fig. 1, Fig. 2 and Fig. 3, the embodiment of AIDS blood purification proposed by the present invention is made detailed Description.

One, the preparation of AIDS blood purification agent

(1) preparation of hybridoma macrophage strain

1, primary cell source

(1) single core blood cell: refer to the lymphocyte separated from blood with density-gradient centrifugation method and monokaryon macrophage Cell.Specific method is: the Cord blood buying the White Blood Cells Concentrate of Blood Center or saving for scientific research takes 2mL sample, PBS 6mL anticoagulation is slowly superimposed on dropper that 4mL lymph has been added is thin by 2~3 times of hemodilution, after mixing well by liquid along tube wall Horizontal centrifugal (400r/min, 20 DEG C) 35min in horizontal centrifuge in the 10mL centrifuge tube of born of the same parents' separating liquid；It is divided into pipe after centrifugation 3 layers, upper layer is blood plasma and PBS liquid, and lower layer is mainly red blood cell and granulocyte, and middle layer is lymphocyte separation medium, in upper, middle layer It is PBMC that, which there be the white cloud and mist layer narrow band based on mononuclearcell in interface, is inserted into cloud and mist layer with capillary syring, is drawn PBMC is placed in another 50mL centrifuge tube, is added 5 times and is centrifuged (300r/min, 20 DEG C) 10min with upper volume PBS, abandons supernatant Cell is resuspended in 50mLPBS, is centrifuged (350r/min, 20 DEG C) 15min, abandons supernatant, and Buffer (PBS+0.5% new life ox blood is added + 2mmol/LEDTA clearly, pH7.2) 2mL resuspension cell, it takes 15uL cell suspension to be added on blood counting chamber and counts 4 under microscope Cell (PBMC) sum in a block plaid.

(2) it single core histocyte: is provided by Zhejiang University's Tissue Engineering Study platform.Substantially belong to from spleen Macrophage, preparation method are: the 1. acquisition and transhipment of spleen tissue: in the approval of the reason committee and patient's informed consent Under, the spleen sample tissue for taking operation to cut off shreds into volume about 1mm immediately³Small tissue blocks, move into equipped with pre-cooling 4 DEG C In sterile sealing bottle, it is transported to cell culture chamber rapidly.2. the preparation of spleen tissue cell suspension: spleen tissue block is moved to Aseptic operating platform, PBS are washed 3 times, and RPMI-1640 is washed 2 times, to remove the blood in tissue and guarantee the sterile of tissue.Machine Tool grinds spleen tissue, at this moment just has a large amount of histocyte is outstanding to be mixed in RPMI-1640 liquid.With 200 mesh stainless steel filtering net mistakes Filter is outstanding to be mixed with histiocytic RPMI-1640 liquid, filtrate be spleen tissue cell suspension (mainly containing red blood cell, lymphocyte, Macrophage etc.).3. the cracking of red blood cell in spleen tissue cell suspension: and then centrifugation is washed with RPMI-1640 liquid (1000r/min, 3min) is added Tris-NH4Cl and acts on 5min, splitting erythrocyte, Quick spin to remove cell debris (1000r/min, 3min), remove supernatant in splitting erythrocyte fragment, PBS washing centrifugation 3 times, RPMI-1640 wash from The heart 1 time, to remove Tris-NH4Cl remaining in suspension, it is avoided to influence the survival of cell, at this point, mainly containing in suspension Spleen tissue macrophage and lymphocyte.4. the adhere-wall culture of spleen tissue macrophage: using aforementioned suspension as culture Cell stoste, Trypan Blue determine vigor and count, and are (3~5) × 10 with RPMI-1640 liquid adjustment cell concentration⁶/ L, will The cell suspension inoculation of concentration is adjusted in glass culture bottle, condition of culture is 37 DEG C, 50mL/LCO₂, 100% humidity, point Not Pei Yang 2~3h, observe form under phase contrast microscope.The digestion of adherent spleen tissue macrophage: adherent spleen tissue macrophage The digestion of cell: sucking culture supernatant, and macrophage is adherent, and PBS blows and beats repeatedly, digests, the washing centrifugation of gained cell suspension (1000r/min, 3min), the macrophage isolated and purified.Further, it is also possible to which the sample discarded after treatment or operation is taken to mention Take preparation, such as cavum peritoneale liquid, alveolar, liver, spleen, peritoneal tissues, small intestinal mucosa.

(3) amniotic fluid, villus cell: Zhejiang University's attached hospital for obstetrics and gynaecology's reproduction heredity laboratory is spare.In reason committee member Can ratify under patient's informed consent, take laboratory diagnosis report after remaining amniotic fluid, villus cell, select logarithmic growth phase cell Continue to employ.

2, cell culture and the adherent preliminary sorting of macrophage

Routinely cell culture, but according to the difference of cellularity, appropriate adjustment incubation time, condition of culture etc., generally Single core blood cell (PBMC) or single core histocyte (macrophage) are placed in containing RPMI-1640 culture medium by adherent method Culture dish in, in 37 DEG C, 5%CO₂Cell incubator (Themo electro corporation CLASS 100, beauty State) in be incubated for 2h, after mononuclearcell is adherent, inhale abandon upper layer suspension cell (cell other than macrophage be not easy it is adherent and Removed with upper liquid), PBs buffer gently washs 3 times, and a small amount of mono- 1640 culture medium of RPMI is added, scrapes patch with cell scraper Parietal cell (predominantly macrophage, but there are also other a small amount of attached cells).1 000r/min is centrifuged 5min, abandons supernatant.Amniotic fluid There is cell growth clone in cell, villus cell culture 1~7 day, cell growth converges the logarithmic growth that rate reaches 60~80% Phase cell, is digested with pancreatin, and PBS cleaning obtains cell suspension, is made into proper cell concentration.

3, cd4 cell sorts

Sort cd4 cell: 1. main agents and instrument using immunomagnetic beads method: (German U.S.A day Ni is biological for CD4 immunomagnetic beads Technology Co., Ltd.)；0.2% Trypan Blue liquid (Shanghai Sheng Gong biotechnology service company)；Newborn bovine serum (Hyclone company)；MiniMACS magnetic separation system (German Mei Tian Ni Bioisystech Co., Ltd).2. cd4 cell is immune Magnetic bead sorting method: cell suspension, which is divided equally to two 1.5mLEppendorf, manages, and is centrifuged (300r/min, 20 DEG C) 10min, discards Supernatant is resuspended the every 80uLBuffer of cell and contains cell number 10⁷It is a, every 10⁷A cell add 20uLCD4MicroBeads or CD8MicroBeads is mixed well, and in 4~8 DEG C of hatching 15min, washs cell with 1mLBuffer, be centrifuged (300r/min, 20 DEG C) 10min, it discards supernatant 500uLBuffer and cell is resuspended, MS splitter is placed in the magnetic field of MACS separator, with 500uLBuffer rinsing is rinsed splitter repetitive operation 3 times by 500uL cell suspension by splitter with 500uLBuffer, Efflux is collected, contains non-cd4 cell in efflux, splitter is taken out from separator, with 1000uLBuffer pressure flush point From column, collection efflux, for cd4 cell, (cell viability detection: taking 15uL cell suspension respectively before and after cell purification and waits bodies for this Product trypan blue solution mixing, the not colored shinny person of microscopically observation are living cells, and the coloring person of swelling is dead cell, calculate 200 The percentage of living cells in a cell).The cell sorted at this time is mainly macrophage.

4, CD14 cell (macrophage) sorts

CD14 is monocyte and the distinctive surface marker of macrophage, theoretically if from single core histocyte, sheep It is sorted in water cell and villus cell, then gained cell is macrophage；If sorted from single core blood cell, gained Cell includes monocyte and macrophage；But because the monocyte service life is short, only survive 1 day in peripheral blood and can not show a candle to macrophage Cell is easy to adherent growth, so removing substantially in cell adhere-wall culture of the invention, the cell sorted out is essentially Macrophage.

