CN114891104A - Monoclonal antibody for identifying AMH and application thereof - Google Patents
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Abstract
The invention belongs to the technical field of biology, and particularly relates to a monoclonal antibody for identifying AMH and application thereof. The monoclonal antibody A and the monoclonal antibody B can both recognize recombinant human proAMH protein, and the monoclonal antibody A can be combined with AMH c Surface, monoclonal antibody B is able to bind to AMH n Surface, monoclonal antibody A and monoclonal antibody B for AMH n 、AMH c The monomers do not have cross reaction; the enzyme linked immunosorbent assay kit developed by combining the monoclonal antibodies has the advantages of high specificity, strong anti-interference capability, high detection sensitivity, good stability and the like, and can stably detect the trace level AMH protein in serum and cell culture medium samples and distinguish the AMH n And AMH c 。
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a monoclonal antibody for identifying AMH and application thereof.
Background
The ovary is a female reproductive organ and has close relation with female reproductive capacity and health condition. The ovary is not only the site where follicles are stored, developed, matured, or ovulate, but also an important endocrine gland in women. Important estrogen hormones including estradiol and progesterone are mainly secreted by follicular granulosa cells and are also regulated by hormones secreted by the hypothalamus and pituitary. Human anti-Mullerian hormone (AMH) is a timer reflecting the function of ovarian reserve and is a protein secreted by granulosa cells of the ovarian antrum preantral and antral follicles, AMH is a homodimeric disulfide-linked glycoprotein with a molecular weight of 140kDa, and is an active AMH protein complex (AMH) N,C ) Divided into AMH n And AMH c Two parts, which are formed by cutting the precursor protein proAMH, and the human body AMH mainly comprises proAMH and AMH N,C These two forms exist, and a small amount of AMH will also exist n And AMH c The monomer (2) of (1).
Polycystic ovarian syndrome (PCOS) patients secrete more AMH, and in addition, AMH is abnormally elevated in some patients with ovarian tumors (e.g., adult granulomatosis), which can be used as a tumor marker to gauge response to treatment and to monitor recurrence. AMH has been shown to be a specific circulating marker for granulocytic tumors, suggesting recurrence of granulocytic tumors 11 months earlier than other clinical markers. The detection of AHM is also of great importance for the diagnosis and prognostic monitoring of the above mentioned diseases.
The existing clinical detection kit can determine the total amount of AMH, but cannot distinguish proAMH from AMH N,C Composite and AMH n And AMH c . Therefore, the AMH is distinguished and detected while the total AMH amount is accurately detected n And AMH c Has potential clinical significance.
Disclosure of Invention
The invention aims to provide a monoclonal antibody for identifying AMH and application thereof, accurately detect the total amount of AMH and distinguish and detect the AMH n And AMH c 。
The invention provides a monoclonal antibody for identifying AMH, which comprises a monoclonal antibody A and/or a monoclonal antibody B;
the monoclonal antibody A has a light chain complementarity determining region CDR3 shown in SEQ ID NO. 7 and a heavy chain complementarity determining region CDR3 shown in SEQ ID NO. 10;
the monoclonal antibody B has the light chain complementarity determining region CDR3 shown in SEQ ID NO. 17 and the heavy chain complementarity determining region CDR3 shown in SEQ ID NO. 20.
Preferably, the light chain complementarity determining region CDR1 of monoclonal antibody A includes the amino acid sequence shown in SEQ ID NO. 5; the light chain complementarity determining region CDR2 of monoclonal antibody A includes the amino acid sequence shown in SEQ ID NO. 6;
the light chain complementarity determining region CDR1 of monoclonal antibody B includes the amino acid sequence shown in SEQ ID NO. 15; the light chain complementarity determining region CDR2 of monoclonal antibody B includes the amino acid sequence shown in SEQ ID NO. 16.
Preferably, the heavy chain complementarity determining region CDR1 of monoclonal antibody A includes the amino acid sequence shown in SEQ ID NO. 8; the heavy chain complementarity determining region CDR2 of monoclonal antibody A comprises the amino acid sequence shown in SEQ ID NO. 9;
the heavy chain complementarity determining region CDR1 of monoclonal antibody B comprises the amino acid sequence shown in SEQ ID NO. 18; the heavy chain complementarity determining region CDR2 of monoclonal antibody B includes the amino acid sequence shown in SEQ ID NO. 19.
Preferably, the variable region of the light chain of the monoclonal antibody A comprises an amino acid sequence shown as SEQ ID NO. 3, and the variable region of the heavy chain of the monoclonal antibody A comprises an amino acid sequence shown as SEQ ID NO. 4;
the variable region of the light chain of the monoclonal antibody B comprises an amino acid sequence shown as SEQ ID NO. 13, and the variable region of the heavy chain of the monoclonal antibody B comprises an amino acid sequence shown as SEQ ID NO. 14.
Preferably, the light chain amino acid sequence of the monoclonal antibody A is shown as SEQ ID NO. 1, and the heavy chain amino acid sequence is shown as SEQ ID NO. 2;
the light chain amino acid sequence of the monoclonal antibody B is shown as SEQ ID NO. 11, and the heavy chain amino acid sequence is shown as SEQ ID NO. 12.
Preferably, the light chain constant regions of monoclonal antibody a and monoclonal antibody B are both kappa chains and the heavy chain constant regions are both IgG1 types.
Preferably, the monoclonal antibody a and the monoclonal antibody B each comprise a rabbit monoclonal antibody.
The invention also provides the application of the monoclonal antibody in the technical scheme in the preparation of an enzyme linked immunosorbent assay kit.
