CN107383198A

CN107383198A - A kind of anti-human CysC antibody and its application

Info

Publication number: CN107383198A
Application number: CN201710784623.6A
Authority: CN
Inventors: 马永; 赵利利; 王安良
Original assignee: ZONHON BIOPHARMA INSTITUTE Inc
Current assignee: ZONHON BIOPHARMA INSTITUTE Inc
Priority date: 2017-09-04
Filing date: 2017-09-04
Publication date: 2017-11-24
Anticipated expiration: 2037-09-04
Also published as: CN107383198B

Abstract

The present invention relates to new anti-human bladder chalone C antibody and its application, belong to immunochemistry field.The present invention is prepared for Multiple Antibodies, and carries out pairing screening, obtains the Antibody Combination (C12 and C14) of sensitivity and specificity energy meet demand；Its convenient a large amount of 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, obtains easy to operate, sensitivity, specificity and coherent detection performance can meet the colloidal gold fast detecting test paper card of the human cystatin C of people's clinical sample detection.

Description

A kind of anti-human CysC antibody and its application

Technical field

The invention belongs to immunochemistry field, is specifically related to two kinds of anti-human bladder chalone C (CysC) antibody and its preparation side The application of method and above-mentioned antibody in human cystatin C detection.

Background technology

Bladder chalone C (Cystatin C, CysC) is made up of, molecular weight 13.3KD 122 amino acid, is a kind of half Guang ammonia Pepsin inhibitor, also referred to as γ-trace of albumin or γ-postglobulin.CysC is produced and deposited with constant speed in vivo In various body fluid, especially using content in cerebrospinal fluid and refining as height.CysC in circulation is only eliminated through glomerular filtration, And in proximal convoluted tubule reabsorption, but blood is not returned to by complete metabolic breakdown after reabsorption, therefore, its blood level is small by kidney Ball filtration rate is determined independent of the influence of any foeign element (such as sex, age and diet), is a kind of reflection glomerulus The ideal marker of filtration rate change.The kidneys such as serum creatinine (Scr), urea (Urea), endogenous creatinine clearance rate (Ccr) are removed Rate index is by many kidney other factors, and such as age, sex, height, muscle mass, diet structure, medicine and renal tubule are to creatinine Secretion influence, and CysC is not influenceed by above-mentioned factor.Research shows, after blood CysC reference concentrations are one-year-old it is stable into People is horizontal, until threescore or so, and asexuality difference.Also there are some researches show compared with other indexs, CysC detects glycosuria The sensitivity of sick nephrosis is 40%, and specificity is 100%, therefore can pass through CysC concentration in periodic detection diabetic's blood Change to see whether the generation of diabetic microvascular complication.In addition, in serum CysC room temperatures can stable in two days, 0 DEG C to 20 It DEG C can stablize 7 days, can stablize 6 months at -80 DEG C, and multigelation does not make significant difference to measured value.To sum up various indexs cause CysC turns into the important sensitive indicator of reflection glomerular filtration situation.

In view of superiority of the CysC in terms of glomerular filtration rate(GFR is reflected, prepares CysC antibody and is quantified for preparing CysC Detection kit has important clinical value.Collaurum quantitative assay is easy to use easy to spread and cost is relatively low, The quantitative detection of disease indicators can be met, be suitable for the real-time test of primary care system.

The content of the invention

The technical problem to be solved in the present invention is to provide can effective, specific binding human cystatin C antibody.More specifically Say on ground：

The first object of the present invention is to provide two kinds of anti-human bladder chalone C antibody.

The first anti-human bladder chalone C antibody (C12),

Its weight chain variable district contains following complementary determining region：Amino acid sequence such as sequence SEQ ID NO：Shown in 1 HCDR1, such as sequence SEQ ID NO：HCDR2 and/or such as sequence SEQ ID NO shown in 2：HCDR3 shown in 3；

And its light-chain variable sequence contains following complementary determining region：Amino acid sequence such as sequence SEQ ID NO：4 Shown LCDR1, such as sequence SEQ ID NO：LCDR2 and/or such as sequence SEQ ID NO shown in 5：LCDR3 shown in 6.

Preferable HCDR1~HCDR3, the nucleotide sequence such as sequence SEQ ID NO of LCDR1~3：Shown in 17~22.

The amino acid sequence such as SEQ ID NO of C12 heavy chain of antibody variable region preferably in the present invention:Shown in 7, light chain The amino acid sequence of variable region such as SEQ ID NO:Shown in 8, weight chain variable district and light chain variable district are directly melted by connecting small peptide Close, its amino acid sequence such as SEQ ID NO:Shown in 35.

It is furthermore preferred that C12 heavy chain of antibody variable region nucleotides sequence such as SEQ ID NO:Shown in 23, the core of light chain variable district Nucleotide sequence such as SEQ ID NO:Shown in 24, weight chain variable district and light chain variable district are directly merged by connecting small peptide, its nucleosides Acid sequence such as SEQ ID NO:Shown in 36.

Second of anti-human bladder chalone C antibody (C14),

Contain following complementary determining region in its heavy chain of antibody variable region：Amino acid sequence such as sequence SEQ ID NO：Shown in 9 HCDR1, such as sequence SEQ ID NO：HCDR2 and/or such as sequence SEQ ID NO shown in 10：HCDR3 shown in 11；

And its antibody light chain variable region sequences contains following complementary determining region：Amino acid sequence such as sequence SEQ ID NO：LCDR1, such as sequence SEQ ID NO shown in 12：LCDR2 and/or such as sequence SEQ ID NO shown in 13：Shown in 14 LCDR3。

Preferable HCDR1~HCDR3, the nucleotide sequence such as sequence SEQ ID NO of LCDR1~3：Shown in 25~30.

The amino acid sequence such as SEQ ID NO of C14 heavy chain of antibody variable region preferably in the present invention:Shown in 15, antibody The amino acid sequence of light chain variable district such as SEQ ID NO:Shown in 16；Weight chain variable district and light chain variable district are straight by connecting small peptide Fusion is connect, its amino acid sequence such as SEQ ID NO:Shown in 35

It is furthermore preferred that C14 heavy chain of antibody variable region nucleotides sequence such as SEQ ID NO:Shown in 31, the core of light chain variable district Nucleotide sequence such as SEQ ID NO:Shown in 32, weight chain variable district and light chain variable district are directly merged by connecting small peptide, its nucleosides Acid sequence such as SEQ ID NO:Shown in 36.

