Rabbit monoclonal antibody based on human epo gene polymorphism c.577del and preparation method thereof

Abstract

The application relates to the technical field of antigen-antibody proteins, in particular to a rabbit monoclonal antibody based on human EPO gene polymorphism c.577del and a preparation method. The method combines antigen preparation and monoclonal antibody preparation to generate a rabbit monoclonal antibody which can specifically recognize VAR-EPO; the rabbit monoclonal antibody has the characteristics of high affinity and high specificity, can specifically and efficiently combine with trace VAR-EPO in human urine and blood samples, does not recognize WT-EPO and rEPO, a two-way immunopurification combined with a protein immunoblotting technology using the antibody can distinguish rEPO and VAR-EPO in human urine and blood samples, directly and accurately detects rEPO in urine and blood samples from various types of people, eliminates the risk of false positive, optimizes the doping detection process, and makes up for the defects in the current world anti-doping field about rEPO detection.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of antigen-antibody proteins, in particular to a rabbit monoclonal antibody based on human EPO gene polymorphism c.577del and a preparation method. BACKGROUND

[0002] Human recombinant erythropoietin (rEPO) has a high proportion of abuse among endurance athletes because it can stimulate erythropoiesis and improve the oxygen-carrying capacity of the human body.

[0003] Recent studies have found that humans EPO The occurrence probability of the human EPO gene polymorphism c.577del in East Asian populations reaches 0.5-1%. The polymorphism can produce a mutant erythropoietin p.Arg193AspfsTer28 (VAR-EPO), and the VAR-EPO contains all the amino acid structures of wild-type erythropoietin (WT-EPO) and is extended by 26 amino acids at the C-terminal end. Therefore, the currently commercially produced EPO monoclonal antibodies can recognize both WT-EPO and rEPO, and VAR-EPO. The molecular weight of VAR-EPO is increased by 3.1 kDa compared with WT-EPO, and is close to that of rEPO. The existing protein immunoblotting doping detection method cannot distinguish rEPO and VAR-EPO, and there is a risk of false positive reports of rEPO in doping detection. For the above reasons, the World Anti-Doping Agency has revised the rEPO detection process and judgment standard, but an effective doping detection method has not been established for the low-dose use of rEPO by people carrying the human EPO gene polymorphism c.577del, which affects the fairness and justice of doping detection. EPO The human EPO gene polymorphism c.577del carries a human population, and an effective doping detection method has not been established for the low-dose use of rEPO by people carrying the human EPO gene polymorphism c.577del, which affects the fairness and justice of doping detection. SUMMARY

[0004] The rabbit monoclonal antibody of the application will be used to establish a bidirectional immunopurification technology as a reverse immunopurification antibody for rEPO confirmation detection of human urine and blood samples, to separate VAR-EPO in the samples and detect the VAR-EPO, so as to determine whether the athlete is a EPO gene c.577del carrier, and further detect rEPO in the sample without VAR-EPO.

[0005] To achieve the above purpose, the application is implemented by the following technical scheme: a preparation method of a rabbit monoclonal antibody based on human EPO EPO gene polymorphism c.577del, which specifically comprises the following steps:

[0006] S1, a rabbit monoclonal antibody based on human EPOThe polypeptide antigen (CHTLPRHSGGGSHTLPRHS) designed and synthesized according to the difference between the amino acid chain (GDDDQVCPPGHIHHLPHQHCLCHTLPRHS) of VAR-EPO encoded by the genetic polymorphism c.577del and WT-EPO was used for rabbit immunization to obtain a rabbit monoclonal antibody specifically recognizing VAR-EPO;

[0007] S2, after five rounds of immunization in S1, the peripheral blood of the rabbit was taken and subjected to flow sorting, and the screening concentrations of VAR-EPO and WT-EPO were set to 10 ng / mL and 100 ng / mL, respectively, and the OD values of the ELISA detection results were set to be greater than or equal to 1.00 and less than or equal to 0.10, respectively, for screening single antigen-specific memory B cells that can secrete antibodies highly recognizing VAR-EPO and not recognizing WT-EPO, and the B cells screened were subjected to subsequent antibody expression and purification.

[0008] S3, for the antibodies obtained by expression and purification in S2, the concentration of VAR-EPO was set to 1 ng / mL, the concentration of the antibody was set to 1 µg / mL, and the OD value of the ELISA detection result was set to be greater than or equal to 0.95, for further screening of rabbit monoclonal antibodies that can efficiently recognize trace VAR-EPO.