The monoclonal antibody A and the monoclonal antibody B can both recognize recombinant human proAMH protein, and after the monoclonal antibody A is combined with the recombinant human proAMH protein, the monoclonal antibody B can still be combined with the recombinant human proAMH protein, and the monoclonal antibody A and the monoclonal antibody B are paired for use in AMH n 、AMH c The monomers have no cross reaction and can be used for distinguishing and detecting proAMH and AMH n Or AMH c A monomer. The monoclonal antibody has the advantages of high specificity, strong anti-interference capability, high detection sensitivity, good stability and the like on the developed enzyme linked immunosorbent assay kit, and can detect the trace level AMH protein and distinguish the AMH n Or AMH c A monomer.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required in the embodiments will be briefly described below.
FIGS. 1-1 to 1-2 show the results of affinity measurement for each of monoclonal antibody A and monoclonal antibody B;
FIG. 2 shows the identification of the epitope bound by monoclonal antibodies A and B on the human AMH protein;
FIG. 3 shows the results of the sensitivity test;
FIG. 4 shows the detection results of the specificity of monoclonal antibodies A and B;
FIG. 5 shows the stability test results of monoclonal antibodies A and B.
Detailed Description
The invention provides a monoclonal antibody for identifying AMH, which comprises a monoclonal antibody A and/or a monoclonal antibody B; the monoclonal antibody A has a light chain complementarity determining region CDR3 shown in SEQ ID NO. 7 and a heavy chain complementarity determining region CDR3 shown in SEQ ID NO. 10; the monoclonal antibody B has the light chain complementarity determining region CDR3 shown in SEQ ID NO. 17 and the heavy chain complementarity determining region CDR3 shown in SEQ ID NO. 20.
In the present invention, the light chain complementarity determining region CDR1 of monoclonal antibody A preferably includes the amino acid sequence shown in SEQ ID NO. 5; the light chain complementarity determining region CDR2 of monoclonal antibody A preferably includes the amino acid sequence shown in SEQ ID NO. 6; the monoclonal antibody A has a light chain complementarity determining region CDR3 shown in SEQ ID NO. 7; the heavy chain complementarity determining region CDR1 of monoclonal antibody A of the present invention preferably includes the amino acid sequence shown in SEQ ID NO. 8; the heavy chain complementarity determining region CDR2 of monoclonal antibody A preferably includes the amino acid sequence shown in SEQ ID NO. 9, and the monoclonal antibody A has the heavy chain complementarity determining region CDR3 shown in SEQ ID NO. 10. The variable region of the light chain of the monoclonal antibody A preferably comprises an amino acid sequence shown as SEQ ID NO. 3; the heavy chain variable region of the monoclonal antibody A preferably comprises an amino acid sequence shown as SEQ ID NO. 4; the light chain amino acid sequence of the monoclonal antibody A is preferably shown as SEQ ID NO. 1, and the heavy chain amino acid sequence is preferably shown as SEQ ID NO. 2; the light chain constant region of the monoclonal antibody A of the invention is preferably a kappa chain, and the heavy chain constant region is preferably of the IgG1 type.
The amino acid sequences of SEQ ID NO 1-10 are as follows:
SEQ ID NO:1：ADVVMTQTPASVSDPVGGTVTIKCQASQTITNFLSWFQQKPGQPPKLLIYNTMTLASGVPSRFKGSGSGTEYTLTISGVECADAATYYCQEGDNWDEVDAVFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC。
SEQ ID NO:2：QSVKESEGGLFKPTDTLTLTCTVSAFSISTYGVSWVRQAPGNGLEYIGWIAATGKVFYASWAKSRSTITRNTYENTVTLKMTSLTVADTATYFCAHVIWATQTFDFWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPMCPPPELPGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPTVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK。
SEQ ID NO:3：ADVVMTQTPASVSDPVGGTVTIKCQASQTITNFLSWFQQKPGQPPKLLIYNTMTLASGVPSRFKGSGSGTEYTLTISGVECADAATYYCQEGDNWDEVDAVFGGGTEVVVK。
SEQ ID NO:4：QSVKESEGGLFKPTDTLTLTCTVSAFSISTYGVSWVRQAPGNGLEYIGWIAATGKVFYASWAKSRSTITRNTYENTVTLKMTSLTVADTATYFCAHVIWATQTFDFWGPGTLVTVSS。
SEQ ID NO:5：QTITNF。
SEQ ID NO:6：NTM。
SEQ ID NO:7：QEGDNWDEVDAV。
SEQ ID NO:8：AFSISTYG。
SEQ ID NO:9：IAATGKV。
SEQ ID NO:10：AHVIWATQTFDF。
the monoclonal antibody A can be combined with recombinant human proAMH protein and specifically combined with AMH c Surface, has an ultra-high affinity for -luxrin-resistant protein, specifically 6.02X 10 -9 M, capable of detecting the levels of anti- lxeridin protein secreted by cells, and anti- lxeridin in serum.
In the present invention, the light chain complementarity determining region CDR1 of monoclonal antibody B preferably includes the amino acid sequence shown in SEQ ID NO. 15; the light chain complementarity determining region CDR2 of monoclonal antibody B preferably includes the amino acid sequence shown in SEQ ID NO. 16, and the monoclonal antibody B has the light chain complementarity determining region CDR3 shown in SEQ ID NO. 17; the heavy chain complementarity determining region CDR1 of monoclonal antibody B preferably includes the amino acid sequence shown in SEQ ID NO. 18; the heavy chain complementarity determining region CDR2 of monoclonal antibody B preferably includes the amino acid sequence shown in SEQ ID NO. 19; the monoclonal antibody B has a heavy chain complementarity determining region CDR3 shown in SEQ ID NO. 20. The variable region of the light chain of the monoclonal antibody B preferably comprises an amino acid sequence shown as SEQ ID NO. 13; the heavy chain variable region of the monoclonal antibody B preferably comprises an amino acid sequence shown as SEQ ID NO. 14; the light chain amino acid sequence of the monoclonal antibody B is preferably shown as SEQ ID NO. 11, and the heavy chain amino acid sequence is preferably shown as SEQ ID NO. 12; the light chain constant region of the monoclonal antibody B of the invention is preferably a kappa chain, and the heavy chain constant region is preferably of the IgG1 type.
