CN109280644B - Anti-human IgG monoclonal antibody, hybridoma cell strain and application thereof - Google Patents
Anti-human IgG monoclonal antibody, hybridoma cell strain and application thereof Download PDFInfo
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Abstract
The invention relates to a hybridoma cell strain and a monoclonal antibody secreted by the same, wherein the antibody can be specifically combined with human IgG. The invention also relates to a kit comprising the hybridoma cell strain or the monoclonal antibody. The monoclonal antibody of the invention has good performances in the aspects of antibody purity, repeatability, antibody titer and stability.
Description
Technical Field
The invention relates to a monoclonal antibody, in particular to an anti-human IgG monoclonal antibody, a hybridoma cell secreting the monoclonal antibody and application of the monoclonal antibody.
Background
Herpes Simplex Virus (HSV) is a virus with an envelope and a double-stranded DNA genome, widely exists in nature, can infect human and many animals, has strong tropism to human skin tissues particularly, and is a common pathogen of skin diseases and venereal diseases. Herpes simplex viruses have two serotypes including: herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2). Genital herpes caused by HSV-1 and HSV-2 has caused an increasingly serious social and public health problem.
The current common laboratory methods for detecting herpes simplex virus include direct immunofluorescence or histochemical staining, virus isolation and culture, antigen latex agglutination experiments, serum antibody detection and PCR technology. The above methods each have advantages and disadvantages. Immunofluorescence staining or immunohistochemical staining is used for directly detecting HSV specific antigen in cells and can be used for a diagnosis experiment. However, the process of collecting the exfoliated cells of the damaged skin mucosa is difficult, and the sensitivity is reduced. The virus isolation culture is the most sensitive diagnosis method in an HSV infection laboratory, and can be used for typing HSV virus isolates. However, cell culture is time-consuming and laborious, which limits the application in clinical tests. The antigen latex agglutination test is to combine specific monoclonal antibody and latex molecule particle into compound, when HSV antigen exists in serum, antigen-antibody-latex polymer is formed, and the agglutination phenomenon visible to naked eyes appears. The method has the advantages of no need of special instruments, simple and quick operation and suitability for screening large-batch specimens. However, the sensitivity is poor, and the detection rate is only 50%. The PCR technology has high detection sensitivity, can detect the virus amount of as little as 3 PFUs, and has preliminarily shown superiority in clinical virus detection. However, the PCR method has high sensitivity, high requirements for operation technology, easy pollution and false positive, and also needs talent technical training, special instruments and imported reagents, which are still difficult to popularize and popularize at present. The detection of HSV serological antibodies is still the most common method for judging HSV infection clinically, and HSV specific antibodies mainly comprise IgG and IgM at present. The concentration of HSV-IgG in serum is higher, and the detection accuracy is higher. Therefore, the developed anti-human IgG monoclonal antibody with better systematic evaluation result has very wide application in the field of in vitro diagnostic reagents.
However, screening monoclonal antibodies for preparing in vitro diagnostic reagents is a complex process, and first a good antigen is obtained to prepare a sufficient amount of antibodies, and then the antibodies are systematically evaluated to obtain candidate antibodies with clinical relevance, and then developed into detection reagents. The antibody titer, the cross reactivity, the stability, the clinical effect and the like are important evaluation factors, for example, the antibody titer reflects the lowest titer of the antigen reaction under a certain concentration, and the lower the titer is, the higher the titer is; antibody cross-reactivity may affect the specificity of the antibody; the stability of the antibody directly influences the reliability of the final result, and the antibody with poor stability has higher requirements on storage conditions and operating conditions, so that the practicability of the antibody as a diagnostic reagent and the reliability of the result are reduced, and the increase of the cost is inevitably caused; clinical trials are used to illustrate the actual effect of the antibodies in diagnosing the corresponding disease or viral infection.
Disclosure of Invention
The invention aims to provide an anti-human IgG monoclonal antibody, a hybridoma cell strain secreting the anti-human IgG monoclonal antibody, a kit containing the monoclonal antibody or the hybridoma cell strain and application of the monoclonal antibody or the hybridoma cell strain in detection of human infection type 1 herpesvirus IgG. Through systematic evaluation, the antibody has better performance in all aspects, so that the antibody is suitable to be used as an immunodiagnostic reagent for preparing an in vitro diagnostic kit.
Therefore, the inventor conducts a great deal of research, immunizes a mouse by human IgG, clones at least 4 times by a limiting dilution method after cell fusion until the monoclonal antibody is reached, screens 1 novel Hybridoma cell strain (Hybridoma) capable of stably secreting the antibody from the obtained clones, marks the Hybridoma cell strain as Hybridoma cell strain 5A3, and stores the Hybridoma cell strain in the China center for type culture collection (CCTCC NO: C2018172) in 2018, 8.23.8.23.8.8.23.g., wuhan university, china, so as to complete the invention.
In a first aspect, the invention provides a hybridoma cell strain which is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of C2018172.
In a second aspect, the invention provides a monoclonal antibody capable of specifically binding to human IgG.
In one embodiment, the monoclonal antibody does not bind to human IgG, murine IgG, rabbit IgG, or bovine IgG human IgM.
In another embodiment, the monoclonal antibody has a median potency of 5 × 10 4 And has better stability and reliability under the conditions of long-term storage and severe thermal acceleration.
