Anti-leptin monoclonal antibody and application thereof

Abstract

The invention discloses an anti-leptin monoclonal antibody and application thereof, and relates to the technical field of biology. According to the present invention, the anti-leptin monoclonal antibodies 2A122H7 and 6B62E3 are developed, and the leptin can be effectively identified; the ELISA kit prepared by using the monoclonal antibody 2A122H7 and 6B62E3 comprises an indirect ELISA kit and a double-antibody sandwich ELISA kit, and has the advantages of strong specificity and high sensitivity on a detection sample, convenient and efficient detection operation and low cost. Effective technical support is provided for research on regulation and control mechanisms of economic characters such as fowl ingestion, fat deposition and reproduction.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to anti-leptin monoclonal antibodies and applications thereof. Background Art

[0002] In the 1960s, scientists discovered a blood circulation factor that was significantly related to weight regulation. Mice that lacked this factor and mice that did not respond to this factor all became obese. Later, Friedman of Rockefeller University confirmed the existence of this circulating factor and named it leptin. In 1994, Zhang et al. first discovered the leptin gene from obese mice through positional cloning technology. Mammalian leptin is a secretory protein composed of 167 amino acid residues, with 21 amino acids at the N-terminus as a signal peptide sequence. Leptin in the blood circulation is a polypeptide hormone containing 146 amino acid residues and a molecular weight of 16kDa. The leptin gene is widely expressed and highly conserved in mammals, amphibians, fish and other species. In 1998, Taouis et al. of the French Poultry Research Laboratory first reported chicken leptin, whose mature peptide consists of 145 amino acid residues and has 96% homology with mouse leptin. However, Friedman-Einat et al. reported that the chicken leptin gene sequence could not be obtained using the RT-PCT method, and Pitel et al. reported that the chicken genome lacked the mammalian leptin gene locus region. So far, the leptin sequence has been controversial, and many scientists have questioned the authenticity of the chicken leptin sequence. However, Japanese scientist Ohkubo discovered in 2008 that chicken serum can activate the signal transduction of leptin receptors, and Lei et al. also found that there are leptin-like substances in chickens in 2014. It was not until 2016 that Israeli scientist Seroussi reported a new chicken leptin sequence and located it on chromosome 1, and the duck leptin gene sequence was also reported.

[0003] Leptin is a polypeptide hormone secreted by animal fat cells, which reflects the signal of body fat and energy storage. Its total serum concentration is proportional to the total body fat mass. The concentration of leptin in the blood circulation is an important indicator to measure the content of adipose tissue in animals. Determining the concentration level of leptin in the blood is very important for studying energy metabolism, nutritional physiology and endocrine regulation.

[0004] Common methods for determining the concentration of leptin in animal blood include radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA). The concentration range of leptin in mammalian serum is generally between 3-50 ng / mL. Depending on the species, the sensitivity of radioimmunoassay for mammals is generally 1.0 ng / mL. In 1996, Ma et al. established a radioimmunoassay method to determine the content of leptin in human blood. On this basis, the radioimmunoassay method can accurately determine the serum leptin content of pigs, sheep, goats and cattle. In 2000, Kauter established an ELISA method to determine the concentration of leptin in sheep serum.

[0005] In 2000, Dridi et al. established a radioimmunoassay for "chicken leptin" using proteins and antibodies constructed from the 'chicken leptin' cDNA sequence cloned by Touis. In 2013, Jiang et al. used a commercial chicken ELISA kit to determine the concentration of leptin-like substances in laying hen serum. However, since the chicken leptin gene sequence reported by Touis in 1998 was incorrect, the recombinant protein and radioimmunoassay obtained based on the sequence were also incorrect.