The amino acid sequences of SEQ ID NO. 11-20 of the invention are as follows:
SEQ ID NO:11：AIEMTQTPSSVSATVGGTVTINCQSSEQINRFLAWYQQKPGQPPKLLIYWGSTLASGVPSRFKGSGSGTDYTLTISGVKCDDAATYYCQSAFYSCSTNTFYLFGGGTEVVVRGDPVAPTVLIFPPSADLVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC。
SEQ ID NO:12：QSVEESGGRLVSPGTPLTLTCTVSAFTLSSRAMSWVRQAPGKGLEWIGIIGVTGQTYYASWAKGRFTISKTSTTVDLKITSPTTEDTATYFCAKDLTYDSFAYAYITDWYGSDLWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPMCPPPELPGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPTVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK。
SEQ ID NO:13：AIEMTQTPSSVSATVGGTVTINCQSSEQINRFLAWYQQKPGQPPKLLIYWGSTLASGVPSRFKGSGSGTDYTLTISGVKCDDAATYYCQSAFYSCSTNTFYLFGGGTEVVVR。
SEQ ID NO:14：QSVEESGGRLVSPGTPLTLTCTVSAFTLSSRAMSWVRQAPGKGLEWIGIIGVTGQTYYASWAKGRFTISKTSTTVDLKITSPTTEDTATYFCAKDLTYDSFAYAYITDWYGSDLWGPGTLVTVSS。
SEQ ID NO:15：EQINRF。
SEQ ID NO:16：WGS。
SEQ ID NO:17：QSAFYSCSTNTFYL。
SEQ ID NO:18：AFTLSSRA。
SEQ ID NO:19：IGVTGQT。
SEQ ID NO:20：AKDLTYDSFAYAYITDWYGSDL。
the monoclonal antibody B can be combined with recombinant human proAMH protein and specifically combined with AMH n Surface, has an ultra-high affinity for -luxrin-resistant protein, specifically 4.7X 10 -11 M, capable of detecting cellsSecreted anti- luxianin protein, and anti- luxianin levels in serum.
The monoclonal antibody A and the monoclonal antibody B are combined for use, and the monoclonal antibody A is specifically bound to AMH c Surface, the monoclonal antibody B specifically binds to AMH n A surface. After the monoclonal antibody A is combined with the recombinant human proAMH protein, the monoclonal antibody B can still be combined with the recombinant human proAMH protein, and the monoclonal antibody A and the monoclonal antibody B can still be used for treating the AMH n 、AMH c The monomers do not have cross reaction and can be distinguished to detect AMH n Or AMH c 。
The method for preparing the monoclonal antibody A and/or the monoclonal antibody B is not strictly required, and the method well known in the field can be selected.
The invention also provides application of the monoclonal antibody combination in preparing an enzyme linked immunosorbent assay kit. The enzyme linked immunosorbent assay kit developed by combining the monoclonal antibodies has the advantages of high specificity, strong anti-interference capability, high detection sensitivity, good stability and the like, can stably detect the trace level AMH protein, and distinguishes the AMH n Or AMH c 。
In order to further illustrate the present invention, the following detailed description of the technical solutions provided by the present invention is made with reference to the accompanying drawings and examples, but they should not be construed as limiting the scope of the present invention.
Example 1
AMH n 、AMH c And preparation of proAMH protein
1. Construction of expression plasmids
Synthesis of coded AMH n 、AMH c And the nucleotide sequence of proAMH (Uniprot SEQ ID NO: P03971), and a Kozac sequence preceded by the initiation codon ATG, to which EcoRI/BamHI enzyme sites encoding AMH were added n 、AMH c And the nucleotide sequence of proAMH is constructed into a pCDNA3.4 mammalian cell expression vector and sequenced, the processes are all carried out by a general biological system (Anhui) limited company, and the sequencing is correct and the construction is successful.
2. Transient transfection and protein expression purification
Expi29 cell culture and transfection adopt an Expi293 expression system, after the Expi293 cell is transiently transfected by a shake flask, the cell is harvested on day 4, and cell supernatant is obtained by centrifugation at 10000 rpm;
separately eluting AMH using 5mL Ni-IMAC affinity pre-packed columns n 、AMH c And proAMH proteins, in particular: the harvested 1L cell supernatant was added in batches to the equilibrated Ni-IMAC affinity pre-packed column using equilibration buffer pH 7.4 (20mM PB, 500mM NaCl and balance deionized water).
Washing 5 column volumes with pH 7.4 buffer (20mM PB, 500mM NaCl, 30mM izole and balance deionized water), collecting the eluate to obtain AMH n A protein;
washing 5 column volumes with pH 7.4 buffer (20mM PB, 500mM NaCl, 50mM izole and balance deionized water), collecting the eluate to obtain AMH c A protein;
washing 5 column volumes with a buffer (20mM PB, 500mM NaCl, 500mM izole and balance deionized water) at pH 7.4, and collecting the eluate to obtain proAMH protein;
example 2
Preparation of human AMH rabbit monoclonal antibody
1) Animal immunization: respectively taking the AMHc and AMHn proteins obtained in the embodiment 1 as immunogens to immunize a New Zealand white rabbit; immunizing each big white rabbit by 200 mu g, mixing immunogen with equal amount of complete Freund adjuvant to prepare an emulsifier by first immunization, carrying out subcutaneous multipoint injection on the abdomen and the back, taking 100 mu g of immunogen at intervals of 3 weeks, mixing the immunogen with equal amount of incomplete Freund adjuvant to prepare the emulsifier, carrying out subcutaneous multipoint injection on the abdomen and the back, carrying out boosting immunization twice, measuring the serum titer by an ELISA (enzyme-linked immuno sorbent assay) method after three times of immunization, taking rabbits with high serum titer, carrying out subcutaneous multipoint injection boosting immunization by 200 mu g of immunogen once, taking spleen after three days, and separating spleen cells.
