Application of human procalcitonin B cell epitope peptide fragment and monoclonal antibody thereof

A monoclonal antibody, human calcitonin technology, applied in the field of immunoassay medicine, can solve the problems of complex preparation process, low product yield and high cost, and achieve the effects of good specificity, high purity and high titer

Active Publication Date: 2012-10-03
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AI-Extracted Technical Summary

Problems solved by technology

[0005] In the prior art, most of the preparation methods of PCT are directly taken from tissue cells, such as parathyroid gland tissue, the cost is high, the preparati...
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Method used

Described screening B cell epitope peptide, wherein the chemically synthesized epitope peptide is: N--DMSSDLERDHRPHV-C (SEQ ID NO: 3), in the chemical synthesis of described B cell epitope peptide A cysteine ​​was introduced at its N-terminus to improve its ability to cross-link with BSA, followed by cross-linking with BSA. Cross-linking rate (>50%), procalcitonin B cell epitope peptide antigen.
Obtain the gene sequence (accession ...
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The invention belongs to the field of immunology in medicine, and particularly relates to an application of a procalcitonin B cell epitope peptide fragment and a monoclonal antibody thereof; the epitope peptide fragment is shown in SEQ ID NO:3, and can be used for preparing hybridoma cells and secreting a corresponding monoclonal antibody; the monoclonal antibody can be used for preparing a diagnostic reagent for procalcitonin detection; the monoclonal antibody has purity of up to above 96%, has a titer of up to 1:256000, and has the advantages of good specificity, suitability for mass preparation, and the like; the monoclonal antibody, multiclonal antibody prepared according to the invention can be used for detection of patient blood procalcitonin content, and determination can be performed by using a double-antibody sandwich ELISA reaction mode, wherein a 'double-antibody sandwich' structure is formed by an enzyme-labeled human anti-PCT monoclonal antibody, an enzyme-label plate coated anti-PCT multiclonal antibody, and a PCT antigen of a sample to be detected.

Application Domain

Technology Topic

TiterB-Cell Epitopes +9


  • Application of human procalcitonin B cell epitope peptide fragment and monoclonal antibody thereof
  • Application of human procalcitonin B cell epitope peptide fragment and monoclonal antibody thereof
  • Application of human procalcitonin B cell epitope peptide fragment and monoclonal antibody thereof


  • Experimental program(5)

