Anti-leptin monoclonal antibodies and their applications

Abstract

This invention discloses anti-leptin monoclonal antibodies and their applications, relating to the field of biotechnology. This invention develops anti-leptin monoclonal antibodies 2A122H7 and 6B62E3, which can effectively recognize leptin. The ELISA kits prepared using these monoclonal antibodies 2A122H7 and 6B62E3 include indirect ELISA and double-antibody sandwich ELISA kits, exhibiting high specificity and sensitivity for the tested samples, and are convenient, efficient, and cost-effective. This invention provides effective technical support for advancing research on the regulatory mechanisms of economic traits such as feed intake, fat deposition, and reproduction in poultry.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to monoclonal antibodies against leptin and their applications. Background Technology

[0002] In the 1960s, scientists discovered a circulating factor significantly related to weight regulation; mice lacking this factor and mice unresponsive to it both became obese. Subsequently, 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 in obese mice using localized cloning technology. Mammalian leptin is a secretory protein composed of 167 amino acid residues, with a 21-amino acid signal peptide sequence at the N-terminus. Circulating leptin is a polypeptide hormone containing 146 amino acid residues with a molecular weight of 16 kDa. 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 shares 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 lacks the mammalian leptin gene locus region. Since then, the leptin sequence has been highly controversial, with many scientists questioning its authenticity. However, in 2008, Japanese scientist Ohkubo discovered that chicken serum could activate leptin receptor signal transduction, and Lei et al. in 2014 also found a leptin-like substance in chickens. Finally, in 2016, Israeli scientist Seroussi reported a new chicken leptin sequence and located it on chromosome 1; the duck leptin gene sequence was also reported at the same time.

[0003] Leptin is a polypeptide hormone secreted by animal adipocytes, which signals the body's fat and energy storage. Its total serum concentration is directly proportional to the amount of body fat. The concentration of leptin in the blood circulation is an important indicator for measuring the content of adipose tissue in animals. Measuring 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 leptin concentration in animal blood include radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA). The concentration of leptin in mammalian serum generally ranges from 3 to 50 ng / mL. Depending on the species, the sensitivity of radioimmunoassay for mammals is typically around 1.0 ng / mL. In 1996, Ma et al. established a radioimmunoassay method for determining leptin levels in human blood. Based on this, radioimmunoassay can accurately determine leptin levels in the serum of pigs, sheep, goats, and cattle. In 2000, Kauter established an ELISA method for determining leptin concentration 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 commercially available chicken ELISA kit to determine the concentration of leptin-like substances in the serum of laying hens. However, because the chicken leptin gene sequence reported by Touis in 1998 was incorrect, the recombinant protein and radioimmunoassay obtained based on that sequence were also incorrect.

[0006] Although the chicken leptin sequence was questionable before 2016, the leptin receptor sequence was correct, and the gene sequence of the leptin receptor is highly conserved across all animals. Based on this, in 2008, Hen et al. developed a leptin-activated cell receptor signal detection system. In this system, the logarithmic processing of leptin concentration showed a linear relationship with the fluorescence signal of receptor activation, allowing the calculation of leptin concentration from the fluorescence signal. This system successfully detected leptin concentrations in the serum of humans, dairy cows, and Xenopus laevis, and also successfully detected leptin concentrations in quail serum, but showed no difference in leptin concentrations among different breeds of chicken. In 2014, Ohkubo in Japan used a similar leptin cell signaling system to observe fluorescence signals in cell nuclei under an electron microscope, suggesting the presence of leptin or leptin-like biological activity in serum. This study only qualitatively observed the biological activity of leptin-like substances and could not determine their concentration. In conclusion, to date, there is no method that can accurately detect leptin concentrations in the serum of chickens and other birds.

[0007] The difficulty in measuring leptin concentration in poultry blood has severely hampered research into its regulatory mechanisms on economic traits such as feeding, fat deposition, and reproduction in poultry. Therefore, it is necessary to develop leptin detection products suitable for poultry. Summary of the Invention

[0008] The purpose of this invention is to provide a monoclonal antibody against leptin and its applications, thereby addressing the problems existing in the prior art. This monoclonal antibody can effectively recognize leptin, thus it can be used to prepare ELISA detection kits for leptin, and it has the advantages of high specificity, high sensitivity, and convenient, efficient, and low-cost detection operation.