In a preferred embodiment, the monoclonal antibody is an antibody secreted by the hybridoma cell line of the invention.
In a third aspect, the invention provides a kit, which comprises the hybridoma cell strain or the monoclonal antibody of the invention.
In a specific embodiment, the kit is a colloidal gold immunoassay kit, a chemiluminescent kit, a radioimmunoassay kit, an enzyme-linked immunoassay kit or a fluorescent immunoassay kit.
In a preferred embodiment, the kit is a chemiluminescent kit.
In another embodiment, the kit is a microfluidic chip.
In a fourth aspect, the invention provides the use of the hybridoma cell strain or monoclonal antibody of the invention in the preparation of a kit.
In one embodiment, the kit is based on immunoassay, preferably, the kit is a colloidal gold immunoassay kit, a chemiluminescent kit, a radioimmunoassay kit, an enzyme-linked immunoassay kit or a fluorescent immunoassay kit.
In a preferred embodiment, the kit is a chemiluminescent kit.
In another embodiment, the kit is a microfluidic chip, preferably, the microfluidic chip is based on immunoassay.
In one embodiment, the kit is for detecting human IgG.
In a preferred embodiment, the human IgG is a herpes simplex virus type 1 IgG.
In addition, the invention also provides application of the hybridoma cell strain or the monoclonal antibody in preparation of a kit for detecting the herpes simplex virus type 1.
The monoclonal antibody has the beneficial effects that firstly, the antibody potency is higher than that of a commercially available human IgG antibody which is usually used in-vitro diagnosis, and the monoclonal antibody has a better immune effect; secondly, the antibody has no cross reaction with human IgG, mouse IgG, rabbit IgG, bovine IgG and human IgM, and has good specific binding capacity; thirdly, it has superior long-term and thermal stability to commercially available human IgG antibodies, that is, it has an extended lifespan and can accept relatively loose storage and operation conditions, thereby greatly saving costs; finally, clinical experiments show that the monoclonal antibody can be used for preparing a chemiluminescence kit for detecting the IgG antibody of the herpes simplex virus type 1, the detection sensitivity can reach 100 percent, and the detection specificity can reach 100 percent.
That is, the monoclonal antibody of the invention shows good performance in various aspects such as antibody titer, cross reactivity, stability and detection effect, so that the invention provides an anti-human IgG monoclonal antibody which can be used for in vitro diagnosis through systematic evaluation and has outstanding comprehensive capacity.
Drawings
FIG. 1 shows a SDS-PAGE electrophoresis of the purified human IgG-Ab5 antibody of the invention;
FIG. 2 shows a titer detection scheme for the antibody human IgG-Ab5 of the invention;
FIG. 3 shows the clinical relevance of the agents of the invention to commercially available agents.
Detailed Description
The present invention will be described in detail below with reference to specific embodiments and examples, and the advantages and various effects of the present invention will be more clearly apparent therefrom. It will be understood by those skilled in the art that these specific embodiments and examples are illustrative of the invention and are not to be construed as limiting the invention.
Throughout the specification, unless otherwise specifically noted, terms used herein should be understood as having meanings as commonly used in the art. Accordingly, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. If there is a conflict, the present specification will control.
Antibodies
As used herein, the term "antibody" refers to immunoglobulin molecules, including but not limited to chimeric antibodies, humanized antibodies, fully human antibodies, CDR-grafted antibodies, and antibody constructs, such as single chain Fv (scFv) or antibody fusion proteins; furthermore, it relates to antibodies produced/synthesized recombinantly or synthetically.
In a preferred embodiment, the antibody is an antibody generated from a hybridoma cell line with the preservation number of CCTCC NO: C2018172.
An "antibody fragment" typically comprises the antigen binding region, light and/or heavy chain variable regions, at least a portion of one or more (e.g., six) CDRs, of the parent antibody that retains at least some of the binding specificity of the parent antibody. In particular, the parent antibody refers to an antibody generated from a hybridoma cell line with a preservation number of CCTCC NO: C2018172. Examples of antibody fragments include, but are not limited to, fab ', F (ab') 2, and Fv fragments; a dimeric molecule; a linear antibody; but chain antibody molecules, e.g., sc-Fv; and multispecific antibodies formed from antibody fragments. Typically, fragments retain at least 50% of the binding activity to C-reactive protein when the activity is expressed on a molar basis. Preferably, the fragment retains at least 60%, 70%, 80%, 90%, 95% or 100% of the binding activity to herpes simplex virus type 1 IgG as compared to the parent antibody.
Preferably, an antibody fragment refers to the antigen binding region, the light and heavy chain variable regions or the six CDRs of an antibody.
"antibody derivatives" refers to antigens including conservative amino acid substitutions (referred to as "conservative variants") of antibodies, which have substantially no altered biological activity compared to the parent antibody.
The invention provides monoclonal forms of the antibodies.
As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially informative antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific for a single antigenic site. Monoclonal antibodies are advantageous because they can be obtained by hybridoma cell culture and are substantially free of contamination by other immunoglobulins.
Reagent kit
The detection kit of the present invention may take various forms, for example, a strip, a kit containing various reagents required for the test, a microfluidic chip, etc., and the kit may be manufactured according to standard procedures known to those skilled in the art.
Kits of the invention may include containers, chips, instructions for use, buffers, immunological aids, and/or other materials, structures, and/or reagents as desired for performing the diagnosis/assay.