Example Embodiment

[0031] Example 1 Preparation of PCT recombinant protein
[0032] On the basis of obtaining the PCT coding gene from GENBANK, we carried out codon preference modification, chemically synthesized the coding gene fragment, cloned into the prokaryotic expression vector, and induced protein expression after DNA sequencing identification. After identification of the nature, the PCT recombinant protein was purified in large quantities. The protein can be used as an immunogen and screening agent for antibody preparation, and can be used as a calibrator when establishing PCT quantitative detection in subsequent experiments. Specifically:
[0033] 1. Gene cloning of PCT recombinant protein:
[0034] Obtain the human PCT protein gene sequence (accession number NM_004102) from GENBANK and submit it to the Graphical codon usage analyzer (http://guca.schoedl.del) to analyze its codon bias; specifically, the human PCT Gene codon usage rate in E. coli The synonymous substitution of <10% is the preferred codon of E. coli to make it easier to express in E. coli. The optimized nucleotide sequence is shown in SEQ ID NO:1:
[0035] gcaccattca ggtctgccct ggagagcagc ccagcagacc cggccacgct cagtgaggac gaagcgcgcc tcctgctggc tgcactggtg caggactatg tgcagatgaa ggccagtgag ctggagcagg agcaagagag agagggctcc agcctggaca gccccagatc taagcggtgc ggtaatctga gtacttgcat gctgggcaca tacacgcagg acttcaacaa gtttcacacg ttcccccaaa ctgcaattgg ggttggagca cctggaaaga aaagggatat gtccagcgac ttggagagag accatcgccc tcatgttagc atgccccaga atgccaacta atag
[0036] The corresponding polypeptide sequence is shown in SEQ ID NO: 2, specifically:
[0037] Ala Pro Phe Arg Ser Ala Leu Glu Ser Ser Pro Ala Asp Pro Ala Thr Leu Ser Glu Asp Glu Ala Arg Leu Leu Leu Ala Ala Leu Val Gln Asp Tyr Val Gln Met Lys Ala Ser Glu Leu Glu Gln Glu Gln Glu Arg Glu Gly Ser Ser Leu Asp Ser Pro Arg Ser Lys Arg Cys Gly Asn Leu Ser Thr Cys Met Leu Gly Thr Tyr Thr Gln Asp Phe Asn Lys Phe His Thr Phe Pro Gln Thr Ala Ile Gly Val Gly Ala Pro Gly Lys Lys Arg Asp Met Ser Ser Asp Leu Glu Arg Asp His Arg Pro His Val Ser Met Pro Gln Asn Ala Asn
[0038] The Chinese and English comparisons of the above amino acids are as follows:
[0039] Glutamine gln Q; glycine gly G; serine ser S; alanine ala A; threonine thr T; valine val V; isoleucine ile I; leucine leu L; tyrosine tyr Y; Phenylalanine phe F; histidine his H; proline pro P; asparagine asn N; methionine met M; glutamic acid glu E; tryptophan trp W; lysine lys K; Cys C; arginine arg R; Asp D
[0040] For effective expression and purification, Sal was added to the 5'- and 3'-end of the sequence respectively , BamH Restriction site, where BamH The GATGATGATGATAAG sequence is added after the site, and its coding Asp-Asp-Asp-Asp-Lys polypeptide sequence is the recognition site of EK enzyme. Then commissioned Shanghai Yingjun Biological Engineering Co., Ltd. to carry out full gene synthesis. The synthesis process is a conventional technology, and you can refer to the book Molecular Cloning: Laboratory Manual.
[0041] Two pET42a-PCT recombinant plasmid construction:
[0042] The pET42a vector and synthetic gene were passed through Sal , BamH Use T after 4 hours 4 DNA ligase 4 ℃ overnight ligation. Add 200μl of the prepared competent DH5α bacteria, ice bath, and add 1μl of the connection product to the tube, transform DH5α bacteria, gently pat and mix, ice bath for 30 minutes, 42℃ water bath for 90 seconds, take out the centrifuge tube and ice bath for 2 minutes, Add 800μl of room temperature 2×YT culture solution and mix well, and culture with shaking at 220 rpm on a 37℃ shaker for 1 hour. Spread 50μl, 200μl and all the remaining transformed bacteria on 3 kanamycin-resistant 2×YT cultures respectively. On the plate, cultured overnight in a constant temperature incubator at 37°C. The next day, the colonies were picked up and inoculated into LB medium for expansion. The plasmids were extracted by alkaline lysis, and the plasmids were taken with Sal , BamH Double enzyme digestion for 4 hours, the digestion system is: pET42a-PCT plasmid DNA 10μl, Sal 1μl, BamH 1μl, 10× buffer K 2μl, ddH 2 O 6μl, and take 10μl of the digested product for identification by 1.5% agarose gel electrophoresis, a fragment consistent with the design value of 378bp can be seen.
[0043] Three PCT recombinant protein induced expression
[0044] The bacteria transformed with pET42a-PCT were expanded culture and induced expression. When the OD value of the bacteria reached 0.6-0.8, IPTG (to a final concentration of 1 mmol/L) was added to induce expression for 6 h. After culturing, each gram of wet bacteria was resuspended in 10 times volume of PBS buffer (pH 7.3), and the bacteria was sonicated after mixing, and centrifuged at 4 ℃ 10,000 rpm for 15 min after the bacteria was broken. The supernatant was filtered with 0.45 μm microporous membrane. filter. Take a small amount of supernatant and precipitate SDS-PAGE electrophoresis to identify the solubility of the target protein. After the identification of the expressed protein, almost all of it exists in the supernatant, which is soluble expression.
[0045] Four PCT recombinant protein purification
