Mouse anti-bovine IgG monoclonal antibody B4 and its application

Abstract

The present invention relates to the technical field of livestock immunology, and in particular to a mouse anti-bovine IgG monoclonal antibody and its application. The mouse anti-bovine IgG monoclonal antibody provided by the present invention can specifically bind to bovine IgG, has no cross reaction with other similar proteins, has high affinity with bovine IgG, and has high stability; based on the antibody, the double antibody sandwich ELISA method is used to detect bovine IgG with high sensitivity, specificity and accuracy, and can realize accurate and high-throughput detection of IgG content in bovine serum or other serum analogs or products containing bovine IgG, and has good application prospects in bovine IgG detection.

Description

Technical Field

[0001] The invention relates to the technical field of livestock immunology, and in particular to a mouse anti-bovine IgG monoclonal antibody and an application thereof. Background Art

[0002] IgG is synthesized and secreted after the body's immune cells recognize antigens, and its content is very high in blood and egg yolk. The level of immunoglobulin is closely related to many diseases and can also indirectly reflect the strength of the body's immunity. Testing the IgG content in the blood of cattle groups can be used to evaluate the health of the group. IgG has a high content in blood and tissues and has strong species specificity. By detecting the type of IgG in the sample, it is possible to identify adulteration of livestock meat. It is also possible to identify whether some multifunctional foods have been added with bovine colostrum. IgG is a very important immune factor in bovine colostrum. The amount of its detection is a quantitative indicator for evaluating the quality of bovine colostrum and its products. At present, bovine colostrum is added to many multifunctional foods to increase immunity, but whether it really contains bovine colostrum and how much it contains requires a very accurate quantitative method to identify the authenticity. It is very important to detect the bovine immunoglobulin (IgG) content in such functional dairy products.

[0003] There are many methods for IgG detection, including single immunodiffusion method, immunoturbidimetry, etc., but these methods are time-consuming, and their accuracy and repeatability are relatively poor. The sensitivity of chemiluminescence method is higher than that of enzyme-linked immunosorbent assay (ELISA), but the instrument required for detection is more expensive than the microplate reader used for ELISA, and it has not yet been widely promoted and used. In view of the defects of the above methods, the ELISA double antibody sandwich method has been widely used due to its high sensitivity and good repeatability.

[0004] Most ELISA kits on the market detect IgG from humans, rats, and mice, but there are relatively few ELISA kits for IgG from pigs, cattle, horses, sheep, rabbits, chickens, and other animals. In particular, the sensitivity and accuracy of ELISA kits for bovine IgG are difficult to guarantee due to the uneven quality of products on the market. In response to this problem, the development of a double antibody sandwich ELISA kit based on monoclonal antibodies is of great significance for improving the detection quality of bovine IgG.

[0005] In view of this, the present invention is proposed. Summary of the invention

[0006] To solve the above technical problems, the present invention uses mouse monoclonal antibody technology to immunize Balb / c mice and then perform fusion screening, thereby obtaining a monoclonal antibody with good specificity and stability. The present invention provides mouse anti-bovine IgG monoclonal antibody and its application.

[0007] Specifically, the technical solution of the present invention is as follows:

[0008] In the first aspect, the present invention provides a mouse anti-bovine IgG monoclonal antibody or an antigen-binding fragment thereof, wherein the amino acid sequences of the complementary determining regions CDR1, CDR2, and CDR3 of the heavy chain variable region of the mouse anti-bovine IgG monoclonal antibody or the antigen-binding fragment thereof are GYAFTSNN, IDPYNGGT, and ARGPYYGNFGPFAY, respectively; and the amino acid sequences of the complementary determining regions CDR1, CDR2, and CDR3 of the light chain variable region are QSLFNSGNQKNF, GAS, and QNDHSYPLT, respectively.

[0009] Preferably, the amino acid sequence of the heavy chain variable region of the mouse anti-bovine IgG monoclonal antibody is as shown in SEQ ID NO: 4, or has at least 80% sequence similarity to SEQ ID NO: 4; the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 3, or has at least 80% sequence similarity to SEQ ID NO: 3.

[0010] In a second aspect, the present invention provides a nucleic acid molecule encoding the mouse anti-bovine IgG monoclonal antibody or its antigen-binding fragment.

[0011] Preferably, the nucleotide sequence encoding the heavy chain variable region is as shown in SEQ ID NO: 2, or has at least 80% sequence similarity to SEQ ID NO: 2; the amino acid sequence encoding the light chain variable region is as shown in SEQ ID NO: 1, or has at least 80% sequence similarity to SEQ ID NO: 1.

[0012] In a third aspect, the present invention provides a biological material, which contains the nucleic acid molecule; the biological material is an expression cassette, a vector or a host cell.