[0006] Although there were doubts about the chicken leptin sequence before 2016, there was no problem with the sequence of the leptin receptor, and the gene sequence of the leptin receptor of all animals is very conservative. Based on this, Hen et al. established a cell receptor signal detection system that can be activated by leptin in 2008. In this system, the leptin concentration can be linearly related to the fluorescence signal of receptor activation after logarithmic processing, so the concentration of leptin can be calculated by the fluorescence signal. The system successfully detected the leptin concentration in the serum of mammals such as humans and cows and clawed frogs, and successfully detected the leptin concentration in quail serum, but there was no difference in the leptin concentration in the serum of different breeds of chickens. In 2014, Ohkubo of Japan used a similar leptin cell signal transduction system to observe fluorescence signals in the cell nucleus under an electron microscope. The author believed that this phenomenon indicated the presence of leptin or leptin-like biological activity in the serum. This study only qualitatively observed the biological activity of leptin-like substances and could not determine their concentration. In short, there is no method that can correctly detect the concentration of leptin in the serum of poultry such as chickens.

[0007] The difficulty in measuring the Leptin concentration in poultry blood has seriously hindered the research on the regulatory mechanism of economic traits such as poultry feeding, fat deposition and reproduction. Therefore, it is necessary to develop leptin detection products suitable for poultry. Summary of the invention

[0008] The purpose of the present invention is to provide an anti-leptin monoclonal antibody and its application to solve the problems existing in the above-mentioned prior art. The monoclonal antibody can effectively identify leptin, so it can be used to prepare an ELISA detection kit for leptin, and has the advantages of strong specificity, high sensitivity, convenient detection operation, high efficiency and low cost.

[0009] To achieve the above object, the present invention provides the following solutions:

[0010] The present invention provides an anti-leptin monoclonal antibody 2A122H7, which comprises CDR-L1, CDR-L2 and CDR-L3 with amino acid sequences as shown in SEQ ID NOs. 3-5, and CDR-H1, CDR-H2 and CDR-H3 with amino acid sequences as shown in SEQ ID NOs. 8-10.

[0011] Furthermore, the amino acid sequence of the heavy chain variable region of the monoclonal antibody 2A122H7 is shown in SEQ ID NO.6, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.1.

[0012] The present invention also provides an anti-leptin monoclonal antibody 6B62E3, which comprises CDR-L1, CDR-L2 and CDR-L3 with amino acid sequences as shown in SEQ ID NOs. 13-15, and CDR-H1, CDR-H2 and CDR-H3 with amino acid sequences as shown in SEQ ID NOs. 18-20.

[0013] Furthermore, the amino acid sequence of the heavy chain variable region of the monoclonal antibody 2A122H7 is shown in SEQ ID NO.16, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.11.

[0014] The present invention also provides a monoclonal antibody combination for detecting leptin, comprising the above-mentioned monoclonal antibody 2A122H7 and monoclonal antibody 6B62E3.

[0015] The present invention also provides a biomaterial, wherein the biomaterial is a substance shown in any one of (1) to (5):

[0016] (1) a gene encoding the monoclonal antibody 2A122H7, which includes a heavy chain variable region encoding gene having a nucleotide sequence as shown in SEQ ID NO.7 and a light chain variable region encoding gene having a nucleotide sequence as shown in SEQ ID NO.2;

[0017] (2) a gene encoding the monoclonal antibody 6B62E3, which includes a heavy chain variable region encoding gene having a nucleotide sequence as shown in SEQ ID NO.17 and a light chain variable region encoding gene having a nucleotide sequence as shown in SEQ ID NO.12;

[0018] (3) a gene expression cassette, the gene expression cassette comprising the coding gene described in (1) or (2);

[0019] (4) a recombinant vector comprising the gene expression cassette;

[0020] (5) A recombinant host cell comprising the recombinant vector.

[0021] The present invention also provides the use of the above-mentioned monoclonal antibody 2A122H7, monoclonal antibody 6B62E3 or monoclonal antibody combination in preparing a kit for detecting leptin.