[0046] The supernatant was filtered and purified on the column with Amersham's AKTAprime. After the Binding buffer was equilibrated, it was linearly eluted with Elution buffer. The main elution peak was collected, diluted 10 times with His-Binding buffer, and then purified by HisTrap HP again. The purified product was purified with Molecular sieve equilibrated with PBS to collect protein. The obtained protein was replaced with HiTrap Desalting buffer system as EK cutting buffer (50 mmol/L Tris-HCl, PH8.0), and EK enzyme was added according to the mass ratio of EK enzyme to protein 1:1000, and cutting was carried out at 4°C at 60 rpm on a shaker for 24 hours. . After cutting, it was diluted with His-Binding buffer and purified by HisTrap HP. It was identified by 15% SDS-PAGE electrophoresis. The molecular weight of the PCT recombinant protein was about 13Kda. The recombinant bacteria not induced by IPTG was used as a negative control, and the recombinant bacteria induced for 6 hours was used as a positive control. After purification, the purity of the target protein reached more than 95%.
[0047] 5. Determination of the concentration and purity of PCT recombinant protein:
[0048] 1 Determination of PCT protein content by Lowry method
[0049] Prepare standard curve
[0051] The kit for Lowry method was purchased from Shanghai Meiji Biotechnology Co., Ltd.
[0052] Reagent A: 1) 10g Na2CO3, 2g NaOH and 0.25g potassium sodium tartrate (KNaC 4 H 4 O 6 ·4H 2 O). Dissolved in 500ml distilled water; 2) 0.5g copper sulfate (CuSO 4 ·5H 2 O) Dissolve in 100 ml of distilled water, mix 50 parts (A) with 1 part (B) before each use, which is reagent A.
[0053] Reagent B: Add 100g of sodium tungstate (Na 2 WO 4 ·2H 2 O), 25 grams of sodium molybdate (Na 2 MoO 4 ·2H 2 O) and 700 ml of distilled water, then add 50 ml of 85% phosphoric acid, 100 ml of concentrated hydrochloric acid, mix well, connect the reflux tube, reflux for 10 hours on a small fire, at the end of the reflux, add 150 g of lithium sulfate (Li 2 SO 4 ), 50 ml of distilled water and a few drops of liquid bromine, continue to boil for 15 minutes at the opening to drive off excess bromine. After cooling, the solution turns yellow (if it is still green, repeat the step of adding liquid bromine again). Dilute to 1 liter, filter, and store the filtrate in a brown reagent bottle. Use standard NaOH titration, phenolphthalein as indicator, and then appropriately dilute, add water about 1 times, so that the final acid concentration is about 1N.
[0054] Standard protein solution: accurately weigh crystalline bovine serum albumin or g-globulin and dissolve it in distilled water with a concentration of about 250 mg/ml. If bovine serum albumin is soluble in water and turbid, you can use 0.9% NaCl solution instead.
[0055] At a wavelength of 650nm, zero the blank tube as a control, and the absorbance of each tube was measured respectively, and the protein concentration was used as the abscissa and the absorbance as the ordinate to prepare a standard curve. After diluting the protein to be tested, the UV spectrophotometer measures A 260 Value and A 280 value. According to the formula, protein concentration C=(1.45×A280-0.75×A260)×dilution factor, calculate the rough concentration of the protein to be tested, and then dilute the protein sample with distilled water to the range of 25~150μg, and react according to the operating procedure in the table above. Measure the absorbance value at 650nm, then find out the corresponding concentration on the standard curve, and multiply it by the dilution factor to get the concentration of the protein to be tested. The average value is calculated by multiple tubes, and the concentration is 1.075g/ml.
[0056] 2 High performance liquid chromatography (HPLC) for purity determination and PCT acquisition calculation
[0057] The purity of the purified protein was analyzed by HPLC. It can be seen that the purity of PCT protein after preliminary purification by SP Sepharose Fast Flow cation exchange column is over 93.25%, and the purity of PCT protein after purification by molecular sieve can reach 98%. It is calculated that 36.5mg of the fusion protein can be obtained per liter of induced bacteria, and about 9.6mg of PCT recombinant protein can be obtained after cutting.
[0058] Six Western blot for specific identification of immune response of PCT recombinant protein
[0059] The final purified procalcitonin protein was identified by 15% SDS-PAGE and analyzed by Western Blot with the anti-PCT monoclonal antibody produced by Abcam. The color was developed by Millipore Immobilon Western Chemiluminescent HRP Subscrate system. The result showed that the anti-PCT monoclonal antibody A cleaning band can be seen at the anti-binding PCT protein.
[0060] Seven stability studies
[0061] Using freeze-dried storage at 4℃ for 180 days, the detection value will decrease <5%, while the calibrator diluent can be stored for one month and it will be reduced by more than 20%, and for 6 months it will be reduced by more than 70%, indicating that long-term storage should be in the form of freeze-drying. In addition, after the lyophilized product is dissolved in the calibrator diluent, the decrease <10%, so it can be used for quantitative detection during this period.

