Anti-pig IgG antibody and its application

Abstract

The present invention belongs to the field of immunological detection, and provides an anti-swine IgG antibody and its application. The antibody against pig IgG provided by the present invention can specifically bind to pig IgG, has no cross reaction with other similar proteins, has high affinity with pig IgG, and has high stability; the double antibody sandwich ELISA method based on the antibody has high sensitivity, specificity and accuracy in detecting pig IgG, and can realize accurate and high-throughput detection of IgG content in pig serum or other serum analogs or products containing pig IgG, and has good application prospects in pig IgG detection.

Description

Technical Field

[0001] The invention belongs to the field of immunological detection, and in particular relates to an anti-pig IgG antibody and an application thereof. Background Art

[0002] In recent years, with the prevalence of African swine fever and new swine infectious diseases, my country's pig farming industry has suffered serious losses. Therefore, it is particularly important to quickly and accurately detect sick pigs. IgG is the most concentrated type of immunoglobulin in animal serum, accounting for about 70%-80% of the immunoglobulin content. It plays a major role in antibody-mediated reactions. At the same time, IgG levels are also a commonly used indicator in clinical immunological tests and humoral immunity research. It is of great significance to fully and systematically understand the immune response of pigs to pathogen infection and take corresponding preventive measures. At the same time, the overall level of immunoglobulins can also indirectly reflect the strength of the body's immunity. The detection of IgG content in the blood of a group can be used to evaluate the health status of the group, and can be used to make auxiliary judgments on the effects of different formulas of feed or nutritional additives.

[0003] Compared with polyclonal antibodies, anti-swine IgG monoclonal antibodies have the advantages of high specificity and stable titer. However, similar imported reagents are expensive, domestic reagents have low titers, large batch differences and insufficient sensitivity. Summary of the invention

[0004] The purpose of the present invention is to provide an anti-pig IgG antibody and application thereof.

[0005] The present invention is based on the principle of hybridoma cell establishment by Kohler and Milstrin, and utilizes PEG fusion technology to establish a hybridoma cell strain that secretes anti-porcine IgG monoclonal antibodies, aiming to provide high-quality, standardized reagents for the detection of porcine IgG.

[0006] In order to achieve the purpose of the present invention, in the first aspect, the present invention provides an anti-swine IgG antibody (monoclonal antibody 4H1) 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 antibody or its antigen-binding fragment are GFTFSNHP, ISTGGSYT, and SRHGYDPERSWYFDV (SEQ ID NOs: 14-16), respectively, and the amino acid sequences of the complementary determining regions CDR1, CDR2, and CDR3 of the light chain variable region are SSVSY (SEQ ID NO: 17), STS, and QQRSRYPYT (SEQ ID NO: 18), respectively.

[0007] Furthermore, the amino acid sequence of the light chain variable region of the antibody or antigen-binding fragment thereof is as shown in SEQ ID NO:3 or has at least 80%, 85%, 90%, 95% or 98% similarity to the sequence shown in SEQ ID NO:3, and the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:4 or has at least 80%, 85%, 90%, 95% or 98% similarity to the sequence shown in SEQ ID NO:4.

[0008] In the present invention, the antibody or antigen-binding fragment thereof includes but is not limited to monoclonal antibody, Fab, Fab', F(ab')2, Fv or single-chain antibody.

[0009] In a second aspect, the present invention provides a nucleic acid molecule encoding the antibody or antigen-binding fragment thereof.

[0010] In a third aspect, the present invention provides biological materials containing the nucleic acid molecule, wherein the biological materials include but are not limited to recombinant DNA, expression cassettes, transposons, plasmid vectors, viral vectors, engineered bacteria or host cells.

[0011] In a fourth aspect, the present invention provides an antibody conjugate, wherein the antibody conjugate is obtained by conjugating the antibody or its antigen-binding fragment with a marker, wherein the marker is selected from one or more of enzyme labeling, biotin labeling, fluorescent dye labeling, chemiluminescent dye labeling, radioactive labeling, etc.

[0012] In a fifth aspect, the present invention provides use of the antibody or antigen-binding fragment thereof, the nucleic acid molecule, or the biological material in the preparation of a product for qualitative or quantitative detection of porcine IgG.

[0013] In a sixth aspect, the present invention provides any of the following uses of the antibody or antigen-binding fragment thereof, the antibody conjugate, or the antibody composition:

[0014] (1) Used for qualitative or quantitative detection of porcine IgG (including non-disease diagnosis and treatment purposes);

[0015] (2) Used for testing vaccine immunogenicity or immune efficacy;

[0016] (3) Used for quality control of products containing porcine IgG.