Basic skills is analogous to cd4 cell, using immunomagnetic beads method.1. reagent: people's CD14 immunomagnetic beads kit (Miltenyi Biotec, Germany), RPMI-1640 culture medium (Hyclone, the U.S.)；2. immunomagnetic beads method: (A) magnetic bead and spy Anisotropic one monocyte of target cell combines: every 1 × 10⁸The magnetic bead and 800uL buffering of 200uL coupling CD14 antibody is added in a PBMC Liquid (contains 10% bovine serum albumin(BSA) 2.5mL and 2mol/L EDTA0.5mL, 4 DEG C of refrigerator pre-coolings), in 15mL centrifuge tube It mixes well, 4 DEG C of incubation 15min, centre can slightly shake 1 time.Take out centrifuge tube after 15min, every 1 × 10⁷A cell is added 1 Buffer is pre-chilled in~2mL, and 1 000r/min is centrifuged 8min, abandons supernatant, and 0.5mL buffer is added and blows and beats into single cell suspension. (B) it collects the monocyte of marked by magnetic bead: cell splitter is placed on MACS magnetic frame, 1mL buffer statocyte is added Splitter drips to no liquid, immediately adds above-mentioned cell suspension in people's cell splitter, rinses cell with 0.5mL buffer Splitter 3 times.After to be rinsed, 1mL buffer is added, the emigrated cells splitter from magnetic frame is quickly pushed with needle column, Go out the cell combined in splitter with one magnetic bead of CD14 antibody, the as macrophage of CD14+.

In addition, following 2 kinds of methods sorting, including 1. adherent method also can be used: PBMC being placed in and is cultivated containing RPMI-1640 In the culture dish of base, in 37 DEG C, contain 5%C0: cell incubator (Themo electro corporation CLASS 100, The U.S.) in be incubated for 2h.It after adherent mononuclear cells, inhales and abandons upper layer suspension cell, PBs buffer gently washs 3 times, is added a small amount of Mono- 1640 culture medium of RPMI, scrapes attached cell with cell scraper.1 000r/min is centrifuged 5min, abandons supernatant.2. fluidic cell Art method: CD14 label: PBMC is taken, with buffer (bovine serum albumin(BSA) 2.5mL and the 2mol/LEDTA 0.5mL containing 10%) Adjusting cell density is 1 × 10⁸CD14+-FITC antibody 100uL is added in/mL in every milliliter of cell suspension, and 4 DEG C are protected from light label 18min, then 1mL streaming buffer is added to terminate dyeing into centrifuge tube, PBs is washed 3 times, with the PBS for containing 2% mycillin Adjustment cell density is 2 × 107/mL.Selected by flow cytometry apoptosis: by the cell of preparation in flow cytometer (BD FAcsAria II, the U.S.) on sort, according to the fluorescence intensity of CD14 antibody, the relative particle of the relative size of cell and cell and interior The complexity of portion's structure collects the cell of CD14+.

5, prepared by CD14 hybridoma cell strain (strain of hybridoma macrophage)

(1) culture medium and main agents: DMEM culture medium, HAT, HT Selective agar medium are purchased from Sigma company, top grade tire ox Serum (FBS) purchases Jinshi City on daytime ocean Hao biological products science and technology responsibility Co., Ltd；DMSO (-- methyl sulfoxide) it is that domestic analysis is pure Reagent.

(2) myeloma cell prepares: fusion the last week takes out the myeloma cell (SP2/ that a pipe freezes out of liquid nitrogen container 0), be immediately placed in hot water thaw (using it is most be Sp2/0 cell strain, the cell strain growth and fusion efficiencies are good, itself is not Any heavy chain immunoglobulin or light chain are secreted, the highest growth scale of cell is 9 × 10⁵/ ml, the doubling time is usually 10~ 15h；Selection homologous cell strain is considered in practical application relevant to human body, if Shanghai Fu Xiang Biotechnology Co., Ltd is to ATCC The NCI-H929 human myeloma cell strain that cell bank is introduced).Appropriate complete culture solution is added after thawing, 1000r/m is centrifuged 3min； It is repeated 1 times.Sediment is moved into Tissue Culture Flask, adds DMEM culture solution, sets CO2 incubator culture, once passed within 3-4 days In generation, expands culture, and fusion adjusts cell state in first 24 hours, guarantees that cellular morphology is good before merging, growth is vigorous.It is added Appropriate basal medium gently beats 1000r/m centrifugation 5-10min after mixing, washes repeatedly cell 2 times into centrifuge tube.

(3) CD14 cell (macrophage) to be hybridized prepares: the mononuclear macrophage that the present invention sorts is with basal medium Total cell number is adjusted to 1 × 10⁸~2 × 10⁸For cell fusion.Blue dyeing phase-contrast microscopy, viable count are expected with platform It should be higher than that 80% is qualified.

(4) cell fusion: CD14 cell (mononuclear macrophage) and myeloma cell are added with 10: 1-5: 1 ratio In centrifuge tube, it is mixed evenly, 1000r/m is centrifuged 5min, discards supernatant, and it gently beats tube bottom to cell grainless and precipitates, weight It is 2 times multiple.Gently rotation preheats centrifuge tube in 37 DEG C of water-baths, by the 50% of 1000 μ L of preheating under aseptic condition after taking-up PEG3000 is added drop-wise in fusion pipe in 60s along tube wall while gently rotating centrifugal pipe, later trains the basis of the 25mL of preheating It supports base to be also added drop-wise in centrifuge tube along tube wall in 3-5min, lightly rotating centrifugal pipe during addition is then allowed to stand In 37 DEG C of water-bath 10min, 1000r/m centrifugation 5min, discards supernatant, 50mL HA T culture medium is added.It is inoculated into after appropriate mixing In 96 well culture plates, 37 DEG C are placed in, is cultivated in 5% CO2 incubator.

(5) the mononuclear macrophage strain with phagocytic function is screened: cell growth status in 96 well culture plates of observation, 7-10 Division can be grown by only having hybridoma after it, discarded HAT culture medium at this time, replaced complete medium.Cell clone growth When area reaches 1/10 cell hole, culture supernatant is gone, selection has the culture hole of the good hybridoma cell strain of growth conditions, shows Position, the size that cell strain growth is marked under micro mirror, draw cell clone in the position of mark using sterile pipette tips has to new In the culture hole of complete medium, then successively doubling dilution to hole is counted below, and 37 DEG C, the interior culture of 5%CO2 incubator one week left The right side, microscopically observation cell growth status take in cell or culture when cell clone is covered with to 1/10 or more hole floor space Clear detection hybridoma macrophage (M φ) strain function.

1. hybridoma macrophage strain swallows bacterium Function detection: macrophage and staphylococcus or Candida albicans are hanged Liquid mixing incubates, and smear is fixed, the dyeing of serge blue liquid, in oily phagocytosis situation under the microscope, counts phagocytosis bacterium and does not gulp down The number of macrophages ratio of bacterium is bitten, to swallow the strong macrophage of bacterium function alternately positive clone strain.

2. hybridoma macrophage strain swallows HIV Function detection: AIDS (AIDS) patient's for taking Disease Control and Prevention Center to save After blood plasma and hybridoma macrophage strain mixed culture, cell strain is separated, PBS is cleaned 3 times, measures the phagocyte strain through cracking The function of swallowing HIV, with specific reference to HIV-1p24 antigen detection kit, (enzyme-linked immunization, Shanghai inspire biotechnology limited Company) operation, with known concentration 0pg/ml, 0.5pg/ml, 1pg/ml, 2.5pg/ml, 5pg/ml, 20pg/ml, 40pg/ml, The p24 antigen of 80pg/ml is lower than 5pg/ml, 0~400pg/ml of measurement range, the range of linearity as control, minimum detection limit 450nm measures absorbance (OD) in 0.5pg/ml~80pg/ml, 15min, and blank control calibration object absorbance value is not higher than 0.050,0pg absorbance value is not less than 1.000 not higher than 0.100,1000pg/ml absorbance, is recognized as absorbance > 0.12 To be positive.Specifically operated by kit specification.

3. hybridoma macrophage strain generates macrophage cytokines detection: with human macrophage migration inhibitory factor (MIF) The operation of ELISA detection kit (hundred stamen Biotechnology Co., Ltd of Shanghai) by specification, detection range are 0~800pg/ml, Susceptibility is 1.0pg/ml, can be under white background, and directly detect by an unaided eye: color is deeper in reacting hole, positive stronger, negative To be colourless or extremely shallow, the depth of the be in color of foundation is indicated with "+", "-" number for reaction.OD value can also be surveyed: in ELISA detector On, at 450nm (if developing the color with ABTS, 410nm), each hole OD value is surveyed after returning to zero with blank control wells, if more than defined 2.1 times of negative control OD value, it is as positive, specifically operated by kit specification.MIF be collection cell factor, growth factor, The multi-effect protein molecular of hormone and enzyme characteristic plays central as inherent immunity and the regulatory factor of inflammatory reaction Effect, it is various infection and active chronic inflammation disease in play panimmunity function.

According to testing result, select the cell clone in the culture hole with stronger macrophage function repeat it is next Wheel dilution culture, repeats 2-3 wheel, and detection function is taken out after stablizing, and is transferred to culture bottle mass propgation.