In the examples, the kit of the present invention is described by taking a fluoroimmunoassay as an example, but the kit of the present invention is not to be construed as being limited to a fluoroimmunoassay.
The kit of the present invention includes an antibody produced from a hybridoma cell line having a preservation number of CCTCC No. C2018172, which may be present in a manner conventional in the art, for example, in a dissolved or dried form in a container, coated on a solid phase carrier (e.g., a film, a plate, a bead, a particle (e.g., a magnetic particle), etc.), and present in a dissolved or dried form in a chamber of a chip, but the present invention is not limited thereto.
Due to objective factors such as transportation and use places, the kit is often suitable for field detection in various complex environments, so that the stability of raw materials is one of important factors for restricting the kit result. As shown in example 10 below, the antibody anti-human IgG monoclonal antibody (denoted as human IgG-Ab 5) of the present invention possesses better stability as a raw material of a chemiluminescent kit than conventional anti-human IgG monoclonal antibodies under extreme conditions, thereby enhancing the reliability of the results of the kit and reducing the cost in phase change.
The antibody of the present invention can be used at a concentration of 0.5 to 10. Mu.g/ml, preferably 5 to 10. Mu.g/ml, and more preferably 6. Mu.g/ml.
Other materials required for diagnosis/detection in the kit of the present invention include, but are not limited to, anti-human IgG antibodies other than the antibody of the present invention, antigens bound to human IgG antibodies, and/or human IgG. The other materials mentioned above may be present in a manner conventional in the art, for example, in dissolved or dried form in a container, coated on a solid support (e.g., a membrane, a plate, beads, particles (e.g., magnetic particles), etc.), in dissolved or dried form in a chamber of a chip, but the present invention is not limited thereto.
Other structures required for performing a diagnostic/test in the kit of the invention include, but are not limited to, structures for sampling, structures for performing controls, and/or structures for observing the results of the test procedure or structure.
Other reagents required for performing the diagnosis/detection in the kit of the present invention include, but are not limited to, detergents, visualization reagents and/or terminating reagents.
In one embodiment, the antibody in the kit of the invention is detectably labeled. Any label and labeling method known to those skilled in the art may be used. For example, the labels that may be used in the present invention include enzymes, radioisotopes, colloidal metals, fluorescent compounds, chemiluminescent compounds, and bioluminescent compounds, but the present invention is not limited thereto.
Commonly used labels may include enzymes (e.g., horseradish peroxidase, beta-galactosidase, alkaline phosphatase, etc.), radioisotopes (e.g., horseradish peroxidase, beta-galactosidase, etc.), and the like 32 P or 125 I) Etc., biotin, digoxigenin, colloidal metals (e.g., colloidal gold, etc.), fluorescent dyes (e.g., fluorescein, rhodamine, texas red, etc.), chemiluminescent compounds or bioluminescent compounds (e.g., dioxetane, luminol or acridinium, etc.). Any labeling step well known in the art may be used, such as covalent coupling of an enzyme or biotin group, iodination, phosphorylation, biotinylation, and the like.
In some embodiments, one or more of the other materials required for diagnosis/detection may also be detectably labeled.
In a preferred embodiment, the kit of the present invention is a kit for detecting herpes simplex virus type 1.
Use of
The anti-human IgG monoclonal antibody or the hybridoma cell strain can be used for any purpose related to the specific reaction of human IgG. Preferably, the antibody or hybridoma cell line can be used for detecting the herpes simplex virus type 1.
The antibody or the hybridoma cell strain can be used for detecting biological samples from human beings.
As used herein, "biological sample" refers to semen, lymph, serum, plasma, urine, synovial fluid, or spinal fluid. In a preferred embodiment, the biological sample is serum or plasma.
The presence of human IgG can be detected quantitatively or qualitatively using immunoassay methods which typically involve incubating or sequentially contacting a biological sample with the antibodies of the invention and/or other materials required for detection and detecting the bound antigen by a variety of techniques well known in the art.
Detection methods include, but are not limited to, autoradiography, fluorescence microscopy, direct and indirect enzymatic reactions, radioisotopic or non-radioisotopic methods, and the like. These methods include, inter alia, western blotting, overlay assays, RIA (radioimmunoassay) and IRMA (immunoradioimmunoassay), GIA (colloidal gold immunoassay), EIA (enzyme immunoassay), ELISA (enzyme-linked immunosorbent assay), FIA (fluorescent immunoassay), and CLIA (chemiluminescent immunoassay).
In one embodiment, the antibody or hybridoma cell line of the invention is suitable for detecting human IgG, thereby diagnosing whether an individual is infected with herpes simplex virus type 1.
The embodiments of the present invention will be described in detail with reference to examples, which do not indicate specific conditions, and which are performed according to conventional conditions or conditions suggested by manufacturers. The reagents or apparatus used are not indicated by the manufacturer, and are conventional products commercially available.