2) B lymphocyte sorting: see patent 201910125091.4 for a method for efficiently isolating single antigen-specific B lymphocytes from spleen cells.
3) Cloning of genes encoding rabbit monoclonal antibodies
The cultured B cell supernatants were identified as positive clones by antigen-coated ELISA. The positive cloned cells are collected, cracked, extracted with RNA, and reverse transcribed into cDNA (reverse transcription 5min at 65 ℃) by using a total RNA extraction kit. Naturally paired rabbit monoclonal antibody light and heavy chain variable region genes (VH and VL) were amplified from the corresponding positively cloned cDNA by PCR and sequenced to determine the sequence.
4) Production and purification of monoclonal antibodies:
respectively loading heavy chain and light chain genes of the rabbit monoclonal antibody in the step 3) on pCDN3.4 expression vectors, and transfecting the plasmid into 293F cells; and transfecting for 72-96 hours to obtain a rabbit monoclonal antibody which contains recombinant recognition human proAMH protein in culture supernatant. Purifying a plurality of recombinant rabbit monoclonal antibodies for recognizing human proAMH protein from the supernatant of the transfected culture medium by using protein A affinity gel resin, sequentially naming the monoclonal antibodies as a monoclonal antibody A and a monoclonal antibody B, identifying the antibodies, subpackaging, and storing at the low temperature of-20 ℃ for later use.
Example 3
Screening and identification of antibodies
1) The affinity of each monoclonal antibody of example 2 was precisely determined using a Biacore 3000 biomolecule interaction analyzer of General Electric (GE) corporation, usa, in which selected monoclonal antibody a and monoclonal antibody B were used at concentrations of 37nM, respectively, and were immobilized on a CM5 chip, and then the monoclonal antibody a and monoclonal antibody B were unbound with recombinant human proAMH proteins at five concentrations of 100nM, 50nM, 33.33nM, 11.11nM, and 3nM, respectively, to obtain an affinity curve, and finally the affinities of monoclonal antibodies a and B were obtained by curve fitting and calculation, the affinity curve being shown in fig. 1-2, and the fitting results being shown in table 1.
TABLE 1 affinity assay results for each monoclonal antibody
| Monoclonal antibodies | Koff(1/s) | Kon(1/Ms) | Affinity (M) |
| A | 1.17×10 -4 | 1.95×10 4 | 6.02×10 -9 |
| B | 7.01×10 -6 | 1.49×10 5 | 4.7×10 -11 |
Note: koff (dissociation rate constant) represents the dissociation rate constant between molecules, and represents the rate of dissociation between molecules; kon (association rate constant) is an intermolecular association rate constant, and represents how fast or slow the intermolecular association is.
As can be seen from Table 1 and FIGS. 1-1 to 1-2, the affinity of monoclonal antibody A was 6.02X 10 -9 M, monoclonal antibody B affinity of 4.7X 10 -11 M。
2) The antigen-recognition epitopes bound to the human AMH protein by the rabbit anti-human proAMH monoclonal antibodies A and B obtained in example 2 were identified using a Gator biomolecule interaction Analyzer from Probe Life, using His-tag recombinant human proAMH protein at a concentration of 50nM, and using the monoclonal antibodies A and B obtained at concentrations of 600nM and 300nM, respectively, as shown in FIG. 2.
As can be seen from FIG. 2, it can be seen from the data obtained by analyzing the pairing between the monoclonal antibodies A and B that the monoclonal antibody B can still bind to the recombinant human proAMH protein after the monoclonal antibody A binds to the recombinant human proAMH protein, and the monoclonal antibodies A and B bind to different sites on the surface of the proAMH protein without interfering with each other. Detecting that the constant region of the light chain of the monoclonal antibody A is a kappa chain, the constant region of the heavy chain is IgG1 type, the amino acid sequences of the complementarity determining regions CDR1, CDR2 and CDR3 on the light chain are respectively shown as SEQ ID NO. 5, SEQ ID NO. 6 and SEQ ID NO. 7, the amino acid sequences of the complementarity determining regions CDR1, CDR2 and CDR3 on the heavy chain are respectively shown as SEQ ID NO. 8, SEQ ID NO. 9 and SEQ ID NO. 10, the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 3, the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 4, the amino acid sequence of the light chain is shown as SEQ ID NO. 1, and the amino acid sequence of the heavy chain is shown as SEQ ID NO. 2; the constant region of the monoclonal antibody B light chain is a kappa chain, the constant region of the heavy chain is IgG1 type, the amino acid sequences of the complementarity determining regions CDR1, CDR2 and CDR3 on the light chain are respectively shown as SEQ ID NO. 15, SEQ ID NO. 16 and SEQ ID NO. 17, the amino acid sequences of the complementarity determining regions CDR1, CDR2 and CDR3 on the heavy chain are respectively shown as SEQ ID NO. 18, SEQ ID NO. 19 and SEQ ID NO. 20, the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 13, the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 14, the amino acid sequence of the light chain is shown as SEQ ID NO. 11, and the amino acid sequence of the heavy chain is shown as SEQ ID NO. 12.
Example 4
1. A double-antibody sandwich enzyme-linked immunoassay method is established based on anti-human AMH protein rabbit monoclonal antibodies A and B, and comprises the following steps:
1) coating the capture antibody, anti-human proAMH protein rabbit monoclonal antibody A, with carbonate buffer (pH9.4, 0.05M), and incubating overnight at 4 ℃; washing the plate with a washing solution;
2) blocking with phosphate buffer (pH7.2, 0.05M) containing 5% bovine serum albumin, 0.05% Tween-20;
3) diluting a standard sample (recombinant human proAMH protein) or a sample to be detected by using a phosphate buffer solution containing 1% of bovine serum albumin and 0.05% of Tween-20, adding the diluted sample into an enzyme label plate, incubating for 1 hour under the normal temperature condition, and washing the plate by using a washing solution;
4) adding a biotin-labeled anti-human proAMH protein rabbit monoclonal antibody B diluted by phosphate buffer solution containing 1% of bovine serum albumin and 0.05% of Tween-20 into a plate, incubating for 1 hour under normal temperature conditions, and washing the plate by using a washing solution;
5) adding avidin-labeled Horse Radish Peroxidase (HRP) diluted by phosphate buffer containing 1% of bovine serum albumin and 0.05% of Tween-20, incubating for 1 hour under normal temperature, and washing the plate by using a washing solution;
6) adding TMB color developing solution, developing at room temperature for 10 min, adding oxalic acid to stop developing, and measuring absorbance at 450nm and 630nm respectively (OD450 minus OD630 is corrected absorbance).