[0009] S4, the antibodies screened in S3 were used for immunoaffinity purification, for treating human urine samples and blood samples to which 150 pg of VAR-EPO was added, respectively, and the purified samples were subjected to Western blotting to detect the signal intensity of VAR-EPO.

[0010] S5, for the antibodies screened in S3, the VAR-EPO coating amount was set to eight levels from 7.81 pg to 1 ng, and the EC50 values were determined by ELISA when the antibody amount was 1 µg and 0.1 µg, respectively.

[0011] S6, according to the results of S4 and S5, TYJ9-R0016 was finally obtained as a rabbit monoclonal antibody that can specifically and efficiently recognize trace VAR-EPO in human urine and blood samples.

[0012] The preparation process of the antibody specifically comprises the following steps:

[0013] S1, preparation of antigen: because VAR-EPO increases 26 amino acids at C terminal compared with WT-EPO on the basis of containing all amino acids and glycosyl structure of WT-EPO, a monoclonal antibody which only recognizes VAR-EPO but does not recognize WT-EPO and rEPO is prepared, considering its immunospecificity, a polypeptide antigen is designed and synthesized based on the difference amino acid chain (GDDDQVCPPGHIHHLPHQHCLCHTLPRHS) of C terminal of VAR-EPO and WT-EPO, and the sequence is: CHTLPRHSGGGSHTLPRHS, after synthesis, the polypeptide is coupled with virus-like particles (VLP) to prepare an antigen for animal immunization;

[0014] S2, preparation of monoclonal antibody:

[0015] 1) animal immunization: two rabbits are immunized, and the immunization scheme is as follows, blood is collected after four immunizations to measure titer, and positive rate of cells before sorting is confirmed, and 20 mL of peripheral blood of the rabbit with qualified titer is taken for flow sorting;

[0016] 2) flow sorting of antigen-specific memory B cells: 4-6 mg of antigen protein is labeled with APC and PE, and cell positive rate is confirmed before sorting; 20 ml of peripheral blood of the rabbit is taken for flow sorting, and antigen-specific memory B cells are obtained by sorting;

[0017] 3) B cell culture and expression supernatant screening and identification: the antigen-specific B cells sorted by flow sorting are cultured, and the Elisa identification of the culture supernatant is performed, the total amount of the supernatant is about 150 ul, and the use amount of 50-100 ul is detected for each antigen;

[0018] 4) single antigen-specific memory B cell variable region amplification and vector construction: the positive B cells sorted by flow sorting are lysed and reverse transcribed to obtain heavy and light chain variable regions, and an expression vector is constructed;

[0019] 5) rapid expression of monoclonal antibody: the antibody light and heavy chain expression vectors are transiently transfected into HEK293 cells for recombinant expression, and the qualified antibody is obtained by purification, and is named as TYJ9-R0016, after SDS polyacrylamide gel electrophoresis combined with coomassie brilliant blue staining, the purity of the obtained antibody is 98.6%, and the molecular weight of the antibody light and heavy chains is 49.90 kDa and 25.99 kDa respectively.

[0020] The application provides a rabbit monoclonal antibody based on EPO The rabbit monoclonal antibody based on gene polymorphism and the amino acid sequence thereof have the following beneficial effects compared with the prior art:

[0021] This rabbit monoclonal antibody, based on the human EPO gene polymorphism c.577del and its amino acid sequence, was designed and synthesized using peptides as antigens based on the C-terminal differential amino acid chains of VAR-EPO and WT-EPO. Antigen-specific memory B cells were obtained using single-B cell technology. After culture, supernatant screening and identification, B cell variable region amplification and vector construction, and monoclonal antibody cloning, expression, and functional verification, a rabbit monoclonal antibody against VAR-EPO was obtained. This antibody is the world's first rabbit monoclonal antibody specifically recognizing VAR-EPO. It can specifically and efficiently bind to trace amounts of VAR-EPO in urine and blood samples, while not recognizing WT-EPO or rEPO. A bidirectional immunopurification technique combined with Western blotting can distinguish between rEPO and VAR-EPO in human urine and blood samples, directly and accurately detecting rEPO from urine and blood samples from various populations, eliminating the risk of false positives, optimizing the doping detection process, and addressing the current deficiencies in rEPO detection in the global anti-doping field.