Example Embodiment

[0063] Example 2 Preparation of procalcitonin B cell epitope peptide
[0064] The sequence of the chemically synthesized B cell epitope peptide segment is derived from the results of bioinformatics analysis, comprehensively analyzing the secondary structure, antigenicity, hydrophobicity, accessibility and flexibility of the PCT protein, and analyzing and scoring each segment. The region with high score is selected as the B cell epitope region. Specifically, use Chou & Fasman to predict β-turn, Emini method to predict antigen surface accessibility, Karplus& Schulz method to predict protein flexibility, Kolaskar & Tongaonkar protein antigenicity analysis, Parker method protein hydrophobicity analysis, and Beppred linear epitope prediction techniques Parameters to obtain potential B cell epitope peptides in human PCT protein;
[0065] Said screening of B cell epitope peptides, wherein the chemically synthesized epitope peptides are: N-- DMSSDLERDHRPHV—C (SEQ ID NO: 3). The chemical synthesis of the B cell epitope peptides is in its N Cysteine ​​is introduced at the end to improve its cross-linking ability with BSA, and then cross-linked with BSA. Crosslinking rate (> 50%) to obtain procalcitonin B cell epitope peptide antigen.
[0066] In addition, the epitope peptide shown in SEQ ID NO: 3 can also be cross-linked with KLH.

