CN105985436B - Anti-human kininogenase antibody and its application - Google Patents
Anti-human kininogenase antibody and its application Download PDFInfo
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- CN105985436B CN105985436B CN201510051431.5A CN201510051431A CN105985436B CN 105985436 B CN105985436 B CN 105985436B CN 201510051431 A CN201510051431 A CN 201510051431A CN 105985436 B CN105985436 B CN 105985436B
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- Peptides Or Proteins (AREA)
Abstract
The present invention relates to anti-human kininogenase antibody and its applications.The present invention is prepared for a variety of monoclonal antibodies, and carries out pairing screening, obtains sensitivity and specificity is able to satisfy the antibody combination (A24 and A32) of demand；It facilitates mass production simultaneously, can meet the needs of larger scale clinical application in the future.The debugging Optimization Work of detection architecture is carried out to above-mentioned antibody combination, it obtains easy to operate, sensitivity, specificity and coherent detection performance can meet the time resolution immunofluorescence chromatography quantitative test card of the enzyme-linked immune quantitative detection reagent box of people's kininogenase of human plasma pattern detection, the colloidal gold immunochromatographimethod quantitative test card of people's kininogenase and people's kininogenase.
Description
Technical field
The invention belongs to immunochemical technique field, be specifically related to anti-human kininogenase antibody and preparation method thereof and
Application of the above-mentioned antibody in the detection of people's kininogenase.
Background technique
Cardiovascular and cerebrovascular disease is the major chronic non-communicable diseases for seriously endangering human health.Wherein coronary heart disease and brain soldier
In be the most common cause of death in the world.In China, with the aging of population, with coronary heart disease, cerebral apoplexy is the heart of representative
The disease incidence of cranial vascular disease, lethality and disability rate show an increasing trend year by year.But 80% cerebral apoplexy is can to prevent
's.Diabetic nephropathy is the most common complication of diabetes, seriously endangers the quality of life and Medical Consumption matter of diabetic
Amount.If not taking positive intervening measure from the height of evidence-based medicine EBM, diabetic nephropathy will develop in a relatively short period of time is
Irreversible end-stage renal disease seriously threatens the existence service life of patient.Therefore, effective method is actively found, is early diagnosed
Cerebral apoplexy, diabetic nephropathy are to carry out effectively protecting the quality of life for being directly related to patient and existence service life.
Kallikrein kinin system (kallikrein-kinin system, KKS) is also known as kinin system, deposits extensively
It is the intracorporal multiple systems of animal, is especially distributed in cardiovascular system more intensive.The system has extensive biology living
Property, and there are close connections with blood coagulation system, renin-angiotensin system and a variety of vasoactive factors systems etc.
And crosstalk, the common pathophysiological process safeguarded the normal physiological function of human body multiple organ and participate in various complexity have
Angiocarpy, kidney, nervous system and glucose metabolism are adjusted, vasodilator participates in inflammatory reaction, pain stimulation and shock instead
It answers.Angiocarpy, kidney, the work in central nervous system disease are concentrated mainly on about the clinical research of kinin system in recent years
With.
Human tissue kallikrein is the most important component part of kinin system, is one group of secreting type serine protease,
Include 15 members.In all known tissue kallikreins, only (pancreas/kidney kassinin kinin discharges human tissue kallikrein 1
Enzyme, Human kallikrein 1, hK1, KLK1, also known as kininogenase, Kininogenase) can effectively hydrolyze low molecular weight swash
Peptide former (LMWK) discharges active kassinin kinin, and then plays the adjustment effect of cardiovascular system and renal function.Basic research
Show in a variety of hypertension animal models it has proven convenient that hK1 mitigates kidney and cardiomegaly and fibrosis with reducing blood pressure
Effect；It is carrying out heart reconstruction, mitigates kidney damage, reduces cerebral infarction incidence and reduces the sides such as neurotrosis harm
The effect in face.Meanwhile by the method for exogenous administration, also demonstrating hK1 is preventing apoplexy, cardiovascular and cerebrovascular and kidney trouble
The effect of aspect.In recent years, further investigations have shown that, mistake of the hK1 level in the occurrence and development of the cardiovascular and cerebrovascular disease of people
There is important clinical meaning in journey, it may be as the index of predicting of stroke rate.The hair of cerebral apoplexy can be predicted in hK1 level
Disease and 5 years can allow patient's early prevention and taking appropriate measures, to reduce brain to a certain extent without event survival rate
The probability of happening of stroke.In addition, hK1 can diagnosis early diabetic nephropathy earlier compared with Urinary Microalbumin Excretion rate.To sum up may be used
See, the horizontal cardiovascular and cerebrovascular disease and diabetic kidney disease for people of hK1 has important predictive value.Therefore, accurately
The level for measuring hK1 all has important meaning in clinical and scientific research.
In view for the treatment of and predicting function of the hK1 in cardiovascular and cerebrovascular disease and diabetic kidney disease, prepares hK1 and quantitatively examine
Test agent box has the application value of important clinical.Has hK1 scientific research immue quantitative detection reagent box: part of reagent both at home and abroad
Box uses competition law, and antibody is polyclonal antibody, and trivial operations calculate cumbersome and easily non-specific binding occur and cause to examine
It is larger to survey resultant error；Part kit uses the sandwich method detection pattern of polyclonal antibody, also there is non-specific binding
It influences；Other kit uses the mode of the double-antibody sandwich of polyclonal antibody and monoclonal antibody, and detection performance has
To further verify.The detection pattern of double antibody sandwich method can effectively avoid the above problem.Therefore, multi-epitope, it is highly sensitive, it is special
The preparation of anisotropic antibody is one of the key factor of people's kininogenase immue quantitative detection reagent box of high quality.Natural hK1 exists
Multiple glycosylation sites, thus recombinantly express hK1 in terms of other conformations such as glycosylation site may with native protein exist compared with
Big difference.Being screened the specific antibody that just will appear screening using recombination hK1 in specificity antibody screening cannot have
Effect identifies the problem of natural hK1.The present invention carries out the screening operation of antibody using naive hK1, can effectively avoid above situation
Generation, and have proven to the binding force of itself and natural hK1.
Meanwhile to meet the different application demand in market, the present invention is prepared for three kinds of hK1 immue quantitative detection reagent boxes: people swashs
Peptide protoenzyme ELISA immue quantitative detection reagent box, can meet the application demand of general small comprehensive hospital, and can develop in the future and upgrade to
Chemoluminescence method quantitative detection (full-automatic) meets the application demand of general hospital automatically detected.People's kininogenase
Colloidal gold quantitative testing test paper card and people's kininogenase fluorogenic quantitative detection test card can meet quickly detection and bedside detection
Demand.Though wherein the sensitivity of colloidal gold quantitative assay and accuracy are not so good as fluorogenic quantitative detection method, its user is easy
In popularization and cost is relatively low, can substantially meet the quantitative detection of disease indicators, the instant detection being suitable in primary care system.
Fluorescent quantitation has higher detection sensitivity, suitable for the higher inspection center of testing requirements or large hospital clinical department
The application demand of bedside diagnosis.