2. Sensitivity detection
Detecting according to the method in the step 1, selecting the coating concentration of the anti-human proAMH protein rabbit monoclonal antibody A to be 2 mug/mL, and the detection concentration of the biomarker anti-human proAMH protein rabbit monoclonal antibody B to be 1 mug/mL; step 3) adding recombinant human proAMH protein as sample, diluting the sample with phosphate buffer solution containing 1% bovine serum albumin and 0.05% Tween-20, wherein the concentration is 10000ng/mL, 5000ng/mL, 2500ng/mL, 1250ng/mL, 625ng/mL, 312.5ng/mL, 156.25ng/mL, 78.125ng/mL, 39.062ng/mL, 19.531ng/mL, 9.765ng/mL, 4.882ng/mL, 2.441ng/mL, 1.220ng/mL, 0.610ng/mL, 0ng/mL, the sample addition amount is 25 muL/well, the logarithm value (Log10) of the human proAMH protein concentration is used as abscissa, the corrected value (OD450-OD630) of the absorbance value is used as ordinate to plot, establishing a standard curve, the lowest human AMH protein concentration with the average absorbance value being three times larger than the blank value is used as the sensitivity of the double-antibody enzyme-linked immunosorbent assay method detection method, the result is shown in FIG. 3, and it can be seen from FIG. 3 that the detection sensitivity of the enzyme-linked immunoassay method using the double antibody sandwich method established by the monoclonal antibodies A and B reaches 1 ng/mL.
3. Detection of the specificity of monoclonal antibodies A and B
Detecting according to the method of step 1, using proAMH protein and AMH respectively n Protein, AMH c The protein is detected as standard protein, wherein all standard proteins (proAMH protein, AMH) n Protein, AMH c Protein) were all 1. mu.g/mL, and the results are shown in FIG. 4, according toFIG. 4 shows that the double-antibody sandwich enzyme-linked immunoassay method based on anti-human proAMH protein rabbit monoclonal antibodies A and B is applied to AMH n 、AMH c None of the proteins was cross-reactive, and anti-human proAMH protein rabbit monoclonal antibodies A and B were highly specific for human proAMH protein.
4. Detection of monoclonal antibodies A and B thermostable
The rabbit monoclonal antibodies A and B of the anti-human proAMH protein are placed in a 37-degree incubator, the samples are respectively taken on days 3, 6, 9 and 14, then the detection is carried out according to the method in the step 1, so as to detect the human proAMH standard protein, and the thermal stability of the rabbit monoclonal antibodies A and B of the anti-human proAMH protein is compared according to a standard curve established by comparing antibody samples treated at 37 degrees for different time periods with antibody samples not treated at 37 degrees, and the result is shown in figure 5. As can be seen from FIG. 5, the two rabbit anti-AMH monoclonal antibodies of the present invention have less than 1% effect on the detection sensitivity and linear range after being treated at 37 ℃ for 14 days, which proves that the rabbit monoclonal antibodies A and B against human AMH protein have strong thermal stability.
The monoclonal antibody A and the monoclonal antibody B provided by the invention have ultrahigh affinity for resisting luxin protein, the monoclonal antibody A specifically detects AMHn, the monoclonal antibody B specifically detects AMHc, and cross reaction does not exist on AMHn or AMHc monomers.
Although the present invention has been described in detail with reference to the above embodiments, it is only a part of the embodiments of the present invention, not all of the embodiments, and other embodiments can be obtained without inventive step according to the embodiments, and the embodiments are within the scope of the present invention.