[0013] In a fourth aspect, the present invention provides a mouse anti-bovine IgG monoclonal antibody conjugate, which is obtained by conjugating the mouse anti-bovine IgG monoclonal antibody or its antigen-binding fragment with a marker, wherein the marker is selected from one or more of an enzyme label, a biotin label, a fluorescent dye label, a chemiluminescent dye label, and a radioactive label.

[0014] In a fifth aspect, the present invention provides a composition of mouse anti-bovine IgG monoclonal antibodies, the composition comprising the mouse anti-bovine IgG monoclonal antibodies or their antigen-binding fragments; and also comprising the following mouse anti-bovine IgG monoclonal antibodies or their antigen-binding fragments: the amino acid sequences of the complementary determining regions CDR1, CDR2, and CDR3 of the heavy chain variable regions are GDSITSGY, ISYSGST, and AREGSTLTTRGFAY, respectively, and the amino acid sequences of the complementary determining regions CDR1, CDR2, and CDR3 of the light chain variable regions are QSVSND, YAS, and QQDYSSPWT, respectively.

[0015] In a sixth aspect, the present invention provides any one of the following (1) to (5) uses of the mouse anti-bovine IgG monoclonal antibody or its antigen-binding fragment, the nucleic acid molecule, the biomaterial, the mouse anti-bovine IgG monoclonal antibody conjugate, or the composition:

[0016] (1) Use in the preparation of a product for detecting the presence or level of mouse anti-bovine IgG monoclonal antibody in a sample;

[0017] (2) Use of the invention in detecting the presence or level of mouse anti-bovine IgG monoclonal antibodies in samples for non-diagnostic and therapeutic purposes;

[0018] (3) Use in the preparation of a product for neutralizing the activity of mouse anti-bovine IgG monoclonal antibodies in a sample;

[0019] (4) Use in the preparation of drugs for neutralizing the activity of mouse anti-bovine IgG monoclonal antibodies in vivo;

[0020] (5) Application in quality control or production of products containing mouse anti-bovine IgG monoclonal antibodies.

[0021] In a seventh aspect, the present invention provides a kit comprising the mouse anti-bovine IgG monoclonal antibody or its antigen-binding fragment, or the antibody conjugate, or the composition.

[0022] Preferably, the kit is an enzyme-linked immunosorbent assay kit, a fluorescent immunoassay kit or a chemiluminescent immunoassay kit.

[0023] Beneficial effects:

[0024] The mouse anti-bovine IgG monoclonal antibody provided by the present invention can specifically bind to bovine IgG, has no cross-reaction with other similar proteins, has high affinity to bovine IgG, and has high stability; based on the antibody, a double antibody sandwich ELISA method is used to detect bovine IgG with high sensitivity, specificity and accuracy, and can accurately and high-throughput detect the IgG content in bovine serum or other serum analogs or products containing bovine IgG, and has good application prospects in bovine IgG detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the drawings required for use in the embodiments or the description of the prior art will be described below.

[0026] Figure 1 This is a standard curve drawn by ELISA Calc software in the double antibody sandwich ELISA method described in Example 2 of the present invention, with the concentration of the standard as the horizontal axis and the absorbance OD value as the vertical axis. DETAILED DESCRIPTION

[0027] The present invention utilizes mouse monoclonal antibody technology, and obtains two monoclonal antibodies with good specificity and stability by fusion screening after immunizing Balb / c mice. The present invention performs antibody pairing, and uses these two highly specific monoclonal antibodies to establish an enzyme-linked immunosorbent assay based on the double antibody sandwich method, and provides a corresponding ELISA kit, which is characterized by good specificity, high sensitivity, and simple operation. The method and kit provided by the present invention can detect the content of bovine IgG in various meat products, bovine dairy products, and serum and blood samples. The method uses an enzyme color development amplification system, and has high sensitivity and strong specificity for identification and detection, which makes up for the various defects of other existing methods.

[0028] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0029] The endpoints and any values ​​of the ranges disclosed in this specification are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0030] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "specific implementations", or "some specific implementations" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the embodiment of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0031] In the examples provided in this specification, if no specific techniques or conditions are specified, the techniques or conditions described in the literature in this field or the product instructions are used. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased through regular channels.

[0032] In the following embodiments, the sequence information involved is as follows.

[0033] The coding gene sequence of the light chain variable region of monoclonal antibody B4 (SEQ ID NO.1):

[0034] GACATTTGTGATGACACAGTCTCCATCCTCCCTGAGTGTGTCAGCAGGAGAGAAGGTCACTATGAGCTGCAAGTCCAGTCAGAGTCTGTTTAACAGTGGAAATCAAAAGAACTTCTTGGCCTGGTACCAGCAGAAACCAGGGCAGCCTCCTAAACTGTTGATCTACGGGGC ATCCACTAGAGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGAACCGATTTCACTCTTACCATCAGCAGTGTGCAGGCTGAAGACCTGGCAGTTTTATTACTGTCAGAATGATCATAGTTATCCGCTCACGTTCGGTGCTGGGACCCAGCTGGAGCTGAAA.