Second purpose of the invention is to provide two kinds of single-chain antibodies, the amino acid sequence such as SEQ ID of the antibody C12 NO:Shown in 33；The amino acid sequence of the antibody C14 such as SEQ ID NO:Shown in 34.

3rd purpose of the invention is to provide the nucleotide sequence of two kinds of above-mentioned single-chain antibodies of coding, the nucleotide sequence Such as SEQ ID NO:37 is shown, such as SEQ ID NO:Shown in 38.

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 bacterium containing above-mentioned expression vector.

6th purpose of the invention is to provide a kind of method for producing above-mentioned single-chain antibody, including：

1) above-mentioned recombinant host bacterium expression antibody is cultivated under suitable conditions；

2) and then from Host Strains purify, collect antibody.

The 7th purpose of the present invention is in providing above-mentioned anti-human bladder chalone C antibody in human cystatin C content is detected Using.

The 8th purpose of the present invention is to provide one group of antibody that can be matched and detected human cystatin C to combination； The antibody is high to the detection sensitivity of combination, and specificity is good.

The 9th purpose of the present invention is to provide a kind of using the anti-human bladder chalone C antibody test human cystatin C Colloidal gold immunochromatographimethod quantitative test card, including sample absorption pad, gold standard pad, reaction film and adsorptive pads；The gold standard pad spraying There is the antibody C12 that colloid gold particle marks, have detection band and quality control band on the reaction film, detection band position is coated with antibody C14, quality control band position are coated with anti-His tag antibodies or Protein L.The preferred nitrocellulose filter of reaction film.It is described anti- The anti-His antibody of the preferred mouse of His tag antibodies.

The present invention is prepared for Multiple Antibodies, and carries out pairing screening, obtains sensitivity and specificity energy meet demand One group of Antibody Combination (C12 and C14)；Its convenient a large amount of production simultaneously, can meet the needs of larger scale clinical application in the future.To upper The debugging Optimization Work that Antibody Combination carries out detection architecture is stated, obtains easy to operate, sensitivity, specificity and coherent detection performance The colloidal gold immunochromatographimethod quantitative test card of the human cystatin C of human plasma pattern detection can be met.

Brief description of the drawings

Fig. 1 antibody M12 and M14 heavy chains and chain variable region gene electrophoretogram.Lane 1 expands for M12 weight chain variable districts PCR Increase gene, Lane 2 is that 500DNA Ladder, Lane 3 are M12 light chain variable district PCR amplification genes, and Lane 4 is M14 heavy chains Variable region PCR amplification genes, Lane 5 are M14 light chain variable district PCR amplification genes.

Fig. 2 single-chain antibody structural representations.V_HRepresent weight chain variabl area sequence, V_LRepresent light-chain variable sequence, His marks Sign as six histidines.

Fig. 3 single-chain antibodies express the agarose gel electrophoresis figure of PCR primer.Fig. 3-a are C12 gene PCR products；Fig. 3-b For C14 gene PCR products.

Fig. 4 single-chain antibody specific detections design sketch (Western Blot).Fig. 4-a are single-chain antibody C12；Fig. 4-b are Single-chain antibody C14.

Fig. 5 colloidal gold immunochromatographimethod quantitative test card result schematic diagrams of the present invention.1 it is sample pad, 2 be reaction film, 3 is Absorption pad, 4 be nature controlling line (C lines), 5 be detection line (T lines), 6 be gold standard pad, 7 be PVC sheet.

Fig. 6 .CysC detection card (CC14-CC12) matched curve figures, wherein calibration curve equation is y=-0.002x²+ 0.120x+0.066, wherein coefficient R²=0.999.

Fig. 7 .CysC detection card (CC14-CC12) range of linearity curve maps, wherein, calibration curve equation y=1.004x+ 0.103, coefficient correlation R²=0.997.

Fig. 8 in the markets obtain the bladder chalone C quantitative detecting reagent of good prestige and method for product comparison chart to be evaluated, Wherein, calibration curve equation y=1.090x-0.517, coefficient correlation R²=0.953, illustrate this kit and commercially available main flow Bladder chalone C quantitative detecting reagent testing result uniformity is preferable.

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 which bifurcated top complementary site (antigen knot bound site) specific binding target antigen immunoglobulin molecules, institute State target antigen such as protein, sugar, polynucleotides, fat, polypeptide, micromolecular compound etc..

" single-chain antibody " (scFv) refers to the weight chain variable district (V of antibody_H) and light chain variable district (V_L) 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 pliability of single-chain antibody.Connected mode can be by V_LN-terminal be connected to V_HC-terminal, Huo Zhexiang Instead.Although eliminating constant region and introducing linker, single-chain antibody still remains specificity of the antibody to 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.Shaping It is the most critical zone of target antigen and antibody binding 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.

The preparation of the anti-human bladder chalone C hybridoma cell strain of embodiment 1.

1. animal immune

It is female according to the immune BALB/c of general immune programme for children to be extracted from the bladder chalone C of human plasma (buying in HyTest companies) Property mouse (being purchased from this experimental animal Co., Ltd of Changzhou Cavan).Specific Immunity referring to《Antibody preparation using experiment with being referred to South》.Immune serum titre is tracked using indirect elisa method, serum titer highest is chosen and mouse is immunized, carry out mice spleen Cell and murine myeloma cell carry out fusion experiment.

2. cell fusion

(1) preparation of spleen cells

By immune mouse, pluck eyeball and take blood, be placed in 75% (v/v) alcohol and soak 10 minutes after disconnected cervical vertebra is put to death, Its spleen is taken out in aseptic operating platform, is placed in cell screen clothes, is fully ground cell, screen cloth is crossed, with sterile 1640 culture medium (being purchased from Gibco companies) centrifuge washing for several times after, cell is resuspended so that single cell suspension is made, and counts, it is standby.