Sequence listing
<110> Yougui saisi (Wuhan) Biotechnology Ltd
<120> monoclonal antibody recognizing AMH and use thereof
<160> 20
<170> SIPOSequenceListing 1.0
<210> 1
<211> 215
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 1
Ala Asp Val Val Met Thr Gln Thr Pro Ala Ser Val Ser Asp Pro Val
1 5 10 15
Gly Gly Thr Val Thr Ile Lys Cys Gln Ala Ser Gln Thr Ile Thr Asn
20 25 30
Phe Leu Ser Trp Phe Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu
35 40 45
Ile Tyr Asn Thr Met Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Lys
50 55 60
Gly Ser Gly Ser Gly Thr Glu Tyr Thr Leu Thr Ile Ser Gly Val Glu
65 70 75 80
Cys Ala Asp Ala Ala Thr Tyr Tyr Cys Gln Glu Gly Asp Asn Trp Asp
85 90 95
Glu Val Asp Ala Val Phe Gly Gly Gly Thr Glu Val Val Val Lys Gly
100 105 110
Asp Pro Val Ala Pro Thr Val Leu Ile Phe Pro Pro Ala Ala Asp Gln
115 120 125
Val Ala Thr Gly Thr Val Thr Ile Val Cys Val Ala Asn Lys Tyr Phe
130 135 140
Pro Asp Val Thr Val Thr Trp Glu Val Asp Gly Thr Thr Gln Thr Thr
145 150 155 160
Gly Ile Glu Asn Ser Lys Thr Pro Gln Asn Ser Ala Asp Cys Thr Tyr
165 170 175
Asn Leu Ser Ser Thr Leu Thr Leu Thr Ser Thr Gln Tyr Asn Ser His
180 185 190
Lys Glu Tyr Thr Cys Lys Val Thr Gln Gly Thr Thr Ser Val Val Gln
195 200 205
Ser Phe Asn Arg Gly Asp Cys
210 215
<210> 2
<211> 440
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 2
Gln Ser Val Lys Glu Ser Glu Gly Gly Leu Phe Lys Pro Thr Asp Thr
1 5 10 15
Leu Thr Leu Thr Cys Thr Val Ser Ala Phe Ser Ile Ser Thr Tyr Gly
20 25 30
Val Ser Trp Val Arg Gln Ala Pro Gly Asn Gly Leu Glu Tyr Ile Gly
35 40 45
Trp Ile Ala Ala Thr Gly Lys Val Phe Tyr Ala Ser Trp Ala Lys Ser
50 55 60
Arg Ser Thr Ile Thr Arg Asn Thr Tyr Glu Asn Thr Val Thr Leu Lys
65 70 75 80
Met Thr Ser Leu Thr Val Ala Asp Thr Ala Thr Tyr Phe Cys Ala His
85 90 95
Val Ile Trp Ala Thr Gln Thr Phe Asp Phe Trp Gly Pro Gly Thr Leu
100 105 110
Val Thr Val Ser Ser Gly Gln Pro Lys Ala Pro Ser Val Phe Pro Leu
115 120 125
Ala Pro Cys Cys Gly Asp Thr Pro Ser Ser Thr Val Thr Leu Gly Cys
130 135 140
Leu Val Lys Gly Tyr Leu Pro Glu Pro Val Thr Val Thr Trp Asn Ser
145 150 155 160
Gly Thr Leu Thr Asn Gly Val Arg Thr Phe Pro Ser Val Arg Gln Ser
165 170 175
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Ser Val Thr Ser Ser Ser
180 185 190
Gln Pro Val Thr Cys Asn Val Ala His Pro Ala Thr Asn Thr Lys Val
195 200 205
Asp Lys Thr Val Ala Pro Ser Thr Cys Ser Lys Pro Met Cys Pro Pro
210 215 220
Pro Glu Leu Pro Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Pro
225 230 235 240
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
245 250 255
Val Asp Val Ser Gln Asp Asp Pro Glu Val Gln Phe Thr Trp Tyr Ile
260 265 270
Asn Asn Glu Gln Val Arg Thr Ala Arg Pro Pro Leu Arg Glu Gln Gln
275 280 285
Phe Asn Ser Thr Ile Arg Val Val Ser Thr Leu Pro Ile Ala His Gln
290 295 300
Asp Trp Leu Arg Gly Lys Glu Phe Lys Cys Lys Val His Asn Lys Ala
305 310 315 320
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Arg Gly Gln Pro
325 330 335
Leu Glu Pro Lys Val Tyr Thr Met Gly Pro Pro Arg Glu Glu Leu Ser
340 345 350
Ser Arg Ser Val Ser Leu Thr Cys Met Ile Asn Gly Phe Tyr Pro Ser
355 360 365
Asp Ile Ser Val Glu Trp Glu Lys Asn Gly Lys Ala Glu Asp Asn Tyr
370 375 380
Lys Thr Thr Pro Thr Val Leu Asp Ser Asp Gly Ser Tyr Phe Leu Tyr
385 390 395 400
Ser Lys Leu Ser Val Pro Thr Ser Glu Trp Gln Arg Gly Asp Val Phe
405 410 415
Thr Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
420 425 430
Ser Ile Ser Arg Ser Pro Gly Lys
435 440
<210> 3
<211> 111
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 3
Ala Asp Val Val Met Thr Gln Thr Pro Ala Ser Val Ser Asp Pro Val
1 5 10 15
Gly Gly Thr Val Thr Ile Lys Cys Gln Ala Ser Gln Thr Ile Thr Asn
20 25 30
Phe Leu Ser Trp Phe Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu
35 40 45
Ile Tyr Asn Thr Met Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Lys
50 55 60
Gly Ser Gly Ser Gly Thr Glu Tyr Thr Leu Thr Ile Ser Gly Val Glu
65 70 75 80
Cys Ala Asp Ala Ala Thr Tyr Tyr Cys Gln Glu Gly Asp Asn Trp Asp
85 90 95
Glu Val Asp Ala Val Phe Gly Gly Gly Thr Glu Val Val Val Lys
100 105 110
<210> 4
<211> 117
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 4
Gln Ser Val Lys Glu Ser Glu Gly Gly Leu Phe Lys Pro Thr Asp Thr
1 5 10 15
Leu Thr Leu Thr Cys Thr Val Ser Ala Phe Ser Ile Ser Thr Tyr Gly
20 25 30
Val Ser Trp Val Arg Gln Ala Pro Gly Asn Gly Leu Glu Tyr Ile Gly
35 40 45
Trp Ile Ala Ala Thr Gly Lys Val Phe Tyr Ala Ser Trp Ala Lys Ser
50 55 60
Arg Ser Thr Ile Thr Arg Asn Thr Tyr Glu Asn Thr Val Thr Leu Lys
65 70 75 80
Met Thr Ser Leu Thr Val Ala Asp Thr Ala Thr Tyr Phe Cys Ala His
85 90 95
Val Ile Trp Ala Thr Gln Thr Phe Asp Phe Trp Gly Pro Gly Thr Leu
100 105 110
Val Thr Val Ser Ser
115
<210> 5
<211> 6
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 5
Gln Thr Ile Thr Asn Phe
1 5
<210> 6
<211> 3
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 6
Asn Thr Met
1
<210> 7
<211> 12
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 7
Gln Glu Gly Asp Asn Trp Asp Glu Val Asp Ala Val
1 5 10
<210> 8