EXAMPLE 1 immunization of mice
Human blood source IgG antigen (Sichuan Michael Biotechnology Co., ltd., lot number 031524) was diluted to 2.0mg/ml with physiological saline, mixed with Freund's complete adjuvant (Sigma Co., cat number SLBF-9338V) in equal volume (100. Mu.g/BALB/c mouse), emulsified into oily emulsion with 1ml syringe until the oily emulsion dropped into water did not disperse, emulsified after administering the emulsion subcutaneously to BALB/c mouse (having reached the center of Master animal, 4 weeks old female, 3) in the first immunization 14 days at a dose of 100. Mu.l/limb axilla, emulsified after taking human IgG and Freund's incomplete adjuvant (Sigma Co., cat number SLBM 9367V) in equal volume (50. Mu.g/BALB/c mouse), in an immunization dose of 50. Mu.l/mouse, after enhancing immunization once every week, tail blood was taken before each immunization, serum was separated, and titer was measured by indirect ELISA. After 3 immunizations, all mice had serum titers greater than 1:10 6 I.e. can be used for fusion. 3 days before the fusion, human IgG was diluted to 2.0mg/ml with physiological saline, and mixed with an equal volume of physiological saline (100. Mu.g/BALB/c mouse) in the tail vein for additional immunization at a dose of 100. Mu.l/mouse.
EXAMPLE 2 preparation of hybridoma cell lines
2-1 preparation of feeder cells
Normal 12-week-old BALB/c mice peritoneal macrophages were used as feeder cells. 1 day before the fusion, BALB/c was used to draw the neck to be killed, 0.1% benzalkonium bromide was soaked for 1 minute, 75% alcohol was added to soak for 1 minute, and the abdominal skin was lifted from the posterior abdomen with a pair of sterilized scissors and forceps in a super clean bench to expose the peritoneum. The peritoneum was sterilized by rubbing with an alcohol cotton ball. 2ml of RPMI1640 medium was injected into the abdominal cavity with a syringe, taking care not to penetrate into the intestinal tract. The syringe was fixed with the right hand, the needle was left in the abdominal cavity, and the abdomen was gently massaged with an alcohol cotton ball with the left hand for 1 minute, followed by aspiration of the injected culture solution. Centrifuging at 1000r/min for 5-10 min, and discarding the supernatant. Resuspending with RPMI1640 culture medium containing 20% newborn calf serum and 1% double antibody, and adjusting cell concentration to 2-5 × 10 5 Number of orml, added to a 96-well plate, 100. Mu.l/well, incubated at 37 ℃ and 5% in CO2.
2-2 preparation of immune splenocytes
The immune serum titer in example 1 was taken to reach 1:10 6 The BALB/c mice (see above) were subjected to blood sampling from the eye, and the serum was isolated as a positive control serum for antibody detection. Meanwhile, a mouse is killed by cervical dislocation, 0.1% benzalkonium bromide is soaked for 1 minute, the mouse is soaked in 75% alcohol for 1 minute, the left abdominal skin is lifted on a sterile plate in a super clean bench, the spleen is visible, the forceps are replaced, the peritoneum is cut off by sterile scissors, the spleen is taken out and placed in a plate filled with 10ml RPMI1640 culture solution, the plate is washed lightly, and the surrounding connective tissues are carefully stripped. The spleen was transferred to another plate containing 10ml of RPMI1640 medium, and gently squeezed by an elbow forceps or a bent needle attached to a 1ml syringe (or the spleen was squeezed by a plunger of the syringe), so that the spleen cells were introduced into the RPMI1640 medium in the plate. The mixture is blown and beaten for several times by a pipette to prepare single cell suspension. To remove large clumps from the spleen cell suspension, filtration through a 200 mesh copper mesh was used. Harvesting the spleen cell suspension, centrifuging for 5-10 minutes at 1000r/min, centrifuging and washing for 1-2 times by using RPMI1640 culture solution, then suspending the cells in 10ml of RPMI1640 culture solution, uniformly mixing, taking the suspension, and adding the phloroglucinol blue dye solution for counting the living cells for later use. Usually 1X 10 per mouse 8 -2.5×10 8 And (4) spleen cells.
Preparation of 2-3 myeloma cells
The manner in which the myeloma cells are maintained prior to fusion is critical to the successful acquisition of hybridoma cells. The goal was to have the cells in logarithmic growth for as long as possible, certainly not less than 1 week before fusion. The cryopreserved cells were not in a state suitable for fusion until 2 weeks after recovery, and the longer myeloma cells were likely to recover for at least 5 days. Myeloma cells that are in logarithmic growth during culture are maintained in medium containing 10% calf serum by inoculating myeloma cells in 10-fold serial dilutions with 12 flasks filled with 5ml of medium. After 1 week, the cells were re-cultured in flasks after the culture reached a relatively dense and non-growing flask. Typical doubling times are 14-16 hours. The preparation method of the myeloma cell suspension comprises the following steps: before fusionMyeloma cells are expanded and cultured for 48 to 36 hours (typically, 2 to 3 cells cultured in 25cm2 flasks are prepared for a fusion experiment using a 96-well plate). On the day of fusion, cells were gently blown down from the vial wall using a glass pipette and collected in a 50ml centrifuge tube. Centrifuging at 1000r/min for 5-10 min, and discarding the supernatant. 30ml of RPMI1640 culture medium was added, and the mixture was washed by centrifugation once. Then, the cells were resuspended in 10ml of RPMI1640 culture medium and mixed well. Taking myeloma cell suspension, adding 0.4% of fetuin blue for counting living cells for later use. When counting cells, 0.1ml of the cell suspension is added into 0.9ml, mixed evenly and counted by a blood counting chamber. Cell density (pieces/ml) = (4 large cell count ÷ 4) × 10 4 X dilution factor.