Summary of the invention
The technical problem to be solved in the present invention is to provide can effective, specific binding people's kininogenase antibody.More
Say to body:
The first object of the present invention is to provide two kinds of anti-human kininogenase antibody.
The first anti-human kininogenase antibody (A24),
Its heavy chain variable region contains complementary determining region below: amino acid sequence is as shown in sequence SEQ ID NO:1
HCDR1, the HCDR2 as shown in sequence SEQ ID NO:2 and/or the HCDR3 as shown in sequence SEQ ID NO:3；
And its light-chain variable sequence contains complementary determining region below: amino acid sequence such as sequence SEQ ID NO:4
Shown in LCDR1, the LCDR2 as shown in sequence SEQ ID NO:5 and/or the LCDR3 as shown in sequence SEQ ID NO:6.
The amino acid sequence of the heavy chain variable region of antibody A 24 preferably in the present invention is as shown in SEQ ID NO:7, gently
The amino acid sequence of chain variable region is as shown in SEQ ID NO:8.
Second of anti-human kininogenase antibody (A32),
Its heavy chain variable region contains complementary determining region below: amino acid sequence is as shown in sequence SEQ ID NO:9
HCDR1, the HCDR2 as shown in sequence SEQ ID NO:10 and/or the HCDR3 as shown in sequence SEQ ID NO:11；
And its light-chain variable sequence contains complementary determining region below: amino acid sequence such as sequence SEQ ID NO:12
Shown in LCDR1, the LCDR2 as shown in sequence SEQ ID NO:13 and/or the LCDR3 as shown in sequence SEQ ID NO:14.
Preferably the present invention in antibody A 32 heavy chain variable region amino acid sequence as shown in SEQ ID NO:15,
The amino acid sequence of light chain variable region is as shown in SEQ ID NO:16.
Second purpose of the invention is to provide two kinds of single-chain antibodies, the amino acid sequence such as SEQ of the single-chain antibody A24
Shown in ID NO:17；The amino acid sequence of the single-chain antibody A32 is as shown in SEQ ID NO:18.Preferably, described single-stranded anti-
Without the HIS label being made of six histidines in body A24, A32.
Third purpose of the present invention is to provide the nucleotide sequence of two kinds of above-mentioned single-chain antibodies of coding, encodes single-chain antibody
The nucleotide sequence of A24 is as shown in SEQ ID NO:19, and the nucleotide sequence such as SEQ ID NO:20 of coding single-chain antibody A32
It is shown.
4th purpose of the invention is to provide a kind of expression vector containing above-mentioned nucleotide sequence.
5th purpose of the invention is to provide a kind of recombinant host cell containing above-mentioned expression vector.Affiliated host cell
It can be Escherichia coli, yeast or mammalian cell, preferably Pichia pastoris.
6th purpose of the invention is to provide a kind of method for producing above-mentioned single-chain antibody, comprising:
1) above-mentioned recombinant host cell expression antibody is cultivated under suitable conditions；
2) it then purified from host cell, collect antibody.
Of the invention the 7th is designed to provide above-mentioned anti-human kininogenase antibody in detection people's kininogenase content
Application.
Of the invention the 8th, which is designed to provide one group, can be matched and be detected the antibody of people's kininogenase to combination；
The antibody is high to combined detection sensitivity, and specificity is good.
Of the invention the 9th is designed to provide a kind of utilization anti-human kininogenase antibody test people's kininogenase
Enzyme-linked immune quantitative detection reagent box, including being coated with the ELISA Plate of A24 or A32 antibody, sample diluting liquid, standard items, having contained
There are detection liquid, cleaning solution, developing solution and the terminate liquid of enzyme labelled antibody A32 or A24.Its detecting step specifically includes that
(1) after Sample dilution being added into the ELISA Plate of coating A24 or A32 antibody, Sample dilution dilution is added
Standard items, negative control and serum or the sample to be examined such as blood plasma；
(2) the diluted detection liquid of Sample dilution is added；
(3) developing solution is added
(4) terminate liquid is added and reads OD value.
The enzyme labelled antibody A32 or A24 is the antibody A 32 or A24 (A32-HRP or A24- of horseradish peroxidase-labeled
) or the antibody A 32 or A24 of alkali phosphatase enzyme mark (A32-AP or A24-AP) HRP；The standard items are Chinese hamster ovary
The purifying protein of cell (CHO) expression recombined human kininogenase.
Of the invention the tenth is designed to provide a kind of utilization anti-human kininogenase antibody test people's kininogenase
Colloidal gold immunochromatographimethod quantitative test card, including sample absorption pad, gold-labelled pad, reaction film and water absorption pad；The gold-labelled pad spray
It is coated with the antibody A 32 or A24 of colloid gold particle label, there is detection band and quality control band on the reaction film, detection band position is coated with
There are antibody A 24 or A32, quality control band position is coated with anti-His tag antibody or Protein L.
The present invention the 11st is designed to provide a kind of utilization anti-human kininogenase antibody test people's kininogenase
Time resolution immunofluorescence chromatograph quantitative test card, including sample absorption pad, fluorescent microsphere pad, reaction film and water absorption pad；Institute
Antibody A described above 32 or A24 that fluorescent microsphere pad is coated with fluorescent microsphere label are stated, has detection band and matter on the reaction film
Control band, detection band position is coated with antibody A 24 described above or A32, quality control band position be coated with anti-His tag antibody or
Protein L。
The preferred nitrocellulose filter of reaction film.The anti-anti- His antibody of the preferred mouse of His tag antibody.
The present invention is prepared for Multiple Antibodies, and carries out pairing screening, obtains sensitivity and specificity is able to satisfy demand
Antibody combination (A24 and A32)；It facilitates mass production simultaneously, can meet the needs of larger scale clinical application in the future.To above-mentioned anti-
Body combination carries out the debugging Optimization Work of detection architecture, obtains easy to operate, sensitivity, and specificity and coherent detection performance can expire
The colloidal gold immunochromatographimethod of the enzyme-linked immune quantitative detection reagent box of people's kininogenase of sufficient clinical sample detection, people's kininogenase
The time resolution immunofluorescence of quantitative test card and people's kininogenase chromatographs quantitative test card.
Detailed description of the invention
Fig. 1 heavy chain of antibody and light-chain variable region gene electrophoretogram.Lane 1 is standard DNA, and Lane 2 is 24 weight of antibody A
Chain variable region DNA, Lane 3 is that 24 light chain variable region DNA, Lane 4 of antibody A is 32 heavy chain variable region DNA, Lane 5 of antibody A
For 32 light chain variable region DNA of antibody A.
Fig. 2 single-chain antibody structural schematic diagram.VHIndicate weight chain variabl area sequence, VLIndicate light-chain variable sequence, His mark
Label are six histidines.
The agarose gel electrophoresis figure of Fig. 3 single-chain antibody expression PCR product.Scheming (a) is A24 gene PCR product；Scheme (b)
For A32 gene PCR product.
Fig. 4 recombinant pichia yeast strain inducing expression supernatant culture solution qualification figure.Fig. 4 (a) finishes red for the recombination of antibody A 24
Yeast strain inducing expression supernatant culture solution qualification figure；Fig. 4 (b) is 32 recombinant pichia yeast strain inducing expression supernatant of antibody A
Culture solution qualification figure.Above-mentioned left figure is SDS-PAGE electrophoretic identification, right figure is Western blot qualification figure.
