CN105949309B - Anti-human C reactive protein antibody and its application - Google Patents

Abstract

The present invention relates to anti-human C reactive protein antibody and its applications.The present invention is prepared for Multiple Antibodies, and carries out pairing screening, obtains sensitivity and specificity is able to satisfy the antibody combination (P24 and P03) 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, obtains easy to operate, sensitivity, and specificity and coherent detection performance can meet the time resolution immunofluorescence chromatography quantitative test card of the Human C-reactiveprotein of people's clinical sample detection.

Description

Anti-human C reactive protein antibody and its application

Technical field

The invention belongs to field of biotechnology, are specifically related to two kinds of anti-human C reactive protein (CRP) antibody and its preparation The application of method and above-mentioned antibody in Human C-reactiveprotein detection.

Background technique

C reactive protein (C reactive protein, CRP) is a member in protein families, is that a kind of acute stage is anti- Albumen is answered, plasma concentration sharply increases in tissue damage and bacterium infection, is the important defense molecule of body, mainly by liver It generates and secretes.CRP is combined into stable disk-like structure with non-covalently bonded by five identical spherical monomers, belongs to just Pentamer family.CRP is symmetrical pentahedron in structure, and monomer is made of 206 amino acid, and molecular weight is about 23KDa, The total molecular weight of CRP is about 118KDa.CRP participates in intracorporal various inflammation and immune response, is coronary atherosclerotic The important symbol object of heart disease (coronary heart disease).A large number of studies show that: high-caliber CRP is peripheral vascular disease, myocardial infarction, brain The independent risk predictive factor of angiosis and vascular death.

CRP content is few in normal human serum, concentration 0.062-8.2mg/L, and half-life period is about 15 hours.Work as generation Start to increase after bacterium infection 2 hours, increase within average 8 hours 1 times, reaches peak within 48 hours, then will not in virus infection It increases.CRP is persistently increased, and prompting body, there are chronic inflammation or autoimmune diseases.CRP changes not by the individual of patient The influence of difference, fuselage state and therapeutic agent.In addition, CRP also plays an important role in other field: 1. by chronic The effect that inflammation causes CRP in the independent hazard factor of cardiovascular disease has been found, and monitors the horizontal variation of CRP in time to the heart The intervention and prognosis of vascular diseases play an important role.Some scholars are it is even contemplated that CRP can be used as the gold mark of cardiovascular danger assessment Standard, and CRP level is higher, the risk that cardiovascular and cerebrovascular occurs is bigger；2. CRP can be used to evaluate the serious journey of acute pancreatitis Degree, when extensive necrosis pancreatitis occurs, the level of CRP may be up to 250mg/L in serum；3. such as CRP and alpha-fetoprotein are joined Application is closed, can be used to identify liver malignancy and benign disease, provides guidance for clinical treatment formulation；In addition, CRP is surveyed Fixed also to have positive meaning to the treatment and prognosis of tumour, malignant tumor patient CRP level mostly increases, after ocal resection CRP level can then decline, and it is very small to carry out the influence of radiotherapy, chemotherapy and corticosteroid therapy to serum CA125, so measuring blood The horizontal variation of clear CRP facilitates the clinical progression for estimating malignant tumour in each tract.4. assessing patient's prognosis: CRP level is higher, prompts disease control bad, prognosis mala.

In view of important function of the CRP in various fields, prepares CRP antibody and be used to prepare CRP immue quantitative detection reagent box With important clinical value.CRP is usually to pass through antibody nephelometry or immunoturbidimetry measures, and their detection Ability is suitable only for the prediction to infection in 3-5mg/L or more, this level, and for coronary artery and cerebrovascular risk prediction It is far from being enough.In addition to above-mentioned technology, using the CRP test card of colloidal gold and fluorogenic quantitative detection technology, it can meet fast The demand detected by speed detection and bed.Wherein fluorogenic quantitative detection has higher detection performance, therefore it is accurate to be suitable for detection Spend the application demand of more demanding inspection center or large hospital clinical department.

Summary of the invention

The technical problem to be solved in the present invention is to provide can effective, specific binding Human C-reactiveprotein antibody.More Specifically:

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

The first anti-human C reactive protein antibody (being denoted as P24),

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 P24 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 C reactive protein antibody (being denoted as P03),

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.