2-4 cell fusion and Selective culture of hybridoma cell lines
Spleen cells and myeloma cells were mixed at a ratio of 1:8.5, adding the mixture into a 50ml centrifuge tube, supplementing the RPMI1640 culture solution to 40ml, and fully and uniformly mixing. Centrifuging at 1000r/min for 5-10 min, and sucking the supernatant as clean as possible. Flicking the fusion tube bottom on the palm to ensure that the precipitated cells are loosened and uniform; preheating in 37 deg.C water bath. Adding 50% PEG (pH 7.4) 1ml preheated to 40 deg.C in a 1ml pipette for about 1 minute (preferably 45 seconds), and mixing by gently shaking. Adding 20-30ml of RPMI1640 culture solution preheated to 37 ℃ within 90 seconds by using a glass dropper; standing at 20-37 deg.C for 10 min. Centrifuging at 700r/min for 5-10 min, and discarding the supernatant. Resuspended in RPMI1640 medium containing 1% HAT and 20% newborn calf serum, and loaded into 10 96-well cell culture plates in equal portions. 37 ℃ C., 5% CO2 culture, and the next day was supplemented with RPMI1640 medium containing 1% HAT and 20% newborn calf serum to 90% of the pore volume. After 5 days, the wells were changed 1/2 of the medium with HAT medium, and after 7 days, the wells were changed 1/2 of the medium again.
Screening of 2-5 Positive hybridoma cell lines
Human blood IgG was diluted to 2ug/ml with 0.06M pH9.6 carbonate buffer, and 100. Mu.l of each well was coated in a 96-well microplate for detection of the fused cell culture supernatant. The plate was washed three times with ELISA wash, patted dry, blocked with 10% calf serum in 0.01M PBS (pH7.2) at 150ul per well at 37 ℃ for 2 hours, patted dry and vacuum-packaged for use. The ninth day after cell fusion, 100 μ L of cell supernatant was taken and put into the above 96-well ELISA plate, 40min was incubated at 37 ℃ for five times, 100 μ L/well of horseradish peroxidase-labeled goat anti-mouse IgG (batch No. 111418, produced by Sichuan Michael Biotech, inc.) was added after washing the plate with ELISA washing solution for five times, 100 μ L of buffer solution containing 0.1% (M/V) o-phenylenediamine, 0.1% (V/V) hydrogen peroxide, pH5.0 citric acid phosphate was added to each well after 30min incubation at 37 ℃ with the plate, 10min was incubated at 37 ℃, 50 μ L of 2M sulfuric acid solution was added to each well to terminate the reaction, and the 450nm absorbance was measured with a multifunction reader. The mouse serum is diluted to 100 times during fusion to be used as a positive control, RPMI1640 complete culture solution is used as a negative control, the OD value of the negative control is less than 0.2, the OD value of the positive control is more than 1.8, the system is effective, when the OD value of the sample is more than or equal to 2 times the OD value of the negative control, the system is positive, and otherwise, the system is negative.
Cloning of 2-6 Positive hybridoma cell lines
Feeder cells were plated according to the method for preparing feeder cells described in example 2-1, a positive hybridoma cell suspension was prepared, diluted with HT medium containing 20% serum to a dilution of 1 cell per ml, and positive wells were selected by cloning 4 or more times in the same manner as in example 2-5 until 100% monoclonals were obtained.
Cryopreservation of 2-7 positive hybridoma cell lines
Transferring 100% of the cells cloned in the example 2-6 and detecting the cells as positive by an indirect method into 24-hole wells for continuous culture, transferring the cells into a cell bottle for expanded culture when the cells are full of 80%, then performing bottle splitting and passage when the cells are full of 80%, dispersing the cells in the cell bottle by using a proper amount of serum-free RPMI-1640 medium when the passage cells grow to a logarithmic phase, collecting the cell suspension in a conical centrifuge tube, recording the volume V of the cell suspension, taking a proper amount of the cell suspension for cell counting to obtain the density (per ml) of the cell suspension, centrifuging the rest at 1000rpm for 5min, discarding supernatant, calculating the number of the precipitated cells according to the cell density and the volume before centrifugation, adding a proper amount of freezing medium into the cell precipitate to adjust the cell density to 2-4 x 10 6 Counting the cells per ml (taking appropriate amount for confirmation, if the number of cells is not in the range, re-centrifuging according to the total cell count amount, and adding appropriate amountFreezing the culture medium to obtain a cell concentration of 2-4 × 10 6 One/ml) and then subpackaged in sterile freezing tubes, wherein each freezing tube is subpackaged with 0.5ml of resuspended cell freezing solution. 1 hybridoma cell strain which can stably secrete the anti-human IgG monoclonal antibody is obtained by cell fusion once, is marked as hybridoma cell strain 5A3 and is preserved in China center for type culture Collection in 2018, 8 and 23 days, and the preservation number is CCTCC NO: C2018172.
EXAMPLE 3 preparation of monoclonal antibodies
Selecting 12-14 weeks healthy BALB/c mice, injecting 0.5mL liquid paraffin (Tianjin Kemi Europe) into abdominal cavity of each mouse, and injecting 2 × 10 into abdominal cavity of each mouse 7 days later 6 And (3) hybridoma cells. Ascites can be generated 7-10 days after inoculating cells, the ascites generation condition of the mice is observed every day, if the abdomen is obviously enlarged and the skin is tense when the mice are touched by hands, the neck can be pulled to kill the mice, the ascites is sucked into a test tube by a dropper, and 1-5mL of ascites can be obtained by one mouse. The collected ascites is centrifuged to take the supernatant, and a small sample is stored in a refrigerator at the temperature of minus 20 ℃.