(2) preparation of feeder cells

Female BAl BIc/c mouse one of 8~10 week old are taken, eyeball is plucked and obtains negative serum, are put to death through disconnected cervical vertebra rearmounted Soaked 10 minutes in 75% (v/v) alcohol；It is sterile to open skin of abdomen, exposure peritonaeum, about 10mL 1640HT are trained with syringe Support base (be purchased from SIGMA companies) injection mouse peritoneal, gently abdomen massage and piping and druming for several times.Draw the culture containing macrophage It is standby in base injection 20%1640HAT culture mediums；

Female BAl BIc/c mouse one of 2~3 week old are taken, is placed in 75% (v/v) alcohol and soaks after disconnected cervical vertebra is put to death 10 minutes；It is sterile to take thymus gland in cell screen clothes, grinding, cross screen cloth, obtain thymocyte be placed in it is above-mentioned containing macrophage It is standby in 20%1640HAT culture mediums.

(3) cell fusions

Murine myeloma cell strain SP2/0 of the selection in exponential phase, collects and counts.Take about 10⁸Individual above-mentioned spleen Cell and 2 × 10⁷Individual above-mentioned SP2/0 cell lines, which are added in fusion pipe, to be mixed, and 1000rpm abandons supernatant (as far as possible after centrifuging 10 minutes Abandon net), fusion pipe is put and gently rubbed back and forth on palm so that precipitation is loose.1mL preheatings are added after elder generation is slow in 60 seconds soon PEG1450 (polyethylene glycol 1450, purchased from SIGMA companies), add 1640HT culture mediums 30mL and terminate, 1000rpm centrifuges 10 points Clock, supernatant is removed, gently friction makes precipitation loose, adds in the 20% 1640HAT culture mediums that step 2 is obtained.

After above-mentioned HAT culture mediums are fully mixed, dispensed with 200 μ L/ holes into 96 porocyte culture plates, put 37 DEG C, 5% CO₂Cell culture incubator in cultivate.20%1640HAT culture mediums are replaced with 10%1640HT culture mediums after one week, are taken after 3 days Detected clearly.

3. anti-human bladder chalone C specific hybrid knurl strain screening

(1) preparation of detection plates：With CB coating buffers dilution CysC (buying in HyTest companies) to 1 μ g/mL, coating 96 Hole ELISA ELISA Plates, 100 μ L/ holes, 2~8 DEG C of coatings overnight, washed once and pat dry；Containing 2% bovine serum albumin(BSA) (Bovine Serum Albumin, BSA) PBST buffer blinds (200ul/ holes), 37 DEG C close 2 hours；Pat dry, it is standby.

(2) screening of positive colonies：The μ L/ holes of cells and supernatant 100 to be checked are added in above-mentioned detection plate, in 37 DEG C Effect was washed and patted dry after 30 minutes, is added the sheep anti-mouse igg of the HRP marks in 100 μ L/ holes, is washed after being acted on 30 minutes in 37 DEG C Wash and pat dry, add the TMB nitrite ions in 100 μ L/ holes, developed the color 15 minutes in 37 DEG C of lucifuges, 50 μ L 2M H are added per hole₂SO₄Eventually Only react, and the reading numerical values at OD450.Positive hole determines principle：OD450 values/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 line.Hybridoma cell strain M12 and M14 are respectively provided with higher potency, subsequently enter then One step carries out antibody variable sequences sequencing analysis to above-mentioned hybridoma cell strain.

The measure of the hybridoma cell strain antibody variable sequences of embodiment 2.

M12 the and M14 antibody variable sequences of above-mentioned hybridoma cell strain secretion are measured.

A.RNA extraction：Reference cell total serum IgE extraction agent box (being purchased from Roche companies) specification is to above-mentioned hybridoma Cell line M12 and M14 carry out Total RNAs extraction and carry out reverse transcription immediately；

B.RNA reverse transcriptions turn into DNA：With reference to Thermo Scientific Reverted First strand cDNA Synthesis Kit (being purchased from Thermo companies) carry out reverse transcription to the total serum IgE extracted in previous step, and cDNA is made, and freeze Be stored in -20 DEG C it is standby；

C. the PCR amplifications and recovery of variable region sequences：So that gained cDNA is template in previous step, with mouse IgG hypotype lists Clonal antibody variable region sequences universal primer is primer, enters performing PCR amplification to the variable region sequences of heavy chain and light chain, PCR is produced Thing is reclaimed through DNA glue reclaims kit (being purchased from TIANGEN companies), sees accompanying drawing 1；

D. the clone of variable region sequences and sequencing：According to cloning vector pMD18-T kit (being purchased from Takara companies) Specification, heavy chain and chain variable region gene are attached with pMD18-T carriers respectively, convert bacillus coli DH 5 alpha, picking Positive colony, Nanjing Genscript Biotechnology Co., Ltd. is transferred to be sequenced.

Sequencing obtains the antibody M12 of hybridoma cell strain secretion heavy chain of antibody variable region amino acid sequence such as SEQ ID NO:7 is shown, chain variable region amino acid sequence such as SEQ ID NO：Shown in 8.The above-mentioned sequence of Vbase2 database analysises, its is heavy The amino acid sequence of each complementary determining region of chain variable region is respectively：Such as sequence SEQ ID NO：HCDR1, such as sequence shown in 1 SEQ ID NO：HCDR2, such as sequence SEQ ID NO shown in 2：HCDR3 shown in 3；Each complementary decision of its light chain variable district The amino acid sequence in area is：Such as sequence SEQ ID NO：LCDR1, such as sequence SEQ ID NO shown in 4：LCDR2 shown in 5, such as Sequence SEQ ID NO：LCDR3 shown in 6.

Sequencing obtains the antibody M14 of hybridoma cell strain secretion heavy chain of antibody variable region amino acid sequence such as SEQ ID NO:15 is shown, chain variable region amino acid sequence such as SEQ ID NO：Shown in 16.The above-mentioned sequence of Vbase2 database analysises, its The amino acid sequence of each complementary determining region of weight chain variable district is respectively：Such as sequence SEQ ID NO：HCDR1, such as sequence shown in 9 Arrange SEQ ID NO：HCDR2, such as sequence SEQ ID NO shown in 10：HCDR3 shown in 11；Each complementation of its light chain variable district Determining the amino acid sequence in area is：Such as sequence SEQ ID NO：LCDR1, such as sequence SEQ ID NO shown in 12：Shown in 13 LCDR2, such as sequence SEQ ID NO：LCDR3 shown in 14.