Fig. 5 single-chain antibody purification effect figure (SDS-PAGE).Scheming (a) is antibody A 24, and figure (b) is antibody A 32.
The Western Blot qualification figure of Fig. 6 antibody A 24 and A32.Swimming lane 1 is Urinary kallidinogenase (UK)；Swimming lane 2 is yeast
Express hK1；Swimming lane 3 is Bacillus coli expression hK1；Swimming lane 4 is that CHO expresses hK1.(a) is schemed for A32 antibody Western Blot knot
Fruit；Scheming (b) is A24 antibody Western Blot result.
Fig. 7 enzyme-linked immunologic detecting kit standard curve of the present invention.Wherein abscissa is protein concentration (ng/mL)；It is vertical to sit
It is designated as detection OD450；R indicates detection related coefficient, is 0.99980322.
Fig. 8 enzyme-linked immunologic detecting kit detection specificity of the present invention.Abscissa is detectable substance concentration (ng/mL)；It is vertical to sit
It is designated as detection OD value.UK indicates Urinary kallidinogenase, is the people's kininogenase extracted from human urine；KLK2 indicates that people organizes kassinin kinin to release
Put enzyme 2；KLK3 indicates human tissue kallikrein 3.
Fig. 9 colloidal gold immunochromatographimethod quantitative test card of the present invention and time resolved immuno fluorometric chromatograph quantitative test card knot
Structure schematic diagram.1 be sample pad, 2 be reaction film, 3 be absorption pad, 4 be nature controlling line (C line), 5 be detection line (T line), 6 be gold mark
Pad or fluorescence bonding pad, 7 be PVC sheet.
Figure 10 colloidal gold immunochromatographimethod quantitative test card standard curve of the present invention.Wherein abscissa is protein concentration (ng/
mL)；Ordinate is T/C value；r2It is 0.992.
Figure 11 time resolution immunofluorescence of the present invention chromatographs quantitative test card standard curve.Wherein abscissa is that albumen is dense
It spends (ng/mL)；Ordinate is detected value；r2It is 0.9952.
Figure 12 time resolution immunofluorescence chromatographs quantitative detection and enzyme linked immunosorbent detection results relevance
Specific embodiment
Definition
" antibody " is also known as immunoglobulin, is a kind of large-scale Y shape protein secreted by bone-marrow-derived lymphocyte, can pass through Y shape
Two of them bifurcated top complementary site (antigen junction coincidence) specific binding target antigen immunoglobulin molecules, institute
State target antigen such as protein, sugar, polynucleotides, rouge, polypeptide, small molecule compound etc..
" single-chain antibody " (scFv) refers to the heavy chain variable region (V of antibodyH) and light chain variable region (VL) pass through 15~20
The single chain fusion protein that amino acid short peptide (linker) connection is formed, the linker for connection are generally rich in glycine and silk
Propylhomoserin, in favor of the stability and flexibility of single-chain antibody.Connection type can be by VLN-terminal be connected to VHC-terminal, Huo Zhexiang
Instead.Although eliminating constant region and introducing linker, single-chain antibody still remains antibody to the specificity of antigen, and it has
Molecular weight is small, penetration power is strong and it is antigenic weak the features such as.
Complementary determining region (complementarity-determining region, CDR), also referred to as hypervariable region.Molding
It is most critical zone of the target antigen in conjunction with antibody in the end of antibody monomer amino acid, in Artificial Immune Network Theory, Mei Gekang
The complementary determining region of body is otherwise known as idiotype or genotype.
Standard items used are that (1mg/mL, preparation method are shown in patent to CHO system expression recombination hK1 in following embodiment
201310746269.X)
The preparation of the anti-human kininogenase hybridoma cell strain of embodiment 1.
1. animal immune
It is pressed with recombined human kininogenase (Chinese hamster ovary cell expression, preparation method are shown in patent 201310746269.X)
BALB/c female mice (being purchased from this experimental animal Co., Ltd of Changzhou Cavan) is immunized according to general immune programme.Specific Immunity
Referring to " Antibody preparation with use experiment guide ".Immune serum titre is tracked using indirect elisa method, chooses serum titer
Mouse boosting cell and murine myeloma cell are carried out fusion experiment by highest immune mouse.
2. cell fusion
(1) preparation of spleen cell
It by immune mouse, plucks eyeball and takes blood, be placed in the alcohol of 75% (v/v) and impregnate 10 minutes through disconnected cervical vertebra execution,
Its spleen is taken out in aseptic operating platform, is placed in cell screen clothes, cell is fully ground, and sieve is crossed, with sterile 1640 culture medium
(be purchased from Gibco company) centrifuge washing for several times after, cell is resuspended so that single cell suspension is made, and count, it is spare.
(2) preparation of feeder cells
Female BAl BIc/c the mouse one for taking 8~10 week old plucks eyeball and obtains negative serum, puts to death postposition through disconnected cervical vertebra
It is impregnated 10 minutes in 75% (v/v) alcohol；Sterile to open skin of abdomen, exposure peritonaeum is trained about 10mL 1640HT with syringe
Base (be purchased from SIGMA company) injection mouse peritoneal is supported, gently abdomen massage and is blown and beaten for several times.Draw the culture containing macrophage
Base injects spare in 20%1640HAT culture medium；
Female BAl BIc/c the mouse one for taking 2~3 week old is placed in 75% (v/v) alcohol through disconnected cervical vertebra execution and impregnates
10 minutes；Sterile to take thymus gland in cell screen clothes, sieve is crossed in grinding, obtain thymocyte be placed in it is above-mentioned containing macrophage
It is spare in 20%1640HAT culture medium.
(3) cell fusion
Selection is in the murine myeloma cell strain SP2/0 of logarithmic growth phase, collects and counts.Take about 108A above-mentioned spleen
Cell and 2 × 107A above-mentioned SP2/0 cell strain, which is added in fusion pipe, to be mixed, and 1000rpm centrifugation abandons supernatant (as far as possible after ten minutes
Abandon net), fusion pipe is set and is gently rubbed back and forth on palm so that precipitating is loose.1mL preheating is added after elder generation is slow in 60 seconds fastly
PEG1450 (polyethylene glycol 1450 is purchased from SIGMA company), is added 1640HT culture medium 30mL and terminates, 1000rpm is centrifuged 10 points
Clock removes supernatant, and gently friction keeps precipitating loose, is added in the 1640HAT culture medium of step 2 obtained 20%.
It after above-mentioned HAT culture medium is mixed well, is dispensed with 200 holes μ L/ into 96 porocyte culture plates, sets 37 DEG C, 5%
It is cultivated in the cell incubator of CO2.20%1640HAT culture medium is replaced with 10%1640HT culture medium after a week, is taken after 3 days
It is detected clearly.
3. anti-human kininogenase specific hybrid tumor cell strain screening
(1) preparation of detection plate: with CB coating buffer dilution Urinary kallidinogenase, (UK, purchase are public in the general biochemical medicine in Guangdong day
Department) to 1 μ g/mL, 96 hole ELISA ELISA Plates, 100 holes μ L/ are coated with, 2~8 DEG C of coatings overnight, washed once and pat dry；Containing 2% junket
The PBST buffer blind (hole 200uL/) of albumen, 37 DEG C are closed 2 hours；It pats dry, it is spare.