For the amino acid sequence of antibody heavy chain variable region preferably in the present invention as shown in SEQ ID NO:15, light chain can Become the amino acid sequence in area 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 P24 Shown in ID NO:19；The amino acid sequence of the single-chain antibody P03 is as shown in SEQ ID NO:21.

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 P24 is as shown in SEQ ID NO:18, and the nucleotide sequence such as SEQ ID NO:20 of coding single-chain antibody P03 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 bacterium expression antibody is cultivated under suitable conditions；

2) it then purified from host strain, collect antibody.

Of the invention the 7th is designed to provide above-mentioned anti-human C reactive protein antibody and contains in detection Human C-reactiveprotein Application in amount.

Of the invention the 8th, which is designed to provide one group, can be matched and be detected the antibody of Human C-reactiveprotein to group It closes；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 C reactive protein antibody test human C-reactive The time resolution immunofluorescence of albumen chromatographs quantitative test card, including sample pad, fluorescence bonding pad, reaction film and water absorption pad；Institute The antibody P03 that fluorescence bonding pad is coated with fluorescent microsphere label is stated, there is detection band and quality control band, detection band position on the reaction film It sets and is coated with antibody P24, quality control band position is coated with anti-His tag antibody or Protein L.The preferred cellulose nitrate of reaction film Plain 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 (P24 and P03)；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, specificity and coherent detection performance Meet the time resolution immunofluorescence chromatography quantitative test card of the Human C-reactiveprotein of people's clinical sample detection.

Detailed description of the invention

Fig. 1 heavy chain of antibody and light-chain variable region gene electrophoretogram.Lane 1 and Lane4 is 200bp DNA Ladder； Lane 2 is antibody P24 heavy chain variable region DNA；Lane 3 is antibody P24 light chain variable region DNA；Lane5 is antibody P03 heavy chain Variable region DNA；Lane 6 is antibody P03 light chain variable region DNA.

Fig. 2 single-chain antibody structural schematic diagram.V_HIndicate weight chain variabl area sequence, V_LIndicate light-chain variable sequence, His mark Label are six histidines.

The agarose gel electrophoresis figure of Fig. 3 single-chain antibody PCR product.

Wherein, Fig. 3-a. is single-chain antibody P24 gene PCR product；Fig. 3-b is single-chain antibody P03 gene PCR product.

Fig. 4 recombinant single chain antibody Pichi strain inducing expression supernatant culture solution qualification figure.

Wherein, Fig. 4-a is recombination single-chain antibody P24 recombinant pichia yeast strain inducing expression supernatant culture solution SDS-PAGE Electrophoretic identification；Fig. 4-b is recombination single-chain antibody P03 recombinant pichia yeast strain inducing expression supernatant culture solution SDS-PAGE electricity Swimming qualification figure.

Fig. 5 .SDS-PAGE electroresis appraisal single-chain antibody purification effect figure.

Wherein, Fig. 5-a is SDS-PAGE electroresis appraisal single-chain antibody P24 purification effect figure；Fig. 5-b is SDS-PAGE electrophoresis Identify single-chain antibody P03 purification effect figure

Fig. 6 is that time resolution immunofluorescence of the present invention chromatographs quantitative test card structural schematic diagram.1 it is sample pad, 2 is anti- Answer film, 3 be water absorption pad, 4 be nature controlling line (C line), 5 be detection line (T line), 6 be fluorescence bonding pad, 7 be PVC sheet.

Fig. 7 time resolution immunofluorescence of the present invention chromatographs the matched curve of quantitative test card detection range.Wherein abscissa is Protein concentration (ng/mL)；Ordinate is detected value；r²It is 0.998.

Fig. 8 time resolution immunofluorescence of the present invention chromatographs the matched curve of the quantitative test card range of linearity.Wherein abscissa is Theoretical concentration (ng/mL)；Ordinate is detected value；r²It is 0.996.

Fig. 9 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 antibody_H) and light chain variable region (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 flexibility of single-chain antibody.Connection type 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 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.

The preparation of the anti-human C reactive protein hybridoma cell strain of embodiment 1.

1. animal immune

BALB/ is immunized according to general immune programme to be extracted from the C reactive protein (purchase is in HyTest company) of human plasma C female mice (is purchased from this experimental animal Co., Ltd of Changzhou Cavan).Specific Immunity is referring to " Antibody preparation and use are tested Guide ".Immune serum titre is tracked using indirect elisa method, chooses the highest immune mouse of serum titer, carries out mouse Splenocyte and murine myeloma cell carry out fusion experiment.