The recombination expression of the single-chain antibody of embodiment 3. and purifying

According to sequencing result in embodiment 2, respectively by hybridoma cell strain M12 and M14 heavy chain of antibody and light chain variable Connection peptide (GGGGS) is added between area₃, six histidines are introduced, and the progress of its full genome is artificial synthesized, it is building up to complete red ferment The recombination expression of single-chain antibody is carried out in mother.Expressed obtained single-chain antibody is respectively designated as antibody C12 and antibody C14, its Structure composition is as shown in Figure 2.The recombination expression of above-mentioned single-chain antibody is specific as follows：

1. the expression plasmid structure of single-chain antibody gene

Artificial synthesized single-chain antibody C12 gene order such as SEQ ID NO:37 is shown, amino acid sequence such as SEQ ID NO:Shown in 33；Artificial synthesized single-chain antibody C14 nucleotide sequence such as SEQ ID NO:38 is shown, amino acid sequence such as SEQ ID NO:Shown in 34.

The fragment upstream of artificial synthesized single-chain antibody C12 and C14 full genome synthesis is introduced into XhoI sequences in pPICZ α A carriers DNA sequence dna after row, downstream introduce XbaI enzyme cutting site, are building up to pUC57 plasmids (by Nanjing Genscript Biotechnology Co., Ltd. There is provided) in, a kind of long-term preservation plasmid is obtained, plasmid is designated as pUC57-C12-scFv- (HIS)₆And pUC57-C14-scFv- (HIS)₆.Enter performing PCR amplification, wherein sense primer P1 is CGCCAGGGTTTTCCCAGTCAC GAC；Anti-sense primer P2 is: AGCGGATAACAATTTCACACAGGA.After Standard PCR program, agarose gel electrophoresis analysis (accompanying drawing 3), two kinds of products are shown Size is consistent with expected size (706bp, 725bp).By PCR obtain gene outcome recovery purifying after, using XhoI (#R0146S, Purchased from New England Biolabs companies) and XbaI (#R0145V, purchased from New England Biolabs companies) double enzymes Cut, be connected to T4 ligases in pPICZ α A (V19520, purchased from Invitrogen) plasmid, be transformed into DH5 α competent cells In, 37 DEG C of overnight incubations in the LB flat boards containing Zeocin (R250-01, purchased from Invitrogen companies).Screen within second day Positive colony bacterium is sequenced, and compares, completely the same with expected sequence, that is, obtains antibody C12 and C14 expression plasmid, be designated as respectively pPICZα-C12-scFv-(HIS)₆And pPICZ α-C14-scFv- (HIS)₆。

2. single-chain antibody gene is in the structure of Pichia pastoris host's engineered strain, screening and expression

YPDS solid mediums are prepared：With reference to Invitrogen company EasySelectPichia Expression Kit Specification；Pichia pastoris competent cell：With reference to EasySelectPichia Expression Kit specifications；BMGY is cultivated Basigamy system：With reference to Invitrogen companies Multi-Copy Pichia Expression Kit specifications；BMMY culture mediums are matched somebody with somebody System：With reference to Invitrogen companies Multi-Copy Pichia Expression Kit specifications.

By pPICZ α-C12-scFv- (HIS)₆And pPICZ α-C14-scFv- (HIS)₆Plasmid, with SacI restriction enzymes Enzyme linearization for enzyme restriction.Linearized vector, respectively electricity conversion are entered X-33 competence yeast cells, applied respectively after ethanol precipitation To the YPDS solid mediums containing Zeocin, 30 DEG C are cultivated 3-5 days cloth, just there is positive colony generation.

The monoclonal of the above-mentioned acquisition of picking is in 5mL BMGY culture mediums, 30 DEG C of cultures to OD₆₀₀When=2.0~6.0, take 1mL preserves strain, and will be transferred to BMMY Small Amount induced expressions after the resuspension of remaining bacterium solution, and it is dense to end to add methanol every 24h Spend for 1% (v/v).After one week, bacterium solution supernatant is collected by centrifugation, passes through Western blot analysis (Western blot), observation Target protein expression.In Western blot primary antibody be anti-HIS-Tag antibody (His-Tag (2A8) Mouse mAb, M20001, it is purchased from Ai Bimate biological medicines (Shanghai) Co., Ltd.).

C12 the and C14 recombination fusion proteins engineering strain of above-mentioned acquisition is inoculated in BMGY culture mediums respectively, 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 one week, fermentation culture is collected.

3. single-chain antibody purifies

Using histidine-tagged affinity column purification of single stranded antibody C12 and C14 fusion protein, prepackage pillar selection is HisTrap HP, are comprised the following steps that：

(1) the removal of impurities pretreatment of zymotic fluid：Above-mentioned expression is obtained into antibody C12 and C14 fusion protein fermented liquid supernatant, from The heart collects supernatant, and adds combination buffer so that supernatant final concentration of 300mM NaCl, 20mM NaH₂PO₄, 10mM Imidazole, adjust pH7.5,0.45 μm of membrane filtration.

(2) HisTrap HP affinity columns purify：With fully-automatic intelligent protein purification system, (AKTA avant150, are purchased from GE healcare companies) affinity purification is carried out to the antibody C12 and C14 fusion protein zymotic fluid that pretreatment obtains, pillar is HisTrap HP (17-5248-02, purchased from GE healcare companies).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 during elution, and collects each eluting peak.By SDS-PAGE electroresis appraisal purity, Merge satisfactory collecting pipe, change buffer solution and be PBS solution and be concentrated by ultrafiltration (1mg/mL) that filtration sterilization is in -20 DEG C of guarantors Deposit standby.

The performance evaluation of the single-chain antibody of embodiment 4.