(6) preservation and recovery of hybridoma macrophage strain: preceding 12 hour adjustment cell growth state is saved, one bottle of life is taken Long vigorous, the good cell of form, is made cell suspension after appropriate digestion, 1000r/min is centrifuged 5min, removes supernatant, flick Tube bottom keeps cell loose, and the 9 parts of complete culture solutions and 1 part of DMSO of 4 DEG C of preservations are added, and dispenses cell cryopreservation tube, and 1mL/ is managed, and -70 Cryopreservation tube, is put into liquid nitrogen container after taking-up and saves backup by DEG C refrigerator overnight.40 DEG C or so of hot water is got out before recovery, it will Cryopreservation tube carefully takes out from liquid nitrogen, and being immediately placed in hot water uniformly to shake makes cell thaw, and is centrifuged after defrosting in 1000r/min 5min opens cryopreservation tube under aseptic condition in superclean bench, the cell after defrosting washed once with complete culture solution, then It is centrifuged 5min in 1000r/min, is discarded supernatant, in case making to expand culture.

6, hybridoma macrophage strain treatment cell preparation

That is the amplification cultivation of hybridoma macrophage strain.Above-mentioned cell precipitation is gently resuspended using complete culture solution and is moved back Enter in culture bottle, sets 37 DEG C, cultivated in 5%CO2 incubator.Amplification cultivation is passed on repeatedly, until required hybridoma cell strain In quantity, every 10 generation of secondary culture positive hybridoma cell strain, detect the function of macrophage hybridoma cell strain, see whether steady It is fixed.Continuation carries out extensive industrialization preparation in several bottles, saves backup.

(2) preparation of HIV-1gp120 antibody

Antibody according to the present invention can entrust professional businessman to prepare, or directly buy from professional businessman, as Shanghai is auspicious The neat units such as Biotechnology Co., Ltd and Shanghai Linc-Bio Science Co., Ltd. all specialize in HIV-1gp120 antibody, The preparation and sale of the various antibody such as gp41 antibody and goat anti-human igg.Method includes hybridoma technology preparation monoclonal antibody, EB Virus Transformation technology preparation monoclonal antibody, hybridoma technology combine preparation monoclonal antibody and base with Epstein-Barr virus transformation technology Because of engineered antibody, specifically it is listed below.

1, it is converted using lymphocyte Epstein-Barr virus and combines preparation HIV-1gp120 monoclonal antibody with hybridoma technology

Specimen origin have it is following it is several by way of: take the lymphocyte strain frozen in Infectious Diseases Lab sample database (through inactivating The immune lymphocyte with EBV transfection of HIV)；The fresh White Blood Cells Concentrate in blood station is bought, inactivation HIV infection strain was then carried out and exempts from The lymphocyte of epidemic disease；It is derived from the cord blood lymphocytes cell (immune through inactivation HIV) saved as scientific research；Directly it is derived from HIV-1 The peripheral blood lymphocytes (being used for itself) of the infected itself, it is thin to separate single core using Histopaque lymphocyte separation medium Born of the same parents (PBMC), adjusting concentration are 2x 10⁶After suitable Epstein-Barr virus (EBV) stoste is added, be placed in 370C, 5%CO2 overnight incubation, B cell to be hybridized is prepared, with the positive hole of ELISA method screening anti-HIV-1 outer membrane protein (gp120), metastatic cells to 24 orifice plates Continue culture 2 weeks, repeats to measure anti-gp120 confirmation positive with ELISA method, continuously clone secondary and massive amplification culture.It will After positive cell strain mixes (3: 1) with the heterogeneous myeloma cell of people mouse (being bought by Zhejiang University's siberian crabapple), 1ml50% is added PEG merges the two, and cell is then resuspended and cultivated liquid in IMDM culture solution, addition Peritoneal Cells of Mice is (by Zhejiang within second day Jiang great Xue siberian crabapple is bought) it is used as trophocyte, anti-gp120 antibody is screened with ELISA after continuing culture 3 weeks, selects strong positive Hole hybrid tumor cell amplification culture, and repeatedly clone is carried out until obtaining stable cell line, with this cell line culture, preparation HIV-1 antibody, using ELISA detection kit, by specification operation measures the Ig subclass of antibody, and with the survey of conventional ELISA method The potency and specificity for determining antibody select the high antibody of high specificity, potency.

2, antibody is prepared using genetic recombination HIV-1gp120 combination hybridoma technology

(1) reagent and recombinant antigen: it is related to reagent: HIV-1gp120 genetic fragment, HIV-1gp120 antigen, HIV antigen Nitrocellulose membrane item by Beijing Wan Tai Pharma Inc. provide BamH I restriction endonuclease Xho I restriction endonuclease, nucleic acid coprecipitator, T4DNALigase is purchased from precious biological Co., Ltd；Liagen plastic recovery kit is purchased from QIAquick company；RPMI 1640 is dry Powder culture medium is purchased from Gibco company；Top grade newborn bovine serum is purchased from bright marine growth Engineering Co., Ltd；SupersignalWest Pico Trial Kit, TMB Substrate Kit are purchased from Pierce company；Mouse Ig subclass detection kit, freund adjuvant And PEG, Hy-poxanthine, Thymidine and Aminoptem are purchased from Sigma company.HIV- antibody diagnosing reagent kit is purchased from Biological Co., Ltd of China of Shanghai section, the goat anti-mouse IgG antibody of horseradish peroxidase (HRP) label are limited purchased from doctor's moral Company.Construction of recombinant plasmid and identification: vector plasmid PEGX-4T-2 BamH I, Xho I digestion, T4DNA Ligase connection Gp120 genetic fragment, recombinant plasmid transformed enter E.colistrain XL1 blue, are sequenced.The inducing expression and mirror of recombinant protein Fixed: recombinant plasmid transformed enters in XL1-Blue Escherichia coli, 25 DEG C under the effect of IPTG inducer, 190r/min concussion, overnight, 4 000r/min are centrifuged 10min, collect bacterium, SDS-PAGE testing goal protein expression situation.Fusion protein purification and identification: Expression product is collected by centrifugation precipitating and hangs through PBS, and after cracking bacterium with 30W Ultrasonic Pulverization instrument, supernatant filtering is collected by centrifugation, Filtrate obtains fusion protein GST-HIV with AKTA PURIFYER100 protein purification instrument, GST column purification, and concentration centrifuge tube carries out Concentration, S21 type biology spectrophotometer measurement concentration, SDS-PAGE identify purifying protein.

(2) animal immune: 6 week old of BALB/c mouse, female, take mouse vein blood by 4 before immune, separation serum, which stays, to be done Negative serum.It is injected intraperitoneally and exempts from after the GST-HIV fusion protein of 50-100 μ g is mixed, emulsified with isometric Freund's complete adjuvant Epidemic disease mouse.After initial immunity, booster immunization mouse after being emulsified every 2 weeks using incomplete Freund's adjuvant and fusion protein is immunized Dosage and approach are the same, repeat to be immunized 2-3 times, the GST-HIV that 50-100 μ g is directly injected intraperitoneally in last time booster immunization melts Hop protein.

(3) foundation of Detection of Monoclonal Antibody: latter all tail vein bloods are immunized for the third time, determine positive serum with square matrix method Best effort concentration and GST-HIV fusion protein best peridium concentration.It operates as follows: according to 1: 1000,1: 500,1: 200,1: 100 four dilution dilutes antigen using coating buffer, longitudinal to be coated with 96 hole elisa Plates, every 100 μ L of hole, 4 DEG C of packets It is stayed overnight, is washed 3 times, every minor tick 3 minutes.Positive serum and negative serum are made into doubling dilution by 1: 1000 respectively, It is laterally loaded onto the 10th hole, every 100 μ L of hole is placed in 37 DEG C of incubation 1h in wet box, washs 3 times, every minor tick 3 minutes.Enzyme mark is anti- Body HRP- sheep anti-mouse igg makees 1: 10000 dilution to specifications, and every hole 100 μ L, 37 DEG C of incubation 1h are washed 3 times.People is added now to match OPD substrate solution, 100 μ L of every hole, 37 DEG C are protected from light appropriate time, and every hole adds 100 μ L terminate liquids to terminate reaction, detects it OD492 value.Positive hybridoma cell screening technique is established.According to experiment condition and method in square matrix method, melted respectively with GST-HIV Hop protein is experimental group, and recombinant bacterium (containing plasmid pET-32a) albumen is control group, screening positive clone after inducing expression.Operation Steps are as follows: with most suitable peridium concentration envelope antigen in 96 hole elisa Plates, 100 holes μ l/, 4 DEG C of refrigerator coatings are overnight.Take out packet It is washed 3 times by cleaning solution is added after plate, washs 3min every time；Every hole adds cell conditioned medium to be measured (1: 5 dilution) 100 μ L, 37 DEG C of temperature Case is incubated for 50min, washs 3 times later, washs 3min every time；Every hole adds-resists 100 μ l, and 37 DEG C of incubation 30min are washed It washs 3 times, washs 3min every time；The 100 μ L of OPD substrate solution now matched is added in every hole, and room temperature is protected from light 10-15min；In every hole 100 μ l of 2mol/L H2SO4 terminate liquid is added for terminating reaction；It will test plate and be placed on survey OD492 value in microplate reader.Control group Set up: positive controls are appropriate diluted positive serum, and negative control group is the unrelated list for having identical dilution with primary antibody Anti- cell conditioned medium.Indirect ELISA the selection result determines.Every group of detection OD492 value, with P (sample value)/N (feminine gender value) >=2.0 Person is judged to positive value.Screening positive clone standard: cell conditioned medium is reacted with positive screening group (purifying rear fusion protein coating) in sun Property, while it is positive sample that the detection hole being negative is reacted with negative selection group (mycoprotein of the plasmid containing pET-32a after induction) Product.