[0017] In a seventh aspect, the present invention provides a pig IgG double antibody sandwich enzyme-linked immunosorbent assay kit, the kit comprising: the antibody 4H1 or its antigen-binding fragment, the anti-pig IgG antibody 9G5 or its antigen-binding fragment, and at least one of an optional substrate color development solution, a blocking solution, and a stop solution.

[0018] The amino acid sequences of the complementary determining regions CDR1, CDR2, and CDR3 of the heavy chain variable region of the antibody 9G5 or its antigen-binding fragment are GYTFTSYV, INPYNDGT, and ARYDGYGFPY (SEQ ID NOs: 9-11), respectively, and the amino acid sequences of the complementary determining regions CDR1, CDR2, and CDR3 of the light chain variable region are QDVGTT (SEQ ID NO: 12), RAS, and QQYTRYPYT (SEQ ID NO: 13), respectively.

[0019] Further, the antibody 4H1 or its antigen-binding fragment is used as the first antibody, and the anti-swine IgG antibody 9G5 or its antigen-binding fragment is used as the second antibody; or,

[0020] The anti-swine IgG antibody 9G5 or an antigen-binding fragment thereof is used as the first antibody, and the antibody 4H1 or an antigen-binding fragment thereof is used as the second antibody.

[0021] In a specific embodiment of the present invention, the antibody 9G5 is fixed on a solid phase carrier, and the solid phase carrier can be selected from ELISA plates, microspheres, nitrocellulose membranes, glass cellulose membranes or nylon membranes, etc., preferably ELISA plates.

[0022] The antibody 4H1 is labeled with an enzyme, and the enzyme labeled with the enzyme can be selected from any one of the following: horseradish peroxidase (HRP), alkaline phosphatase (AP), glucose oxidase, β-galactosidase, lysozyme, malate dehydrogenase, etc., preferably horseradish peroxidase.

[0023] Furthermore, the kit also includes at least one of a substrate color developing solution, a blocking solution or a stop solution.

[0024] Commonly used substrates include, but are not limited to, o-phenylenediamine (OPD), tetramethylbenzidine (3,3',5,5'-tetramethylbenzidine, TMB) and ABTS [2,2'-azino-di-(3-ethylbenziazobinesulfonate-6)], etc., preferably TMB.

[0025] The blocking solution may be a 2% BSA solution, and the stopping solution may be a 1.5-2.5 M (preferably 2 M) sulfuric acid solution.

[0026] The principle of detecting the content of pig IgG by using the enzyme-linked immunosorbent assay technology based on the double antibody sandwich method is as follows: anti-pig IgG monoclonal antibody 1 is coated on the ELISA plate; gradient dilution standard and pre-diluted sample are added respectively, and the pig IgG in the standard and sample will fully bind to the coated antibody on the ELISA plate; after washing the plate, HRP-labeled anti-pig IgG antibody 2 is added, and the antibody will specifically bind to the pig IgG in the standard and sample captured by the coated antibody on the plate; after washing the plate, the color developer substrate TMB is added, and if there are different concentrations of pig IgG in the sample in the reaction well, HRP will turn the colorless TMB into different shades (positively correlated) of blue substances, and the reaction well will turn yellow after adding the stop solution; finally, the absorbance of the sample in the reaction well is measured at λmax=450nm (OD 450nm ), the concentration of pig IgG in the sample is proportional to OD, and the concentration of pig IgG in the sample can be calculated through the standard curve. This method uses an enzyme colorimetric amplification system, which has a high detection sensitivity and can detect samples with relatively low content. The detection range is 0.4-25ng / mL.

[0027] By means of the above technical solution, the present invention has at least the following advantages and beneficial effects:

[0028] The porcine IgG antibody provided by the present invention can specifically bind to porcine IgG, has no cross-reaction with other similar proteins, has high affinity to porcine IgG, and has high stability; the double antibody sandwich ELISA method based on the antibody has high sensitivity, specificity and accuracy for detecting porcine IgG, can realize accurate and high-throughput detection of IgG content in porcine serum or other serum analogs or products containing porcine IgG, and has good application prospects in porcine IgG detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The figure is a diagram showing the results of SDS-PAGE electrophoresis of purified monoclonal antibodies 4H1 and 9G5 in a preferred embodiment of the present invention; wherein M is a protein molecular weight marker, in KDa.