[0035] The coding gene sequence of the heavy chain variable region of monoclonal antibody B4 (SEQ ID NO.2):

[0036] GAGATCCAGCTGCAGCAGTCTGGACCTGAGCTGGTGAAGCCTGGGGCTTCAGTGAAGGTGTCCTGCAAGGCTTCTGGTTATGCATTCACTAGCAACAACATGTACTGGGTGAAGCAGAGCCATGGAAAGAGCCTTGAGTGGATTGGATATATTGATCCTTACAATGGTGGTACTAGGTACAA CCAGAAGTTCAAGGGCAAGGCCACATTGACTGTTGACAAGTCCTCCAGCACAGCCTACATGCATGTCAACAGCCTGACATCTGAGGACTCTGCAGTTTATTACTGTGCAAGGGGGCCCTACTATGGTAACTTCGGCCCGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA.

[0037] The amino acid sequence of the light chain variable region of monoclonal antibody B4 (SEQ ID NO.3):

[0038] DIVMTQSPSSLSVSAGEKVTMSCKSSQSLFNSGNQKNFLAWYQQKPGQPPKLLIYGASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDHSYPLTFGAGTQLELK.

[0039] The amino acid sequence of the heavy chain variable region of monoclonal antibody B4 (SEQ ID NO.4):

[0040] EIQLQQSGPELVKPGASVKVSCKASGYAFTSNNMYWVKQSHGKSLEWIGYIDPYNGGTRYNQKFKGKATLTVDKSSSTAYMHVNSLTSEDSAVYYCARGPYYGNFGPFAYWGQGTLVTVSA.

[0041] The coding gene sequence of the light chain variable region of monoclonal antibody C11 (SEQ ID NO.5):

[0042] AGTATTGTGATGACCCAGACTCCCAAATTCCTGCTTGTATCAGCAGGAGACAGGGTTACCATAACCTGCAAGGCCAGTCAGAGTGTGAGTAATGATGTAGCTTGGTACCAACAGAAGCCAGGGCAGTCTCCTAAACTGCTGATATACTATGCATCCAATCGCTACACTGGAGTCCCTGATCGCTTCACTGGCAGTGGATATGGGACGGATTTCACTTTCACCATCAACACTGTGCAGGCTGAAGACCTGGCAGTTTATTTCTGTCAGCAGGATTATAGCTCTCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA。

[0043] Coding gene sequence of the heavy chain variable region of monoclonal antibody C11 (SEQ ID NO.6):

[0044] GAGGTGCAGCTTCAGGAGTCAGGACCTAGCCTCGTGAAACCTTCTCAGACTCTGTCCCTCACCTGTTCTGTCACTGGCGACTCCATCACCAGTGGTTACTGGAACTGGATCCGGAAATTCCCAGGGAATAAACTTGACTACATGGGGTACATAAGCTACAGTGGTAGCACTTACTACAATCCATCTCTCAAAAGTCGAATCTCCATCACTCGAGACACATCCAAGAACCAGTACTACCTGCAGTTGAATTCTGTGACTACTGAGGACACAGCCACATATTACTGTGCAAGAGAGGGATCTACTTTGACTACGAGAGGTTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA。

[0045] Amino acid sequence of the light chain variable region of monoclonal antibody C11 (SEQ ID NO.7):

[0046] SIVMTQTPKFLLVSAGDRVTITCKASQSVSNDVAWYQQKPGQSPKLLIYYASNRYTGVPDRFTGSGYGTDFTFTINTVQAEDLAVYFCQQDYSSPWTFGGGTKLEIK。

[0047] The amino acid sequence of the heavy chain variable region of monoclonal antibody C11 (SEQ ID NO.8):

[0048] EVQLQESGPSLVKPSQTLSLTCSVTGDSITSGYWNWIRKFPGNKLDYMGYISYSGSTYYNPSLKSRISITRDTSKNQYYLQLNSVTTEDTATYYCAREGSTLTTRGFAYWGQGTLVTVSA.

[0049] Complementarity determining region CDR1 of the light chain variable region of monoclonal antibody B4 (SEQ ID NO.9):

[0050] QSLFNSGNQKNF.

[0051] Complementarity determining region CDR2 of the light chain variable region of monoclonal antibody B4 (SEQ ID NO.10):

[0052] GAS.

[0053] Complementarity determining region CDR3 of the light chain variable region of monoclonal antibody B4 (SEQ ID NO.11):

[0054] QNDHSYPLT.

[0055] Complementarity determining region CDR1 of the heavy chain variable region of monoclonal antibody B4 (SEQ ID NO.12):

[0056] GYAFTSNN.

[0057] Complementarity determining region CDR2 of the heavy chain variable region of monoclonal antibody B4 (SEQ ID NO.13):

[0058] ID P Y N G T .