EXAMPLE 4 purification of monoclonal antibodies
The frozen human IgG-Ab5 ascites at the temperature of below-20 ℃ is thawed overnight in a refrigerator at the temperature of 2-8 ℃ one day in advance. The next day, ascites fluid was mixed, centrifuged at 12000rpm for 20 minutes at 2-8 ℃, degreased and precipitated, the supernatant diluted 5-10 times with a Mab Loading Buffer, and filtered with a 0.22 μm filter. The filtered solution was loaded into 5mL-mabselectsure media and the breakthrough was collected using AKTA purifiers. And (3) after the sample loading is finished, balancing the chromatographic column by using a balance liquid until the baseline is stable, eluting the target protein by using an eluent, collecting an elution peak of more than 100mAu, cleaning the chromatographic column by using 0.1M sodium hydroxide after the elution is finished, and then storing the chromatographic column. The eluted target protein was neutralized, and 0.1mL of a neutralizing solution was added dropwise to each mL of the eluate. After mixing and neutralizing, the protein was dialyzed in 5L of dialysis solution, and the solution was changed every two hours for 3 times. The dialyzed target protein was centrifuged at 12000rpm for 20 minutes, and the supernatant was used as a final product, and subjected to electrophoresis (as shown in FIG. 1).
EXAMPLE 5 purity test of monoclonal antibody
The electrophoresis glass plate is assembled, and SDS-PAGE gel of 12% separating gel at the lower layer and 5% concentrated gel at the upper layer is prepared. The gel electrophoresis tank is assembled, and a proper amount of 1 × electrophoresis buffer solution is added. After the antibody concentration was measured, a small amount of the antibody was diluted with 20mM PBS to a concentration of about 1mg/mL, 40. Mu.l of the diluted antibody was added to 10. Mu.l of 5 Xsample buffer, mixed well, boiled for 10 minutes, and centrifuged at 5000rpm for 10 minutes for use. 10ul of supernatant was removed and the gel was electrophoresed at 70V until the boundary between the concentrated gel and the separation gel (about 15 min) and electrophoresed at 140V until bromophenol blue was about to run out of the gel. After electrophoresis, the SDS-PAGE gel is stripped from the electrophoresis glass plate, put into a staining solution, and the glass plate is cleaned and dried for later use. The SDS-PAGE gel was dip-stained with Coomassie Brilliant blue stain for 30 minutes and then eluted with Coomassie Brilliant blue stain until the background was colorless (which was suitably heat-destained). And (3) taking a picture of the PAGE gel by using a gel imager, and analyzing the gray level of the image by using software to estimate the purity of the antibody.
Example 6 detection of the titer of culture supernatant of hybridoma cell lines
Human blood IgG (Sichuan Mike New biological Material technology Co., ltd., batch No. 031524) was diluted to μ g/ml with 0.06M carbonate buffer solution pH9.6, and each well of a 96-well plate was coated with 100 μ l. Placing the mixture in a refrigerator for overnight at 2-8 ℃, discarding liquid in holes the next day, washing the mixture by an ELISA plate washer for three times, patting the mixture to be dry, sealing the mixture for 2 hours at 37 ℃ by PBS (phosphate buffer solution) containing 10% calf serum and having a pH value of 7.2 and a concentration of 150 mu l/hole, and patting the mixture to be dry for detecting cell culture supernatant, ascites and antibody titer. Cell supernatant titer was measured by 2-fold stepwise dilution from well 1 to well 12 in 0.01M PBS buffer (pH7.2). The mouse serum is diluted to 100 times during fusion to be used as a positive control, RPMI1640 complete culture solution is used as a negative control, the OD value of the negative control is less than 0.2, the OD value of the positive control is more than 2.5, the detection system is effective, when the OD value is more than or equal to 2 multiplied by the OD value of the negative control, the detection system is positive, otherwise, the detection system is negative. The dilution ratio corresponding to the lowest positive hole is the titer of the cell culture supernatant, and the titer of the hybridoma cell strain culture supernatant can reach 1:4096 or more. The culture supernatant titer is shown in table 1.