[0030] Figure 2 This is the standard curve established for detecting pig IgG by the double antibody sandwich ELISA method in a preferred embodiment of the present invention. DETAILED DESCRIPTION

[0031] The present invention aims to provide a kit capable of accurately, rapidly and in large quantities detecting the content of pig IgG in samples.

[0032] The present invention adopts the following technical solution:

[0033] The present invention provides an ELISA plate coated with anti-swine IgG monoclonal antibody 9G5; the light chain and heavy chain variable region DNA sequences of antibody 9G5 are SEQ ID NO: 5 and SEQ ID NO: 6, respectively. The light chain and heavy chain variable region amino acid sequences are SEQ ID NO: 7 and SEQ ID NO: 8, respectively.

[0034] The present invention provides an anti-swine IgG monoclonal antibody 4H1 labeled with HRP, wherein the light chain and heavy chain variable region DNA sequences are SEQ ID NO: 1 and SEQ ID NO: 2, respectively, and the light chain and heavy chain variable region amino acid sequences are SEQ ID NO: 3 and SEQ ID NO: 4, respectively.

[0035] The present invention also provides a kit for detecting pig IgG, wherein the kit is configured with a pig IgG detection reagent, preferably comprising an ELISA plate coated with anti-pig IgG monoclonal antibody pig IgG-9G5, an HRP-labeled anti-pig IgG monoclonal antibody pig IgG-4H1 solution, an HRP colorimetric substrate, and a standard.

[0036] The present invention also provides a method for using a kit for detecting pig IgG, comprising adding a test sample containing pig IgG to an ELISA plate coated with anti-pig IgG monoclonal antibody pig IgG-9G5, adding an HRP-labeled anti-pig IgG monoclonal antibody pig IgG-4H1 solution for reaction, then adding an HRP color development substrate, and calculating the content of pig IgG in the test sample according to the color development OD value.

[0037] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0038] Example 1 Preparation of anti-swine IgG monoclonal antibody

[0039] 1. Animal immunization

[0040] Female Balb / c mice aged 6-8 weeks were selected and immunized with naturally extracted porcine IgG antigen emulsified with an equal volume of Freund's adjuvant. The immunization cycle was two weeks. Blood was collected after three immunizations to measure the titer, and the immunization was boosted again three days before fusion.

[0041] 2. Cell fusion

[0042] The mice were killed by cervical dislocation, and the spleen was removed aseptically. The spleen was 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 the fusion promoter polyethylene glycol (PEG) was added. Under the action of polyethylene glycol, various lymphocytes can fuse with myeloma cells to form hybridoma cells. The specific operation is as follows:

[0043] Myeloma (SP2 / 0) cell activation:

[0044] Thaw and resuscitate commercial SP2 / 0 cells, then suspend in nutrient solution (RPMI-1640, supplemented with 10% calf serum), and culture in an incubator at 37°C and 5% CO2; subculture after 3-5 days, and replace the culture medium one day before fusion. On the day of fusion, discard all the culture medium in the culture dish and replace it with serum-free medium. After blowing the cells off, place them in a 50mL centrifuge tube, add serum-free medium 1640 to 50mL, count, and centrifuge for later use.

[0045] Preparation of immune splenocytes:

[0046] Take a Balb / c mouse that has been boosted with immunization, bleed it from its orbits to kill it (collect serum, i.e., positive serum), soak it in 75% alcohol for 5-10 minutes for disinfection, then fix it on a dissecting board for dissection, remove the spleen and cut it, grind it in a sterile sieve, filter the ground spleen cells once with a sieve, add 1640 to 50 mL of serum-free culture medium, count it, and set it aside after centrifugation.

[0047] Preparation of feeder cells:

[0048] Take an unimmunized Balb / c mouse, bleed it from its orbit, and collect the negative serum. Inject 10 mL of the HAT-prepared culture medium 1640 into the mouse's peritoneal cavity, then aspirate and place it in another centrifuge tube for later use. The fluid contains peritoneal macrophages.