[0059] Complementarity determining region CDR3 of the heavy chain variable region of monoclonal antibody B4 (SEQ ID NO.14):

[0060] ARGPYYGNFGPFAY.

[0061] The complementary determining region CDR1 of the light chain variable region of monoclonal antibody C11 (SEQ ID NO.15):

[0062] QSVSND.

[0063] Complementarity determining region CDR2 of the light chain variable region of monoclonal antibody C11 (SEQ ID NO.16):

[0064] YAS.

[0065] The complementary determining region CDR3 of the light chain variable region of monoclonal antibody C11 (SEQ ID NO.17):

[0066] QQDYSSPWT.

[0067] The complementarity determining region CDR1 of the heavy chain variable region of the monoclonal antibody C11 (SEQ ID NO. 18): GDSITSGY.

[0068] The complementarity determining region CDR2 of the heavy chain variable region of the monoclonal antibody C11 (SEQ ID NO. 19): ISYSGST.

[0069] Complementarity determining region CDR3 of the heavy chain variable region of monoclonal antibody C11 (SEQ ID NO.20):

[0070] AREGSTLTTRGFAY.

[0071] Example 1

[0072] Preparation of anti-bovine IgG monoclonal antibodies.

[0073] 1. Animal immunization.

[0074] Female Balb / c mice aged 6-8 weeks were selected and immunized with purified bovine IgG emulsified with an equal volume of Freund's adjuvant. The mice were immunized once every two weeks for a total of three times, and the immunization was boosted again three days before fusion.

[0075] 2. Preparation of feeder cells.

[0076] Take an unimmunized BALB / c mouse, bleed it from the eye socket and kill it. Collect the negative serum. Inject 2-3 mL of 1640 basal medium into the mouse's peritoneal cavity, aspirate it after blowing and place it in another centrifuge tube for later use. The liquid contains peritoneal macrophages. Prepare spleen cell suspension in the same way as above and place it in the peritoneal macrophage tube. Centrifuge at 1000r / min for 10min to remove the supernatant. After the cells are suspended with HAT medium, place them in a 37°C, 5% CO2 incubator for later use.

[0077] 3. Myeloma (SP2 / 0) cell culture.

[0078] Commercial SP2 / 0 cells were thawed and revived, then resuspended in cell culture medium (RPMI-1640, supplemented with calf serum) and cultured in an incubator at 37°C and 5% CO2. After 3-5 days, the cells were subcultured and expanded until the number of cells reached about 1×10 7 -2×10 7 After that, the cells were washed twice with RPMI-1640 basal medium and resuspended in 5 mL 1640 basal medium, and counted for later use.

[0079] 4. Preparation of immune spleen cells.

[0080] A BALB / c mouse that had been boosted with immunization was taken and sacrificed by bleeding from the orbits, and the serum was collected as positive serum), which was then disinfected by soaking in 75% alcohol for 5-10 minutes, and then fixed on a dissecting board for dissection, and the spleen was removed and cut, and squeezed and ground in a sterilized dish. After filtering out the ground immune cells with a filter, the cells were washed twice with RPMI-1640 basal medium, and then resuspended in 10 mL 1640 basal medium, counted and set aside.

[0081] 5. Fusion.

[0082] 1×10 7 -2×10 7 SP2 / 0 and 1×10 8 Mix the immune cells in a 50mL centrifuge tube and centrifuge at 1000r / min for 8min. After discarding the supernatant, place the centrifuge tube containing the cell mixture in a 37℃ water bath, then add 0.8 mL (sigma) of 50% PEG preheated to 37℃, stir and let stand for 30s. After standing, add 10ml of RPMI-1640 basal medium preheated at 37℃. After mixing, centrifuge at 1000r / min for 5min, discard the supernatant and let stand at 37℃ for 5-8min. After resuspending with HAT medium, mix with feeder cell suspension, seed in 96-well culture plates, 250ul / well, and culture in a 37℃, 5% CO2 incubator. On the 4th day after fusion, change to HT medium and continue to culture. When the fused cell colonies grow to 7-14 days, there are obvious cell clusters under the microscope, and the culture medium turns slightly yellow, perform antibody detection.

[0083] 6. Screening of hybridoma positive clones.

[0084] Coating: Dilute bovine IgG to 1-10 μg / ml with coating buffer; add 100 μl to each microwell and incubate at 4°C overnight or at 37°C for 1 hour; shake off the liquid in the well (try to drain the liquid in the well); wash 3 times, 1 minute each time, and pat dry.

[0085] Blocking: Add 250 μl of blocking solution (2% OVA) to each microwell, shake gently, and incubate at 37°C for 1 hour; shake off the liquid in the wells; wash 3 times, 1 minute each time, and pat dry.

[0086] Sample addition: Take 50 μl of the supernatant of each well of the hybridoma to be tested and add it to the enzyme-labeled wells in order, shake gently, incubate at 37℃ for 1 hour, wash 3 times, 1 minute each time, and pat dry.