(2) screening of positive colony: 100 hole μ L/ of cells and supernatant to be checked is added in above-mentioned detection plate, in 37 DEG C
Effect was washed and is patted dry after 30 minutes, and the sheep anti-mouse igg of the HRP label in 100 holes μ L/ is added, washes after acting on 30 minutes in 37 DEG C
It washs and pats dry, the TMB developing solution in 100 holes μ L/ is added, colour developing 15 minutes is protected from light in 37 DEG C, the 2M H of 50 μ L is added in every hole2SO4Eventually
It only reacts, and the reading numerical values at OD450.Positive hole determines principle: OD450 value/negative control value >=2.1.Choose positive gram
Grand strain carries out Cell-cloned screening.After three to four-wheel cloning screening, monoclonal cell strain positive rate 100% is i.e. true
It is set to stable cell line, singling is carried out to cell strain.Hybridoma cell strain C24 and C32 all have higher potency, it is then subsequent into
One step carries out antibody variable sequences sequencing analysis to above-mentioned hybridoma cell strain.
The measurement of 2. hybridoma cell strain antibody variable sequences of embodiment
Above-mentioned hybridoma cell strain C24 and the antibody variable sequences C32 are measured.
The extraction of a.RNA: reference cell total serum IgE extraction agent box (being purchased from Roche company) specification is to above-mentioned hybridoma
Cell strain C24 and C32 carry out Total RNAs extraction and carry out reverse transcription immediately；
B.RNA reverse transcription becomes DNA: referring to Thermo Scientific Reverted First strand cDNA
Synthesis Kit (being purchased from Thermo company) carries out reverse transcription to total serum IgE extracted in previous step, and cDNA is made, freezes
Be stored in -20 DEG C it is spare；
C. the PCR amplification and recycling of variable region sequences: using gained cDNA is template in previous step, with mouse IgG hypotype list
Clonal antibody variable region sequences universal primer is primer, carries out PCR amplification to the variable region sequences of heavy chain and light chain, PCR is produced
Object is recycled through DNA plastic recovery kit (being purchased from TIANGEN company), sees attached drawing 1；
D. the clone of variable region sequences and sequencing: according to cloning vector pMD18-T kit (being purchased from Takara company)
Heavy chain and light-chain variable region gene are attached with pMD18-T carrier by specification respectively, convert bacillus coli DH 5 alpha, picking
Positive colony transfers to InvitrogenTMCompany is sequenced.
Sequencing obtain the antibody heavy chain variable region amino acid sequence of hybridoma cell strain C24 as shown in SEQ ID NO:7, it is light
Chain variable region amino acid sequence is as shown in SEQ ID NO:8.The above-mentioned sequence of Vbase2 database analysis, heavy chain variable region it is each
The amino acid sequence of complementary determining region is respectively: the HCDR1 as shown in sequence SEQ ID NO:1, such as sequence SEQ ID NO:2 institute
The HCDR2 and/or the HCDR3 as shown in sequence SEQ ID NO:3 shown；The amino acid of each complementary determining region of its light chain variable region
Sequence is: the LCDR1 as shown in sequence SEQ ID NO:4, the LCDR2 as shown in sequence SEQ ID NO:5 and/or such as sequence
LCDR3 shown in SEQ ID NO:6.
Sequencing obtain the antibody heavy chain variable region amino acid sequence of hybridoma cell strain C32 as shown in SEQ ID NO:15,
Chain variable region amino acid sequence is as shown in SEQ ID NO:16.The above-mentioned sequence of Vbase2 database analysis, heavy chain variable region
The amino acid sequence of each complementary determining region be respectively: HCDR1, such as sequence SEQ ID as shown in sequence SEQ ID NO:9
HCDR2 shown in the NO:10 and/or HCDR3 as shown in sequence SEQ ID NO:11；Each complementary determining region of its light chain variable region
Amino acid sequence be: the LCDR1 as shown in sequence SEQ ID NO:12, the LCDR2 as shown in sequence SEQ ID NO:13 and/
Or the LCDR3 as shown in sequence SEQ ID NO:14.
The recombinant expression and purifying of 3. single-chain antibody of embodiment
According to sequencing result in embodiment 2, respectively by the heavy chain of antibody and light chain variable of hybridoma cell strain C24 and C32
Link peptide (GGGGS) is added between area3, six histidines are introduced, and by its full genome according to the inclined of pichia yeast expression system
The method that love property carries out codon optimization, carries out the recombinant expression of single-chain antibody.Expressed obtained antibody is respectively designated as resisting
Body A24 and antibody A 32, structure composition is as shown in Fig. 2.The recombinant expression of above-mentioned single-chain antibody has as follows:
1. the expression plasmid of antigen-4 fusion protein gene constructs
The gene order of antibody A 24 after codon optimization is as shown in SEQ ID NO:19, amino acid sequence such as SEQ ID
Shown in NO:17；The nucleotide sequence of antibody A 32 after codon optimization is as shown in SEQ ID NO:20, amino acid sequence such as SEQ
Shown in ID NO:18.The segment upstream of antibody A 24 and the synthesis of A32 full genome after optimization is introduced into XhoI in pPICZ α A carrier
DNA sequence dna after sequence, downstream introduce XbaI enzyme cutting site, are building up to pMD19-T Simple Vector plasmid and (are purchased from
Invitrogen company) in, a kind of long-term preservation plasmid is obtained, plasmid is denoted as pMD19-A24, pMD19-A32.Carry out PCR expansion
Increase, wherein upstream primer P1 is CGCCAGGGTTTTCCCAGTCAC GAC；Downstream primer P2 are as follows:
AGCGGATAACAATTTCACACAGGA.After Standard PCR program, agarose gel electrophoresis analyzes (attached drawing 3), shows two kinds of products
Size and expected size (790bp, 800bp) are consistent.By PCR obtain gene product recovery purifying after, using XhoI (#R0146S,
Purchased from New England Biolabs company) and XbaI (#R0145V is purchased from New England Biolabs company) double enzymes
It cuts, is connected in pPICZ α A (V19520 is purchased from Invitrogen) plasmid with T4 ligase, is transformed into DH5 α competent cell
In, 37 DEG C of overnight incubations in the LB plate containing Zeocin (R250-01 is purchased from Invitrogen company).It screens within second day
Positive colony bacterium sequencing, compares, completely the same to get to the expression plasmid of antibody A 24 and A32 with expected sequence, is denoted as respectively
pPICZα-A24、pPICZα-A24。
2. antigen-4 fusion protein gene is in the building, screening and expression of Pichia pastoris host's engineered strain
Pichia pastoris competent cell, YPDS solid medium, BMGY culture medium, BMMY culture medium: it is purchased from
Invitrogen company.
By pPICZ α-A24 and pPICZ α-A32 plasmid, linearized with SacI digestion with restriction enzyme.After ethanol precipitation
By linearized vector, electrotransformation enters X-33 competence yeast cells respectively, and it is solid to be respectively coated the YPDS containing Zeocin
Body culture medium, 30 DEG C are cultivated 3-5 days, just there is positive colony generation.