[0022] This rEPO doping test method can prevent... EPO The c.577del gene polymorphism carrier population sample was misdiagnosed as positive for the doping substance rEPO, which can also make up for the deficiency of existing technology in that it cannot detect low doses of rEPO in this population. It solves the key problem of rEPO detection in doping testing laboratories around the world. In addition, this monoclonal antibody can also be used to prepare VAR-EPO test kits for preliminary and rapid diagnosis of the presence of EPO gene polymorphism c.577del. Attached Figure Description

[0023] Figure 1 A comparison diagram of (1) the marker and (2) the rabbit monoclonal antibody provided for this invention. Detailed Implementation

[0024] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0025] Please see Figure 1 This invention provides a technical solution: a rabbit monoclonal antibody based on the human EPO gene polymorphism c.577del and its amino acid sequence, specifically including the following steps:

[0026] S1, Adopting a human-based approach EPOThe polypeptide antigen (CHTLPRHSGGGSHTLPRHS) designed and synthesized according to the difference between the amino acid chain (GDDDQVCPPGHIHHLPHQHCLCHTLPRHS) of VAR-EPO encoded by the genetic polymorphism c.577del and WT-EPO is used for rabbit immunization to obtain a rabbit monoclonal antibody specifically recognizing VAR-EPO;

[0027] S2, after five rounds of immunization in S1, the peripheral blood of the rabbit is taken and subjected to flow sorting, the screening concentrations of VAR-EPO and WT-EPO are set to 10 ng / mL and 100 ng / mL respectively, the ELISA detection result OD value is set to be greater than or equal to 1.00 and less than or equal to 0.10 respectively, and the single antigen-specific memory B cells secreting the antibody highly recognizing VAR-EPO and not recognizing WT-EPO are screened, and the B cells screened are subjected to subsequent antibody expression and purification.

[0028] S3, for the antibody expressed and purified in S2, the concentration of VAR-EPO is set to 1 ng / mL, the concentration of the antibody is set to 1 µg / mL, and the ELISA detection result OD value is set to be greater than or equal to 0.95, and the rabbit monoclonal antibody highly recognizing trace VAR-EPO is further screened.

[0029] S4, the antibody screened in S3 is used for immunoaffinity purification, and is used for treating human urine samples and blood samples respectively added with 150 pg VAR-EPO, and the purified samples are subjected to protein immunoblotting to detect the signal intensity of VAR-EPO.

[0030] S5, for the antibody screened in S3, the VAR-EPO coating amount is set to eight levels from 7.81 pg to 1 ng, and the EC50 value is determined by ELISA when the antibody amount is 1 µg and 0.1 µg respectively.

[0031] S6, according to the results of S4 and S5, TYJ9-R0016 is finally obtained as a rabbit monoclonal antibody specifically and efficiently recognizing trace VAR-EPO in human urine and blood samples.

[0032] The preparation process of the antibody specifically comprises the following steps:

[0033] S1, Preparation of antigen: Since VAR-EPO has 26 amino acids added to the C-terminus of WT-EPO based on the complete amino acid and glycosyl structure of WT-EPO, in order to prepare monoclonal antibodies that recognize only VAR-EPO but not WT-EPO and rEPO, a polypeptide antigen was designed and synthesized based on the difference in the C-terminal amino acid chain (GDDDQVCPPGHIHHLPHQHCLCHTLPRHS) between VAR-EPO and WT-EPO, taking into account immunogenicity, specificity, hydrophobicity, surface accessibility, and spatial conformation. The polypeptide antigen was designed and synthesized by Beijing Yiqiao Godz, and the sequence was: CHTLPRHSGGGSHTLPRHS. The synthesized polypeptide was coupled with virus-like particles (VLP) to prepare the antigen for animal immunization.

[0034] S2, Preparation of monoclonal antibodies:

[0035] 1) Animal immunization: Two rabbits were immunized, and the immunization scheme is as follows. After four immunizations, the titer was measured by blood sampling, and the positive rate of the cells was confirmed before sorting. Peripheral blood 20 mL of the rabbit with qualified titer was taken for flow sorting:

[0036] 2) Flow sorting of antigen-specific memory B cells: 4-6 mg of antigen protein was labeled with APC and PE, and the cell positive rate was confirmed before sorting. Rabbit peripheral blood 20 ml was taken for flow sorting to obtain antigen-specific memory B cells;

[0037] 3) B cell culture and expression supernatant screening and identification: The antigen-specific B cells sorted by flow cytometry were cultured and the Elisa of the culture supernatant was identified. The total amount of supernatant was about 150 μl, and the amount of each antigen detection was 50-100 μl;

[0038] 4) Single antigen-specific memory B cell variable region amplification and vector construction: The positive B cells sorted by flow cytometry were lysed and reverse transcribed to obtain heavy and light chain variable regions, and expression vectors were constructed;