[0009] To achieve the above objectives, the present invention provides the following solution:

[0010] The present invention provides a monoclonal antibody against leptin, 2A122H7, which comprises amino acid sequences CDR-L1, CDR-L2 and CDR-L3 as shown in SEQ ID NO. 3-5, and amino acid sequences CDR-H1, CDR-H2 and CDR-H3 as shown in SEQ ID NO. 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 a monoclonal antibody against leptin, 6B62E3, comprising amino acid sequences CDR-L1, CDR-L2 and CDR-L3 as shown in SEQ ID NO. 13-15, and amino acid sequences CDR-H1, CDR-H2 and CDR-H3 as shown in SEQ ID NO. 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 combination of monoclonal antibodies 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 any one of the substances shown in (1)-(5):

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

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

[0018] (3) A gene expression cassette, wherein the gene expression cassette contains the encoding gene described in (1) or (2);

[0019] (4) A recombinant vector containing the gene expression cassette;

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

[0021] The present invention also provides the application of the above-mentioned monoclonal antibody 2A122H7, monoclonal antibody 6B62E3 or combination of monoclonal antibodies in the preparation of 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 kit for detecting leptin, comprising the above-described monoclonal antibody combination.

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

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

[0026] The anti-leptin monoclonal antibodies 2A122H7 and 6B62E3 provided in this invention can effectively recognize leptin. The ELISA kits prepared using these monoclonal antibodies 2A122H7 and 6B62E3 include indirect ELISA kits and double-antibody sandwich ELISA kits, exhibiting high specificity and sensitivity for the tested samples, and are convenient, efficient, and cost-effective. This invention provides effective technical support for advancing research on the regulatory mechanisms of economic traits such as feed intake, fat deposition, and reproduction in poultry. Attached Figure Description

[0027] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0028] Figure 1The graph shows the detection results of leptin concentration in different samples. Detailed Implementation

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

[0030] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0031] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0032] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

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

[0034] Example 1

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

[0036] The previously expressed and purified goose leptin recombinant protein was used to immunize eight SPF-grade female BALB / c mice. Blood samples were collected before immunization as a negative control. Immunization protocol: On day 1, the first immunization was performed by adding 1 mL of Freund's complete adjuvant to 0.5 mL of antigen (0.5 μg / mL), emulsifying, and injecting subcutaneously at multiple points (at least 8 points) on the back of the neck. On day 15, the second immunization was performed by adding 1 mL of incomplete adjuvant to 1 mL of antigen (0.5 μg / mL), emulsifying, and injecting subcutaneously at multiple points (at least 8 points) on the back of the neck. The third and fourth immunizations were then performed on days 29 and 36, using the same method as on day 15. On day 50, blood was collected intravenously to detect antibody titers. A total of eight Balb / c mice were immunized in two batches during this phase. Serum titer tests showed that all mice met the requirements, therefore, the next step, hybridoma cell fusion experiments, was planned.

[0037] Mice were euthanized with carbon dioxide gas, and the spleens were aseptically removed and crushed in a petri dish to prepare a spleen cell suspension. Prepared syngeneic myeloma cells were mixed with mouse spleen cells in a specific ratio and fused using a combination of semi-solid and liquid methods to form hybridoma cells. The cell culture supernatant after fusion was detected by indirect ELISA. Cell lines with high titers and affinity were selected for the first subcloning. Five days later, the cell culture supernatant of the first subcloning was detected by indirect ELISA, and cell lines with high titers and affinity were selected for the second subcloning. Five days later, the cell culture supernatant of the second subcloning was detected by indirect ELISA, and cell lines with high titers and affinity were selected for the third subcloning. Eight positive cell lines were obtained. The eight 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 low-concentration protein coating test, 8 cell lines with high titers were selected for subcloning. After subcloning, the subtype identification results of the 8 cell lines showed that they were all single subtypes. Ascites fluid was prepared from 2 cell lines with high titers and purified to prepare antibodies 1A41A3, 2B111E5, 3A111A8, 6F11F12, 6B62E3, 2A122H7, 4H122C11 and 6C93H4. The purity of all of them was verified by SDS and was above 90%. The antibody quality met the requirements, so the next step of monoclonal antibody pairing experiment was arranged.

[0042] 2. Monoclonal antibody pairing assay for leptin protein in poultry

[0043] An indirect ELISA method was used to detect the labeled antibody, and then an indirect competitive ELISA method was used to detect different epitopes of the antibody.

[0044] (1) Detection by indirect ELISA method

[0045] The main method is as follows: Goose leptin antigen was directly coated with a diluent at a concentration of 1 μg / mL. The primary antibody was a monoclonal antibody, serially diluted. The secondary antibody was goat anti-mouse HRP. The reaction time was: primary antibody 1 h + secondary antibody 40 min + color development 15 min.

[0046] The steps for an indirect ELISA experiment 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 incubate overnight at 4°C.

[0048] 2) Washing: Discard the coating liquid the next day and wash once with 200 μL of washing solution per well.