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 8
Ala Phe Ser Ile Ser Thr Tyr Gly
1 5
<210> 9
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 9
Ile Ala Ala Thr Gly Lys Val
1 5
<210> 10
<211> 12
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 10
Ala His Val Ile Trp Ala Thr Gln Thr Phe Asp Phe
1 5 10
<210> 11
<211> 216
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 11
Ala Ile Glu Met Thr Gln Thr Pro Ser Ser Val Ser Ala Thr Val Gly
1 5 10 15
Gly Thr Val Thr Ile Asn Cys Gln Ser Ser Glu Gln Ile Asn Arg Phe
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile
35 40 45
Tyr Trp Gly Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Lys Gly
50 55 60
Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Gly Val Lys Cys
65 70 75 80
Asp Asp Ala Ala Thr Tyr Tyr Cys Gln Ser Ala Phe Tyr Ser Cys Ser
85 90 95
Thr Asn Thr Phe Tyr Leu Phe Gly Gly Gly Thr Glu Val Val Val Arg
100 105 110
Gly Asp Pro Val Ala Pro Thr Val Leu Ile Phe Pro Pro Ser Ala Asp
115 120 125
Leu Val Ala Thr Gly Thr Val Thr Ile Val Cys Val Ala Asn Lys Tyr
130 135 140
Phe Pro Asp Val Thr Val Thr Trp Glu Val Asp Gly Thr Thr Gln Thr
145 150 155 160
Thr Gly Ile Glu Asn Ser Lys Thr Pro Gln Asn Ser Ala Asp Cys Thr
165 170 175
Tyr Asn Leu Ser Ser Thr Leu Thr Leu Thr Ser Thr Gln Tyr Asn Ser
180 185 190
His Lys Glu Tyr Thr Cys Lys Val Thr Gln Gly Thr Thr Ser Val Val
195 200 205
Gln Ser Phe Asn Arg Gly Asp Cys
210 215
<210> 12
<211> 448
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 12
Gln Ser Val Glu Glu Ser Gly Gly Arg Leu Val Ser Pro Gly Thr Pro
1 5 10 15
Leu Thr Leu Thr Cys Thr Val Ser Ala Phe Thr Leu Ser Ser Arg Ala
20 25 30
Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly
35 40 45
Ile Ile Gly Val Thr Gly Gln Thr Tyr Tyr Ala Ser Trp Ala Lys Gly
50 55 60
Arg Phe Thr Ile Ser Lys Thr Ser Thr Thr Val Asp Leu Lys Ile Thr
65 70 75 80
Ser Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala Lys Asp Leu
85 90 95
Thr Tyr Asp Ser Phe Ala Tyr Ala Tyr Ile Thr Asp Trp Tyr Gly Ser
100 105 110
Asp Leu Trp Gly Pro Gly Thr Leu Val Thr Val Ser Ser Gly Gln Pro
115 120 125
Lys Ala Pro Ser Val Phe Pro Leu Ala Pro Cys Cys Gly Asp Thr Pro
130 135 140
Ser Ser Thr Val Thr Leu Gly Cys Leu Val Lys Gly Tyr Leu Pro Glu
145 150 155 160
Pro Val Thr Val Thr Trp Asn Ser Gly Thr Leu Thr Asn Gly Val Arg
165 170 175
Thr Phe Pro Ser Val Arg Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser
180 185 190
Val Val Ser Val Thr Ser Ser Ser Gln Pro Val Thr Cys Asn Val Ala
195 200 205
His Pro Ala Thr Asn Thr Lys Val Asp Lys Thr Val Ala Pro Ser Thr
210 215 220
Cys Ser Lys Pro Met Cys Pro Pro Pro Glu Leu Pro Gly Gly Pro Ser
225 230 235 240
Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
245 250 255
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Asp Asp Pro
260 265 270
Glu Val Gln Phe Thr Trp Tyr Ile Asn Asn Glu Gln Val Arg Thr Ala
275 280 285
Arg Pro Pro Leu Arg Glu Gln Gln Phe Asn Ser Thr Ile Arg Val Val
290 295 300
Ser Thr Leu Pro Ile Ala His Gln Asp Trp Leu Arg Gly Lys Glu Phe
305 310 315 320
Lys Cys Lys Val His Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
325 330 335
Ile Ser Lys Ala Arg Gly Gln Pro Leu Glu Pro Lys Val Tyr Thr Met
340 345 350
Gly Pro Pro Arg Glu Glu Leu Ser Ser Arg Ser Val Ser Leu Thr Cys
355 360 365
Met Ile Asn Gly Phe Tyr Pro Ser Asp Ile Ser Val Glu Trp Glu Lys
370 375 380
Asn Gly Lys Ala Glu Asp Asn Tyr Lys Thr Thr Pro Thr Val Leu Asp
385 390 395 400
Ser Asp Gly Ser Tyr Phe Leu Tyr Ser Lys Leu Ser Val Pro Thr Ser
405 410 415
Glu Trp Gln Arg Gly Asp Val Phe Thr Cys Ser Val Met His Glu Ala
420 425 430
Leu His Asn His Tyr Thr Gln Lys Ser Ile Ser Arg Ser Pro Gly Lys
435 440 445
<210> 13
<211> 112
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 13
Ala Ile Glu Met Thr Gln Thr Pro Ser Ser Val Ser Ala Thr Val Gly
1 5 10 15
Gly Thr Val Thr Ile Asn Cys Gln Ser Ser Glu Gln Ile Asn Arg Phe
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile
35 40 45
Tyr Trp Gly Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Lys Gly
50 55 60
Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Gly Val Lys Cys
65 70 75 80
Asp Asp Ala Ala Thr Tyr Tyr Cys Gln Ser Ala Phe Tyr Ser Cys Ser
85 90 95
Thr Asn Thr Phe Tyr Leu Phe Gly Gly Gly Thr Glu Val Val Val Arg
100 105 110
<210> 14
<211> 125
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 14
Gln Ser Val Glu Glu Ser Gly Gly Arg Leu Val Ser Pro Gly Thr Pro
1 5 10 15
Leu Thr Leu Thr Cys Thr Val Ser Ala Phe Thr Leu Ser Ser Arg Ala
20 25 30
Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly
35 40 45
Ile Ile Gly Val Thr Gly Gln Thr Tyr Tyr Ala Ser Trp Ala Lys Gly
50 55 60
Arg Phe Thr Ile Ser Lys Thr Ser Thr Thr Val Asp Leu Lys Ile Thr
65 70 75 80
Ser Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala Lys Asp Leu
85 90 95
Thr Tyr Asp Ser Phe Ala Tyr Ala Tyr Ile Thr Asp Trp Tyr Gly Ser
100 105 110
Asp Leu Trp Gly Pro Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 15
<211> 6
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 15
Glu Gln Ile Asn Arg Phe
1 5