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% CO₂Cell incubator in cultivate.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 C reactive protein specific hybrid tumor strain screening

(1) with CB coating buffer dilution CRP (purchase is in HyTest company) to 1 μ g/mL, 96 holes the preparation of detection plate: are coated with ELISA ELISA Plate, 100 holes μ L/, 2~8 DEG C of coatings overnight, washed once and pat dry；Containing 2% bovine serum albumin(BSA) (Bovine Serum Albumin, BSA, buy in Yancheng Sai Bao Biotechnology Co., Ltd) PBST buffer blind (hole 200ul/), 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 M24 and M03 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 M24 and the antibody variable sequences M03 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 M24 and M03 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 SynthesisKit (being purchased from Thermo company) carries out reverse transcription to total serum IgE extracted in previous step, and cDNA is made, freezes It is spare in -20 DEG C；

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 Nanjing Genscript Biotechnology Co., Ltd. to be sequenced.

Sequencing obtain the antibody heavy chain variable region amino acid sequence of hybridoma cell strain M24 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 the HCDR3 as shown in sequence SEQ ID NO:3 shown；The amino acid sequence of each complementary determining region of its light chain variable region Column are: the LCDR1 as shown in sequence SEQ ID NO:4, the LCDR2 as shown in sequence SEQ ID NO:5 and such as sequence SEQ ID LCDR3 shown in NO:6.

Sequencing obtain the antibody heavy chain variable region amino acid sequence of hybridoma cell strain M03 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 HCDR3 as shown in sequence SEQ ID NO:11；Each complementary determining region of its light chain variable region Amino acid sequence is: the LCDR1 as shown in sequence SEQ ID NO:12, the LCDR2 as shown in sequence SEQ ID NO:13 and such as LCDR3 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 hybridoma cell strain M24 and M03 heavy chain of antibody and light chain variable region Between be added link peptide (GGGGS)₃, six histidine tag SEQ ID NO:17 are introduced, and its full genome is merged into histidine The method that label carries out codon optimization according to the preferences of pichia yeast expression system, carries out the recombinant expression of single-chain antibody. Expressed obtained antibody is respectively designated as antibody P24 and antibody P03, structure composition are as shown in Fig. 2.Above-mentioned single-chain antibody Recombinant expression it is specific as follows:

A) the expression plasmid building of antigen-4 fusion protein gene

The nucleotide sequence of antibody P24 after codon optimization is as shown in SEQ ID NO:18, amino acid sequence such as SEQ Shown in ID NO:19；The nucleotide sequence of antibody P03 after codon optimization as shown in SEQ ID NO:20, amino acid sequence such as Shown in SEQ ID NO:21.The segment upstream of the antibody P24 and P03 full genome synthesis after optimization pPICZ α A is introduced respectively to carry XhoI restriction enzyme site in body, downstream introduce XbaI enzyme cutting site, are building up in pUC57 plasmid, obtain a kind of long-term preservation plasmid, Plasmid is denoted as pUC57-P24 and pUC57-P03 (being provided by Nanjing Jin Sirui Science and Technology Ltd.).PCR amplification is carried out, wherein on Swim primer P1 are as follows: 5'-TGT AAA ACG ACG GCC AGT-3'；Downstream primer P2 are as follows: 5'-CAG GAA ACA GCT ATG AC-3'.After Standard PCR program, agarose gel electrophoresis analyzes (attached drawing 3-a, Fig. 3-b), shows two kinds of primer sizes and expection Size (800bp, 780bp) is consistent.After PCR is obtained gene product recovery purifying, using XhoI, (#R0146S is purchased from New England Biolabs company) and XbaI (#R0145V is purchased from New England Biolabs company) double digestion, connected with T4 It connects enzyme to be connected in pPICZ α A (V19520 is purchased from Invitrogen) plasmid, is transformed into DH5 α competent cell, is containing 37 DEG C of overnight incubations in the LB plate of Zeocin (R250-01 is purchased from Invitrogen company).Second day screening positive clone bacterium Sequencing compares, completely the same to get the expression plasmid for arriving single-chain antibody P24 and P03 with expected sequence, is denoted as pPICZ α-respectively P24 and pPICZ α-P03.