Example Embodiment

[0068] Example 3 Preparation and identification of monoclonal antibodies
[0069] 1. Immunization of Balb/c mice with a derivative of procalcitonin B cell epitope peptide
[0070] The procalcitonin B cell epitope peptide antigen obtained in Example 2 was taken out from the refrigerator at -80°C as an antigen, dissolved and filtered.
[0071] 6-week-old female Balb/c mice weighing about 20g were selected for immunization. Antigen emulsification uses the double syringe mutual push method. For the first immunization, the procalcitonin B cell epitope peptide antigen shown in SEQ ID NO: 4 was emulsified and mixed with an equal volume of Freund's complete adjuvant to obtain an antigen mixture. Each mouse was skinned in an amount of 100 μg. Add intraperitoneal injection at more points. The second and third immunizations were performed on the 14th and 28th days. The adjuvant was changed to incomplete Freund's adjuvant. The antigen amount, injection volume and route were unchanged. After the third immunization, the titer was determined by indirect ELISA. Three days before fusion, a booster immunization was performed. Each mouse was intraperitoneally injected with 100 μg PCT without adjuvant, and the cells were fused three days later.
[0072] 2. Determination of serum titer of immunized Balb/c mice
[0073] 10 days after the third immunization, blood was taken from the tail vein of the mouse to detect the serum antibody titer. Soak the newly purchased ELISA plate in double distilled water overnight, dry it for later use; use the coating solution (0.05mol/L sodium carbonate buffer: 0.16g Na 2 CO 3 , 0.293g NaHCO 3 , 0.02g NaN 3 , Add deionized water to dissolve 100ml) Dilute the PCT recombinant protein antigen obtained in Example 1 to the best working concentration of 5μg/ml, add 100μl of antigen diluent to each well, incubate at 37°C for 1 hour, seal with tape, Overnight at 4℃, pour out the liquid in the wells, absorb the remaining reaction liquid in the wells, fill up the washing liquid once, then fill the washing liquid slowly and shake for 2min, decanted, repeat five times, and finally put the reaction plate upside down to absorb water On the paper, make the washing liquid drain out of the holes. After natural drying, it is sealed with tape. This is an ELISA plate coated with PCT recombinant protein antigen, add 300μl blocking solution, incubate at 37°C for 1.5 hours, wash 5 times; blood collection and diluted serum: pinch the tail of the rat and disinfect with 75% alcohol Cut a gap in the tail vein with scissors, take 20μl of blood, centrifuge at 2000rpm for 30min, take 1μl of the supernatant and add 999μl of antibody diluent to mix, and perform volume-fold dilution from 1:100 to 1:3200, and the diluted test Add 100μl of serum to each well, and at the same time take mouse pre-immune serum diluted 1:100 as negative control, and antibody dilution as blank control. Incubate at 37°C for 1 to 1.5 hours, wash 5 times; Dilute horseradish peroxidase goat anti-mouse IgG (Shanghai Yixin Biotechnology Co., Ltd.) to 1:10000, add 100μl to each well, incubate at 37°C for 1.5 hours, Wash 5 times; add 100μl of o-phenylenediamine solution per well, 15 minutes in the dark at room temperature, add 100μl of stop solution to each well. Observe the result. The product after OPD oxidation is orange-red. Use an enzyme-linked immunoassay to record the 492nm reading. After the control hole is adjusted to zero, the OD value of each hole is measured to be greater than 2.1 times of the negative control OD value, that is, it is positive. Serum titer reaches 1:3200 and can be used for cell fusion.
[0074] 3. Preparation of mouse spleen cell suspension and SP2/0 cell suspension
[0075] Take the immunized Balb/c mice, remove the eyeballs of the mice and let them be put to death. After centrifugation of the eye blood, the serum is collected as a positive control for ELISA. The spleen is taken out aseptically and placed in a glass dish containing 10ml of incomplete medium, and washed , Carefully peel off the surrounding connective tissue and adipose tissue, change a glass dish, remove the spleen, place it in a 200-mesh stainless steel net, grind it with the inner core of a syringe, and rinse with incomplete medium from time to time to allow the spleen cells to pass through When the mesh enters the solution, the spleen cells are transferred to a 10ml glass centrifuge tube, centrifuged horizontally at 800rpm for 10min, and the supernatant is removed. In the same way, wash the cells once with 10ml of incomplete medium, collect the precipitated cells by centrifugation, resuspend the cells in 10ml of incomplete medium and mix, the cell count is about 1×10 8 Cells.