<210> 16
<211> 3
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 16
Trp Gly Ser
1
<210> 17
<211> 14
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 17
Gln Ser Ala Phe Tyr Ser Cys Ser Thr Asn Thr Phe Tyr Leu
1 5 10
<210> 18
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 18
Ala Phe Thr Leu Ser Ser Arg Ala
1 5
<210> 19
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 19
Ile Gly Val Thr Gly Gln Thr
1 5
<210> 20
<211> 22
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 20
Ala Lys Asp Leu Thr Tyr Asp Ser Phe Ala Tyr Ala Tyr Ile Thr Asp
1 5 10 15
Trp Tyr Gly Ser Asp Leu
20
Claims (8)
1. Monoclonal antibodies recognizing AMH, comprising monoclonal antibody A and/or monoclonal antibody B;
the monoclonal antibody A has a light chain complementarity determining region CDR3 shown in SEQ ID NO. 7 and a heavy chain complementarity determining region CDR3 shown in SEQ ID NO. 10;
the monoclonal antibody B has the light chain complementarity determining region CDR3 shown in SEQ ID NO. 17 and the heavy chain complementarity determining region CDR3 shown in SEQ ID NO. 20.
2. The monoclonal antibody of claim 1, wherein the light chain complementarity determining region CDR1 of monoclonal antibody A comprises the amino acid sequence set forth in SEQ ID NO. 5; the light chain complementarity determining region CDR2 of monoclonal antibody A includes the amino acid sequence shown in SEQ ID NO. 6;
the light chain complementarity determining region CDR1 of monoclonal antibody B includes the amino acid sequence shown in SEQ ID NO. 15; the light chain complementarity determining region CDR2 of monoclonal antibody B includes the amino acid sequence shown in SEQ ID NO. 16.
3. The monoclonal antibody of claim 1, wherein the heavy chain complementarity determining region CDR1 of monoclonal antibody A comprises the amino acid sequence shown in SEQ ID NO. 8; the heavy chain complementarity determining region CDR2 of monoclonal antibody A comprises the amino acid sequence shown in SEQ ID NO. 9;
the heavy chain complementarity determining region CDR1 of monoclonal antibody B comprises the amino acid sequence shown in SEQ ID NO. 18; the heavy chain complementarity determining region CDR2 of monoclonal antibody B includes the amino acid sequence shown in SEQ ID NO. 19.
4. The monoclonal antibody of claim 1, wherein the variable region of the light chain of monoclonal antibody A comprises the amino acid sequence shown in SEQ ID NO. 3, and the variable region of the heavy chain of monoclonal antibody A comprises the amino acid sequence shown in SEQ ID NO. 4;
the variable region of the light chain of the monoclonal antibody B comprises an amino acid sequence shown as SEQ ID NO. 13, and the variable region of the heavy chain of the monoclonal antibody B comprises an amino acid sequence shown as SEQ ID NO. 14.
5. The monoclonal antibody of claim 1, wherein the light chain amino acid sequence of monoclonal antibody A is shown as SEQ ID NO. 1, and the heavy chain amino acid sequence is shown as SEQ ID NO. 2;
the light chain amino acid sequence of the monoclonal antibody B is shown as SEQ ID NO. 11, and the heavy chain amino acid sequence is shown as SEQ ID NO. 12.
6. The monoclonal antibody of claim 1, wherein the light chain constant regions of both monoclonal antibody a and monoclonal antibody B are kappa chains and the heavy chain constant regions are both of the IgG1 type.
7. The monoclonal antibody of claim 1, wherein the monoclonal antibody a and the monoclonal antibody B each comprise a rabbit monoclonal antibody.
8. Use of the monoclonal antibody of claims 1-7 in the preparation of an enzyme linked immunosorbent kit.
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|---|---|---|---|---|
| EP3257867A1 (en) * | 2016-06-17 | 2017-12-20 | Biomérieux | Method for preparing anti-amh antibodies and uses thereof |
| CN110128535A (en) * | 2019-03-27 | 2019-08-16 | 浙江工业大学 | A kind of AMH monoclonal antibody and its preparation and application |
| CN110407937A (en) * | 2018-04-26 | 2019-11-05 | 江苏正大天创生物工程有限公司 | A kind of preparation and its application of anti-AMH monoclonal antibody |
| WO2020103691A1 (en) * | 2018-11-20 | 2020-05-28 | 厦门万泰凯瑞生物技术有限公司 | Specific antibody for amh, and uses thereof |
| CN111217907A (en) * | 2020-01-21 | 2020-06-02 | 四川大学 | anti-AMH monoclonal antibody and application thereof |
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| EP3257867A1 (en) * | 2016-06-17 | 2017-12-20 | Biomérieux | Method for preparing anti-amh antibodies and uses thereof |
| CN110407937A (en) * | 2018-04-26 | 2019-11-05 | 江苏正大天创生物工程有限公司 | A kind of preparation and its application of anti-AMH monoclonal antibody |
| WO2020103691A1 (en) * | 2018-11-20 | 2020-05-28 | 厦门万泰凯瑞生物技术有限公司 | Specific antibody for amh, and uses thereof |
| CN110128535A (en) * | 2019-03-27 | 2019-08-16 | 浙江工业大学 | A kind of AMH monoclonal antibody and its preparation and application |
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