[0022] The present invention also provides an indirect ELISA kit for detecting leptin, comprising the above-mentioned monoclonal antibody 2A122H7 or monoclonal antibody 6B62E3.

[0023] The present invention also provides a double-antibody sandwich ELISA detection kit for detecting leptin, comprising the above-mentioned monoclonal antibody combination.

[0024] Furthermore, the double antibody sandwich ELISA detection kit also includes an enzyme-labeled plate, an enzyme-labeled secondary antibody, a washing solution, a color developing solution and a stop solution.

[0025] The present invention discloses the following technical effects:

[0026] The anti-leptin monoclonal antibodies 2A122H7 and 6B62E3 provided by the present invention can effectively identify leptin. The ELISA kits prepared by the present invention using the monoclonal antibodies 2A122H7 and 6B62E3, including indirect ELISA kits and double-antibody sandwich ELISA kits, have strong specificity and high sensitivity for detecting samples, and the detection operation is convenient, efficient and low-cost. The present invention provides effective technical support for promoting the research on the regulatory mechanism of economic traits such as poultry feeding, fat deposition and reproduction. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0028] Figure 1This is a graph showing the detection results of leptin concentrations in different samples. DETAILED DESCRIPTION

[0029] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0030] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0031] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0032] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to the skilled artisan. The present invention description and examples are exemplary only.

[0033] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0034] Example 1

[0035] 1. Preparation of monoclonal antibodies against poultry leptin protein

[0036] The previously expressed and purified goose leptin recombinant protein was used to immunize 8 SPF female BALB / c mice, and blood was collected before immunization as a negative control. Immunization process: On the first day, the first immunization was performed, 0.5mL of antigen (0.5μg / mL) was added to 1mL of Freund's complete adjuvant, emulsified, and injected subcutaneously at multiple points (at least 8 points) on the back of the neck; on the 15th day, the second immunization was performed, 1mL of antigen (0.5μg / mL) was added to 1mL of Freund's incomplete adjuvant, emulsified, and injected subcutaneously at multiple points (at least 8 points) on the back of the neck; then the third and fourth immunizations were performed on the 29th and 36th days, and the immunization method was the same as on the 15th day; on the 50th day, venous blood was collected to detect the antibody titer. In this stage, a total of 8 Balb / c mice were immunized in two batches, and the titer test results of the mouse serum showed that they all met the requirements, so the next step of hybridoma cell fusion experiment was arranged.

[0037] Mice were killed by carbon dioxide gas, and the spleen was removed aseptically, squeezed and ground in a plate to prepare a spleen cell suspension. The prepared syngeneic myeloma cells were mixed with mouse spleen cells in a certain proportion, and fused by a combination of semi-solid and liquid to form hybridoma cells. The cell culture supernatant after fusion was detected by indirect ELISA, and cell lines with higher titer and affinity were selected for the first subcloning; 5 days later, the cell culture supernatant of the first subclone was detected by indirect ELISA, and cell lines with higher titer and affinity were selected for the second subcloning; 5 days later, the cell culture supernatant of the second subclone was detected by indirect ELISA, and cell lines with higher titer and affinity were selected for the third subcloning. The cell culture supernatant of the third subclone was detected by indirect ELISA, and 8 positive cell lines were obtained. The 8 positive cell lines were expanded and cultured, and serum was collected for subtype identification. The identification results are shown in Table 1.

[0038] Table 1 Subtype detection results

[0039]

[0040]

[0041] A total of 33 cell lines were screened in this stage. After the low-concentration protein coating test, 8 cell lines with higher titers were selected for subcloning. After subcloning, the subtype identification results showed that all of the 8 cell lines were of a single subtype. Two cell lines with higher titers were selected to prepare ascites, and after purification, antibodies 1A41A3, 2B111E5, 3A111A8, 6F11F12, 6B62E3, 2A122H7, 4H122C11 and 6C93H4 were prepared. The purity verified by SDS was above 90%, and the antibody quality met the requirements, so the next step of monoclonal antibody pairing experiment was arranged.