[0087] Add enzyme-labeled anti-antibody: dilute HRP-labeled goat anti-mouse IgG (Solarbio, catalog number: SE131) to 1:10000 with antibody diluent, add 100 μl to each well, shake gently, and incubate at 37°C for 1 hour; shake off the liquid in the well; wash 3 times, 1 minute each time, and pat dry.

[0088] Add color developing solution: Add 100 μl TMB color developing solution to each well, shake gently, and incubate at 37°C for 10 min.

[0089] Stop reaction: add 50 μl of stop solution (2 M sulfuric acid solution) to each well.

[0090] Determination result: OD of microplate reader 450nm The reading was performed at 3 times that of the negative well, which was considered positive.

[0091] 7. Cloning of hybridoma cells (limiting dilution method).

[0092] Prepare mouse feeder cell layer before cloning (same method as above).

[0093] Gently blow the hybridoma cells to be cloned from the culture wells and count the number of live cells; dilute the cells with culture medium to 10 cells / ml; add the cell suspension to the prepared 96-well culture plates of feeder cells, 100μl / well, so that each well contains 1 cell. Add 100μl / well of liquid on the 4th day of culture, and carefully observe the growth of cells in each well on the 5th-6th day and record it.

[0094] 8. Detection of specific antibodies.

[0095] On the 7th to 9th day after cloning, when the cell clones have grown to cover 1 / 3 to 1 / 2 of the visual field, they can be tested; the cells in the positive wells can be moved to a 24-well culture plate, and when the cells in the 24-well plate grow well, they can be intraperitoneally inoculated into mice to collect ascites.

[0096] 9. Determination of variable region sequences of monoclonal antibodies B4 and C11.

[0097] The number of hybridoma cells collected is greater than 10 6 , and sent to Shanghai Biotechnology for sequencing.

[0098] The amino acid sequences of the light chain and heavy chain variable regions of monoclonal antibody B4 are shown in SEQ ID NO:3 and SEQ ID NO:4, respectively, and the encoding gene sequences are shown in SEQ ID NO:1 and SEQ ID NO:2, respectively.

[0099] The amino acid sequences of the light chain and heavy chain variable regions of monoclonal antibody C11 are shown in SEQ ID NO:7 and SEQ ID NO:8, respectively, and the encoding gene sequences are shown in SEQ ID NO:5 and SEQ ID NO:6, respectively.

[0100] 10. Large-scale preparation of monoclonal antibodies B4 and C11.

[0101] After the establishment of the hybridoma cells, the mouse peritoneal cavity was injected, and the ascites was collected about 7 days later. The collected ascites was centrifuged at 4000rpm, 4℃ for 20 minutes, and the supernatant was collected. The ascites supernatant was diluted 4 times with the equilibration buffer and filtered with a 0.45µm filter; 0.5ml rprotein G was loaded into the chromatography column, and the peristaltic pump, recorder and detector were turned on; the chromatography column was equilibrated with the equilibration buffer, and then the sample was loaded and the flow-through was collected; the column was washed with the equilibration buffer to completely remove the impurities, and the elution buffer was replaced, and the elution peak was collected with a collection tube containing an appropriate amount of neutralization buffer. The purified antibody was dialyzed with an excess of 0.1M PBS, pH7.4. The dialyzed antibody was concentrated to a concentration of >1mg / ml.

[0102] Example 2

[0103] Establishment of double antibody sandwich ELISA detection method.

[0104] 1. Determination of relative affinity constant.

[0105] Dilute bovine IgG to 2μg / ml with coating buffer; add 100μl to each well of the microplate and coat overnight at 4℃; shake off the liquid in the well and add 200μl of blocking solution to each well of the microplate, shake gently, and incubate at room temperature for 2h; wash 3 times, 1 minute each time, and pat dry. Sample addition: dilute the monoclonal antibody to be tested to saturation concentration, take 100μl per well and add it to the enzyme-labeled well, and incubate at room temperature for 2h. After washing the plate with washing solution, add 2.5, 3.0, 3.5, 4.0, 5.0, 0mol / L NaSCN solution 60μl / well in turn, stand at room temperature for 15min, wash 3 times, 1 minute each time, and pat dry. Add HRP-labeled goat anti-mouse IgG, stand at room temperature for 45min, wash 3 times, 1 minute each time, and pat dry. Add TMB color development solution 100μl / well, color development at room temperature for 10min. Add 50μl of stop solution to each well. Measure the OD value at 450nm and 630nm using an enzyme marker. 450nm -OD 630nm The sodium thiocyanate concentration corresponding to the decrease to 50% of the uneluted is the relative affinity constant of the antibody, expressed in mol / l. The results in Table 1 show that all B4 and C11 monoclonal antibodies have high affinity (>2.7 mol / L).