[0049] Fusion:

[0050] 1×10 7 -2×10 7 SP2 / 0 and 10 8Mix the immune cells in a 50mL centrifuge tube, 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 1mL (sigma) of 50% PEG preheated to 37℃, stir and let stand for 30s. After standing, add 1640 serum-free medium preheated at 37℃. After mixing, centrifuge at 1000r / min for 5min, discard the supernatant and let stand at 37℃ for 5-8min. Then mix with the feeder cell suspension, seed in a 96-well culture plate, 200μL / well, and culture in a 37℃, 5% CO2 incubator. On the 4th day after fusion, change to HAT medium and continue to culture. When the fused cell colony grows to 1 / 4 of the culture well and the culture medium turns slightly yellow, perform antibody detection.

[0051] 3. Screening of hybridoma positive clones and cell cloning

[0052] The purpose of selective culture is to screen fused hybridoma cells, using HAT selective culture medium. In HAT culture medium, unfused myeloma cells die because they lack hypoxanthine-guanine-phosphoribosyltransferase and cannot synthesize DNA using the salvage pathway. Although unfused spleen cells have hypoxanthine-guanine-phosphoribosyltransferase, they cannot survive in vitro for a long time and gradually die. Only fused hybridoma cells can survive and proliferate in HAT culture medium because they have obtained hypoxanthine-guanine-phosphoribosyltransferase from spleen cells and have the characteristics of myeloma cells that can proliferate indefinitely. The specific operation is as follows:

[0053] The steps for screening positive hybridoma cells using indirect ELISA are as follows:

[0054] Coating antigen: dilute the antigen to 1-10 μg / mL with coating buffer; add 100 μL to each microwell, shake gently, and store in a 4°C refrigerator overnight or at 37°C for 2 hours; shake off the liquid in the well (tap dry as much as possible); wash 3 times, 2-3 minutes each time.

[0055] Block the uncoated positions in the ELISA wells: add 200 μL of blocking solution (5% skim milk powder or 2% BSA) to each microwell, shake gently, and incubate at 37℃ for 1 hour; discard the liquid in the wells; fill each well with washing buffer, let stand for 2-3 minutes, discard the liquid in the wells, pat dry, and use this method to wash 3 times with washing buffer. Sample addition: add 50 μL of supernatant from each well of the hybridoma to be tested to the ELISA wells in order, shake gently. Incubate at 37℃ for 1 hour, wash, and pat dry.

[0056] Add enzyme-labeled anti-antibody: First dilute the enzyme-labeled secondary antibody to an appropriate working concentration with diluent according to the instructions, add 100μL to each well, shake gently, and place at 37℃ for 30min; then wash and pat dry. Add color development solution: Add 100μL of freshly prepared color development solution to each well, shake gently, and place at 37℃ for 10min. Stop the reaction: Add 50μL of stop solution to each well.

[0057] Determination result: OD of microplate reader 450 The reading was performed at 3 times that of the negative well, which was considered positive.

[0058] Cloning of hybridoma cells (limiting dilution method):

[0059] Prepare a mouse feeder cell layer before cloning; gently blow the hybridoma cells to be cloned from the culture well and count the number of live cells using a hemocytometer; dilute the cells to 5, 10, or 30 cells / mL with complete culture medium;

[0060] Add the three concentrations of cell suspension to the prepared 96-well culture plate of feeder cells, 100 μL / well, so that each well contains 0.5, 1 and 3 cells, respectively. Add one drop 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;

[0061] Detection of specific antibodies: On the 7th to 9th day after cloning, when the cell clones cover 1 / 3-1 / 2 of the visual field, they can be detected; after three clonings using the same method, the cells with positive cells in a single well can be selected for the last time and transferred to a 24-well culture plate. When the cells in the 24-well plate grow well, they can be intraperitoneally inoculated into mice to collect ascites.

[0062] 4. Sequence determination of variable regions of monoclonal antibodies 4H1 and 9G5

[0063] The number of hybridoma cells collected was greater than 10 6: The total RNA extraction kit of Beijing Solebow Technology Co., Ltd. was used to extract the total RNA of the two cell lines according to the instructions; the first chain of cDNA was synthesized and specifically amplified using the reverse transcription kit of Beijing Solebow Technology Co., Ltd., and the amplified DNA was constructed into a T vector and sent to Qingke Bio for sequencing. The gene sequencing results were obtained: the light chain variable region sequence of the 4H1 cell line is 315bp long, encoding 107 amino acids, the DNA sequence is shown in SEQ ID NO:1, and the protein sequence is shown in SEQ ID NO:3; the heavy chain variable region sequence is 366bp long, encoding 122 amino acids, the DNA sequence is shown in SEQ ID NO:2, and the protein sequence is shown in SEQ ID NO:4. The light chain variable region sequence of the 9G5 cell line is 324bp long, encoding 108 amino acids, the DNA sequence is shown in SEQ ID NO:5, and the protein sequence is shown in SEQ ID NO:7; the heavy chain variable region sequence is 351bp long, encoding 117 amino acids, the DNA sequence is shown in SEQ ID NO:6, and the protein sequence is shown in SEQ ID NO:8.