(4) cell fusion: myeloma cell prepares: it is thin that fusion the last week takes out the myeloma that a pipe freezes out of liquid nitrogen container Born of the same parents are immediately placed in hot water defrosting.Appropriate complete culture solution is added after thawing, 1000r/min is centrifuged 3min；It is repeated 1 times.It will precipitating Object moves into Tissue Culture Flask, adds DMEM culture solution, sets CO2 incubator culture, is once passed on or expanded culture within 3-4 days, Fusion adjusts cell state in first 24 hours, guarantee that cellular morphology is good before merging, growth is vigorous.Appropriate pancreatin is used before fusion It is collected after digestion using centrifuge tube, appropriate basal medium is added into centrifuge tube, 1000r/min is centrifuged after gently beaing mixing 5-10min is washed repeatedly cell 2 times.Splenocyte prepares: before fusion, taking a Balb/c mouse, wins eyeball and take blood, bloodletting Complete post-tensioning neck is put to death, and is soaked in 75% ethyl alcohol.It takes out layback and puts and be fixed in dissection plate, spleen is taken under gnotobasis, it will Spleen moves into plate.Then 1640 basal medium of 10mL RPMI is added in plate, is repeatedly extruded with flat mouth tweezers broken Afterwards, it aspirates piping and druming repeatedly using asepsis injector, single cell suspension is made.1000r/min is centrifuged 10min after counting viable count, Basal medium is added and adjusts total cell number to 1 × 10⁸~2 × 10⁸For cell fusion.Cell fusion: by splenocyte and marrow Oncocyte is added in centrifuge tube with 10: 1-5: 1 ratio, is mixed evenly, and 1000r/min is centrifuged 5min, is discarded supernatant, is gently struck It beats tube bottom to cell grainless to precipitate, be repeated 2 times.Gently rotation preheats centrifuge tube, sterile item after taking-up in 37 DEG C of water-baths The 50%PEG3000 of 1000 μ L of preheating is added drop-wise in fusion pipe in 60s along tube wall under part while gently rotating centrifugal pipe, The basal medium of the 25mL of preheating is also added drop-wise in centrifuge tube along tube wall in 3-5min later, it is light during addition Lightly rotating centrifugal pipe is then allowed to stand in 37 DEG C of water-bath 10min, 1000r/m centrifugation 5min, discards supernatant, 50mL HA T is added Culture medium.It is inoculated into 96 well culture plates after appropriate mixing, is placed in 37 DEG C, is cultivated in 5% CO2 incubator.

(5) screening of positive clone strain: it is thin to only have hybridoma for cell growth status in 96 well culture plates of observation after 7-10 days Born of the same parents can grow division, discard HAT culture medium at this time, replace complete medium.Cell clone growth area reaches 1/10 thin When hilum, culture supernatant is gone, positive hybridoma cell clone is screened by the monoclonal antibody screening technique established before.Using improved Limiting dilution assay --- gradient limiting dilution assay continuously carries out 3-4 wheel to the positive cell hole that indirect ELISA preliminary screening goes out Subclone.Selection has the culture hole of the good positive hybridoma cell strain of growth conditions, marks cell strain growth under microscope Position, size are drawn in cell clone to the new culture hole for having complete medium using sterile pipette tips in the position of mark, so Successively doubling dilution to hole is counted below afterwards, and 37 DEG C, the interior culture one week or so of 5%CO2 incubator, microscopically observation cell is grown Situation takes cells and supernatant to carry out antibody inspection side when cell clone is covered with to 1/10 or more hole floor space.To testing result Repeat next round dilution culture for the cell clone in positive culture hole, 2-3 wheel is repeated, after detecting supernatant titer plateaus It takes out, is transferred to culture bottle mass propgation.

(6) preservation of hybridoma cell strain and secondary culture: saving and recovery: saving preceding 12 hour adjustment cell and grows shape State.Take one bottle of growth vigorous, cell suspension is made in the good cell of form after appropriate digestion, 1000r/m is centrifuged 5min, goes Clear liquid, flicking tube bottom keeps cell loose, and the 9 parts of complete culture solutions and 1 part of DMSO of 4 DEG C of preservations are added, and dispenses cell cryopreservation tube, Cryopreservation tube is put into liquid nitrogen container after taking-up and saves backup by 1mL/ pipe, -70 DEG C of refrigerator overnights.40 DEG C of left sides are got out before recovery Right hot water, cryopreservation tube is carefully taken out from liquid nitrogen, and being immediately placed in hot water uniformly to shake makes cell thaw, after defrosting 1000r/m is centrifuged 5min, opens cryopreservation tube under aseptic condition in superclean bench, the cell after defrosting is washed with complete culture solution It washs once, is then centrifuged 5min in 1000r/m, discards supernatant, cell precipitation moves into training after being gently resuspended using complete culture solution It supports in bottle, sets 37 DEG C, cultivated in 5%CO2 incubator.Secondary culture positive hybridoma cell continuously passed for 10 generations, using indirect The method of ELISA measures culture supernatant antibody titer, observes the variation of potency, whether observe this positive hybridoma cell strain can be steady Determine secretory antibody.

(7) preparation of monoclonal antibody mouse ascites: low-speed centrifugal collects the hybridoma after culture, dilute by basal medium Releasing cell number is 1 × 10⁷/mL.Mouse peritoneal injects 0.2mL/ only, and mouse ascites production is observed after injection, bright to abdomen Aobvious distension rises, and punctures abdominal cavity with syringe needle, centrifuge tube collects ascites.Acquisition finishes, and injects appropriate basal medium to mouse peritoneal, Every 2-3 days, same method took ascites again.The ascites being collected into, 10000r/m are centrifuged 5min, and Aspirate supernatant dispenses, -20 DEG C It saves.

(8) CHARACTERISTICS IDENTIFICATION of monoclonal antibody: specificity: Weston-Blotting experiment is carried out referring to " molecular cloning " method, and half Dry method transfer, program is as follows: first with recombination mycoprotein after the CD4 fusion protein of purifying and induction through 12%SDS-PAGE, one Group is used as control, and one group is used as transfer.Electrophoresis finishes, and after glue is cut and an equal amount of 6 filter paper is put into Cathode buffer In；By NC film first with ethyl alcohol impregnate 3-5min, be then placed in deionized water, after 1-3min again with 6 onesize big filters Paper is put into togerther in anolyte；The cathode plate of electrophoresis tank is smeared to wet filter in taking-up Cathode buffer with Cathode buffer Paper and gel are successively placed on cathode plate, gently extrude bubble.NC film in anode buffer liquid and 6 filter paper are taken from anolyte again It is successively layered on gel out, gently extrudes bubble.Finally gently cover electrophoresis tank anode plate.After powering on, according to NC film Area, 2mA/cm2 size of current transfer 2h；After transfer, glue is dyed after taking out, dilute with PBS after transfer membrane takes out The 5% skimmed milk power closing released, 4 DEG C of refrigerators are stood overnight.After closing overnight, confining liquid is discarded, washs 3 with washing buffer It is secondary, each 5min.After washed, 1: 10 diluted monoclonal antibody cell conditioned medium is added, the jog on shaking table reacts at room temperature 60min.It abandons Primary antibody is removed, then is washed 3 times with washing buffer, each 5min.1: 5000 dilution is added in the condition groped according to Dot-ELISA The secondary antibody of degree, the jog on shaking table react at room temperature 50min.Secondary antibody is discarded, then is washed 3 times with washing buffer, each 5min. The NC film for having protein band is marked, DAB color developing agent is added, is terminated instead after reacting appropriate time with deionized water flushing It answers.CD4 fusion protein of the potency to purify measures hybridoma supernatant and list using indirect ELISA method for detection antigen The potency of anti-ascites.Monoclonal antibody subgroup identification selects mouse monoclonal Ig class/subgroup identification ELIAS secondary antibody to use kit measurement, presses It is operated according to kit operational manual, steps are as follows: the appropriate diluted fusion protein of CD4 after purification is coated in ELISA Plate, Every hole 100uL sets 4 DEG C of refrigerator overnights.Coating buffer in ELISA Plate is patted dry, is then washed once with washing buffer, 3min.So Afterwards by Hybridoma Cell Culture supernatant adding hole to be measured, every 100 μ L of hole sets 37 DEG C of incubators and incubates 30min.With washing after patting dry Buffer is washed to wash five times, 3min/ times.6 kinds of enzyme markers in this kit are separately added into hole, every 100 μ L of hole is placed in 37 DEG C incubate 30min.Continue to be washed five times, 3min/ times with washing buffer.It is added eventually after adding OPD substrate solution to be protected from light colour developing 15 minutes Only liquid detects OD492 value with microplate reader, and the type that OD492 value is obviously higher by other holes is HIV-1gp120 monoclonal antibody Ig classification.