[0042] 2. Poultry Leptin protein monoclonal antibody pairing experiment

[0043] The labeled antibody was detected by indirect ELISA, and the different epitopes of the antibody were detected by indirect competitive ELISA.

[0044] (1) Indirect ELISA method detection

[0045] The main method is as follows: the antigen goose leptin is directly coated with diluent, the coating concentration is 1μg / mL, the primary antibody is a monoclonal antibody, which is graded and diluted, and the secondary antibody is goat anti-mouse-HRP. Experimental reaction time: primary antibody 1h + secondary antibody 40min + color development 15min.

[0046] The indirect ELISA experimental steps are as follows:

[0047] 1) Antigen coating: dilute the antigen to the specified concentration with coating solution (5% skim milk powder), add 100 μL / well to a polystyrene 96-well reaction plate, and place at 4°C overnight.

[0048] 2) Washing: The next day, discard the coating solution and wash once with 200 μL / well of washing solution.

[0049] 3) Blocking: Add 200 μL / well blocking solution and place at 37°C for 2 h.

[0050] 4) Washing: discard the liquid, and wash three times with 200 μL / well of washing solution (0.01 M PBST buffer, pH 7.4, containing 0.5% Tween 20).

[0051] 5) Add the sample to be tested (primary antibody): add 100 μL of the sample to be tested to each well (if the sample needs to be diluted, dilute it according to the proportion) and place it at 37°C for 1 hour.

[0052] 6) Washing: discard the sample to be tested, wash 3 times with 200 μL / well of washing solution.

[0053] 7) Add enzyme-labeled secondary antibody: Add HRP-labeled goat anti-mouse IgG (1:10000, diluted with enzyme diluent), 100 μL / well, and place at 37°C for 40 min.

[0054] 8) Washing: discard the enzyme-labeled secondary antibody, wash 5 times with 200 μL / well of washing solution.

[0055] 9) Color development: Add 90 μL / well of freshly prepared color development solution (TMB solution) and place in the dark at 37°C for 15 min.

[0056] 10) Stop reaction and colorimetry: After adding 50 μL / well of stop solution (2 mol / L H2SO4 solution), measure the absorbance of each well at 450 nm using an ELISA reader.

[0057] The test results are shown in Table 2.

[0058] Table 2 Indirect ELISA method test results

[0059]

[0060] (2) Indirect competitive ELISA method detection

[0061] The indirect competitive ELISA method was used to detect different epitopes of the antibody. The antigen coating concentration was 1 μg / mL. The primary antibody detection was: 50 μL labeled antibody + 50 μL detection antibody, the negative control was 50 μL labeled antibody + 50 μL autoantibody, and the positive control was 50 μL labeled antibody + 50 μL SP2 / 0 culture supernatant. The test results are shown in Table 3. The test results showed that 6B62E3 and 2B111E5 were obviously different epitopes from the labeled antibody 2A122H7.

[0062] Table 3 Indirect competitive ELISA method detection results

[0063] Antibody strain to be tested 2A122H7-Bio labeled antibody 1 / 100000 1A41A3 0.0259 2B111E5 0.5313 3A111A8 0.021 4H122C11 0.0214 6B62E3 0.4821 6F11F12 0.0191 Negative control 0.0487 Positive Control 0.5559

[0064] 3. Monoclonal Antibody Sequencing

[0065] We commissioned a biological company to sequence antibodies 2A122H7 and 6B62E3, and the results are as follows:

[0066] The amino acid sequence of the light chain variable region of antibody 2A122H7 is shown in SEQ ID NO.1, the nucleotide sequence of the gene encoding it is shown in SEQ ID NO.2, and the amino acid sequences of CDR-L1, CDR-L2 and CDR-L3 are shown in SEQ ID NOs.3-5, respectively; the amino acid sequence of the heavy chain variable region of antibody 2A122H7 is shown in SEQ ID NO.6, the nucleotide sequence of the gene encoding it is shown in SEQ ID NO.7, and the amino acid sequences of CDR-H1, CDR-H2 and CDR-H3 are shown in SEQ ID NOs.8-10, respectively.