The monoclonal of the above-mentioned acquisition of picking is in 5mL BMGY culture medium, 30 DEG C of cultures to OD600When=2.0~6.0, take
1mL saves strain, and will be transferred to BMMY Small Amount inducing expression after the resuspension of remaining bacterium solution, dense to end every adding methanol for 24 hours
Degree is 1% (v/v).After a week, supernatant of bacteria solution is collected by centrifugation, passes through PAGE gel electrophoresis and Western blot analysis
(Western blot), object observing protein expression situation (attached drawing 4).Primary antibody is anti-HIS-Tag antibody in Western blot
(His-Tag (2A8) Mouse mAb, M20001, be purchased from Ai Bimate biological medicine (Shanghai) Co., Ltd.).
A24 the and A32 recombination fusion protein engineering strain of above-mentioned acquisition is inoculated in respectively in BMGY culture medium, 30
DEG C, 220rpm is cultivated to cell density to OD600=2.0~6.0, methanol was added every 24 hours to final concentration of 1.0% (v/
v).After a week, fermentation culture is collected.
3. fusion protein purification
Using 32 fusion protein of histidine tag affinity column antibody purification A24 and antibody A, pre-installs pillar and be selected as
HisTrap HP, the specific steps are as follows:
(1) the removal of impurities pretreatment of fermentation liquid: obtaining antibody A 24 and A32 fusion protein fermented liquid supernatant for above-mentioned expression, from
The heart collects supernatant, and combination buffer is added, so that supernatant final concentration of 300mM NaCl, 20mM NaH2PO4,
10mMImidazole adjusts pH7.5,0.45 μm of membrane filtration.
(2) the affine column purification of HisTrap HP: with fully-automatic intelligent protein purification system, (AKTA avant150, is purchased from
GE healcare company) affinity purification, pillar are carried out to the antibody A 24 and 32 fusion protein fermentation liquid of antibody A of pretreatment acquisition
For HisTrap HP (17-5248-02 is purchased from GE healcare company).Combination buffer is 300mM NaCl, 20mM
NaH2PO4, 10mM Imidazole, pH7.5, elution buffer is 300mM NaCl, 20mM NaH2PO4, 500mM
Imidazole, pH7.5.Linear elution is carried out when elution, and collects each eluting peak.By SDS-PAGE electroresis appraisal purity,
By attached drawing 5 it is found that two kinds of purity of protein after purification reach 95% or more；Merge satisfactory collecting pipe, replacement buffering
Liquid is PBS solution and is concentrated by ultrafiltration (1mg/ml) that filtration sterilization is saved backup in -20 DEG C.
As known to those skilled in the art, recombinant protein can not tape label, other shapes can also can also be added with other labels
The link peptide of formula.Regardless of whether tape label or with various forms of labels all can be used Capto L purifying.
The performance evaluation of 4. antibody of embodiment
1. the Western blot of antibody A 24 and A32 are identified
A. polyacrylamide gel electrophoresis: 12% separation gel of configuration, 5% concentration glue, respectively loading standard protein, You Rui
Crin (UK), Pichia anomala expression hK1, Bacillus coli expression hK1 and CHO system expression hK1, electrophoresis 1 hour under constant pressure；
B. transferring film: transferring film 1 hour under the conditions of constant current (35mA/ film), by the protein on two blocks of polyacrylamide gels point
It is not transferred on two nitrocellulose filters.Coomassie brilliant blue G250 dyes the SDS-PAGE glue for completing transferring film, observes
The residual condition of albumen；
C. close: the TBST buffer blind (confining liquid) containing 5% defatted milk, 4 DEG C overnight；Cleaning solution after closing (TBST,
It is detailed in TaKaRa company's T BST buffer) it washed once, 10 minutes；
D. antigen-antibody reaction: confining liquid dilution (pressing 1:400 volume ratio) horseradish peroxidase-labeled A24 (A24-
HRP, 1mg/mL, our company is using classical Over-voltage protection label, similarly hereinafter) and horseradish peroxidase-labeled A32 (A32-HRP,
1mg/mL, our company is using classical Over-voltage protection label, similarly hereinafter), it is separately added into above-mentioned two nitrocellulose filters, room temperature
Reaction 1 hour；TBST is washed 5 times, every time 10 minutes；
E. it develops the color and takes pictures: blotting residual liquid on nitrocellulose filter, it is steady that every nitrocellulose filter is separately added into 2mL
Sizing Peroxidase Solution (1mL) and luminol/enhancing agent solution (1mL) mixed liquor (purchase is in Thermo company),
The surface of even wetting nitrocellulose filter, room temperature are clapped after being protected from light one minute in gel imaging system (purchase is in GE company)
According to leaving and taking result.
Experimental result (attached drawing 6) shows that two kinds of antibody of the invention can be reacted with the hK1 in four kinds of sources, it was demonstrated that this hair
The reactivity of bright two kinds of antibody, and prove that it has preferable react with natural hK1.In addition, experimental result is as it can be seen that four kinds of sources
The molecular weight of hK1 is consistent with theoretical value: UK is natural hK1, degree of glycosylation highest, therefore its molecular weight is maximum；Escherichia coli
It is minimum to express hK1 degree of glycosylation, therefore molecular weight is minimum；CHO system expression hK1 degree of glycosylation is slightly less than native protein but height
In Yeast expression hK1, therefore its molecular weight is between UK and Yeast expression hK1.
2. antibody A 24 and A32 are in the performance evaluation of ELISA detection platform
Antibody in above-mentioned preparation example is subjected to combinations of pairs, carries out pairing detection respectively as coated antibody or labelled antibody
Standard items, detecting step are as follows:
1) using the PB buffer of 20mM PH7.4, by Y0, (irrelevant single-chain antibody, as negative control, preparation method is joined
Add applicant's earlier application: 201410020156.6), A24 or A32 (as coated antibody) be diluted to 8ug/mL respectively, dispense
Into ELISA Plate (hole 100uL/), 4 DEG C, overnight (16 hours)；
2) PBST (the 20mM PB7.4 containing 0.05%Tween-20, similarly hereinafter) is patted dry after washed once (hole 200uL/)；
3) confining liquid (PBST for being 2%BSA containing mass volume ratio) closing (hole 200uL/), 37 DEG C, 2 hours；Discard envelope
It is patted dry after closing liquid；
4) Sample dilution dilution standard product, until concentration (ng/mL) is 200,100 and 0.Above-mentioned strength solution is added respectively
Enter in the ELISA Plate for being coated with A24 and A32 obtained to step 3) (hole 100uL/), 37 DEG C are reacted 1 hour；PBST is washed 5 times
It is patted dry after (hole 200uL/)；
5) (anti-as label according to 1:2000 volume ratio dilution HRP label A24 (A24-HRP) or A32 (A32-HRP)
Body)；Diluted A32-HRP or A24-HRP is separately added into the obtained ELISA Plate of step 4), 37 DEG C are reacted 30 minutes；PBST is washed
It is patted dry after washing 5 times；
6) TMB developing solution (purchase is in Huzhou Ying Chuan company) is added, the hole 100uL/, 37 DEG C are reacted 15 minutes；
7) 2M H is added2SO4OD450 is read in microplate reader (purchase is in Thermo company) immediately in terminate liquid, the hole 50uL/.