(9) HIV-1gp120 monoclonal antibody is after further refining using (professional businessman can be entrusted to complete).

(3) preparation of HIV-1gp41 antibody

With the preparation of HIV-1gp120 antibody

(4) preparation of goat-anti people-Ig

It is antigen by made HIV-1gp120 antibody and gp41 antibody, immune sheep prepares antibody.

1, experimental animal: immune is selected with experimental animal is the white goat of animal institute of Zhejiang University hybridization, 30 jin of weight The between twenty and fifty ewe two of the health of left and right, is smeared at the back of animal with dyestuff before immune, makes specific label.Using doing as everybody else does The feeding manner of stable breeding guarantees that sheep has to suitable movement daily, and it is left to move general half an hour at dusk every time for run duration The right side, is conducive to healthy and strong in this way, avoids sheep overfertilization, increases the immunity of body, and drinking-water is sufficient, and give suitable fine fodder, Green grass, corn, wheat bran, wheat, vitamin etc. keep its nutrition balanced.Often check experiment sheep health status.

2, HIV-1gp120 antibody and gp41 antibody are antigen: HIV-1gp120 antibody and gp41 antibody prepared by the present invention (IgG) concentration is respectively 2.5mg/mL (can be provided by businessman), mixes 0.1mL antigen, 1.9mL sterile PBS, 2mL not before being inoculated with It is spare that immunogen emulsion is made in family name's adjuvant completely (or not exclusively).

3, goat is immune: choose two goats, be labeled as goat A, goat B, antigen inoculation (HIV-1gp120 antibody and HIV-1gp41 antibody).Immune position is front and back groin, 2 point injections of every place's groin point, a total of 8 injection points.Note Mode is penetrated as subcutaneous injection, every goat per injection 4mL immunogene contains 250 μ g antigens；Each injection point injecting immune is former 0.5mL, containing about 32 μ g antigens.Preceding two goats are immunized to draw blood respectively 10mL, are labeled as 0dP1, -20 DEG C of preservations；It is immune for the first time I.e. 1, exempt from immunogene: the sterile PBS+ Freund's complete adjuvant 2mL (CFA) of 0.1mL antigen+1.9mL；Draw blood 10mL after 7 days immune, Serum is separated, 7dP1 is labeled as, ELISA detects serum titer, -20 DEG C of remaining serum preservations.Start within 3~4 weeks to be immunized for second, Two immunogenes: the sterile PBS+2mL incomplete Freund's adjuvant (IFA) of 0.1mL antigen+1.9mL, draw blood 10mL after 7 days immune, separation Serum, is labeled as 7dP2, and ELISA detects serum titer, -20 DEG C of remaining serum preservations.Third time immunization time be 6-8 weeks after, Three exempt from immunogene: the sterile PBS+2mL incomplete Freund's adjuvant (IFA) of 0.1mL antigen+1.9mL, and draw blood 10mL after 7 days immune, point From serum, it is labeled as 7dP3, ELISA detects serum titer, -20 DEG C of remaining serum preservations.If serum titer does not reach 1 at this time ∶10⁶More than, then it needs to exempt from again primary；If serum titer is up to 10⁶Do not have to then be immunized again above, draw blood every other week later 50mL separates serum, -20 DEG C of preservations.

4, prepared by serum: general that rear can detect in sheep jugular vein blood collection for 7~10 days is immunized every time.By assistant Baoding Animal keeps standing position it, after neck cropping, sterile cotton balls cleaning disinfection, searches out the blood sampling of jugular vein hand syringes, The fixation of syringe position is taken into blood 5-10mL.Bioactivity is carried out after isolating serum.7~10 days after the third immunization, warp It once can use blood 30-50mL after bioactivity is qualified.Serum is aseptically separated, in plate or triangular flask to be collected in After blood clotting, 37 are put into after clot and bottle wall removing in gnotobasis (such as superclean bench) with sterile dropper DEG C, 1~2h is put into 4 DEG C overnight after taking out, so that serum is sufficiently precipitated and (cannot be freezed, otherwise generate haemolysis), through centrifugation point Serum out puts low temperature refrigerator preservation into.It must dispense again and save backup after signing is qualified before.

5, the measurement of antiserum titre: antiserum titre is using ELISA measuring method: being to be coated with sample when coating After liquid is diluted to 1: 1000 concentration, every hole adds 100 μ L on ELISA Plate, is then placed in aluminium box and is put into 4 DEG C of refrigerators overnight. The next morning, which takes out, pats dry coating buffer, is washed three times with PBST, and every minor tick five minutes pats dry enzyme with gauze for the last time The confining liquid of 10% serum is added in target, and every hole adds 100UL, is put into 37 DEG C, 1~2h of water-bath.Then it takes out again and pats dry closing Liquid washs 3 every minor tick of ELISA Plate five minutes with PBST, is patted dry for the last time with gauze, and 100 hole μ L/ primary antibodies (1: 2000 are added Dilution is diluted with the PBST of 4% cow's serum, is put into 37 DEG C, 1~2h of water-bath).Then it takes out again and pats dry primary antibody liquid, PBST is washed Wash ELISA Plate three times, every minor tick 5 minutes pats dry ELISA Plate with gauze for the last time, it is added secondary antibody liquid (rabbit-anti sheep 1: 1000), Every hole adds 100 μ L, is put into 37 DEG C, 1~2h of water-bath.It takes out again and pats dry secondary antibody liquid and wash the every minor tick five of ELISA Plate 3 with PBST Minute, it is patted dry for the last time with gauze, the H SO of 2M is added in every hole after adding 50 μ L substrate developing solutions, black out to develop the color 10-20 minutes 50 μ L of solution terminate reaction, after with microplate reader survey OD value (in half an hour).

Two, the preparation of AIDS blood purification

1, the preparation of blood purification cell column

Clean hybridoma macrophage prepared by the present invention with sterile saline, 1000r/min centrifugation, after cleaning again with 1000r/min is centrifuged 5min, and cell precipitation is taken to be packed into 200ml hydrostatic column made of acrylate etc high molecular material It is interior, it is fills up to 4/5, cell column is sealed spare.

2, the outfit of blood purification gel antibody

Gp120 and gp41 antibody is mixed with goat-anti gp120 and gp41 antibody respectively, makes the goat-anti gp120 in mixed liquor It is fully tied with gp41 antibody, but the surplus free gp120 and gp41 antibody for having enough high titres, then will contain combination The mixed antibody of type and sequestered gp120 antibody and mixed antibody containing mating type and sequestered gp41 antibody again with etc. Potency mixing, is made into final mixed antibody, prepares 5 kinds of gel mixed antibodies respectively with the agarose C1-4B solution of gradient concentration, The potency for making HIVgp120 and gp41 antibody is 1: 700,1: 500,1: 300,1: 200,1: 100 and corresponding agarose concentration It is followed successively by 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, by antibody titer from low to high spare blood to be perfused in equal volume Liquid daf molecule column makes gel form antibody titer from high to low from top to bottom and the layering of agarose concentration from low to high Distribution.Agar gel is formed by filter opening and reduces with increasing for agarose concentration, and clarifier entrance agarose concentration is low, Filter opening is just big, is conducive to the association reaction of plasma perfusion and high titre antibody or cell and HIV；And exit concentration is high, filter opening It is just small, it is easy to detention HIV or Large molecular conjugates.Clarifier is combined with a variety of special and non-specific HIV and removes composition, in order to avoid Extraordinary strain is because of the futile treatment caused by immunity difference.