TABLE 1 hybridoma cell supernatant titer results
Example 7 ascites titer test
ELISA was performed as in example 6. The specific method comprises the following steps: the ascites fluid in the 1 st well is diluted 10 times from the 2 nd well to the 7 th well and 2 times from the 8 th well to the 10 th well in PBS buffer solution of 0.01M pH7.2. The 11 th hole uses the serum of the mouse diluted to 100 times during fusion as a positive control, the 12 th hole uses the complete culture solution of RPMI1640 as a negative control, the OD value of the negative control is less than 0.2, the OD value of the positive control is more than 1.8, the detection system is effective, when the OD value is more than or equal to 2 multiplied by the OD value of the negative control, the detection system is positive, otherwise, the detection system is negative. The dilution ratio corresponding to the lowest positive hole is the ascites titer, the detection result of the anti-human IgG monoclonal antibody (marked as human IgG-Ab 5) prepared from the hybridoma cell strain (marked as hybridoma cell strain 5A 3) is shown in Table 2, and the ascites titer can reach 1 as shown in Table 2: 1.6X 10 6 。
TABLE 2 ascites titer test results
Example 8 antibody Titer detection
The purified antibody prepared in example 4 was diluted to 1mg/ml in 0.01M PBS buffer (pH7.2), and the antibody was diluted 10-fold stepwise from well 1 to well 4 and 2-fold stepwise from well 5 to well 11. Antibody titer determination standard: taking log (dilution) as an abscissa and OD value as an ordinate to make a curve, and taking the curve equation as y = min + (max-min)/(1 +10^ ((logEC 50-x) × Hillslope)), fitting the curve by sigmaplot software, and taking the titer =10logEC50. The results show that the antibody titer secreted by the hybridoma cell strain (5A 3) is more than 5 multiplied by 10 4 . Two commercial anti-human IgG monoclonal antibodies B and C were tested against the same assay, and by fitting, the median titers were all below 5X 10 4 Lower than the antibodies secreted by the hybridoma cells of the invention. The titer assay results are presented in table 3 and figure 2.
TABLE 3 antibody titer test
| Number of enzyme-labeled well | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | Positive control |
| Dilution factor of antibody | 10 | 100 | 1×10 3 | 1×10 4 | 2×10 4 | 4×10 4 | 8×10 4 | 16×10 4 | 32×10 4 | 64×10 4 | 128×10 4 | 3.1254 |
| Log value of |
1 | 2 | 3 | 4 | 4.301 | 4.6021 | 4.9031 | 5.2041 | 5.5051 | 5.8062 | 6.10721 | 3.0364 |
| Human IgG-Ab5 | 3.0058 | 2.6899 | 2.5993 | 2.383 | 2.3103 | 2.0085 | 1.628 | 1.2697 | 0.9856 | 0.7672 | 0.62766 | Negative control |
| Commercial B | 1.7556 | 1.5325 | 1.3441 | 1.1943 | 0.9887 | 1.0077 | 0.5463 | 0.4141 | 0.3335 | 0.3165 | 0.23698 | 0.037281 |
| Commercial C | 3.5828 | 3.1533 | 2.72 | 2.6099 | 2.4823 | 2.1493 | 1.6787 | 1.1787 | 0.8462 | 0.5699 | 0.4415 | 0.038583 |
Example 9 Cross-reactivity assay
(1) HRP-labeled human IgG-Ab5
Solution preparation:
a.2mg/ml HRP:1mgHRP in 0.5ml purified water (ready to use)
60mM sodium metaperiodate: 80.2mg of sodium metaperiodate is dissolved in 6.25ml of purified water (for use in the spot)
c.160mM ethylene glycol: 8.93ul ethylene glycol was added to 1ml of purified water (ready for use)
d.1mg/ml sodium borohydride: 1mg sodium borohydride dissolved in 1ml purified water (ready for use)
e. Saturated ammonium sulfate
f.0.05M carbonate buffer (pH 9.6): sodium carbonate 1.59g/L, sodium bicarbonate 2.93g/L
Slowly dripping 0.5ml of sodium metaperiodate into 0.5ml of HRP, uniformly mixing while adding, standing in a dark place at 2-8 ℃ for 30min, slowly adding 0.5ml of ethylene glycol into the solution while adding, uniformly mixing while adding, reacting in a dark place at room temperature for 30min, slowly adding the activated HRP into 0.5mg of antibody (the label ratio is 1.
(2) Human IgG, mouse IgG, rabbit IgG, bovine IgG and human IgM (2.5 ug/ml) were coated at 100ul per well overnight at 4 ℃.10mM Tris-HCl (7.4) +0.5% casein blocking, 150ul per well, 37 degrees 2h. Labeling anti-human IgG-Ab5 according to the method, diluting the labeled enzyme-labeled antibody by 1000, 2000 and 4000 times respectively, reacting with five coating antigens respectively, adding 50ul enzyme-labeled antibody, incubating for 30min at 37 ℃, washing a plate, developing color, and detecting results show that no cross reaction exists between the human IgG-Ab5 and the five antigens.
Example 10 stability verification
The human IgG-Ab5 monoclonal antibody can be applied to indirect method for detecting the herpes simplex virus type 1 infection serum antibody, the human IgG-Ab5 antibody is marked with HRP according to example 9 and then diluted according to a certain proportion, and is subjected to accelerated treatment in 37 ℃ water bath for 6 days, and two samples with different concentration gradients are detected after the accelerated treatment for 6 days. The results in table 4 show that the signal retention rate of two gradient samples detected after the antibody is subjected to 37-degree water bath for 6 days is more than 90%, and the requirements of a reagent platform are met.
Table 4 stability verification
Example 11 clinical diagnosis
The human IgG-Ab5 monoclonal antibody can be applied to indirect method for detecting herpes simplex virus type 1 infection serum antibody, the antibody is applied to a chemiluminescence kit of the company and used as an enzyme-labeled antibody, the result of detecting a clinical sample has more than 95% of correlation with the result of detecting the clinical sample by the kit which is currently sold in the company, the detection result is listed in Table 5, and the correlation is compared in figure 3.
TABLE 5 sample testing
It is to be understood that the invention disclosed is not limited to the particular methodology, protocols, and materials described, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims.
Those skilled in the art will also recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.