[0106] Table 1

[0107]

[0108] 2. HRP labeling of monoclonal antibody C11.

[0109] Add monoclonal antibody C11 to the dialysis bag and immerse it in 0.01M CB buffer for overnight dialysis at 4℃. Take 10mg HRP and dissolve it in 2ml water. Prepare a fresh NaIO4 solution, 0.1M, add 0.4ml 0.1M NaIO4 to 2ml HRP solution, mix well, and dialyze it in 10mM NaAc buffer at 4℃ overnight at room temperature and away from light for 45min. Take out the antibody and HRP solution that have been dialyzed overnight and add 0.1ml, 0.2M pH9.5 carbonate buffer to adjust the pH. Then immediately add the antibody that has been taken out to the HRP solution and stir gently for 2 hours at room temperature and away from light. Weigh 0.04g NaBH4 and dissolve it in 10ml water, take 0.4ml and add it to the reaction solution, mix well, and place it at 4℃ for 2 hours. Take it out and put it in the dialysis bag, dialyze it in 0.01M PBS, change the solution once after 2 hours, and dialyze it at 4℃ overnight. The labeled solution dialyzed overnight was taken out, an equal volume of glycerol was added, and the solution was stored at -20°C.

[0110] 3. Preparation of monoclonal antibody B4 ELISA plate.

[0111] Dilute monoclonal antibody B4 to 2μg / mL with CB; add 100μl to each well of the microplate and coat overnight at 4℃; discard the liquid in the well, add 200μl of blocking solution to each well, shake gently, and incubate at room temperature for 2h; discard the liquid in the well, place in a dry room to dry for 16~18h. Place in an aluminum foil bag containing desiccant, vacuum seal, and store at 4℃.

[0112] 4. Establishment of double antibody sandwich ELISA method.

[0113] 30 minutes before the experiment, take out the ELISA plate coated with monoclonal antibody B4, return to room temperature, wash the plate 3 times and spin dry. Add 100μl of bovine IgG standard with different dilution concentrations, the dilution concentrations are 50ng / ml, 25ng / ml, 12.5ng / ml, 6.25ng / ml, 3.12ng / ml, 1.56ng / ml, 0.78ng / ml and set up a blank control. After sealing the plate, incubate in a room temperature incubator for 2h, wash the plate 4 times and pat dry. Add 100μl enzyme-labeled antibody HRP-C11 working solution to the reaction well, seal the plate, incubate in a room temperature incubator for 45min, wash the plate 4 times and pat dry. Add 100 μl of chromogenic substrate TMB to the reaction wells, seal the plate and color develop for 15 min at room temperature in the dark, add 50 μl of stop solution, use a microplate reader to measure the OD value at the maximum absorption wavelength of 450 nm and the reference wavelength of 630 nm, and subtract the OD value at 630 nm from the OD value at 450 nm. The concentration of the standard is the horizontal axis (ng / ml) and the absorbance OD value is the vertical axis (OD 450nm -OD 630nm ), and the standard curve was drawn using ELISA Calc software (using four-parameter fitting). The results showed that the detection range was 0.78-50 ng / ml, R 2 is 0.9992 ( Figure 1 ).

[0114] 5. Double antibody sandwich ELISA sensitivity test.

[0115] The average value of the OD of 20 zero standard concentrations was added with two standard deviations to calculate the corresponding detectable concentration, and the sensitivity was 16.487 pg / ml (Table 2).

[0116] Table 2

[0117]

[0118] 6. Double antibody sandwich ELISA specific detection.

[0119] 30 minutes before the experiment, take out the ELISA plate coated with monoclonal antibody B4, return to room temperature, wash the plate 3 times and pat dry. Add 100 μl of bovine IgG standard or bovine IgG structural similar proteins at different dilutions, including duck IgY, chicken IgY, rabbit IgG, bovine IgA, goat IgG, pig IgG, human IgG, mouse IgG, rat IgG, duck IgA, goose IgA, duck IgM, goose IgM. After sealing the plate, incubate in a room temperature incubator for 2 hours, wash the plate 4 times and pat dry. Add 100 μl enzyme-labeled antibody HRP-C11 working solution to the reaction well, seal the plate and incubate at room temperature for 45 minutes, wash the plate 4 times and pat dry. Add 100 μl of chromogenic substrate TMB to the reaction wells, seal the plate and color develop at room temperature in the dark for 15 min, add 50 μl of stop solution, use an ELISA reader for dual wavelength detection, measure the OD value at the maximum absorption wavelength of 450 nm and the reference wavelength of 630 nm, and subtract the OD value at 630 nm from the OD value at 450 nm. Establish a standard curve and calculate the results. The results show that the antibody does not react with other similar proteins (Table 3).

[0120] Table 3

[0121]

[0122] 7. Double antibody sandwich ELISA stability test.

[0123] The stability was investigated by 37°C accelerated test.