[0067] SEQ ID NO.1:

[0068] DIVLTQSPASLAVSLGQRATISCRASESVDNYGISFMNWFQQRPGQPPKILIY AASNQGS GVPARFSGSGSGTDFSLNIHPMEEDDTAMYFC QQSKEVPFT FGSGTKLEIKRA.

[0069] SEQ ID NO.2:

[0070] GACATTGTGCTGACCCAATCTCCAGCTTCTTTGGCTGTGTCTCTAGGGCAGAGGGCCACCATTTCCTGTAGAGCCAGCGAAAGTGTTGATAATTATGGCATTAGTTTTATGAACTGGTTCCAACAGAGACCAGGACAGCCACCCAAAATCCTCATCTATGCTGCATCCAACCAAGGATCCGGGGTCCCTGCCAGGTTTAGTGGCAGTGGGTCTGGGACAGACTTCAGCCTCAACATCCATCCTATGGAGGAGGATGATACTGCAATGTATTTCTGTCAGCAAAGTAAGGAGGTTCCATTCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAACGGGCT。

[0071] SEQ ID NO.3:RASESVDNYGISFMN。

[0072] SEQ ID NO.4:AASNQGS。

[0073] SEQ ID NO.5:QQSKEVPFT。

[0074] SEQ ID NO.6:

[0075] QVQLQQSGPELVKPGASLKISCKASGYAFSGSWMNWVKQRPGQGLEWIGRIYPGYGN TNYNDKFKGKATLTADKSSNTAYMQLTSLTSVDSAVYFCARSGNYGNDYYYAMDYWGQG TSVTVSS。

[0076] SEQ ID NO.7:

[0077] CAGGTCCAACTGCAGCAGTCTGGACCTGAGCTGGTGAAGCCTGGGGCCTCATTGAAGATTTCCTGCAAAGCTTCTGGCTACGCATTCAGTGGCTCTTGGATGAACTGGGTGAAGCAGAGGCCTGGACAGGGTCTTGAGTGGATTGGACGGATTTATCCTGGATATGGAAATACTAACTACAATGA TAAATTCAAGGGCAAGGCCACACTGACTGCAGACAAATCCTCCAACACAGCCTACATGCAGCTCACCAGCCTGACCTCTGTGGACTCTGCGGTCTATTTCTGTGCAAGATCGGGGAACTATGGTAACGACTATTACTATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA.

[0078] SEQ ID NO.8: GSWMN

[0079] SEQ ID NO.9: RIYPGYGNTNYNDKFKG.

[0080] SEQ ID NO. 10: SGNYGNDYYYAMDY.

[0081] The amino acid sequence of the light chain variable region of antibody 6B62E3 is shown in SEQ ID NO.11, the nucleotide sequence of the gene encoding it is shown in SEQ ID NO.12, and the amino acid sequences of CDR-L1, CDR-L2 and CDR-L3 are shown in SEQ ID NOs.13-15, respectively; the amino acid sequence of the heavy chain variable region of antibody 6B62E3 is shown in SEQ ID NO.16, the nucleotide sequence of the gene encoding it is shown in SEQ ID NO.17, and the amino acid sequences of CDR-H1, CDR-H2 and CDR-H3 are shown in SEQ ID NOs.18-20, respectively.

[0082] SEQ ID NO.11:

[0083] NIVLTQSPASLAVSLGQRATISCRASESVDSYGNSFMYWYQQKPGQPPKLLIYLASKLE SGVPARFSGSGSRTDFTLTIDPVEADDAATYYCHQNNEDPLTFGAGTKLELKRA.