1. single-chain antibody C12 and C14 Western blot identifications

A. polyacrylamide gel electrophoresis：12% separation gel, 5% concentration glue are configured, respectively loading standard protein and day Right CysC albumen (buying in HyTest companies), electrophoresis 1 hour under constant pressure；

B. transferring film：Transferring film 1 hour under the conditions of constant current (35mA/ films), by the protein on two blocks of polyacrylamide gels point It is not transferred on two nitrocellulose filters.Coomassie brilliant blue G250 dyes to the SDS-PAGE glue for completing transferring film, observes The residual condition of albumen；

C. close：TBST buffer blinds (confining liquid) containing 5% defatted milk, 4 DEG C overnight；Cleaning solution after closing (TBST, Refer to TaKaRa company's T BST buffer) it washed once, 10 minutes；

D. antigen-antibody reaction：Confining liquid dilution (presses 1:400 volume ratios) horseradish peroxidase-labeled C12 (C12- HRP, 1mg/mL, our company are marked using classical Over-voltage protection, similarly hereinafter) and horseradish peroxidase-labeled C14 (C14-HRP, 1mg/mL, our company are marked using classical Over-voltage protection, 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. develop the color and take pictures：Residual liquid on nitrocellulose filter is blotted, it is steady that every nitrocellulose filter is separately added into 2mL The mixed liquor (buying in Thermo companies) of sizing Peroxidase Solution (1mL) and luminol/enhancing agent solution (1mL), The surface of even wetting nitrocellulose filter, room temperature lucifuge is reacted one minute takes pictures after gel imaging system (buying in GE companies) (Fig. 4-a, Fig. 4-b), leaves and takes result.

Testing result is visible, and C12 and C14 have preferably specificity, can specific detection CysC albumen.

2. single-chain antibody C14 and C12 is in the evaluation of collaurum detection platform

The antibody purified in embodiment 3 is subjected to combinations of pairs, matched respectively as coated antibody or labelled antibody Bladder chalone C (CysC) is detected, detecting step is as follows：

1) C14 or C12 are diluted to 1mg/mL with antibody coating buffer, lined on nitrocellulose filter；

2) it is laid on after the C14 of colloid gold label or C12 being diluted into 3 times with 0.01M PB buffer solutions on pad；

3) pad pasting as shown in accompanying drawing 5, slitting, is loaded and (specifically prepares referring to embodiment 5)

4) CysC standard items (many red self-controls) are diluted with Sample dilution, is 7mg/L, 1mg/L to concentration, the two are dense Degree standard items and zero standard's product (i.e. Sample dilution) add 50ul (C12 coatings-C14 into colloidal-gold detecting-card respectively Mark or C14 coatings-C12 marks), detection card is placed on readout instrument after 10min and carries out reading.As a result it is as shown in the table：

From the above results, it is seen that the double antibody sandwich method that C14 forms as coated antibody, C12 as labelled antibody Detecting system can be applied to collaurum detection platform, carry out CysC detection.

The preparation of the colloid gold immune detection card of 5 anti-human bladder chalone C of embodiment

1st, solution is prepared

1) prepared by 0.01M PB buffer solutions：Weigh Na₂HPO₄·12H₂O 3.22g, NaH₂PO₄·2H₂O 0.15g, add pure Change water 1000mL, rotor is stirred to dissolving, and its pH7.4 ± 0.1,0.45um membrane filtrations are determined with pH meter.

2) prepared by confining liquid：Bovine serum albumin(BSA) (BSA) 5g is weighed, adds 0.01M PB solution (PH7.4 ± 0.1) 50mL, Rotor is stirred to dissolving.

3) prepared by antibody coating buffer：0.2mL isopropanols are taken, add 9.8mL 0.01M PB solution (PH7.4 ± 0.1), are turned Son stirring 5-10min.

4) gold labeling antibody is redissolved liquid and prepared：1g bovine serum albumin(BSA)s are weighed, 5g trehaloses, it is molten to add 100mL 0.01M PB Liquid (pH7.4 ± 0.1), rotor is stirred to dissolving, adds 25ul Tween-20, continues to stir 5-10min with rotor.

5) prepared by Sample dilution：Weigh 12.5g bovine serum albumin(BSA)s add 500mL 0.01M PB solution (PH7.4 ± 0.1), rotor is stirred to dissolving, adds 0.5mL Proclin300, continues to stir 5-10min with rotor.

2nd, prepared by human cystatin C colloidal-gold detecting-card

1) mark of collaurum

Antibody C12 colloid gold label (by taking 1mL colloidal gold solutions mark as an example)：Use K₂CO₃It is (every to adjust collaurum pH value 4ul 0.2M K are added in 1mL collaurums₂CO₃), 5-10 minutes are stirred, it is (every that antibody C12 is slowly added into colloidal gold solution 25ug antibody C12 are added in 1mL collaurums), stirring at low speed 30 minutes；Confining liquid is added to its final concentration of 10% (quality hundred Divide ratio), stir 20 minutes；12000rpm centrifuges 30min after standing 30min；Go supernatant 100ul gold labeling antibodies to redissolve liquid to redissolve Precipitate the C12 antibody for producing colloid gold label.

2) gold standard pad and reaction film preparation

After C12 gold labeling antibodies are diluted into 3 times, it is sprayed in gold standard pad 6, drying for standby；

After antibody C14 is diluted into 0.8mg/mL with antibody diluent, the T of reaction film 2 (nitrocellulose filter) is coated in The position of line 5；After anti-His tag antibodies are diluted into 0.2mg/mL with antibody diluent, (the nitrocellulose of reaction film 2 is coated in Film) the position of C lines 4, reaction film drying for standby.

3) pad pasting, cut film, assembling

Sample pad 1, gold standard pad 6, the nitrocellulose filter 2 for being coated with antibody, adsorptive pads 3 are set gradually from left to right (as shown in Figure 5), and should contact a little between any two, the T lines 5 of the nitrocellulose filter for being coated with antibody are in left, C lines 4 Cut on the right side, and according to shell size, load shell, it is standby to complete detection blocking.

4) kit assembles

The detection card that will be assembled, drier are fitted into aluminium foil bag, heat sealing machine sealing, labelling；

Sample dilution is dispensed by 1mL/ pipes, and loads valve bag, labelling according to kit specification；

According to trimmed size, by the interior bag of certain number, 1 valve bag containing Sample dilution, 1 part of specification, 1 conjunction Case marker label load in packing box, and label is posted outside packing box.

3rd, human cystatin C colloidal-gold detecting-card application method

1) outer packing is opened, detection card is taken out from sealing aluminium foil bag, is placed on flat table.