[0124] Place the monoclonal antibody B4-coated ELISA plate, HRP-labeled monoclonal antibody C11, and bovine IgG standard at 37°C for accelerated stability testing for 13 days (equivalent to about 19.5 months at 4°C). Then take out the test. The test method is to take out the ELISA plate, wash the plate 3 times and pat dry. Add 100μl of different dilution concentrations of standard, the concentration is 50ng / ml, 25ng / ml, 12.5ng / ml, 6.25ng / ml, 3.12ng / ml, 1.56ng / ml, 0.78ng / ml and set a blank control. After sealing the plate, incubate at room temperature for 2h, wash the plate 4 times and pat dry. Add 100μl of enzyme-labeled antibody HRP-C11 working solution to the reaction well, seal the plate, incubate at room temperature for 45min, wash the plate 4 times and pat dry. Add 100μl of chromogenic substrate TMB to the reaction wells, seal the plate and color for 15min at room temperature in the dark, add 50μl of stop solution, use an ELISA reader for dual wavelength detection, measure the OD value at the maximum absorption wavelength of 450nm and the reference wavelength of 630nm, and subtract the OD value at 630nm from the OD value at 450nm. Draw a standard curve with different concentrations of standards as the horizontal axis and the corresponding OD value as the vertical axis to establish a regression equation. The results showed that on the 13th day, the OD value of the high point of the standard curve (50ng / ml) was still greater than 2.3, and the OD value gradient of the standard curve was good (Table 4).

[0125] Table 4

[0126]

[0127] Example 3

[0128] Determination of IgG content in bovine serum.

[0129] 30 minutes before the experiment, take out the ELISA plate coated with monoclonal antibody B4, return to room temperature, wash the plate 3 times and pat dry. Add 100 μl of bovine IgG standard and bovine serum samples with different dilution multiples. The dilution concentrations of bovine IgG standard are 50ng / ml, 25ng / ml, 12.5ng / ml, 6.25ng / ml, 3.12ng / ml, 1.56ng / ml, 0.78ng / ml, and set up a blank control. After sealing the plate, incubate at room temperature for 2 hours, wash the plate 4 times and pat dry. Add HRP-C11 working solution to the reaction wells, seal the plate, incubate at room temperature for 45 minutes, wash the plate 4 times and pat dry. Add 100μl of chromogenic substrate TMB to the reaction wells, seal the plate and color for 15min at room temperature in the dark, add 50μl of stop solution, use an ELISA reader for dual wavelength detection, measure the OD value at the maximum absorption wavelength of 450nm and the reference wavelength of 630nm, and subtract the OD value at 630nm from the OD value at 450nm. According to literature reports, the IgG content in normal bovine serum ranges from 17 to 27mg / ml (depending on age and individual content), and the test results show that the content determined by this kit is basically consistent with the report, and the test results show that this method can accurately determine the content of bovine serum IgG (Table 5).

[0130] Table 5

[0131]

[0132] Example 4

[0133] Comparison with a foreign brand of antibody against dairy samples.

[0134] The antibody pair of foreign brand A (GENORISE, USA) was selected for analogy testing, and the experiment was carried out in the most appropriate way for each brand (operated in full accordance with its instructions). The results were compared by measuring the sample content: 8 freshly collected bovine whey samples were randomly selected for simultaneous measurement. The measurement results showed that the content measured by this kit was higher, which was consistent with the literature report, and the content measured by brand A was lower (Table 6).

[0135] Table 6

[0136]

[0137] Example 5

[0138] Determination of IgG content in liquid milk and milk powder.

[0139] 30 minutes before the experiment, take out the ELISA plate coated with monoclonal antibody B4, return to room temperature, wash the plate 3 times and pat dry. Add 100 μl of bovine IgG standards and samples at different dilution multiples. The dilution concentrations of bovine IgG standards are 50 ng / ml, 25 ng / ml, 12.5 ng / ml, 6.25 ng / ml, 3.12 ng / ml, 1.56 ng / ml, and 0.78 ng / ml, and set up a blank control. After sealing the plate, incubate at room temperature for 2 hours, wash the plate 4 times and pat dry. Add HRP-C11 working solution to the reaction wells, seal the plate, incubate at room temperature for 45 minutes, wash the plate 4 times and pat dry. Add 100μl of chromogenic substrate TMB to the reaction wells, seal the plate and color for 15min at room temperature in the dark, add 50μl of stop solution and use a microplate reader for dual wavelength detection, measure the OD value at the maximum absorption wavelength of 450nm and the reference wavelength of 630nm, and subtract the OD value at 630nm from the OD value at 450nm. Randomly select 2 common brand milk samples (one of which is fresh milk) and 1 brand milk powder sample on the market for simultaneous determination. The results are shown in Table 7. The IgG content in bovine colostrum powder is much greater than that in ordinary milk powder, which is consistent with literature reports.