[0084] SEQ ID NO.12:

[0085] AACATTGTGCTGACCCAATCTCCAGCTTCTTTGGCTGTGTCTCTAGGGCAGAGGGCCACCATATCCTGCAGAGCCAGTGAAAGTGTTGATAGTTATGGCAATAGTTTTATGTACTGGTACCAGCAGAAACCAGGACAGCCACCCAAACTCCTCATCTATCTTGCATCCAAGTTAGAATCTGGGGTCCCTGCCAGGTTCAGTGGCAGTGGGTCTAGGACAGACTTCACCCTCACCATTGATCCTGTGGAGGCTGATGATGCTGCAACATATTACTGTCACCAAAATAATGAGGATCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAACGGGCT。

[0086] SEQ ID NO.13:RASESVDSYGNSFMY。

[0087] SEQ ID NO.14:LASKLES。

[0088] SEQ ID NO.15:HQNNEDPLT。

[0089] SEQ ID NO.16:

[0090] EVQLQQSGPELVKPGASVKISCKTSGYTFTEYTMHWVKQSHGKSLEWIGGINPNSGVI SYNQRFQGKATLTVDKSSSTAYMELRSLTSEDSAVYYCTRLSKWYYFDYWGQGTTLSVSS。

[0091] SEQ ID NO.17:

[0092] GAGGTCCAGCTGCAACAGTCTGGACCTGAGCTGGTGAAGCCTGGGGCTTCAGTGAAGATATCCTGCAAGACTTCTGGATACACATTCACTGAATACACCATGCACTGGGTGAAGCAGAGCCATGGAAAGAGCCTTGAGTGGATTGGAGGTATCAATCCTAACAGTGGTGTTATTAGT TATAATCAGAGGTTCCAGGGCAAGGCCACATTGACTGTAGACAAGTCCTCCAGCACAGCCTACATGGAGCTCCGCAGCCTGACATCTGAGGATTCTGCAGTCTATTACTGTACAAGATTGAGTAAATGGTACTACTTTGACTACTGGGGCCAAGGCACCACTCTCTCAGTCTCCTCA.

[0093] SEQ ID NO.18: EYTMH.

[0094] SEQ ID NO. 19: GINPNSGVISYNQRFQG.

[0095] SEQ ID NO. 20: LSKWYYFDY.

[0096] Example 2

[0097] A double antibody sandwich ELISA kit for detecting leptin, comprising the following components:

[0098] The ELISA plate was coated with antibody 2A122H7 (capture antibody) at a coating concentration of 2 μg / mL and the coating method was the same as in Example 1;

[0099] Detection antibody: 6B62E3 antibody;

[0100] Enzyme-labeled secondary antibody: avidin-HRP;

[0101] Washing solution: 0.01% PBST buffer;

[0102] Color development solution: TMB substrate color development solution;

[0103] Stop solution: 2mol / LH2SO4 solution.

[0104] Example 3

[0105] A double antibody sandwich ELISA kit for detecting leptin, comprising the following components:

[0106] The ELISA plate was coated with antibody 6B62E3 (capture antibody) at a coating concentration of 2 μg / mL and the coating method was the same as in Example 1;

[0107] Detection antibody: 2A122H7 antibody;

[0108] Enzyme-labeled secondary antibody: avidin-HRP;

[0109] Washing solution: 0.01% PBST buffer;

[0110] Color development solution: TMB substrate color development solution;

[0111] Stop solution: 2mol / LH2SO4 solution.

[0112] Example 4

[0113] The double antibody sandwich ELISA detection kits of Example 2 and Example 3 were used to detect leptin protein solutions of different concentrations, and the results are shown in Tables 4 and 5. The results show that the sensitivity of the paired detection of antibodies 2A122H7 and 6B62E3 is high.