B) building, screening and expression of the single-chain antibody gene in Pichia pastoris host's engineered strain

YPDS solid medium is prepared: referring to Invitrogen company EasySelect Pichia Expression Kit Specification；Pichia pastoris competent cell preparation: referring to EasySelect Pichia Expression Kit specification；BMGY Culture medium is prepared: referring to Invitrogen company Multi-Copy Pichia Expression Kit specification；BMMY culture Basigamy system: referring to Invitrogen company Multi-Copy Pichia Expression Kit specification.

By pPICZ α-P24 and pPICZ α-P03 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 electroresis appraisal, object observing albumen table Up to situation (attached drawing 4-a, Fig. 4-b).

P24 the and P03 recombination engineered strain of above-mentioned acquisition is inoculated in respectively in 500mL 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.

C) single-chain antibody purifies

Using histidine tag affinity column antibody purification P24 and P03 single-chain antibody, pre-installs pillar and be selected as HisTrap HP, the specific steps are as follows:

Step 1: the removal of impurities of fermentation liquid pre-processes: above-mentioned expression is obtained on antibody P24 and P03 fusion protein fermentation liquid Clearly, supernatant is collected by centrifugation, and combination buffer is added, so that supernatant final concentration of 300mM NaCl, 20mM NaH₂PO₄, 10mM Imidazole adjusts pH7.5,0.45 μm of membrane filtration.

Step 2: the affine column purification of HisTrap HP: with fully-automatic intelligent protein purification system (AKTA avant150, Purchased from GE healcare company) affinity purification, pillar are carried out to antibody P24 and P03 the fusion protein fermentation liquid that pretreatment obtains 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-a and Fig. 5-b it is found that two kinds of purity of protein after purification reach 95% or more, replacement buffer be PBS solution simultaneously It is concentrated by ultrafiltration (1mg/ml), 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 identification of antibody P24 and P03

A. 12% non-denaturing polyacrylamide gel and SDS- polyacrylamide polyacrylamide gel electrophoresis: is respectively configured Gel；Loading standard protein and natural CRP albumen (purchase is in HyTest company) respectively, electrophoresis 1 hour under constant pressure；

B. transferring film: transferring film 1 hour under the conditions of constant current (35mA/ film), the protein on polyacrylamide gel is turned respectively It moves on nitrocellulose filter.Coomassie brilliant blue G250 dyes the polyacrylamide gel for completing transferring film, observes albumen Residual condition；

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 P24 (P24- HRP, 1mg/mL, our company is using classical Over-voltage protection label, similarly hereinafter) and horseradish peroxidase-labeled P03 (P03-HRP, 1mg/mL, our company is using classical Over-voltage protection label, similarly hereinafter), it is separately added into above-mentioned nitrocellulose filter, reacts at room temperature 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.

The experimental results showed that the natural CRP that two kinds of antibody of the invention can be extracted with denaturation and non denatured blood reacts, demonstrate,prove The binding force of two kinds of antibody and natural CRP of the present invention is illustrated.

2, evaluation of the single-chain antibody P24 and P03 in time-resolved fluorescence detection platform

P24 antibody is diluted to 1mg/ml with the PBS of 0.05mol/L pH7.2, is lined on nitrocellulose filter；With The P03 antibody that the borate buffer of 0.05mol/L pH8.0 marks time-resolved fluorescence microballoon dilutes 10 times, is sprayed at combination On pad；By pad pasting shown in attached drawing 6, cut, be loaded (specific preparation is referring to embodiment 5).It will test card detectable concentration content respectively For the PBS of natural CRP albumen (purchase is in HyTest company) and 0.05mol/L pH7.2 of 400,200ng/ml, testing result It is as follows:

From the above results, the double-antibody sandwich detection system of P24, P03 composition can be applied to time-resolved fluorescence inspection Platform is surveyed, certain density natural CRP albumen is able to detect.

The preparation of the time-resolved fluoroimmunoassay detection card of embodiment 5.C- reactive protein

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.

The preparation of fluorescence antibody dilution: contain 1%BSA, 10% trehalose, 0.025% Tween-20,0.05mol/L pH8.0 Borate buffer, with 0.22U film filter, be placed in 4 DEG C it is spare, validity period 7 days.

The preparation of coated antibody dilution: containing 3% methanol, 0.025% Tween-20, and the PBS of 0.05mol/L pH7.2 is used 0.22U film filtering, be placed in 4 DEG C it is spare, validity period 7 days.

Sample dilution: contain 0.5%BSA, 0.025% Tween-20,0.05% antipyrine, 0.05%proclin300, The borate buffer of 0.05mol/L pH8.0,2-28 DEG C preservation validity period 1 year.