[0076] Remove the SP2/0 cells from the liquid nitrogen, quickly put them in a 37°C water bath, and shake them until the cell solution is completely dissolved. Transfer the cells to a 10ml centrifuge tube and centrifuge horizontally at 800rpm for 10min. Discard the supernatant and 10ml complete culture medium. Resuspend the pellet, transfer the cell suspension to a 50ml culture flask and place it in a 37°C, 5% CO2 incubator. After the cells have grown well, select the cells with 8-AG-containing selective medium for a week; 2 days before fusion, transfer 1 flask of cells to 4 flasks, then the cells are in the logarithmic growth phase on the day of fusion, the viability is just right, and the cell size is uniform. Round and translucent, on the day of fusion, use an elbow dropper to gently blow down the SP2/0 cells from the tube wall, collect them in a centrifuge tube, centrifuge, discard the supernatant, and wash the pellet with incomplete medium, 10ml is incomplete Resuspend the medium and count the cells, about 5×10 7.
[0077] Four preparation of trophoblast
[0078] Take an unimmunized Balb/c mouse, remove the eyeballs and let them bloodlett, put them to death, soak in 75% ethanol for 5 minutes, cut the skin of the mouse, lift the peritoneum with tweezers, cut a small mouth with scissors, and take the pre-cooled dropper with an elbow dropper. Flush the abdominal cavity with complete medium, and aspirate the lotion into a 50ml centrifuge tube. In the same way, the abdominal cavity was washed with incomplete medium for 3 times, the washing liquid was collected, and the supernatant was removed by horizontal centrifugation at 1000 rpm at room temperature for 10 minutes. The cells were resuspended in 10 ml of incomplete medium and counted.
[0079] Five fusion of myeloma cells and spleen B lymphocytes
[0080] Before fusion, place PEG1500 in a 37℃ incubator to pre-warm, and draw 1×10 7 Myeloma cell suspension and 1×10 8 Put a spleen B lymphocyte suspension (cell number 1:10) into a 50ml centrifuge tube, add 30ml incomplete medium, mix well, centrifuge at 1000rpm for 10min, discard the supernatant, flick the bottom of the tube to loosen the cell mass Make a paste, put the centrifuge tube in a 37°C water bath, use a dropper to draw 0.8ml of pre-warmed 50% PEG1500 solution, slowly add cells to the cells along the tube wall about 2cm from the bottom of the tube, and rotate the centrifuge tube while adding it for 1 min After adding left and right, then let stand for 90s, add 30ml of pre-warmed incomplete medium at 37℃ drop by drop to stop the fusion, add it within 3min, the speed will be slow first and then fast, the action will be gentle, put the centrifuge tube in the 37℃ incubator Set aside for 5 minutes, take out the centrifuge tube, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, add 10ml HAT medium to resuspend the cells, gently pipette, mix, inoculate the fused cells into a 96-well cell culture plate with trophoblasts, press 100μl/well, each culture plate leaves 6 wells to inoculate SP2/0 cells as a negative control for HAT selection, set at 37℃, 5% CO 2 Cultivate in an incubator.
[0081] 6. Selective cultivation of fusion cells and screening of hybridoma cells
[0082] On the 5th day after the fusion, the growth of the cells can be observed under an inverted microscope and supplemented with 100μl of HAT medium, and cultured with HT medium on the 14th day. 10 to 14 days after fusion, when the cells grow to the bottom of 1/2 of the full culture well, the culture supernatant is detected by indirect ELISA to screen positive clones; the purified PCT recombinant protein prepared in Example 1 is coated with an enzyme label Plate (0.5μg/well), overnight at 4℃, wash 5 times with washing buffer, 5min each time, pat dry the liquid, add 300μl blocking solution to each well, incubate at 37℃ for 2h, add 100μl cell culture supernatant, select a small positive control Mouse immune serum, select SP2/0 culture supernatant for negative control, wash solution for blank control, incubate at 37°C for 2h; wash the plate: add 100μl 1:10000 diluted HRP-labeled goat anti-mouse IG antibody to each well, Incubate at 37°C for 2h; wash, pat