2) 2.5 μ l serum/plasma samples are drawn, adds in Sample dilution, is sufficiently mixed.

3) draw samples of the 50ul after processing to add in the well of detection card, stand 10min at room temperature.

4) detection card is put into immunochromatography quantitative analysis instrument, presses " quick detection " key and start to detect, instrument will be automatic Detection card is scanned.

5) reading/printing testing result from the display screen of immunochromatography quantitative analysis instrument.

4th, human cystatin C colloidal-gold detecting-card Detection results are assessed

1) accuracy：By C14 (coating)-C12 (mark) detections card by detection card application method detection 1,7mg/L CysC Each 10 replications of reference material, detection card precision is calculated after rejecting outlier.Experimental result shows three Concentration Testing knots Fruit coefficient of variation CV<10%.

Concentration point (mg/L)	1	7
			CV	9.43%	9.75%

2) detection range：By C14 (coating)-C12 (mark) detect card detection various concentrations CysC recombinant proteins 0.15, 0.3rd, 0.625,1.25,2.5,5,10mg/L, matched curve and detection range are 0.2-10mg/L (such as accompanying drawing 6).

3) range of linearity：High level sample and Sample dilution are configured to 5 series concentration samples according to a certain percentage, used C14 (coating)-C12 (mark) detection card detections, each pattern detection 3 times, carry out regression calculation by result and theoretical concentration, sentence Break whether linear in the concentration range.The range of linearity is 0.2-10mg/L (such as accompanying drawing 7).It is 0.2mg/ to detect card sensitivity L。

4) degree of accuracy：By C14 (coating)-C12 (mark) detections card by detection card application method detection 1,7mg/L CysC Each 3 repetitions of reference material, calculate average value and the relative deviation of theoretical value, and experimental result shows that three Concentration Testing results are relative Deviation B<5%.

Concentration point (mg/L)	1	7
			B	3.4%	4.14%

5th, the degree of accuracy-methodology compares：

Guangzhou Wondfo Biotech. Co., Ltd.'s Guang of good prestige is obtained in selection like product in the market Chalone C quantitative detecting reagents (immunochromatographic method) compare product comparison checking.30 parts of clinical patient samples are selected, by 1 to 20 Serial number, with reference product and C14 to be evaluated (coating)-C12 (mark) colloidal-gold detecting-card simultaneously tested, It is measured according to 1,2,3......28,29,30,30,29,28......3,2,1 sample order.Control and production to be evaluated The testing result coefficient R of product²=0.953, illustrate that two methods testing result has preferable correlation (such as accompanying drawing 8).

6th, recipe determination

In addition to above-mentioned optimal preparation example 1, applicant also attempts a variety of preparation schemes, for example, several groups of detection blockings below it is standby and Using result such as following table：

SEQUENCE LISTING

<110>Jiangsu Zhonghong Biopharma Institute Inc.

<120>A kind of anti-human CysC antibody and its application

<130>A kind of anti-human CysC antibody and its application

<160> 38

<170> PatentIn version 3.3

<210> 1

<211> 8

<212> PRT

<213> mouse

<400> 1

Gly Tyr Thr Phe Thr Ser Tyr Trp

1 5

<210> 2

<211> 8

<212> PRT

<213> mouse

<400> 2

Val Asp Pro Gly Ser Gly Thr Thr

1 5

<210> 3

<211> 6

<212> PRT

<213> mouse

<400> 3

Thr Ala Gly Phe Asp Tyr

1 5

<210> 4

<211> 6

<212> PRT

<213> mouse

<400> 4

Gln Asn Ile Asn Val Trp

1 5

<210> 5

<211> 3

<212> PRT

<213> mouse

<400> 5

Lys Ala Ser

1

<210> 6

<211> 9

<212> PRT

<213> mouse

<400> 6

Gln Gln Gly Gln Thr Tyr Pro Leu Thr

1 5

<210> 7

<211> 113

<212> PRT

<213> mouse

<400> 7

Gln Val Gln Leu Gln Gln Pro Gly Ser Glu Leu Val Arg Pro Gly Thr

1 5 10 15

Ser Val Lys Leu Ser Cys Arg Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Trp Met His Trp Val Lys Gln Arg His Gly Gln Gly Leu Glu Trp Leu

35 40 45

Gly Asn Val Asp Pro Gly Ser Gly Thr Thr Asn Tyr Glu Glu Asn Phe

50 55 60

Lys Thr Lys Gly Thr Leu Thr Val Asp Thr Ser Ser Ser Thr Val Tyr

65 70 75 80

Met His Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Thr Ala Gly Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser

100 105 110

Ala

<210> 8

<211> 107

<212> PRT

<213> mouse

<400> 8

Asp Ile Gln Met Asn Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Asp Thr Ile Thr Ile Thr Cys His Ala Ser Gln Asn Ile Asn Val Trp

20 25 30

Leu Gly Trp Phe Gln Gln Lys Pro Glu Asn Ile Pro Lys Leu Leu Ile

35 40 45

Tyr Lys Ala Ser Thr Leu His Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Val Phe Thr Leu Thr Ile Ser Ser Pro Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Gly Gln Thr Tyr Pro Leu

85 90 95

Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 9

<211> 8

<212> PRT

<213> mouse

<400> 9

Gly Tyr Thr Phe Thr Asp Phe Tyr

1 5

<210> 10

<211> 8

<212> PRT

<213> mouse

<400> 10

Ile Trp Pro Gly Ser Gly Asn Thr

1 5

<210> 11

<211> 11

<212> PRT

<213> mouse

<400> 11

Ala Arg Gly Thr Gly Thr Gly Tyr Phe Asp Val

1 5 10

<210> 12

<211> 7

<212> PRT

<213> mouse

<400> 12

Gln Val Gln Ile Phe Ser Asn

1 5

<210> 13

<211> 3

<212> PRT

<213> mouse

<400> 13

Ala Ala Lys

1

<210> 14

<211> 9

<212> PRT

<213> mouse

<400> 14

Gln His Phe Trp Gly Thr Pro Tyr Thr

1 5

<210> 15

<211> 118

<212> PRT

<213> mouse

<400> 15

Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Phe

20 25 30

Tyr Leu Asn Trp Val Lys Gln Arg Thr Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Trp Pro Gly Ser Gly Asn Thr Tyr Tyr Asn Glu Lys Phe