2, C reactive protein fluorescence detection blocking is standby

1) time-resolved fluorescence microballoon marks

P03 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 P03 antibody 75ul of concentration 1mg/ml is added in microballoon after activation, 250r/min25 DEG C of isothermal vibration reacts 2h； 58ul 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) spraying of fluorescence bonding pad

6 specking method of P03 antibody fluorescence bonding pad is as follows: being resisted the P03 fluorescence of above-mentioned preparation with fluorescence antibody dilution Body dilutes 10 times, is sprayed on whole bonding pad (long 300mm, the glass fibre of wide 12mm).

3) coating of nitrocellulose filter

Nitrocellulose filter method for coating is as follows: taking the P24 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 P24 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 6 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.

3, the application method of C reactive protein 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, C reactive protein fluorescence detection card detection effect is assessed

1) accuracy: by P24 (coating)-P03 (label) detection card by detection card application method detection 1,10,50mg/l Each 25 replications of CRP reference material (being purchased from hytest) calculate detection card precision after rejecting outlier.Experimental result is shown Three Concentration Testing result coefficient of variation CV < 15%.

Concentration point (mg/l)	1	10	50
				CV	12%	8%	4%

2) detection range: P24 (coating)-P03 (label) the CRP native protein for detecting card detection various concentration (is purchased from Hytest) 0.5,1,2,4,8,16,32,64,128mg/l, matched curve and detection range are 0.5-120mg/L (such as attached drawing 7).

3) high level sample and Sample dilution the range of linearity: are configured to 5 series of concentrations samples according to a certain percentage (0.5,1,10,50,100mg/ml) detects card detection, each pattern detection 3 times, by result with P24 (coating)-P03 (label) Regression calculation is carried out with theoretical concentration, is judged whether linear in the concentration range.The range of linearity be 0.5-100mg/L (such as Attached drawing 8).Sensitivity is 0.5mg/L

4) accuracy-rate of recovery: by the detection card of P24 (coating)-P03 (label) detection additive amount be respectively 1,10, The natural CRP albumen of 50mg/l, testing result such as following table.

Concentration point (mg/l)	1	10	50
				The rate of recovery	115%	93%	102%

5, accuracy-methodology compares:

The above results show that the CRP detection card performance of P24 (coating)-P03 (label) is more excellent, select in similar product at present Guangzhou Wondfo Biotech. Co., Ltd.'s whole process C reactive protein (hsCRP+ routine CRP) of good prestige is obtained in the market Immue quantitative detection reagent box (immunochromatographic method) compares product comparison verifying.Select 20 parts of clinical patient samples, by 1 to 20 it is suitable Sequence number is tested simultaneously with the CRP fluorescence detection card of reference product and P24 to be evaluated (coating)-P03 (label), is pressed It is measured according to 1,2,3......18,19,20,20,19,18......3,2,1 sample order.Control and product to be evaluated Testing result coefficient R²=0.98, illustrate that two methods testing result has preferable correlation (such as attached drawing 9).

6, recipe determination:

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:

Claims (8)

1. anti-human C reactive protein antibody, comprising:

Heavy chain variable region contains complementary determining region below: amino acid sequence HCDR1 as shown in sequence SEQ ID NO:1, such as HCDR2 shown in the sequence SEQ ID NO:2 and HCDR3 as shown in sequence SEQ ID NO:3；

And light-chain variable sequence contains complementary determining region below: amino acid sequence is as shown in sequence SEQ ID NO:4 LCDR1, the LCDR2 as shown in sequence SEQ ID NO:5 and the LCDR3 as shown in sequence SEQ ID NO:6.

2. anti-human C reactive protein antibody described in claim 1, it is characterized in that the amino acid sequence of heavy chain variable region such as SEQ Shown in ID NO:7, the amino acid sequence of light chain variable region is as shown in SEQ ID NO:8.

3. anti-human C reactive protein antibody described in claim 1, it is characterized in that amino acid sequence is as shown in SEQ ID NO:19.

4. encoding the nucleotide sequence of antibody described in claim 3, which is characterized in that nucleotide sequence such as SEQ ID NO:18 institute Show.

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 3, comprising:

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

2) it then purified from host cell, collect antibody.

8. application of the anti-human C reactive protein antibody of any one of claim 1-3 in detection Human C-reactiveprotein content.