Formula one: gp120 and gp41 antibody is kept the temperature after 100 DEG C dissolve at 39~42 DEG C with carrier function 0.7%, 0.8%, 0.9%, 1.0%, 1.1% agarose C1-4B physiological saline is made into reversed corresponding 1: 700,1: 500,1 : 300, the gel antibody of 1: 200,1: 100 gradient titre successively takes 40ml gel antibody that blood is added by low titre to high titre In liquid daf molecule column, it is desirable that under the gel antibody being formerly added is cooled to 37 DEG C as just then adding after semi-solid agar gel Once, so that the blood purification cell column is formed antibody titer from high to low (from import to outlet) from top to bottom and from low to high The layer distributed of agarose concentration, wherein in conjunction with goat-anti HIV (gp120 and gp41) antibody and unbonded gp120 antibody, Gp41 antibody and hybridoma macrophage, which are fixed in gel, to be play a part of to adsorb HIV.

Formula two: gp120 and gp41 antibody is cooled to 39~42 DEG C after 100 DEG C dissolve with carrier function After the agarose C1-4B of 1% content is made into 1: 50~1000 titre gradient by multiple proportions, Reperfu- sion blood purification cell column makes Blood purification cell column forms antibody titer from high to low and agarose from low to high (from import to outlet) from top to bottom The layer distributed of concentration, wherein in conjunction with goat-anti HIV (gp120 and gp41) antibody and unbonded gp120 antibody, gp41 are anti- Body and hybridoma macrophage, which are fixed in gel, to be play a part of to adsorb HIV.

Formula three: antibody prepared by different HIV infection strains and its corresponding titre being made by multiple proportions are indicated respectively The valence value of gradient, such as 1 titre 1: 100 of HIV infection strain pipe, 1: 200 pipe ...；2 titre 1: 100 of HIV infection strain pipe, 1: 200 pipes ..., and so on, it then will infect the liquid agarose C1- of 1 titre 1: 100 of strain pipe with 10ml39~42 DEG C heat preservation 4B is put into blood purification cell column after mixing, it is to be placed be cooled to semi-solid gel after, then will infection 1 titre 1: 200 of strain pipe with The liquid agarose C1-4B of 10ml39~42 DEG C heat preservation is mixed, and mixing liquid is put into the gel upper layer of blood purification cell column, according to this Analogize.It to be prepared in practical applications by combination, for example the HIV infection strain in somewhere period has totally 3 plants of A, B, C, preparation Antibody has the pipe of A strain 1: 100,1: 200 pipe ...；The pipe of B strain 1: 100,1: 200 pipe ...；The pipe of C strain 1: 100,1: 200 pipe ...；Through Be exactly after combination ABC strain 1: 100 pipe, ABC strain 1: 200 manage ... ABC strain 1: 1000 manage, then by ABC strain 1: 1000 manage and The liquid agarose C1-4B of 10ml39~42 DEG C heat preservation is put into blood purification cell column after mixing, to be placed to be cooled to semisolid After gel, then by the liquid agarose C1-4B mixing of the pipe of ABC strain 1: 900 and 10ml39~42 DEG C heat preservation, mixing liquid is put into blood Semisolid gel upper layer has been cooled in daf molecule column, and so on, centre can be spaced selection, for example then select ABC 1: 500 pipe of strain and the liquid agarose C1-4B of 10ml39~42 DEG C heat preservation are mixed, and mixing liquid is put into blood purification cell column It is cooled to semisolid gel upper layer, the dosage of liquid agarose C1-4B can also adjust in 1ml~10ml (to be made by At blood purification cell column, form gradient antibody content from low to high from import to outlet, only have in view of antigen and antibody Immune response can be just played in proper ratio, forms precipitation line and prevents moving ahead for comparator antibody or antigen, so difference HIV contains When the blood plasma of amount passes through cleanser, HIV is just adsorbed detention in different levels, and HIV infection strain of the invention can be with In conjunction with goat-anti Ig and unbonded gp120 and gp41 antibody in the almost all of infection strain at that time of letter lid and adsorbent It is all the HIV aglucon being fixed in Agar Gel in advance, HIV antibody is tied especially in conjunction with the HIV antibody and HIV for having goat-anti HIV It is bigger that conjunction is formed by immune complex molecular weight, is entirely capable of being delayed in Agar Gel and being removed, about along with aperture For 85nm Agar Gel inherently can detention diameter be 100~120nm HIV, hardly had so theoretically inferring Treat invalid AIDS patient).

3, the specification and material of blood purification

Blood purification cell column or blood purification are the cylinder or rectangular, infundibulate that bottom diameter is small, top diameter is big, volume For 200~300ml, inlet and outlet are equipped with cell screen clothes, and entrance top diameter sieve mesh number is 800 mesh；Exit bottom diameter sieve mesh Number be 2.0~5.0 mesh (2.5~5.0 mesh are equivalent to 0.1~0.2 micron or 100~200 nanometers), specifically can be made into 2.0 mesh, 2.5 mesh, 3.0 mesh, 3.5 mesh, 4.0 mesh, 4.5 mesh and 5.0 mesh 7 kinds of different sizes, to stop 120 nanometers inhibition of HIV or Bigger bacterium；The cell strainer that mesh number is 100 mesh (being equivalent to 4 microns) is arranged in liquid outlet, may filter out to stop Cell；It is equipped with buffer area between liquid entrance and mesh screen, is conducive to the stability of system circulation.

Blood purification selects the high molecular material of acrylate etc, it is desirable that good biocompatibility, hardly activation are mended Body, the change for not causing inflammatory reaction, not causing leucocyte, blood platelet, blood oxygen pressure, complement C 3, C5a.Can by covalently, The methods of grafting, polymerization improve the structure of material, the inhomogeneities for adjusting surface, hydrophily, reduction to blood coagulation and oxidative stress Influence, to improve biocompatibility, reduce complication generation.Add hydrophilic gel in clarifier inner surface, by 2 methyl-props Alkene acyloxyethyl phosphocholine-butyl methacrylate is solidificated in cellulose acetate film, by controlling wet-spinning procedure, is produced CA/PMB30, CA/PMB80 and CA/PMB30-80, blood and cell compatibility with higher.There is anticoagulation by certain Substance be solidificated on the material of carrier or clarifier inner surface, can inhibit blood clotting, improve biocompatibility, can also reduce Heparin dosage, and be possible to realize no-rod tractor.Such as heparin is aggregated on polyacrylonitrile-polyethyleneimine film, effect may Can be more preferable, and the allergic reaction during purification can be reduced, the polyacrylonitrile surface for solidifying chitosan and heparin covalent object is also shown Good blood compatibility, and can inhibit the activity of pseudomonas aeruginosa, reduce cell-cytotoxic reaction.Heparin covalent is combined To polyether sulfone surface, the mechanical property of polyether sulfone was not only maintained, but also the anticoagulation function of clarifier inner surface can be improved.In acetic acid Covalent immobilisation linoleic acid film on tunica fibrosa, or the linoleic acid for being covalently bound to polyacrylic acid is grafted onto polysulfones film surface, all may be used To have better histocompatbility and anticoagulant effect.With the continuous development of high molecular material and nanotechnology, with mankind's blood The close material of endothelial tube will will appear in the near future.

Three, the preparation of plasma separator

1, it preparation principle: is prepared according to the molecular size of haemocyte and blood plasma components.Such as visible component (blood in blood of human body Cell) size are as follows: normocyte is about 7 microns (μm), is the discoid cell of concave-concave；Leucocyte is divided into 5 kinds, neutrality About 12 μm of granulocyte, eosinophil is more bigger, and basophilic granulocyte and neutrophil leucocyte are close, and 6-8 μm of small lymphocyte, Approximate with red blood cell, monocyte is maximum, and about 15-20 μm.Blood platelet is disc, 1~4 micron of diameter to 7~8 microns not Platelet mean diameter Deng, people is 2-4 micron, 0.5~1.5 micron of thickness.

2, material: poly-vinegar non-woven fabrics, acetate fiber, absorbent cotton etc. can be selected, it is desirable that good biocompatibility hardly activates Complement, the change for not causing inflammatory reaction, not causing leucocyte, blood platelet, blood oxygen pressure, complement C 3, C5a.It can be by altogether The methods of valence, grafting, polymerization improve the structure of material, the microinhomogeneities for adjusting surface, hydrophily, reduction to blood coagulation and oxygen Change stress influence, the generation to improve sieving adequacy and biocompatibility, reduce complication.