[0114] Table 4 Results of testing leptin protein solutions of different concentrations using the double antibody sandwich ELISA detection kit of Example 2

[0115] Protein (ng / mL) OD value 1000 3.7962 500 3.469 250 2.5953 125 2.3156 62.5 1.3863 31.25 0.6988 15.625 0.293 7.8125 0.1360 3.90625 0.1014 1.8 0.0826 0.9 0.0554 0 0.0415

[0116] Table 5 Results of testing leptin protein solutions of different concentrations using the double antibody sandwich ELISA detection kit of Example 3

[0117]

[0118]

[0119] Example 5

[0120] The double antibody sandwich ELISA detection kit of Example 2 was used to detect the leptin concentration in the serum of quail, high-fat yellow-feathered broiler, low-fat yellow-feathered broiler, Landes goose, duck, Muscovy duck and 70-day-old goose. The results are shown in Figure 1 The results showed that the double antibody sandwich ELISA detection kit developed by the present invention can effectively detect the leptin concentration in serum samples.

[0121] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. An anti-leptin monoclonal antibody 2A122H7, characterized in that: It comprises CDR-L1, CDR-L2 and CDR-L3 whose amino acid sequences are shown in SEQ ID NOs. 3-5, and CDR-H1, CDR-H2 and CDR-H3 whose amino acid sequences are shown in SEQ ID NOs. 8-10.

2. The monoclonal antibody 2A122H7 according to claim 1, characterized in that The amino acid sequence of the heavy chain variable region of the monoclonal antibody 2A122H7 is shown in SEQ ID NO.6, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.

1.

3. An anti-leptin monoclonal antibody 6B62E3, characterized in that: It comprises CDR-L1, CDR-L2 and CDR-L3 whose amino acid sequences are shown in SEQ ID NOs. 13-15, and CDR-H1, CDR-H2 and CDR-H3 whose amino acid sequences are shown in SEQ ID NOs. 18-20.

4. The monoclonal antibody 6B62E3 according to claim 3, characterized in that The amino acid sequence of the heavy chain variable region of the monoclonal antibody 2A122H7 is shown in SEQ ID NO.16, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.

11.

5. A monoclonal antibody combination for detecting leptin, characterized in that: It comprises the monoclonal antibody 2A122H7 according to claim 1 or 2 and the monoclonal antibody 6B62E3 according to claim 3 or 4.

6. A biomaterial, characterized in that The biological material is a substance shown in any one of (1) to (5): (1) The gene encoding the monoclonal antibody 2A122H7 according to claim 2, comprising a heavy chain variable region encoding gene having a nucleotide sequence as shown in SEQ ID NO.7 and a light chain variable region encoding gene having a nucleotide sequence as shown in SEQ ID NO.2; (2) The gene encoding the monoclonal antibody 6B62E3 according to claim 4, comprising a heavy chain variable region encoding gene having a nucleotide sequence as shown in SEQ ID NO.17 and a light chain variable region encoding gene having a nucleotide sequence as shown in SEQ ID NO.12; (3) a gene expression cassette, the gene expression cassette comprising the coding gene described in (1) or (2); (4) a recombinant vector comprising the gene expression cassette; (5) A recombinant host cell comprising the recombinant vector.

7. Use of the monoclonal antibody 2A122H7 according to claim 1 or 2, the monoclonal antibody 6B62E3 according to claim 3 or 4, or the monoclonal antibody combination according to claim 5 in the preparation of a kit for detecting leptin.

8. An indirect ELISA kit for detecting leptin, characterized in that: It comprises the monoclonal antibody 2A122H7 according to claim 1 or 2 or the monoclonal antibody 6B62E3 according to claim 3 or 4.

9. A double antibody sandwich ELISA detection kit for detecting leptin, characterized in that: Comprising the monoclonal antibody combination according to claim 5.

10. The double antibody sandwich ELISA detection kit according to claim 9, characterized in that: The double antibody sandwich ELISA detection kit also includes an enzyme-labeled plate, an enzyme-labeled secondary antibody, a washing solution, a color developing solution and a stop solution.

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