Claims (12)
1. A hybridoma cell strain 5A3 is preserved in China center for type culture Collection with the preservation number of CCTCC NO: C2018172.
2. A monoclonal antibody secreted by the hybridoma cell strain of claim 1.
3. A kit comprising the hybridoma cell strain of claim 1 or the monoclonal antibody of claim 2.
4. The kit according to claim 3, which is a colloidal gold immunoassay kit, a chemiluminescent kit, a radioimmunoassay kit, an enzyme-linked immunoassay kit, a fluorescent immunoassay kit or a microfluidic chip.
5. The kit of claim 4, which is a chemiluminescent kit.
6. Use of the hybridoma cell strain of claim 1 or the monoclonal antibody of claim 2 in the preparation of a kit.
7. The use according to claim 6, said kit being an immunoassay-based kit.
8. The use according to claim 7, wherein the kit is a colloidal gold immunoassay kit, a chemiluminescent kit, a radioimmunoassay kit, an enzyme-linked immunoassay kit, a fluorescent immunoassay kit or a microfluidic chip.
9. The use according to claim 8, said kit being a chemiluminescent kit.
10. The use according to claim 6, the kit being for the detection of human IgG.
11. The use of claim 10, wherein the human IgG is herpes simplex virus type 1 IgG.
12. Use of the hybridoma cell strain of claim 1 or the monoclonal antibody of claim 2 in the preparation of a kit for detecting herpes simplex virus type 1.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008014390A1 (en) * | 2006-07-28 | 2008-01-31 | Idexx Laboratories, Inc. | Identification of leucocytes bearing diagnostic markers for transmissible spongiform encephalopathies |
| CN101430329A (en) * | 2008-09-24 | 2009-05-13 | 王贤理 | Immunoglobulin G detection reagent |
| WO2011048043A1 (en) * | 2009-10-19 | 2011-04-28 | F. Hoffmann-La Roche Ag | NON-CROSS-REACTIVE ANTI IgG ANTIBODIES |
| CN102353777A (en) * | 2011-07-05 | 2012-02-15 | 深圳市卫武光明生物制品有限公司 | Kit and method for testing human cytomegalovirus IgG antibody |
| CN107635581A (en) * | 2015-02-13 | 2018-01-26 | 赛诺菲 | Stable liquid preparation for monoclonal antibody |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE3581400D1 (en) * | 1984-09-28 | 1991-02-21 | Teijin Ltd | MOUSE / HUMAN HYBRIDOMA WITH GENERATION OF ANTIVIRAL HUMAN ANTIBODIES, THEIR PRODUCTION AND ANTIVIRAL HUMAN MONOCLONAL ANTIBODIES. |
| CN101113459A (en) * | 2007-07-16 | 2008-01-30 | 东莞太力生物工程有限公司 | Recombination duplicating deficient virus, pharmaceutical composition containing the virus and uses thereof |
| CN104650236B (en) * | 2013-06-28 | 2018-02-23 | 英科隆生物技术（杭州）有限公司 | A kind of heterologous cross-linking agent and its application in rubella virus detects |
| CN108588030B (en) * | 2018-03-30 | 2020-07-14 | 四川迈克生物新材料技术有限公司 | Anti-human IgM monoclonal antibody, hybridoma cell strain and application thereof |
| CN108330106B (en) * | 2018-03-30 | 2020-07-14 | 四川迈克生物新材料技术有限公司 | Anti-human IgM monoclonal antibody, hybridoma cell strain and application thereof |
-
2018
- 2018-10-16 CN CN201811201438.0A patent/CN109280644B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008014390A1 (en) * | 2006-07-28 | 2008-01-31 | Idexx Laboratories, Inc. | Identification of leucocytes bearing diagnostic markers for transmissible spongiform encephalopathies |
| CN101430329A (en) * | 2008-09-24 | 2009-05-13 | 王贤理 | Immunoglobulin G detection reagent |
| WO2011048043A1 (en) * | 2009-10-19 | 2011-04-28 | F. Hoffmann-La Roche Ag | NON-CROSS-REACTIVE ANTI IgG ANTIBODIES |
| CN102656460A (en) * | 2009-10-19 | 2012-09-05 | 弗·哈夫曼-拉罗切有限公司 | Non-cross-reactive anti IgG antibodies |
| CN102353777A (en) * | 2011-07-05 | 2012-02-15 | 深圳市卫武光明生物制品有限公司 | Kit and method for testing human cytomegalovirus IgG antibody |
| CN107635581A (en) * | 2015-02-13 | 2018-01-26 | 赛诺菲 | Stable liquid preparation for monoclonal antibody |
Non-Patent Citations (4)
| Title |
|---|
| A Rapid Method to Characterize Mouse IgG Antibodies and Isolate Native Antigen Bingding IgG B Cell Hybridoma;Haolin Liu等;《PlosOne》;20150828(第0136613期);全文 * |
| 抗人IgG（Fc特异性）单克隆抗体的制备、鉴定及应用;李文等;《空军总医院学报》;19931231;第9卷(第3期);全文 * |
| 抗人免疫球蛋白G多克隆荧光抗体的制备及在肾活检中的;段春光等;《医学研究生学报》;20151220;第18卷(第12期);全文 * |
| 抗人免疫球蛋白IgG单克隆抗体制备及其特性鉴定;王香菊等;《中国法医学杂志》;19960630;第14卷(第2期);全文 * |
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