50 55 60

Lys Asp Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met His Phe Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys

85 90 95

Ala Arg Gly Thr Gly Thr Gly Tyr Phe Asp Val Trp Gly Ala Gly Thr

100 105 110

Thr Val Thr Val Ser Ala

115

<210> 16

<211> 109

<212> PRT

<213> mouse

<400> 16

Arg Trp Glu Leu Ser His Met Val Asp Leu Gln Ala Ala Ala Asn Ser

1 5 10 15

Leu Val Tyr Pro Arg Phe Thr Met Asp Phe Gln Val Gln Ile Phe Ser

20 25 30

Asn Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu

35 40 45

Val Tyr Ala Ala Lys Asn Leu Ala Asp Gly Val Pro Ser Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Gln Tyr Ser Leu Lys Ile Asn Ser Leu Gln

65 70 75 80

Ser Glu Asp Phe Gly Ser Tyr Tyr Cys Gln His Phe Trp Gly Thr Pro

85 90 95

Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg

100 105

<210> 17

<211> 24

<212> DNA

<213> mouse

<400> 17

ggctacacat tcaccagcta ctgg 24

<210> 18

<211> 24

<212> DNA

<213> mouse

<400> 18

gttgatcctg gtagcggtac tact 24

<210> 19

<211> 18

<212> DNA

<213> mouse

<400> 19

accgcggggt ttgactac 18

<210> 20

<211> 18

<212> DNA

<213> mouse

<400> 20

cagaacatta atgtttgg 18

<210> 21

<211> 9

<212> DNA

<213> mouse

<400> 21

aaggcttcc 9

<210> 22

<211> 27

<212> DNA

<213> mouse

<400> 22

caacagggtc aaacttatcc gctcacg 27

<210> 23

<211> 339

<212> DNA

<213> mouse

<400> 23

caggtccaac tgcagcaacc tgggtctgag ctggtgaggc ctggaacttc agtgaagctg 60

tcctgcaggg cttctggcta cacattcacc agctactgga tgcactgggt gaagcagagg 120

catggacaag gccttgagtg gctaggaaat gttgatcctg gtagcggtac tactaactac 180

gaggagaatt tcaagaccaa gggcacactg actgtggaca cttcctccag tacagtctac 240

atgcacctca gcagcctgac atctgaggac tctgcggtct attattgtac cgcggggttt 300

gactactggg gccaaggcac cactctcact gtctctgca 339

<210> 24

<211> 321

<212> DNA

<213> mouse

<400> 24

gacatccaga tgaaccagtc tccatccagt ctgtctgcat cccttggaga cacaattacc 60

atcacttgcc atgccagtca gaacattaat gtttggttag gctggttcca gcagaaacca 120

gaaaatattc ctaaactatt gatctataag gcttccacct tgcacacagg cgtcccatca 180

cggtttagtg gcagtggatc tggaacagtt ttcacattaa ccatcagcag cccgcagcct 240

gaagacattg ccacttacta ctgtcaacag ggtcaaactt atccgctcac gttcggtgct 300

gggaccaagt tggaaatcaa a 321

<210> 25

<211> 24

<212> DNA

<213> mouse

<400> 25

ggctacacct tcactgactt ctat 24

<210> 26

<211> 24

<212> DNA

<213> mouse

<400> 26

atttggcctg gaagtggtaa tact 24

<210> 27

<211> 33

<212> DNA

<213> mouse

<400> 27

gcaagaggga ctgggacggg gtacttcgat gtc 33

<210> 28

<211> 21

<212> DNA

<213> mouse

<400> 28

caagtgcaga ttttcagtaa t 21

<210> 29

<211> 9

<212> DNA

<213> mouse

<400> 29

gctgcaaaa 9

<210> 30

<211> 27

<212> DNA

<213> mouse

<400> 30

tgtcaacatt tttggggtac tccgtac 27

<210> 31

<211> 354

<212> DNA

<213> mouse

<400> 31

caggttcagc tgcagcagtc tggagctgag ctggcgaggc ccggggcttc agtgaagctg 60

tcctgcaagg cttctggcta caccttcact gacttctatc taaactgggt gaagcagagg 120

actggacagg gccttgagtg gattggagag atttggcctg gaagtggtaa tacttactac 180

aatgagaagt tcaaggacaa ggccacactg actgcagaca aatcctccag cacagcctac 240

atgcacttca gcagcctgac atctgaggac tctgcagtct atttctgcgc aagagggact 300

gggacggggt acttcgatgt ctggggcgca gggaccacgg tcaccgtctc cgca 354

<210> 32

<211> 324

<212> DNA

<213> mouse

<400> 32

cgttgggagc tctcccatat ggtcgacctg caggcggccg cgaattcact agtgtatcca 60

agattcacca tggattttca agtgcagatt ttcagtaatt tagcatggta tcagcagaaa 120

cagggaaaat ctcctcagct cctggtctat gctgcaaaaa acttagcaga tggtgtgcca 180

tcaaggttca gtggcagtgg atcaggcaca cagtattccc tcaagatcaa tagcctgcag 240

tctgaagatt ttgggagtta ttactgtcaa catttttggg gtactccgta cacgttcgga 300

ggggggacca agctggaaat caaa 324

<210> 33

<211> 235

<212> PRT

<213> mouse

<400> 33

Gln Val Gln Leu Gln Gln Pro Gly Ser Glu Leu Val Arg Pro Gly Thr

1 5 10 15

Ser Val Lys Leu Ser Cys Arg Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Trp Met His Trp Val Lys Gln Arg His Gly Gln Gly Leu Glu Trp Leu

35 40 45

Gly Asn Val Asp Pro Gly Ser Gly Thr Thr Asn Tyr Glu Glu Asn Phe

50 55 60

Lys Thr Lys Gly Thr Leu Thr Val Asp Thr Ser Ser Ser Thr Val Tyr

65 70 75 80

Met His Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Thr Ala Gly Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser

