CN118684769A

CN118684769A - Anti-pig IgG antibody, antibody composition and application thereof

Info

Publication number: CN118684769A
Application number: CN202411189023.1A
Authority: CN
Inventors: 苑晓松; 陈新新; 潘悦; 王河川; 路轲; 王芳; 马玉岭; 王永明; 石元朔
Original assignee: Shanghai Solaibao Biochemical Technology Co ltd; Beijing Solarbio Technology Co ltd
Current assignee: Shanghai Solaibao Biochemical Technology Co ltd; Beijing Solarbio Technology Co ltd
Priority date: 2024-08-28
Filing date: 2024-08-28
Publication date: 2024-09-24

Abstract

The invention belongs to the field of immunological detection, and provides an anti-pig IgG antibody, an antibody composition and application thereof. The antibody of the pig IgG provided by the invention can be specifically combined with the pig IgG, has no cross reaction with other similar proteins, has higher affinity with the pig IgG, and has higher stability; the detection of the pig IgG by the double-antibody sandwich ELISA method based on the antibody has higher sensitivity, specificity and accuracy, can realize accurate and high-flux detection of the IgG content in pig serum or other serum analogues or products containing the pig IgG, and has better application prospect in pig IgG detection.

Description

Anti-pig IgG antibody, antibody composition and application thereof

Technical Field

The invention belongs to the field of immunological detection, and particularly relates to an anti-pig IgG antibody, an antibody composition and application thereof.

Background

In recent years, along with the epidemic of African swine fever and new infectious diseases of pigs, serious losses are caused to the pig raising industry in China, so that the rapid and accurate detection of sick pigs is particularly important. IgG is the highest concentration immunoglobulin in animal serum, and accounts for 70% -80% of the content of the immunoglobulin, and plays a main role in antibody-mediated reaction, and meanwhile, the level of IgG is also a common index for clinical immunoassay and humoral immunity research, so that the pig immune response to pathogen infection is comprehensively and systematically known, and the important significance is achieved by adopting corresponding prevention measures. Meanwhile, the integral content of the immunoglobulin can also indirectly reflect the intensity of the immunity of the organism. The content of IgG in the group detection blood can be used for evaluating the health condition of the group, and the effect of feeds or nutritional additives with different formulas can be evaluated in an auxiliary way.

Compared with polyclonal antibody, monoclonal antibody of anti-pig IgG has the advantages of high specificity, stable titer and the like, but similar imported reagents have the problems of high price, low titer of domestic reagents, large inter-batch difference and insufficient sensitivity.

Disclosure of Invention

The invention aims to provide an anti-pig IgG antibody, an antibody composition and application thereof.

According to the principle of construction of the hybridoma cells of Kohler and Milstrin, the invention utilizes the PEG fusion technology to establish the hybridoma cell strain secreting the anti-pig IgG monoclonal antibody, and aims to provide high-quality and standardized reagent for detection of pig IgG.

To achieve the object of the present invention, in a first aspect, the present invention provides an anti-pig IgG antibody (monoclonal antibody 9G 5) or an antigen-binding fragment thereof, wherein the amino acid sequences of the complementarity determining regions CDR1, CDR2, CDR3 of the heavy chain variable region of the antibody or antigen-binding fragment thereof are GYTFTSYV, INPYNDGT, ARYDGYGFPY (SEQ ID NO: 9-11), and the amino acid sequences of the complementarity determining regions CDR1, CDR2, CDR3 of the light chain variable region are QDVGTT (SEQ ID NO: 12), RAS, QQYTRYPYT (SEQ ID NO: 13), respectively.

Further, the amino acid sequence of the light chain variable region of the antibody or antigen binding fragment thereof is shown as SEQ ID NO. 7 or has at least 80%, 85%, 90%, 95% or 98% similarity to the sequence shown as SEQ ID NO. 7, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 8 or has at least 80%, 85%, 90%, 95% or 98% similarity to the sequence shown as SEQ ID NO. 8.

In the present invention, the antibody or antigen binding fragment thereof includes, but is not limited to, monoclonal antibodies, fab ', F (ab') 2, fv, or single chain antibodies.

In a second aspect, the invention provides nucleic acid molecules encoding said antibodies or antigen binding fragments thereof.

In a third aspect, the invention provides biological materials comprising the nucleic acid molecules, including but not limited to recombinant DNA, expression cassettes, transposons, plasmid vectors, viral vectors, engineering bacteria or host cells.