[0064] 5. Large-scale preparation of monoclonal antibodies 4H1 and 9G5

[0065] After the establishment of hybridoma cells, the cells were injected into the peritoneal cavity of mice, and the ascites was collected after about 7 days. The antibodies were purified by Protein G affinity chromatography ( Figure 1 ).

[0066] Example 2 Construction of a double antibody sandwich ELISA kit for detecting pig IgG content

[0067] 1. Antibody affinity constant detection

[0068] Prepare the ELISA plate according to the method of (indirect ELISA screening of positive hybridoma cells), dilute the purified antibody to the same concentration, 100 μL / well, and incubate at 37°C for 1 hour. After washing the plate with PBST, use different concentrations of sodium thiocyanate to elute, add 0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0 mol / L solution 60 μL / well in turn, let stand at room temperature for 15 minutes, wash the plate with PBST, add secondary antibody (sheep anti-mouse IgG-HRP, 1:5000), and detect with color. Result determination: OD after elution 450 The sodium thiocyanate concentration corresponding to the decrease to 50% of the uneluted concentration is the relative affinity constant of the antibody, expressed in mol / L (Table 1).

[0069] Table 1

[0070] NaSCN(mol / L) 9G5 4H1 5 0.448 0.677 4.5 0.433 0.531 4 0.479 0.514 3.5 0.506 0.477 3 1.033 0.359 2.5 1.392 0.628 2 1.955 1.591 1.5 2.174 2.18 1 2.298 2.328 0.5 2.267 2.523 0 2.264 2.471 blank 0.103 0.087 Relative affinity constant 3 2.25

[0071] 2. HRP labeling of monoclonal antibody (anti-swine IgG antibody 2) derived from cell line 4H1

[0072] Add anti-swine IgG antibody 2 to the dialysis bag and immerse it in 0.01M CB buffer at 4℃ for overnight dialysis. Take 10mg HRP and dissolve it in 2mL water. Prepare a fresh NaIO4 solution of 0.1mol / L, add 0.4mL 0.1mol / L 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 of 0.2mol / L carbonate buffer at pH 9.5. Then immediately add the antibody that has been taken out to the HRP solution and stir gently for 2h 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 2h. Take it out and put it in the dialysis bag, dialyze it in 0.01mol / L PBS, change the solution once after two 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.

[0073] 3. Preparation of ELISA plate of monoclonal antibody (anti-swine IgG antibody 1) derived from cell line 9G5

[0074] Dilute the anti-swine IgG antibody 1 to 2 μg / mL with 0.05M pH9.6 carbonate coating buffer. Add 100 μL to the reaction wells of a 96-well polystyrene ELISA plate and incubate at 4°C overnight. The next day, discard the solution in the wells and wash three times with washing buffer, 3 minutes each time. After the above steps, block each well of the reaction plate with 2% BSA solution, 250 μL per well, and block at room temperature for 2 hours. Discard the solution in the wells, dry in a drying room, evacuate, and store at 4°C.

[0075] 4. Establishment of double antibody sandwich ELISA method and determination of sensitivity

[0076] 30 minutes before the experiment, take out the ELISA plate coated with anti-swine IgG antibody 1, return to room temperature, wash the plate 3 times and spin dry. Add 100 μL of different dilution concentrations of swine IgG standard, the swine IgG dilution concentrations are 400ng / mL, 200ng / mL, 100ng / mL, 50ng / mL, 25ng / mL, 12.5ng / mL, 6.25ng / mL and set up blank controls. At the same time, add 100 μL of blank diluent (PBST solution, i.e., 0.1mol / L phosphate buffer containing 0.05% Tween 20) to 20 blank wells. After sealing the plate, incubate in a room temperature incubator for 2 hours, wash the plate 4 times and spin dry. Add 100 μL of enzyme-labeled antibody HRP-4H1 working solution to the reaction well, seal the plate, incubate in a room temperature incubator for 30 minutes, wash the plate 4 times and spin dry. Add 100 μl of chromogenic substrate TMB to the reaction wells, seal the plate and color for 15 minutes at room temperature in the dark, add 50 μl of stop solution 2M sulfuric acid solution, and immediately measure the OD value at 450nm wavelength using an ELISA reader (within 5 minutes). Use different concentrations of pig IgG standards as the horizontal axis and the corresponding OD values ​​as the vertical axis to draw a standard curve and establish a regression equation. The results show that its detection range is 0.4-25ng / mL, R 2 is 0.9998( Figure 2 ). The average value of OD of 20 zero standard concentrations was added with two standard deviations to calculate the corresponding detectable concentration. The results showed that the sensitivity of the double antibody sandwich ELISA method was 1.373586 ng / ml (Table 2).