The result is as follows:
From the above results, it is flat to can be applied to enzyme linked immunosorbent detection for the double-antibody sandwich detection system of A32, A24 composition
Platform, and two kinds of pairings of A24 (coating)-A32 (label), A32 (coating)-A24 (label) have preferable detection effect.With non-phase
Antibody is closed without nonspecific reaction.
3. antibody A 24 and A32 are in the evaluation of colloidal gold detection platform
Be 200ng/ml, 100ng/ml with Sample dilution dilution standard product to concentration, by the two concentration and
The PBS of 0.025mol/L pH7.5 adds 50uL (A24 coating-A32 label or A32 coating-into colloidal-gold detecting-card respectively
A24 label, specific preparation is referring to embodiment 6), it will test card in 10-15min and be placed on readout instrument and detected.Testing result
It is as follows:
The above results as it can be seen that A32, A24 composition double-antibody sandwich detection system can be applied to colloidal gold detection platform, and
Two kinds of pairings of A24 (coating)-A32 (label), A32 (coating)-A24 (label) have preferable detection effect.
4. antibody A 24 and A32 are in the evaluation of time-resolved fluorescence detection platform
A24 or A32 are diluted to 1mg/ml with the PBS of 0.025mol/L pH7.5, lined on nitrocellulose filter；With
The A32 or A24 that the borate buffer of 0.05mol/L pH8.0 marks time-resolved fluorescence microballoon dilute 20 times, and specking is in knot
It closes on pad；By pad pasting shown in attached drawing 9, cut, be loaded (specific preparation is referring to embodiment 7).Will test card, detectable concentration contains respectively
Amount is the PBS of 200, the standard items of 100ng/ml and 0.025mol/L pH7.5, and testing result is as follows:
From the above results, the double-antibody sandwich detection system of A32, A24 composition can be applied to time-resolved fluorescence inspection
Platform is surveyed, and two kinds of pairings of A24 (coating)-A32 (label), A32 (coating)-A24 (label) have preferable detection effect.
The application of 5. antibody A 24 of embodiment and A32 in enzyme-linked immune quantitative detection reagent box
This detection method uses the principle of double antibody sandwich method.
1. material:
It is coated with buffer: the PB buffer of 0.02M PH7.4；Confining liquid: containing 2%BSA (mass volume ratio, similarly hereinafter)
PBST buffer；Cleaning solution: PBST buffer；Sample dilution: containing 1%BSA, 0.5% casein, 5% lowlenthal serum,
The phosphate buffer (PH7.4) of 0.02%Procline 300 and 0.05%Tween-20；Standard items: CHO system expression weight
Group hK1 (1mg/mL, preparation method are shown in patent 201310746269.X)；Developing solution: TMB (purchase is in Huzhou Ying Chuan company)；Eventually
Only liquid: 2M H2SO4。
2. instrument:
Microplate reader (purchase is in Thermo company), universal microbiological incubator (purchase is in Thermo company), automatically
Board-washing machine (purchase is in Shenzhen Hui Song company)
3. method:
(1) preparation of detection plate: antibody A 24 is diluted to 8ug/mL by coating buffer, and the hole 100uL/ is added to ELISA Plate
In, 4 DEG C stand overnight (16 hours)；PBST is patted dry after washed once；The hole 200uL/ confining liquid closing, after 37 DEG C are placed 2 hours
It pats dry, drains overnight (16 hours), aluminium foil bag vacuum packaging, 4 DEG C save backup；
(2) preparation of Sample dilution: contain 1%BSA, 0.5% casein, 5% lowlenthal serum, 0.02%Procline
The phosphate buffer (20mM, PH7.4) of 300 and 0.05%Tween-20, according to mentioned component proportional arrangement Sample dilution,
It is saved backup in 4 DEG C；
(3) preparation of standard items: 10uL standard items are added into 19.99mL Sample dilution, in 4 DEG C after mixing well
It saves backup；
(4) it detects the preparation of liquid: horseradish peroxidase (HRP) is coupled to antibody A 32 (A32-HRP, 1mg/ml)；It will
5uL A32-HRP is added into the Sample dilution of 9.995mL, saves backup in 4 DEG C after mixing well
4. the application method of people's kininogenase enzyme-linked immunologic detecting kit
(1) vacuum-packed detection ELISA Plate is taken out in 4 DEG C and related reagent is spare after equilibrium at room temperature；
(2) dilution of standard items: 10uL standard items are added into 490uL Sample dilution, after mixing well to obtain the final product
10ng/mL standard solution；By 10ng/mL standard solution doubling dilution, obtain respectively final concentration (ng/mL) be 5,2.5,
1.25,0.625,0.3125,0.15625 and 0.078125 standard solution；Using Sample dilution as negative control, i.e. 0ng/
mL；
(3) Sample dilution in the hole 50uL/ is separately added into ELISA Plate；It is right that standard items, feminine gender is added into ELISA Plate again
According to and sample to be examined (hole 50ul/)；37 DEG C are placed 60 minutes；PBST is patted dry after washing 5 times；
(4) detection liquid is added in detection hole, the hole 100uL/, 37 DEG C are placed 30 minutes；PBST is patted dry after washing 5 times；
(5) developing solution is added in detection hole, the hole 100uL/, 37 DEG C are placed 15 minutes；
(6) terminate liquid is added in detection hole, OD450 is read in microplate reader immediately in the hole 50uL/.
According to the corresponding OD450 readings of each concentration of standard items, use 1.3 software of CurveExpert with concentration for horizontal seat
Mark, OD value are the drafting that ordinate carries out standard curve, obtain calibration curve equation；The OD value that will test sample substitutes into standard song
The concentration (ng/mL) of sample to be examined is calculated after line equation.
5. detection performance is evaluated:
The performance evaluation of detection method of the present invention is carried out according to above-mentioned testing conditions.
(1) TIANZHU XINGNAO Capsul:
The rate of recovery is to verify the index of detection accuracy, and the present invention adds the detection kit based on A24 and A32 then
The verifying of add-back yield.
Pooled plasma is obtained after 20 parts of blood plasma are mixed, using the hK1 content in method measurement pooled plasma B of the invention
For C0；The standard items (A liquid) that concentration is respectively 5ng/mL, 2.5ng/mL, 1.25ng/mL and 0.625ng/mL are added to mixed
It closes in blood plasma B, the volume ratio between A liquid and blood plasma B be added is 1:9, and the recycling of each additive amount is calculated according to formula (1)
Rate.
In formula: R is the rate of recovery；
V is the volume that A liquid is added；
V0For the volume of pooled plasma sample B；
C is the detectable concentration that pooled plasma sample B is added after A liquid；
C0For the detectable concentration of plasma sample B；
CSFor the concentration of A liquid.