3, type and spec: for the shape of separator, filter core preparation can be made with materials such as acetate fiber or absorbent cotton The shapes such as flat structure are prepared into as filter core at column construction, with materials such as poly-vinegar non-woven fabrics；By haemocyte and blood to be separated The molecular size of slurry composition determines aperture.It is plasma separator according to the present invention property stabilization, good biocompatibility, penetrating Property high high molecular polymer hollow fibre type filter is made, hollow-fiber film diameter is 270~370 μm, and film thickness is 50 μm, Aperture is 0.2~0.6 μm, and fibre length is 13.5~26 μm.The hole only permit blood plasma filtration, but can stop all cells at Point.

Four, the application component and purposes of AIDS blood purification

1, key member: (1) plasma separator: for separating mononuclear blood cell and blood plasma；(2) it blood purification: is used for HIV in adsorbed plasma.

2, additional member: including blood pump, heparin pump, sound pulse pressure and air monitering, temperature control system, off gas system, The parts such as monitored conductivity system, ultrafiltration monitoring and leakage blood monitoring form.

(1) blood pump (Blood Pump): for pushing blood circulation to maintain going on smoothly for blood purification treatment, usually Blood pump part often has rotary test speed function, and to monitor the blood circumstance of patient, therefore blood pump runner and groove spacing are set It is accurate, and need often adjustment, the case where according to bloody path pump line, spacing is generally set as 3.2~3.3mm, can not be too loose, Otherwise it is inaccurate to will cause blood flow detection；Also can not be too tight, it otherwise will cause pipe breakage.

(2) heparin pump (Heparin Pump): heparin pump is equivalent to the micro-injection pump clinically applied, to continue to Injecting heparin in sieving pipeline (patient blood) contacts with air since the blood of patient recycles in vitro, is easy to happen blood coagulation Phenomenon anticoagulative can be occurred using heparin pump.

(3) sound pulse pressure monitors: the main stopping state to dynamic monitoring blood separator micropore of arterial blood pressure monitoring, separately Outside to monitor extracorporal circulatory system thrombus, solidification and the variation of pressure.When blood flow deficiency, angiosthenia will be reduced；When have blood coagulation, When thrombosis, especially separator blockage of the micro orifice, angiosthenia will be increased；Vein pressure monitoring is used to monitor pipeline blood reflux Pressure, when separator blockage of the micro orifice, blood coagulation, thrombosis, blood flow is insufficient and venous return syringe needle falls off when, vein pressure It will decline, if bloody path return pipe distortion blocking or reflux syringe needle block, vein pressure will be increased.

(4) air monitering (Air Detector): the air bubble for monitoring blood pathway generally uses ultrasonic listening Principle, in order to avoid patient occur air embolism and be arranged.When having monitored air bubble, detection system can drive it is dynamic, Vein bloody path folder carrys out blocking blood flow, prevents dangerous generation.

In short, Import computer regulates and controls and is made the people of operation on the basis of key member of the present invention and additional member Property, the personalization for the treatment of, the safety of design and modularization, automatic monitoring and regulation, liquid crystal display, voluntarily judgement is warned Report the blood purifying therapeutical instrument of the micro computers such as reason and ring off signal processing.

Five, the connecting path and application method of AIDS blood purification

1, it installs: such as Fig. 1, with sterile working connecting components, including separator, clarifier and each circulation line.

2, it is vented: with sterile saline filling liquid separator, clarifier and each circulation line, excluding separator, clarifier And its gas, bubble in circulation line, it goes through, confirmation after gas, bubble without using.

3, lead to liquid: arterial blood line pipe (1) being connected to the arteries of AIDS patient, in operation the row of going through again Whether gas is complete, and whether liquid stream is unobstructed, and flow liquid in pipe is avoided to pollute.

4, anticoagulant: to inject anti-coagulants (heparin) into liquid stream from heparin pump (2), be for the first time 2500U or 20~30U/kg.

5, start: one end of arterial blood line pipe (1) is connected with arteries, venous line (5) are connected into venous blood Pipe, then open blood pump (2), blood flow be 100~150ml/min, such as Fig. 1, when arterial blood through arterial blood line pipe (1) into Entering plasma separator (4), the blood plasma separated reaches clarifier (8) through circulation line (7) under the action of blood plasma pump (6), Wait be full of blood plasma, about 10 minutes, blood plasma is begun releasing, is flowed out through circulation line (10), it is synchronous that blood plasma is perfused to clarifier (9), When blood plasma in clarifier (8) has nearly flowed, perfusion blood plasma is started again at, clarifier (9) begins releasing blood plasma at this time, and two Clarifier (8), the clarifier 9 of parallel connection) alternately, purified blood plasma is through circulation line (10) and plasma separator (4) institute Isolated haemocyte is fed back after converging through venous line (5).Such as Fig. 2, when blood to be separated enters the interior of plasma separator (1) When chamber (2), the effect through valve (8) can enter the exocoel (6) of separator by the small molecule blood plasma components (5) of micropore (3), Then it flows out, and cannot be flowed out through valve (8) by the haemocyte (4) of micropore (3) through plasma outlet port (7).Such as Fig. 3, when containing When the blood plasma of HIV (1) enters clarifier, HIV (1) therein is fixed on HIV antibody (2) in agar gel (6), huge respectively Phagocyte (4) is combined into antigen antibody complex (3), macrophage phagosome (5), and the HIV after being combined no longer moves down, In addition the HIV for 100~120nm not being combined is again by under smaller about 1% concentration of 85nm in aperture due to concentration is higher Layer agar gel molecular sieve detention is at (7).HIV is so removed, until the plasma circulation amount (usually 9L) being previously set, is controlled Treatment just end, entire therapeutic process is controlled by computer, and can detect working condition at any time, it is easy to use, automate and Safety.

Six, the verifying of AIDS blood purification effect

1, the verifying of HIV-1gp120 antibody, gp41 cleaning antibody HIV effect

The present inventor's basic skills according to the invention has done following simple confirmatory experiment: take HIV-1gp120 antibody, Gp41 antibody (Shanghai Rui Qi Biotechnology Co., Ltd) is added to and keeps the temperature after 100 DEG C dissolve in 50 DEG C of 1.0% spare fine jades In lipolysaccharide C1-4B, titre is 1: 300~500 after mixing, takes 2.5 × 300mm Westergren's blood sedimentation tube 5 of sterilizing, draws respectively 1.0% agarose C1-4B solution is to 200mm scale, and agarose becomes semisolid after cooling.Ling Qu Disease Control and Prevention Center and infectious disease are real Test the sample of 5 AIDS patients of room sample database preservation, each about 10mL of blood plasma after removing cell, blood before respectively taking 9mLAIDS to filter Slurry injects blood sedimentation tube upper end blank pipe in batches, the 1.0% agarose C1-4B antibody-containing of blood sedimentation tube lower layer to be flowed through and from blood In immersed tube after outflow, efflux, blood plasma after referred to as AIDS filter are collected.Blood plasma and blood plasma after filter before taking AIDS to filter, according to HIV- 1p24 antigen detection kit (enzyme-linked immunization, Shanghai inspire Biotechnology Co., Ltd) operation, with known concentration 0pg/ml, The p24 antigen of 0.5pg/ml, 1pg/ml, 2.5pg/ml, 5pg/ml, 20pg/ml, 40pg/ml, 80pg/ml are minimum as control Detection limit is lower than 5pg/ml, 0~400pg/ml of measurement range, and 450nm is surveyed in the range of linearity 0.5pg/ml~80pg/ml, 15min Determine absorbance (OD), blank control calibration object absorbance value is not higher than 0.100,1000pg/ not higher than 0.050,0pg absorbance value Ml absorbance is not less than 1.000, is considered as the positive as absorbance > 0.12, and testing result (table 1) illustrates, the filter of AIDS blood plasma After crossing simple purification device, part HIV is adsorbed by corresponding antibodies, and after the 1st filtration, HIV total body clearance is 20.01%, After the 2nd filtration, total body clearance 27.99%, after the 3rd filtration, total body clearance 37.36%.Illustrate with filtration The increase HIV of number can be removed constantly, to reach treatment AIDS purpose.1 AIDS blood plasma of table filters simple purification device Front and back p24 testing result (pg/ml)