[0077] Table 2

[0078]

[0079]

[0080] 5. Double antibody sandwich ELISA specificity detection

[0081] 5.1 Cross-testing with standards from other species

[0082] 30 minutes before the experiment, take out the ELISA plate coated with anti-swine IgG antibody 1, return to room temperature, wash the plate 3 times and spin dry. Add 100 μL of different dilution multiples of swine IgG standard (from Solarbio, catalog number SP037) or high concentration of swine IgG structure-similar protein, among which all samples of swine IgM, swine IgE, bovine IgG, rabbit IgG, chicken IgG, chicken IgM, duck IgG, mouse IgG, chicken IgY, and human IgG were diluted to high concentrations, 320 ng / mL, 160 ng / mL, 80 ng / mL, and 40 ng / mL, respectively. After sealing the plate, incubate in a room temperature incubator for 2 hours, wash the plate 4 times and spin dry. Add 100 μL of enzyme-labeled antibody HRP-4H1 working solution to the reaction well, incubate in a room temperature incubator for 45 minutes after sealing the plate, wash the plate 4 times and spin dry. Add 100 μl of chromogenic substrate TMB to the reaction well, seal the plate and color develop at room temperature in the dark for 15 min, add 50 μl of stop solution 2M sulfuric acid solution, and immediately measure the OD value at 450 nm wavelength using an ELISA reader (within 5 minutes). The results showed that the antibody did not react with other similar proteins (Table 3).

[0083] Table 3

[0084]

[0085] 5.2 Cross-detection with IgG in meat from other species

[0086] 30 minutes before the experiment, take out the ELISA plate coated with anti-swine IgG antibody 1, return to room temperature, wash the plate 3 times and spin dry. Add 100 μL of the same dilution multiple of the sample, and all the samples of pork, beef, mutton, chicken, duck and goose are homogenized, and the dilution multiple is 1:100. After sealing the plate, incubate in a room temperature incubator for 2 hours, wash the plate 4 times and spin dry. Add 100 μL of enzyme-labeled antibody HRP-4H1 working solution to the reaction well, incubate in a room temperature incubator for 45 minutes after sealing the plate, wash the plate 4 times and spin dry. Add 100 μl of chromogenic substrate TMB to the reaction well, color at room temperature and avoid light for 15 minutes after sealing the plate, add 50 μl of stop solution 2M sulfuric acid solution, and immediately measure the OD value (within 5 minutes) at a wavelength of 450nm using an ELISA reader. The results showed that the antibody did not react with other meats. According to the standard curve, the IgG content in pork homogenate was calculated to be 13.66935 μg / mL (Table 4).

[0087] Table 4

[0088]

[0089]

[0090] 6. Double antibody sandwich ELISA stability test

[0091] 6.1 37℃ Accelerated Stability Test

[0092] The monoclonal antibody 9G5-coated ELISA plate, HRP-labeled monoclonal antibody 4H1, and standard pig IgG were placed at 37°C for accelerated stability testing and placed for 15 days (about 24 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 spin dry. Add 100μL of pig IgG standard with different dilution concentrations, the pig IgG dilution concentrations are 400ng / mL, 200ng / mL, 100ng / mL, 50ng / mL, 25ng / mL, 12.5ng / mL, 6.25ng / 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 spin dry. Add 100μL of enzyme-labeled antibody HRP-4H1 working solution to the reaction well, incubate in a room temperature incubator for 45min after sealing the plate, wash the plate 4 times and spin dry. Add 100 μL of chromogenic substrate TMB to the reaction well, seal the plate and color for 15 minutes at room temperature in the dark, add 50 μL of stop solution 2M sulfuric acid solution, and immediately measure the OD value at 450nm wavelength with an ELISA reader (within 5 minutes). Use different concentrations of pig IgG standards as the horizontal axis and the corresponding OD value as the vertical axis to draw a standard curve and establish a regression equation. The results show that the kit has good stability (Table 5).