[0140] Table 7

[0141]

[0142] Example 6

[0143] Determination of bovine IgG content in various meat products.

[0144] 30 minutes before the experiment, take out the ELISA plate coated with monoclonal antibody B4, return to room temperature, wash the plate 3 times and pat dry. Add 100 μl of bovine IgG standards and samples at different dilution multiples. The dilution concentrations of bovine IgG standards are 50 ng / ml, 25 ng / ml, 12.5 ng / ml, 6.25 ng / ml, 3.12 ng / ml, 1.56 ng / ml, and 0.78 ng / ml, and set up a blank control. After sealing the plate, incubate at room temperature for 2 hours, wash the plate 4 times and pat dry. Add HRP-C11 working solution to the reaction wells, seal the plate, incubate at room temperature for 45 minutes, wash the plate 4 times and pat dry. Add 100 μl of chromogenic substrate TMB to the reaction wells, seal the plate and color develop at room temperature in the dark for 15 min, add 50 μl of stop solution and use a microplate reader for dual wavelength detection, measure the OD value at the maximum absorption wavelength of 450 nm and the reference wavelength of 630 nm, and subtract the OD value at 630 nm from the OD value at 450 nm. From the results, it can be seen that the kit can specifically identify IgG in highly diluted (5000-fold dilution) bovine tissues, but has no reaction with proteins from other species (Table 8).

[0145] Table 8

[0146]

[0147] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A mouse anti-bovine IgG monoclonal antibody or an antigen-binding fragment thereof, characterized in that: The amino acid sequences of the complementary determining regions CDR1, CDR2 and CDR3 of the heavy chain variable region of the mouse anti-bovine IgG monoclonal antibody or its antigen-binding fragment are GYAFTSNN, IDPYNGGT and ARGPYYGNFGPFAY respectively; the amino acid sequences of the complementary determining regions CDR1, CDR2 and CDR3 of the light chain variable region are QSLFNSGNQKNF, GAS and QNDHSYPLT respectively.

2. The mouse anti-bovine IgG monoclonal antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The amino acid sequence of the heavy chain variable region of the mouse anti-bovine IgG monoclonal antibody is shown in SEQ ID NO:4; the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

3.

3. A nucleic acid molecule, characterized in that It encodes the mouse anti-bovine IgG monoclonal antibody or its antigen-binding fragment as described in claim 1 or 2.

4. The nucleic acid molecule according to claim 3, characterized in that The nucleotide sequence encoding the heavy chain variable region is shown in SEQ ID NO: 2; the amino acid sequence encoding the light chain variable region is shown in SEQ ID NO:

1.

5. Biomaterial, characterized in that It contains the nucleic acid molecule according to claim 3 or 4; the biological material is an expression cassette, a vector or a host cell.

6. A mouse anti-bovine IgG monoclonal antibody conjugate, characterized in that: The invention is obtained by coupling the mouse anti-bovine IgG monoclonal antibody or the antigen-binding fragment thereof according to claim 1 or 2 with a label, wherein the label is selected from one or more of enzyme labeling, biotin labeling, fluorescent dye labeling, chemiluminescent dye labeling and radioactive labeling.

7. A composition of mouse anti-bovine IgG monoclonal antibodies, characterized in that: The composition comprises the mouse anti-bovine IgG monoclonal antibody or its antigen-binding fragment according to claim 1 or 2; and further comprises the following mouse anti-bovine IgG monoclonal antibody or its antigen-binding fragment: the amino acid sequences of the complementary determining regions CDR1, CDR2, and CDR3 of the heavy chain variable region are GDSITSGY, ISYSGST, and AREGSTLTTRGFAY, respectively, and the amino acid sequences of the complementary determining regions CDR1, CDR2, and CDR3 of the light chain variable region are QSVSND, YAS, and QQDYSSPWT, respectively.

8. Use of the mouse anti-bovine IgG monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2, the nucleic acid molecule according to claim 3 or 4, the biomaterial according to claim 5, the mouse anti-bovine IgG monoclonal antibody conjugate according to claim 6, or the composition according to claim 7 in the preparation of a product for detecting the presence or level of bovine IgG in a sample.

9. Any of the following uses of the mouse anti-bovine IgG monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2, the mouse anti-bovine IgG monoclonal antibody conjugate according to claim 6, or the composition according to claim 7: (1) Use in detecting the presence or level of bovine IgG in a sample for non-diagnostic and therapeutic purposes; (2) Application in quality control or production of products containing bovine IgG.

10. A kit, characterized in that It comprises the mouse anti-bovine IgG monoclonal antibody or its antigen-binding fragment according to claim 1 or 2, or the antibody conjugate according to claim 6, or the composition according to claim 7.

11. The kit according to claim 10, characterized in that: It is an enzyme-linked immunosorbent assay kit, a fluorescent immunoassay kit or a chemiluminescent immunoassay kit.

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