Examination criteria curve (attached drawing 7) of the present invention is as it can be seen that the detection has good linear relationship (r=0.99) and has
Highly sensitive (156pg/mL) and negative background values (OD=0.0525).According to above-mentioned TIANZHU XINGNAO Capsul calculation method, knot is calculated
Fruit see the table below:
In table as it can be seen that aforementioned four concentration TIANZHU XINGNAO Capsul average value be 101.17%, each concentration TIANZHU XINGNAO Capsul with return
Yield mean value difference is less than or equal to 8.29%, it was demonstrated that this detection method accuracy is high.
(2) specific:
It compares through protein sequence as it can be seen that people's kininogenase organizes kassinin kinin to discharge with the people in human tissue kallikrein family
The homology of enzyme 2 (KLK2) and human tissue kallikrein 3 (KLK3) respectively reaches 66% and 60%, this experiment then is mainly investigated
Detection specificity of the detection method established by the present invention to KLK2 and KLK3.Detection method is as described above, be separately added into sample
Urinary kallidinogenase (UK) after dilution gradient dilution, KLK2 (purchase is in R&D company) and KLK3 (purchase is in R&D company), finally
Detect the specificity of the detection method.Testing result (attached drawing 8) is as it can be seen that people's kininogenase enzyme linked immunological of the present invention is quantitative
Detection method specificity is good.
(3) repeated
Three parts of representative plasma samples are repeated into detection 10 times respectively, calculate the average value M of 10 measurement results
With standard deviation SD, coefficient of variation CV is obtained according to formula CV=SD/M × 100%, as a result as follows:
For the above results as it can be seen that three parts of blood plasma detect CV < 10%, ELISA detection kit of the present invention has preferable repeat
Property.
6. the present invention attempts plurality of reagents box preparation method, following table shows that several different preparation methods are (unlisted in table
Step, parameter are equal described in the present embodiment) and corresponding reagent box detection performance.
The preparation of the colloid gold immune detection card of the anti-human kininogenase of embodiment 6.
1. the label of colloidal gold
The colloid gold label of antibody A 32: K is used2CO3It adjusts colloidal gold pH value and (5uL 0.2M is added in every 1ml colloidal gold
K2CO3), it is slowly added to (it is anti-that 30ug be added in every 1ml colloidal gold with the diluted antibody A 32 of the PBS of 0.2M into colloidal gold solution
Body A32), it stirs at low speed 30 minutes；Confining liquid (1%BSA) is added to its final concentration of 10% (mass percent), stirs 20 points
Clock；12000rpm is centrifuged 30min after standing 30min；Supernatant gold labeling antibody is gone to redissolve liquid 0.01M PB PH7.4+1%BSA+
0.025%Tween 20+5% sucrose) redissolve A32 antibody up to colloid gold label.
2. gold-labelled pad and reaction film preparation
After A32 gold labeling antibody is diluted well, it is sprayed in gold-labelled pad 6, drying for standby；Antibody A 24 and anti-His label are resisted
After body uses coated antibody dilution (3% methanol+25mM PBS buffer solution (pH7.5)) dilution good respectively, it is coated on reaction respectively
The T line 5 of film 2 (nitrocellulose filter), 4 position of C line are spare after dry.
3. pad pasting cuts film, assembling
Sample pad 1, gold-labelled pad 6, the nitrocellulose filter 2 for being coated with antibody, water absorption pad 3 are set gradually from left to right
(as shown in Figure 9), and should contact a little between any two, the T line 5 of the nitrocellulose filter for being coated with antibody is in left, C line 4
It is cut on the right side, and according to shell size, is packed into shell, it is standby to complete detection blocking.
4. kit assembles
Assembled detection is blocked, desiccant, dropper is fitted into aluminium foil bag, heat sealing machine sealing, labelling.
5. the application method of the colloid gold immune detection card of anti-human kininogenase
1) Sample dilution (the PB buffer of 10mM PH7.4) restores to room temperature, and concussion mixes spare；
2) it detects Sample Dilution: 1mL Sample dilution, then accurate absorption 10 μ L serum/blood is added in clean centrifuge tube
Sample is starched, is added in centrifuge tube, oscillation mixes well.
3) sample-adding and interpretation: the sample after drawing 50 μ L dilution with liquid-transfering gun is slowly added into well, beginning timing, and 10
It will test card in~15 minutes and be placed on readout instrument and detected, instrument will be scanned detection card, and will as the result is shown
On the screen of readout instrument.Determine more than 15min clock, as a result in vain.
6. the colloid gold immune detection card detection effect assessment of anti-human kininogenase
1) range of linearity detects
The standard items (0.125,0.25,0.5,1,2,4,8,16ng/ml) of various concentration are measured, standard curve, line are drawn
Property detection range, as a result as shown in Fig. 10.Detection card sensitivity is 0.125ng/ml.
2) repeatability detection
Same batch detection card carries out repeating to detect 10 times to 1ng/ml, 4ng/ml sample, calculates coefficient of variation CV,
CV=standard deviation SD/ average M × 100%
| Concentration (ng/ml) | 1 | 4 |
| CV | 5.4% | 8.9% |
3) accuracy detection (TIANZHU XINGNAO Capsul)
The ability that pure analyte is added for assessing the detection kit Accurate Determining.It is identical that pooled serum is divided into volume
3 parts, standard items are added in wherein 2 parts, 5ng/ml, the recycling sample of 1.25ng/ml concentration, in another sample is made
The PB buffer of the PH7.4 without measured object of same amount is added, basic sample is made.
| Concentration (ng/ml) | 1.25 | 5 |
| The rate of recovery | 92.3% | 89% |
7. in addition to above-mentioned optimal preparation method, the present invention has also been attempted a variety of preparation methods, such as prepared by 4 kinds of following table
Example:
The preparation of the time-resolved fluoroimmunoassay detection card of 7. people's kininogenase of embodiment
This detection method uses the principle of double-antibody sandwich immunochromatographic method.
1, solution is prepared
Prepared by the borate buffer of 0.05mol/L pH8.0: taking the H of 0.1mol/L3BO370ml, with 0.025mol/L's
Na2B4O7·10H2O adjusts pH to 8.0, and is settled to 100ml, be placed in 4 DEG C it is spare, validity period 3 months.
1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride (EDC is purchased from SIGMA company) solution preparation:
1.5gEDC be added 100ml deionized water, be made into aqueous solution be placed in 4 DEG C it is spare, validity period 3 months.
The preparation of confining liquid: (percentage is mass volume ratio) containing 10%BSA, the boron of 0.05mol/L pH8.0
Acid buffer, with 0.22um membrane filtration, be placed in 4 DEG C it is spare, validity period 7 days.
Fluorescence antibody dilution preparation: contain 1%BSA, 10% sucrose, 0.025% Tween-20,0.05mol/L pH8.0's
Borate buffer, with 0.22um film filter, be placed in 4 DEG C it is spare, validity period 7 days.
The preparation of coated antibody dilution: contain 1% sucrose, the PBS of 3% methanol 0.025mol/L pH7.5, with 0.22U film mistake
Filter, be placed in 4 DEG C it is spare, validity period 7 days.
Sample dilution: contain 0.1%BSA, 0.025% Tween-20,0.01% antipyrine, 0.02%proclin300,
The borate buffer of 0.05mol/L pH8.0,10-30 DEG C preservation validity period 1 year.