[0093] Table 5

[0094] 37℃ test time / day 0 3 7 11 15 400ng / ml 2.733 2.542 2.292 2.228 2.502 200ng / ml 1.66 1.508 1.278 1.253 1.487 100ng / ml 0.961 0.791 0.729 0.697 0.803 50ng / ml 0.471 0.444 0.389 0.375 0.455 25ng / ml 0.275 0.236 0.214 0.21 0.244 12.5ng / ml 0.149 0.131 0.118 0.125 0.134 6.25ng / ml 0.092 0.077 0.072 0.078 0.077 0 0.029 0.024 0.017 0.019 0.018

[0095] 6.2 4℃ stability test

[0096] The ELISA plate coated with monoclonal antibody 9G5, HRP-labeled monoclonal antibody 4H1, and standard pig IgG were placed at 4°C for stability test, and taken out at 0 days, 6 months, 12 months, 18 months, and 24 months, and then taken out for testing. The detection method is to take out the ELISA plate, wash the plate 3 times and spin dry, add 100μL of pig IgG standard with different dilution concentrations, and the pig IgG dilution concentration is 400ng / mL, 200ng / mL, 100ng / mL, 50ng / mL, 25ng / mL, 12.5ng / mL, 6.25ng / mL and set a blank control. After sealing the plate, incubate in a room temperature incubator for 2h, wash the plate 4 times and spin dry. Add 100μL of enzyme-labeled antibody HRP-4H1 working solution to the reaction well, seal the plate, incubate in a room temperature incubator for 45min, wash the plate 4 times and spin dry. Add 100 μL of chromogenic substrate TMB to the reaction wells, seal the plate and color for 15 minutes at room temperature in the dark, add 50 μL of stop solution 2M sulfuric acid solution, and immediately measure the OD value at 450nm wavelength with an ELISA reader (within 5 minutes). Use different concentrations of pig IgG standards as the horizontal axis and the corresponding OD values ​​as the vertical axis to draw a standard curve and establish a regression equation. The results show that the stability of the kit remains good after long-term storage at 4°C (Table 6).

[0097] Table 6

[0098] Pig IgG, ng / mL Day 0 June December 18 months 24 months 400 2.406 2.359 2.432 2.438 2.431 200 1.374 1.325 1.416 1.433 1.429 100 0.791 0.778 0.807 0.818 0.83 50 0.424 0.413 0.439 0.463 0.449 25 0.247 0.244 0.249 0.261 0.258 12.5 0.244 0.128 0.137 0.141 0.132 6.25 0.087 0.085 0.089 0.09 0.075 0 0.021 0.021 0.023 0.026 0.021

[0099] 7. Spike recovery rate

[0100] High, medium and low concentrations of pig IgG were added to the selected healthy pig plasma. If the content in the sample is high, it can be used for appropriate dilution. Table 7 shows the recovery rate of pig IgG spike. The test results show that the recovery rate of spike is within the normal range of 70%-130%.

[0101] Table 7

[0102]

[0103] 8. Linear Dilution

[0104] The known high concentration of pig IgG was added to the selected 4 healthy pig plasmas, and the dilutions were 1:2, 1:4, 1:8, and 1:16, and the recovery experiments were performed to evaluate the linearity. The linear recovery rate of pig IgG is shown in Table 8.

[0105] Table 8

[0106]

[0107]

[0108] Comparison of the test results of Example 3 with other foreign brands of the same type of kit

[0109] We selected a foreign Abnova (ab291065) brand for analogy testing and conducted the experiment in the most appropriate way for each brand (following the instructions in full). We compared the performance of the kits in the following two aspects.

[0110] a. Comparison of standard curve: The results show that the fitting curve of this kit (R 2 =0.99996) is better than the comparison brand kit (R 2 =0.99757), and the background performance of this kit was excellent (zero well value was 0.02), which was lower than that of its brand kit (zero well value was 0.043) (Table 9).

[0111] Table 9

[0112]

[0113] b. Comparison of sample test results: 30 pig serum samples were randomly selected for simultaneous testing (unit: mg / mL). According to literature reports, the IgG content in normal pig serum ranges from 3 to 20 mg / mL (depending on age and individual content). The test results show that the content of this kit is consistent with that of foreign brands, ranging from 3 to 20 mg / mL (Table 10).