[0049] 3) Blocking: Add 200 μL of blocking solution per well and incubate at 37°C for 2 hours.

[0050] 4) Washing: Discard the liquid and wash 3 times with 200 μL / well of washing buffer (0.01 M PBST buffer containing 0.5% Tween 20 at pH 7.4).

[0051] 5) Add the test sample (primary antibody): Add 100 μL of the test sample to each well (if the sample needs to be diluted, dilute it according to the ratio), and incubate at 37℃ for 1 h.

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

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

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

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

[0056] 10) Termination of reaction and colorimetric analysis: After adding 50 μL of stop solution (2 mol / L H2SO4 solution) per well, 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 Results of indirect ELISA method

[0059]

[0060] (2) Detection using indirect competitive ELISA method

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

[0062] Table 3. Results of the indirect competitive ELISA method

[0063] Antibody strain number 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] A biotechnology company was commissioned to sequence antibodies 2A122H7 and 6B62E3. 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 its encoding gene 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 NO.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 its encoding gene 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 NO.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 its encoding gene 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 NO.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 its encoding gene 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 NO.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 comprises the following components:

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

[0099] Antibody detected: 6B62E3 antibody;

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

[0101] Washing buffer: 0.01% PBST buffer;

[0102] Developing solution: TMB substrate developing solution;

[0103] Termination solution: 2 mol / L H2SO4 solution.

[0104] Example 3

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

[0106] The ELISA plate coated with antibody 6B62E3 (capture antibody) was coated at a 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 buffer: 0.01% PBST buffer;

[0110] Developing solution: TMB substrate developing solution;

[0111] Termination solution: 2 mol / L H2SO4 solution.

[0112] Example 4

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

[0114] Table 4 shows the results of detecting different concentrations of leptin protein solutions using the double-antibody sandwich ELISA kit from 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 shows the results of detecting different concentrations of leptin protein solutions using the double-antibody sandwich ELISA kit from Example 3.

[0117]

[0118]

[0119] Example 5

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

[0121] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A monoclonal antibody 2A12 2H7 against leptin, characterized in that, The monoclonal antibody 2A12 2H7 comprises a CDR-L1, a CDR-L2 and a CDR-L3 having the amino acid sequences as shown in SEQ ID NO. 3-5, respectively, and a CDR-H1, a CDR-H2 and a CDR-H3 having the amino acid sequences as shown in SEQ ID NO. 8-10, respectively.

2. The monoclonal antibody 2A12 2H7 according to claim 1, characterized in that, The monoclonal antibody 2A12 2H7 comprises a CDR-L1, a CDR-L2 and a CDR-L3 having the amino acid sequences as shown in SEQ ID NO. 3-5, respectively, and a CDR-H1, a CDR-H2 and a CDR-H3 having the amino acid sequences as shown in SEQ ID NO. 8-10, respectively.

3. A combination of monoclonal antibodies for detecting leptin, characterized in that, The monoclonal antibody 6B6 2E3 and the monoclonal antibody 2A12 2H7 of claim 1 or 2; The monoclonal antibody 6B6 2E3 comprises a CDR-L1, a CDR-L2 and a CDR-L3 having the amino acid sequences as shown in SEQ ID NO. 13-15, respectively, and a CDR-H1, a CDR-H2 and a CDR-H3 having the amino acid sequences as shown in SEQ ID NO. 18-20, respectively.

4. A biomaterial, characterized by, The biological material is any one of the substances as shown in (1)-(4): (1) a coding gene of the monoclonal antibody 2A12 2H7 of claim 2, comprising a heavy chain variable region coding gene having the nucleotide sequence as shown in SEQ ID NO. 7 and a light chain variable region coding gene having the nucleotide sequence as shown in SEQ ID NO. 2; (2) a gene expression cassette, comprising the coding gene of (1); (3) a recombinant vector comprising the gene expression cassette of (2); (4) a recombinant host cell comprising the recombinant vector of (3).

5. Use of the monoclonal antibody 2A12 2H7 of claim 1 or 2 or the combination of monoclonal antibodies of claim 3 in the preparation of a kit for detecting leptin.

6. An indirect ELISA kit for detecting leptin, characterized by, The monoclonal antibody 2A12 2H7 of claim 1 or 2.

7. A double antibody sandwich ELISA test kit for detecting leptin, characterized by, The combination of monoclonal antibodies of claim 3.

8. The double antibody sandwich ELISA test kit according to claim 7, characterized in that, The double-antibody sandwich ELISA detection kit further comprises an enzyme-labeled plate, an enzyme-labeled secondary antibody, a washing solution, a color developing solution and a termination solution.

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