2, the verifying of HIV effect is removed in the strain of hybridoma macrophage

The effect of removing HIV for check cross tumor macrophage strain, the present invention devises easy test method: taking and goes out 2.5 × 300mm Westergren's blood sedimentation tube of bacterium 5 draws the macrophage hybridization for being centrifuged (1000r/min, 5min) precipitating respectively Tumor cell strain is then drawn and is kept the temperature after 100 DEG C dissolve in 39~42 DEG C of 0.9% spare agarose C1- to 200mm scale 4B reaches about 10mm long scale, set blood sedimentation tube it is cooling after, agarose becomes semisolid, can prevent blood sedimentation tube inner cell flow out but The substance of the water and chemical analysis etc that will not prevent small molecule passes through.Ling Qu Disease Control and Prevention Center and Infectious Diseases Lab sample database are protected 5 blood plasma of AIDS (AIDS) patient deposited, respectively about 10mL, respectively takes 9mLAIDS to filter preceding blood plasma and injects blood sedimentation tube (letter in batches Easy purification device) upper end blank pipe, wait flow through the hybridoma macrophage strain layer of blood sedimentation tube lower layer and out of blood sedimentation tube after outflow, receipts Collect efflux, blood plasma after referred to as AIDS filter.Blood plasma and blood plasma after filter before taking AIDS to filter, with human macrophage migration inhibitory factor (MIF) ELISA detection kit (hundred stamen Biotechnology Co., Ltd of Shanghai) pairing detection, by specification operation, detection range , can be under white background for 0~800pg/ml, susceptibility 1.0pg/ml, directly detect by an unaided eye: color is got in reacting hole Deep, positive stronger, to be colourless or extremely shallow, the depth of the be in color of foundation is indicated negative reaction with "+", "-" number.OD can also be surveyed Value: on ELISA detector, at 450nm (if developing the color with ABTS, 410nm), each hole OD is surveyed after returning to zero with blank control wells Value, it is as positive if more than 2.1 times of defined negative control OD value.As a result such as table 1, MIF testing result is equal in blood plasma before filtering For negative (or because content causes degradation etc. lower than detection sensitivity, blood plasma long-term preservation), and testing result is in blood plasma after filtering It is positive.Illustrate that macrophage hybridoma cell strain produces MIF cell factor in this process.MIF be collection cell factor, growth because Son, the multi-effect protein molecular of hormone and enzyme characteristic, as in inherent immunity and the performance of the regulatory factor of inflammatory reaction The effect of pivot plays panimmunity function in various infection and active chronic inflammation disease.Making the filtration letter of AIDS blood plasma Before and after easy clarifier while MIF pairing detection, the present invention has also done the pairing detection of HIV-1p24, according to HIV-1p24 antigen Detection kit (Biotechnology Co., Ltd is inspired in enzyme-linked immunization, Shanghai) operation, with known concentration 0pg/ml, 0.5pg/ The p24 antigen of ml, 1pg/ml, 2.5pg/ml, 5pg/ml, 20pg/ml, 40pg/ml, 80pg/ml are as control, minimum detection limit Lower than 5pg/ml, 0~400pg/ml of measurement range, 450nm measures extinction in the range of linearity 0.5pg/ml~80pg/ml, 15min It spends (OD), blank control calibration object absorbance value is not higher than 0.100,1000pg/ml extinction not higher than 0.050,0pg absorbance value Degree is not less than 1.000, is considered as the positive as absorbance > 0.12, and as a result (table 2) illustrates the simple purification of AIDS blood plasma filtration After device, part HIV is swallowed by macrophage hybridoma cell strain to be adsorbed, and the blood plasma HIV after filtration is significantly reduced, through the 1st After secondary filtration, HIV clearance rate is 20.55%, and after the 2nd filtration, HIV clearance rate is 42.83%, p < 0.01, is had obvious Effect, illustrate with filtration number increase HIV can constantly be removed, thus reach treatment AIDS purpose.

1 AIDS blood plasma of table filter MIF testing result before and after the simple purification device of the macrophage containing hybridoma (it is quantitative: pg/ml)

2 AIDS blood plasma of table filters the simple purification device front and back p24 testing result (p24:pg/ of the macrophage containing hybridoma ml)

Above-mentioned simple experiment show the HIV in blood plasma can by blood purification agent (HIVgp120 antibody, HIVgp41 antibody, The strain of hybridoma macrophage, agar gel micropore) absorption removing, it is significant to show that AIDS blood purification of the invention has Remove the therapeutic efficiency of blood plasma inhibition of HIV.

Claims (10)

1. a kind of AIDS blood purification for medical domain, which is characterized in that bottom is arranged in the exit of the clarifier Diameter cell screen clothes, inside perfusion agar gel, the agar gel is interior to be furnished with the strain of hybridoma macrophage, HIV antibody and combination The HIV antibody of goat-anti Ig, make the entrance of the clarifier to exit form antibody titer from high to low and from as low as The layer distributed of high agarose concentration but hybridoma macrophage strain accounts for the 4/5 of overall perfusion object；The clarifier and it can divide Purging in vitro device is constituted from the separator of blood plasma and haemocyte, for removing the HIV in blood plasma；The hybridoma macrophage Strain is prepared with CD14 cell and myeloma cell's fusion；The HIV antibody using HIV as antigen prepare, including gp120 antibody and Gp41 antibody；The HIV antibody of the goat-anti Ig is prepared by antigen of HIV antibody, including goat-anti gp120 antibody and goat-anti gp41 resist Body.

2. AIDS blood purification according to claim 1, which is characterized in that the clarifier by its exit sieve Net plays fixed and molecular sieve agar gel, preparation hybridoma macrophage strain therein, HIV antibody, combines goat-anti The HIV antibody of Ig collectively forms molecular sieve mechanical removal and cell and antibody mediated immunity removes the barrier of blood plasma HIV.

3. according to claim 1,2 any AIDS blood purification, which is characterized in that the volume of the clarifier is 200~300ml, entrance top diameter cell screen clothes mesh number are 800 mesh, and exit bottom diameter cell screen clothes mesh number is 2.0~5.0 mesh.

4. AIDS blood purification according to claim 1, which is characterized in that clarifier exit bottom diameter cell screen clothes 7 kinds of different sizes of 2.0 mesh, 2.5 mesh, 3.0 mesh, 3.5 mesh, 4.0 mesh, 4.5 mesh and 5.0 mesh are made in mesh number.

5. AIDS blood purification according to claim 1, which is characterized in that the antibody titer from high to low Layer distributed refers to the mixed antibody potency from the entrance of clarifier to exit successively with 1: 700,1: 500,1: 300,1: 200,1: 100 point 5 layers.

6. AIDS blood purification according to claim 1, which is characterized in that the agarose concentration from low to high Layer distributed refer to the agarose concentration from the entrance of clarifier to exit successively with 0.7%, 0.8%, 0.9%, 1.0%, 1.1% point 5 layers.

7. according to claim 1,2,5 any AIDS blood purification, which is characterized in that the HIV antibody includes HIV-1gp120 antibody, HIV-1gp41 antibody.

8. AIDS blood purification according to claim 1, which is characterized in that the hollow-fiber film of the separator Diameter is 270~370 μm, and film thickness is 50 μm, and aperture is 0.2~0.6 μm, and fibre length is 13.5~26 μm, can stop institute Some cell filtrations.

9. a kind of preparation of the AIDS blood purification cleanser for medical domain, which is characterized in that with sterile physiological salt The hybridoma macrophage strain prepared is merged with sp2/0 myeloma cell in water cleaning by human monocytemacrophage, 1000r/min from The heart is centrifuged 5min again after cleaning with 1000r/min, and cell precipitation is taken to be packed into 200ml cylinder made of high-biocompatibility material Describe in device, be fills up to 4/5, cell column is made, separately by gp120 antibody and gp41 antibody respectively with goat-anti gp120 antibody and sheep The mixing of anti-gp41 antibody, is fully tied goat-anti gp120 antibody and goat-anti the gp41 antibody in mixed liquor, but it is surplus have it is free Gp120 antibody and gp41 antibody, then by the mixed antibody containing mating type and sequestered gp120 antibody and containing combine The mixed antibody of type and sequestered gp41 antibody again with etc. potency mixing, final mixed antibody is made into, respectively with gradient concentration Agarose C1-4B solution prepare 5 kinds of gel mixed antibodies, make the total titer of the HIVgp120 antibody of mating type and sequestered with And the total titer of the gp41 antibody of mating type and sequestered is 1: 700,1: 500,1: 300,1: 200,1: 100 and corresponding fine jade Lipolysaccharide concentration is followed successively by 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, by antibody titer from low to high with isometric perfusion Spare cell column makes gel therein form antibody titer from high to low from top to bottom and agarose concentration from low to high Layer distributed.

10. any AIDS blood purification of claim 1-8 is preparing the application in purging in vitro device, feature Be, the connection structure of the purging in vitro device are as follows: one end of arterial blood line pipe (1) through heparin pump (2) and blood pump (3) with Plasma separator (4) be connected, plasma separator (4) through blood plasma pump (6) and circulation line (7) clarifier in parallel with two (8, 9) it is connected, then successively converges through circulation line (10), venous line (5).