2, people's kininogenase fluorescence detection blocking is standby
1) time-resolved fluorescence microballoon marks
A32 antibody labeling method is following (by taking 500uL reaction system as an example): 400uL borate buffer solution being added to be centrifuged in 2ml
The unloaded fluorescent microsphere (being purchased from Thermo company) of 1% partial size 200nm of 100uL concentration is added in Guan Zhong, and vortex oscillation mixes.Again
10uLEDC solution, shaken at room temperature 15min is added.10 DEG C of centrifugation 10min of 14000rpm, remove supernatant, and precipitating is slow with 0.5ml boric acid
Fliud flushing dissolution, ultrasonic disperse, power 100W, time 1min (ultrasonic 3s interval 3s).
The A32 antibody 50uL of concentration 1mg/ml is added in microballoon after activation, 250r/min20 DEG C of isothermal vibration reacts 2h；
55uL confining liquid is added, isothermal vibration reacts 4h.10 DEG C of centrifugation 15min of 14000rpm are washed 2 times, remove supernatant, and precipitating is used
The dissolution of 0.5ml borate buffer, last time centrifuged deposit is dissolved with fluorescence antibody dilution and ultrasonic disperse, is placed in 4 DEG C
Constant temperature saves.
2) specking of fluorescence bonding pad
A32 antibody fluorescence bonding pad specking method is as follows: with fluorescence antibody dilution by the A32 fluorescence antibody of above-mentioned preparation
20 times of dilution, specking is on whole bonding pad.
3) coating of nitrocellulose filter
Nitrocellulose filter method for coating is as follows: taking the A24 antibody of 0.5ml concentration 4mg/ml, is added to 5ml graduated centrifuge tube
In, add coated antibody dilution to 1ml, is coated in 5 position of T line of nitrocellulose filter 2.Take 0.5ml concentration anti-for 4mg/ml
HIS antibody, is added in centrifuge tube, adds coated antibody dilution to 1ml, is coated in 4 position of C line of nitrocellulose filter 2.
4) pad pasting, cut, be loaded
Sample pad 1, fluorescence bonding pad 6, the nitrocellulose filter (reaction film) 2 for being coated with A24 antibody, by water absorption pad 3 from
Left-to-right is set gradually, and a little contact between any two, and the T line 5 of the nitrocellulose filter is in left, C line 4 in right (such as attached drawing 9
It is shown), and cut according to the size that gets stuck, loading is got stuck, and it is standby to complete detection blocking.
5) kit assembles
Take aluminium foil bag and desiccant；Open heat sealing machine, preheating；It will test card, desiccant is fitted into aluminium foil bag；Use heat sealing machine
Seal aluminium foil bag；It is labelled.
Meanwhile applicant is also used A24 antibody to do fluorescent marker, is coated on nitrocellulose filter (reaction using A32 antibody
Film) at 2T line, it is standby to carry out fluorescence detection blocking for remaining step, parameter constant.
3, the application method of people's kininogenase fluorescence detection card
1) it dilutes sample to be tested: 1mL Sample dilution, then accurate absorption 10 μ L serum/blood is added in clean centrifuge tube
Sample is starched, is added in centrifuge tube, oscillation mixes well.
2) sample-adding and interpretation: the sample after drawing 50 μ L dilution with liquid-transfering gun is slowly added into well, beginning timing, and 10
Result is quantitatively judged with fluorescence immune chromatography instrument in~15 minutes.Determined more than 15 minutes, as a result in vain.
4, people's kininogenase fluorescence detection card detection effect is assessed
1) A32 (coating)-A24 (label) accuracy: is detected to each 25 weights of standard items of card detection 0.25,1,4ng/ml
Repetition measurement is fixed, calculates detection card precision after rejecting outlier.Experimental result show three Concentration Testing result coefficient of variation CV <
15%.
| Concentration point (ng/ml) | 0.25 | 1 | 4 |
| CV | 14% | 13% | 9% |
A24 (coating)-A32 (label) is detected into card and repeats above-mentioned experiment, as a result such as following table,
| Concentration point (ng/ml) | 0.25 | 1 | 4 |
| CV | 18% | 16% | 14% |
2) detection is linear: A32 (coating)-A24 (label) being detected to the standard items of card detection various concentration: 0.0625,
0.125,0.25,0.5,1,2,4,8,16,32ng/ml, standard curve and linear relationship (such as attached drawing 11).Detection card detection is sensitive
Degree is 0.0625ng/ml.
It is 0.0625ng/ml that A24 (coating)-A32 (label), which detects card sensitivity,.
3) accuracy-rate of recovery: it is respectively 1,5,20ng/ that A32 (coating)-A24 (label), which is detected card detection additive amount,
The standard items of ml, testing result such as following table.
| Concentration point (ng/ml) | 1 | 5 | 20 |
| The rate of recovery | 112% | 94% | 104% |
A24 (coating)-A32 (label) is detected into card and repeats above-mentioned experiment, as a result such as following table,
| Concentration point (ng/ml) | 1 | 5 | 20 |
| The rate of recovery | 116% | 108% | 89% |
5, accuracy methodology compares:
The above results show that the detection card performance of A32 (coating)-A24 (label) is more excellent, therefore are made methodology comparison
Verifying.20 parts of clinical patient samples are selected, by 1 to 20 serial number, with ELISA (preparation of embodiment 5) and fluorescence detection
It is tested, is measured according to 1,2,3......18,19,20,20,19,18......3,2,1 sample order simultaneously.
The coefficient R of ELISA and fluorescence detection testing result2=0.98, illustrate fluorescence detection and ELISA testing result have compared with
Good correlation (such as attached drawing 12).
6, the comparison being formulated:
In addition to above-mentioned optimal preparation example 1, applicant also attempts a variety of preparation methods, for example, 5 groups of detection blockings below it is standby and
Using the result is as follows:
In this description, the present invention is described with reference to its specific embodiment.But it is clear that can still make
Various modifications and alterations are without departing from the spirit and scope of the invention.Therefore, the description and the appended drawings should be considered as illustrative
And not restrictive.
Claims (8)
1. anti-human kininogenase antibody A 24, it is characterized in that the amino acid sequence of heavy chain variable region is as shown in SEQ ID NO:7, gently
The amino acid sequence of chain variable region is as shown in SEQ ID NO:8.
2. anti-human kininogenase antibody A 24 as described in claim 1, it is characterized in that amino acid sequence such as SEQ ID NO:17 institute
Show.
It is made of 3. not contained in the amino acid sequence of anti-human kininogenase antibody A 24 as claimed in claim 2 six histidines
HIS label.
4. the nucleotide sequence of antibody described in claim 2 is encoded, as shown in SEQ ID NO:19.
5. the expression vector containing nucleotide sequence described in claim 4.
6. the recombinant host cell containing expression vector described in claim 5.
7. the method for producing antibody described in claim 2, comprising:
1) recombinant host cell described in claim 6 is cultivated under suitable conditions express antibody；
2) it then purified from host cell, collect antibody.
8. the anti-human kininogenase antibody A 24 of any one of claim 1-2 is in the kit of preparation detection people's kininogenase content
Application.
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