[0039] 5) Rapid expression of monoclonal antibodies: The antibody light and heavy chain expression vectors were transiently transfected into HEK293 cells for recombinant expression, and the purified antibody meeting the conditions was obtained, named TYJ9-R0016. After SDS polyacrylamide gel electrophoresis combined with coomassie brilliant blue staining, the purity of the obtained antibody was 98.6%, and the molecular weight of the antibody light and heavy chains was 49.90 kDa and 25.99 kDa, respectively. The related comparison results are shown in Figure 1 , and the light and heavy amino acid sequences are as follows:

[0040] GATGTCGTGATGACCCAGACTGCATCCCCCGTGTCTGCGGCTGTTGGAGGCACAGTCACCATCAATTGCCAGTCCAGTCAGAGTGTTTATGATAACGACAATTTAGACTGGTTTCAGCAGAAACCAGGGCAGCCTCCCAAGCTCCTGATCTACGATGCATCCAAACTGGCATCTGGGGTCCCATCGCGGTTCAGCGGCAGTGGATCTGGGACACAGTTCACTCTCACCATCAGCGACGTGCAGTGTGACGATGCTGCCACTTACTACTGTCTAGGCGGTTATGATTGTAGTCGTGTTGATTGTGGTACTTTCGGCGGAGGGACCGAGGTGGTGGTCAAAGGTGATCCAGTTGCACCTACTGTCCTCATCTTCCCACCAGCTGCTGATCAGGTGGCAACTGGAACAGTCACCATCGTGTGTGTGGCGAATAAATACTTTCCCGATGTCACCGTCACCTGGGAGGTGGATGGCACCACCCAAACAACTGGCATCGAGAACAGTAAAACACCGCAGAATTCTGCAGATTGTACCTACAACCTCAGCAGCACTCTGACACTGACCAGCACACAGTACAACAGCCACAAAGAGTACACCTGCAAGGTGACCCAGGGCACGACCTCAGTCGTCCAGAGCTTCAATAGGGGTGACTGTTAA

[0041] DVVMTQTASPVSAAVGGTVTINCQSSQSVYDNDNLDWFQQKPGQPPKLLIYDASKLASGVPSRFSGSGSGTQFTLTISDVQCDDAATYYCLGGYDCSRVDCGTFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC-

[0042]

[0043] QSVKESGGRLVTPGTPLTLTCTVSGFSLSRYDMVWVRQAPGEGLKWIGFIYASGI TY Y AS W AK GRFTISKTSTTVDLSITGPTTEDTATYFCARPWGIWGPGTLVTVSSGQ PKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQS SGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPS VFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQ F NSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMG PPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSK LSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK-

[0044] Based on the C-terminal difference amino acid chain of VAR-EPO and WT-EPO, a peptide segment is designed and synthesized as an antigen, and antigen-specific memory B cells are obtained by single B cell technology. After culture and supernatant screening and identification, B cell variable region amplification and vector construction, monoclonal antibody cloning expression and function verification, a rabbit monoclonal antibody of VAR-EPO is obtained. Through the light and heavy amino acid sequences of the above antibody, trace VAR-EPO in urine and blood samples can be specifically and efficiently combined, while WT-EPO and rEPO are not recognized. The two-way immunopurification technology combined with protein immunoblotting technology established by using the antibody can distinguish rEPO and VAR-EPO in human urine and blood samples, directly and accurately detect rEPO in urine and blood samples from various populations, eliminate the risk of false positive, optimize the process of stimulant detection, and make up for the defects in the current world anti-stimulant field about rEPO detection.

[0045] Meanwhile, the contents not described in detail in the specification all belong to the prior art known to those skilled in the art.

[0046] It has to be noted that, in the present document, the terms "first", "second", etc. merely serve to identify different entities or actions and do not necessarily require or imply any actual relationship or order between these entities or actions, and the term "comprises", "comprising", or any other variant is intended to cover non-exclusive inclusions, such that processes, methods, articles, or apparatuses that comprise a list of elements are not required to comprise only those elements but can include other elements not expressly listed or inherent to such processes, methods, articles, or apparatuses.

[0047] While embodiments of the present application have been shown and described with reference to particular embodiments thereof, it will be understood by those skilled in the art that various changes in form and details can be made therein without departing from the spirit and scope of the application. The scope of the application is defined by the appended claims and their equivalents.

Claims

1. A human-based EPO Rabbit monoclonal antibody against gene polymorphism c.577del, characterized in that, The rabbit monoclonal antibody comprises a light chain as shown in sequence SEQ ID NO.3 and a heavy chain as shown in sequence SEQ ID NO.

4.

2. The application of the monoclonal antibody as described in claim 1, characterized in that, The application utilizes the monoclonal antibody as described in claim 1 to detect human recombinant erythropoietin agonists.

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