100 105 110

Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

115 120 125

Asp Ile Gln Met Asn Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly

130 135 140

Asp Thr Ile Thr Ile Thr Cys His Ala Ser Gln Asn Ile Asn Val Trp

145 150 155 160

Leu Gly Trp Phe Gln Gln Lys Pro Glu Asn Ile Pro Lys Leu Leu Ile

165 170 175

Tyr Lys Ala Ser Thr Leu His Thr Gly Val Pro Ser Arg Phe Ser Gly

180 185 190

Ser Gly Ser Gly Thr Val Phe Thr Leu Thr Ile Ser Ser Pro Gln Pro

195 200 205

Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Gly Gln Thr Tyr Pro Leu

210 215 220

Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile Lys

225 230 235

<210> 34

<211> 241

<212> PRT

<213> mouse

<400> 34

Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Phe

20 25 30

Tyr Leu Asn Trp Val Lys Gln Arg Thr Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Trp Pro Gly Ser Gly Asn Thr Tyr Tyr Asn Glu Lys Phe

50 55 60

Lys Asp Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met His Phe Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys

85 90 95

Ala Arg Gly Thr Gly Thr Gly Tyr Phe Asp Val Trp Gly Ala Gly Thr

100 105 110

Thr Val Thr Val Ser Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

115 120 125

Gly Gly Gly Gly Ser Arg Trp Glu Leu Ser His Met Val Asp Leu Gln

130 135 140

Ala Ala Ala Asn Ser Leu Val Tyr Pro Arg Phe Thr Met Asp Phe Gln

145 150 155 160

Val Gln Ile Phe Ser Asn Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys

165 170 175

Ser Pro Gln Leu Leu Val Tyr Ala Ala Lys Asn Leu Ala Asp Gly Val

180 185 190

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Gln Tyr Ser Leu Lys

195 200 205

Ile Asn Ser Leu Gln Ser Glu Asp Phe Gly Ser Tyr Tyr Cys Gln His

210 215 220

Phe Trp Gly Thr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile

225 230 235 240

Lys

<210> 35

<211> 15

<212> PRT

<213> mouse

<400> 35

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210> 36

<211> 45

<212> DNA

<213> mouse

<400> 36

ggtggtggtg gatccggagg tggtggttct ggtggtggtg gttct 45

<210> 37

<211> 705

<212> DNA

<213> mouse

<400> 37

caggtccaac tgcagcaacc tgggtctgag ctggtgaggc ctggaacttc agtgaagctg 60

tcctgcaggg cttctggcta cacattcacc agctactgga tgcactgggt gaagcagagg 120

catggacaag gccttgagtg gctaggaaat gttgatcctg gtagcggtac tactaactac 180

gaggagaatt tcaagaccaa gggcacactg actgtggaca cttcctccag tacagtctac 240

atgcacctca gcagcctgac atctgaggac tctgcggtct attattgtac cgcggggttt 300

gactactggg gccaaggcac cactctcact gtctctgcag gtggtggtgg atccggaggt 360

ggtggttctg gtggtggtgg ttctgacatc cagatgaacc agtctccatc cagtctgtct 420

gcatcccttg gagacacaat taccatcact tgccatgcca gtcagaacat taatgtttgg 480

ttaggctggt tccagcagaa accagaaaat attcctaaac tattgatcta taaggcttcc 540

accttgcaca caggcgtccc atcacggttt agtggcagtg gatctggaac agttttcaca 600

ttaaccatca gcagcccgca gcctgaagac attgccactt actactgtca acagggtcaa 660

acttatccgc tcacgttcgg tgctgggacc aagttggaaa tcaaa 705

<210> 38

<211> 723

<212> DNA

<213> mouse

<400> 38

caggttcagc tgcagcagtc tggagctgag ctggcgaggc ccggggcttc agtgaagctg 60

tcctgcaagg cttctggcta caccttcact gacttctatc taaactgggt gaagcagagg 120

actggacagg gccttgagtg gattggagag atttggcctg gaagtggtaa tacttactac 180

aatgagaagt tcaaggacaa ggccacactg actgcagaca aatcctccag cacagcctac 240

atgcacttca gcagcctgac atctgaggac tctgcagtct atttctgcgc aagagggact 300

gggacggggt acttcgatgt ctggggcgca gggaccacgg tcaccgtctc cgcaggtggt 360

ggtggatccg gaggtggtgg ttctggtggt ggtggttctc gttgggagct ctcccatatg 420

gtcgacctgc aggcggccgc gaattcacta gtgtatccaa gattcaccat ggattttcaa 480

gtgcagattt tcagtaattt agcatggtat cagcagaaac agggaaaatc tcctcagctc 540

ctggtctatg ctgcaaaaaa cttagcagat ggtgtgccat caaggttcag tggcagtgga 600

tcaggcacac agtattccct caagatcaat agcctgcagt ctgaagattt tgggagttat 660

tactgtcaac atttttgggg tactccgtac acgttcggag gggggaccaa gctggaaatc 720

aaa 723

Claims

1. anti-human bladder chalone C antibody, including：

Weight chain variable district contains following complementary determining region：Amino acid sequence such as sequence SEQ ID NO：HCDR1 shown in 9, such as Sequence SEQ ID NO：HCDR2 and/or such as sequence SEQ ID NO shown in 10：HCDR3 shown in 11；

And light-chain variable sequence contains following complementary determining region：Amino acid sequence such as sequence SEQ ID NO：Shown in 12 LCDR1, such as sequence SEQ ID NO：LCDR2 and/or such as sequence SEQ ID NO shown in 13：LCDR3 shown in 14.

2. the anti-human bladder chalone C antibody described in claim 1, it is characterized in that the amino acid sequence of weight chain variable district such as SEQ ID NO:Shown in 15, the amino acid sequence such as SEQ ID NO of light chain variable district:Shown in 16.

3. the anti-human bladder chalone C antibody described in claim 1, it is characterized in that amino acid sequence such as SEQ ID NO:Shown in 34.

4. the nucleotide sequence of antibody described in claim 3 is encoded, such as SEQ ID NO:Shown in 38.

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 antibody described in claim 3 is produced, including：

1) recombinant host cell expression antibody described in claim 6 is cultivated under suitable conditions；

2) and then from host cell purify, collect antibody.

8. application of the anti-human bladder chalone C antibody of any one of claim 1-3 in bladder chalone C content in detecting sample.