dry the liquid, add 100μl of freshly prepared o-phenylenediamine solution per well, react in the dark at room temperature for 10-15 minutes, add 100μl of stop solution per well to stop the reaction, and detect the absorbance at 450nm with a microplate reader. The result was that BALB/C mice were immunized with PCT epitope peptides as antigens. After successful fusion, cloned and ELISA screened, hybridoma cell lines secreting PCT monoclonal antibodies were obtained, and the cell culture supernatant titer reached 1:6400. This hybridoma cell has been cryopreserved several times and can still secrete antibodies stably after subculturing in vitro for more than 3 months.
[0084] Seven cloning of positive hybridoma cells
[0085] After screening the positive clones, the positive hybridoma cells were cloned and cultured immediately by the limiting dilution method, feeder cells were prepared, resuspended in 10ml incomplete medium, and positive clones were collected and counted, and the positive clones were counted with incomplete medium Dilute to 100 cells/20ml, take a 96-well cell culture plate pre-filled with trophoblasts, add 200μl cell suspension, transfer the remaining positive cloned cells to a 24-well plate for expansion, collect the cells and cryopreserve in liquid nitrogen , And put the culture plate at 37℃, 5% CO 2 Incubate in an incubator, observe the cell growth under the microscope after 3 days, detect the titer by ELISA after 10 days, and clone the strongest positive clone again until the positive rate of the cells reaches 100%, then the strain can be determined; After the titer of the culture supernatant of the hybridoma cell line of the strain, the hybridoma cell of the fixed strain is expanded and cultured and sent to the China Type Culture Collection (Wuhan University Collection Center) for preservation. The preservation number is CCTCC NO: C201137. Stable secretion of the monoclonal antibody.
[0086] 8. Preparation of mouse ascites, purification of antibody and determination of titer
[0087] Choose 10 healthy female Balb/c mice, intraperitoneally inject 0.5ml of sterilized paraffin oil/mouse, 1-2 weeks later, each mouse intraperitoneally inject 0.5×10 6 ~1×10 6 Hybridoma cells were injected with 0.25 ml of a mixture of liquid paraffin and incomplete Freund’s adjuvant at the same time. After the mice had obvious ascites, they were killed by pulling their necks. The ascites were taken out from the abdominal cavity with a straw, centrifuged at 4°C for 15 minutes, and the clear ascites fluid in the middle section was separated and collected. Use HiTrap rProtein A HP column to access AKTA Explorer purified antibody, see figure 1 , The purity is greater than 95% by SDS-PAGE. The titer of the purified antibody detected by the indirect ELISA method reached 1:256000, which preliminarily shows that the obtained monoclonal antibody has a high binding ability to the PCT molecule, and it is packed and cryopreserved for use in Example 5.
[0088] Nine Anti-PCT monoclonal antibody affinity determination
[0089] In order to test the binding ability of anti-PCT monoclonal antibodies to the PCT antigen, the affinity constant (Kd) detection method of monoclonal antibodies based on the principle of antigen/antibody competitive binding was used to determine the affinity of the obtained monoclonal antibodies. Dissolve the purified PCT antigen in 0.05mol/l carbonic acid buffer (pH9.5), adjust the final concentration of PCT to 1μg/ml, add 100μl to each well of the ELISA plate, and seal the strip with tape at 4℃ overnight . The liquid in the wells was patted dry the next day, and each well was sealed with 1% BSA in PBS solution for 2 hours, washed and dried, and stored at 4°C for later use. The antigen-antibody reaction system was established according to the measurement principle and method. The initial reaction concentration of anti-PCT monoclonal antibody was diluted to 40ng/ml, and the initial concentration of PCT antigen was diluted to 360mg/ml. The antigen concentration is diluted by 30, 15, 7.5, 3.75, 1.875, 0.938, 0.469, 0.235 (unit is 10 -12 mol/l) to calculate the affinity constant of PCT monoclonal antibody. The results show that it has high affinity, Kd=5.3×10 -8 mol/L.
[0090] For the experimental procedures of this embodiment, please refer to "Modern Immunological Experimental Technology" (edited by Shen Yan and Zhou Rulin).
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