[0114] Table 10

[0115]

[0116]

[0117] Example 4 Determination of pig serum IgG content

[0118] 30 minutes before the experiment, take out the ELISA plate coated with monoclonal antibody 9G5, return to room temperature, wash the plate 3 times and spin dry. Select the slaughterhouse to purchase 4 serums (numbered 1, 2, 3, and 4), add 100 μL of pig serum samples / standards with different dilutions, the sample dilution multiples are 50,000 times and 100,000 times, the dilution concentrations of pig IgG standards are 400 ng / mL, 200 ng / mL, 100 ng / mL, 50 ng / mL, 25 ng / mL, 12.5 ng / mL, 6.25 ng / mL and set up blank controls. After sealing the plate, incubate in a room temperature incubator for 2 hours, wash the plate 4 times and spin dry. Add 100 μL of enzyme-labeled antibody HRP-4H1 working solution to the reaction well, incubate in a room temperature incubator for 45 minutes after sealing the plate, wash the plate 4 times and spin dry. Add 100 μL of chromogenic substrate TMB to the reaction well, seal the plate and color at room temperature in the dark for 15 minutes, add 50 μL of stop solution 2M sulfuric acid solution, and immediately measure the OD value at 450nm wavelength with an enzyme marker (within 5 minutes). The test results show that this method can accurately determine the content of porcine serum IgG, and has a good linear relationship within a certain range (Table 11).

[0119] Table 11

[0120]

[0121] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. An anti-swine IgG 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 antibody or its antigen-binding fragment are GFTFSNHP, ISTGGSYT, and SRHGYDPERSWYFDV, respectively, and the amino acid sequences of the complementary determining regions CDR1, CDR2, and CDR3 of the light chain variable region are SSVSY, STS, and QQRSRYPYT, respectively.

2. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The amino acid sequence of the light chain variable region of the antibody or antigen-binding fragment thereof has at least 80% similarity to the sequence shown in SEQ ID NO:3, and the amino acid sequence of the heavy chain variable region has at least 80% similarity to the sequence shown in SEQ ID NO:

4.

3. The antibody or antigen-binding fragment thereof according to claim 2, characterized in that: The amino acid sequence of the light chain variable region of the antibody or antigen-binding fragment thereof is shown in SEQ ID NO:3, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:

4.

4. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, characterized in that: The antibody or antigen-binding fragment thereof is a monoclonal antibody, Fab, Fab', F(ab')2, Fv or a single-chain antibody.

5. A nucleic acid molecule encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4.

6. A biological material containing the nucleic acid molecule according to claim 5, characterized in that: The biological material is recombinant DNA, expression cassette, transposon, plasmid vector, virus vector, engineered bacteria or host cell.

7. An antibody conjugate, characterized in that: The antibody conjugate is obtained by conjugating the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4 with a label, and the label is selected from one or more of enzyme labeling, biotin labeling, fluorescent dye labeling, chemiluminescent dye labeling, and radioactive labeling.

8. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, the nucleic acid molecule according to claim 5, the biomaterial according to claim 6, or the antibody conjugate according to claim 7 in the preparation of a product for qualitative or quantitative detection of porcine IgG.

9. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4 or the antibody conjugate according to claim 7 in the qualitative or quantitative detection of porcine IgG for purposes other than disease diagnosis and treatment.

10. A pig IgG double antibody sandwich enzyme-linked immunosorbent assay kit, characterized in that: The kit comprises: the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, the anti-swine IgG antibody 9G5 or the antigen-binding fragment thereof, and at least one of a substrate color developing solution, a blocking solution, and a stopping solution; The amino acid sequences of the complementary determining regions CDR1, CDR2, and CDR3 of the heavy chain variable region of the antibody 9G5 or its antigen-binding fragment are GYTFTSYV, INPYNDGT, and ARYDGYGFPY, respectively, and the amino acid sequences of the complementary determining regions CDR1, CDR2, and CDR3 of the light chain variable region are QDVGTT, RAS, and QQYTRYPYT, respectively.

11. The kit according to claim 10, characterized in that The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4 is used as the first antibody, and the anti-swine IgG antibody 9G5 or the antigen-binding fragment thereof is used as the second antibody; or The anti-swine IgG antibody 9G5 or an antigen-binding fragment thereof is used as the first antibody, and the antibody or an antigen-binding fragment thereof according to any one of claims 1 to 4 is used as the second antibody.

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