In a fourth aspect, the present invention provides an antibody conjugate obtained by coupling the antibody or an antigen binding fragment thereof to a label selected from one or more of an enzyme label, a biotin label, a fluorescent dye label, a chemiluminescent dye label, a radiolabel, and the like.

In a fifth aspect, the invention provides an anti-pig IgG antibody composition comprising the antibody 9G5 or antigen-binding fragment thereof and anti-pig IgG antibody 4H1 or antigen-binding fragment thereof.

The amino acid sequences of the complementarity determining regions CDR1, CDR2 and CDR3 of the heavy chain variable region of the antibody 4H1 or antigen binding fragment thereof are GFTFSNHP, ISTGGSYT, SRHGYDPERSWYFDV (SEQ ID NOS: 14-16), respectively, and the amino acid sequences of the complementarity determining regions CDR1, CDR2 and CDR3 of the light chain variable region are SSVSY (SEQ ID NO: 17) and STS, QQRSRYPYT (SEQ ID NO: 18), respectively.

In a sixth aspect, the invention provides the use of said antibody or antigen binding fragment thereof or said nucleic acid molecule or said biological material or said antibody conjugate or said antibody composition for the preparation of a product for the qualitative or quantitative detection of pig IgG.

In a seventh aspect, the invention provides any one of the following uses of the antibody or antigen binding fragment thereof or the antibody conjugate or the antibody composition:

(1) For qualitative or quantitative detection of pig IgG (including for non-disease diagnosis and therapeutic purposes);

(2) The method is used for detecting the immunogenicity or immune effect of the vaccine;

(3) Is used for quality control of pig IgG-containing products.

In an eighth aspect, the invention provides a pig IgG double-antibody sandwich enzyme-linked immunosorbent assay kit, wherein the antibody 9G5 or an antigen-binding fragment thereof is used as a first antibody, and the anti-pig IgG antibody 4H1 or an antigen-binding fragment thereof is used as a second antibody; or alternatively

The anti-pig IgG antibody 4H1 or antigen-binding fragment thereof is used as a first antibody, and the antibody 9G5 or antigen-binding fragment thereof is used as a second antibody.

In one embodiment of the present invention, the antibody 9G5 is immobilized on a solid support, which may be selected from the group consisting of an elisa plate, a microsphere, a nitrocellulose membrane, a glass cellulose membrane, a nylon membrane, and the like, preferably an elisa plate.

The antibody 4H1 is labeled with an enzyme, which may be selected from any one of the following: horseradish peroxidase (HRP), alkaline Phosphatase (AP), glucose oxidase, β -galactosidase, lysozyme, malate dehydrogenase, and the like, with horseradish peroxidase being preferred.

Further, the kit also comprises at least one of a substrate chromogenic solution, a blocking solution, a stopping solution and the like.

Common substrates include, but are not limited to, O-phenylenediamine (O-PHENYLENEDIAMINE, OPD), tetramethylbenzidine (3, 3', 5' -tetramethylbenzidine, TMB), and ABTS [2,2' -azino-di- (3-ethylbenziazobine sulfonate-6) ] and the like, with TMB being preferred.

The blocking solution is, for example, a 2% BSA solution, and the stop solution is, for example, a 1.5-2.5M (preferably 2M) sulfuric acid solution.

The principle of detecting the pig IgG content by adopting an ELISA detection technology based on a double-antibody sandwich method is as follows: coating an anti-pig IgG monoclonal antibody 1 on an ELISA plate; respectively adding a gradient diluted standard substance and a pre-diluted sample, wherein pig IgG in the standard substance and the sample can be fully combined with a coating antibody on an ELISA plate; after washing the plate, adding HRP-labeled anti-pig IgG antibody 2, wherein the antibody can specifically bind with pig IgG in a standard product and a sample captured by the coated antibody on the plate; after washing the plate, adding a chromogenic agent substrate TMB, if pig IgG with different concentrations exists in the sample in the reaction hole, the HRP can change colorless TMB into blue substances with different depths (positive correlation), and after adding a stopping solution, the reaction hole can change into yellow; finally, the absorbance (OD _450nm) of the reaction well sample was measured at λmax=450 nm, the concentration of pig IgG in the sample was proportional to OD, and the concentration of pig IgG in the sample was calculated from the standard curve. The method uses an enzyme chromogenic amplifying system, has higher detection sensitivity, can detect samples with lower content, and has a detection range of 0.4-25ng/mL.

By means of the technical scheme, the invention has at least the following advantages and beneficial effects:

the antibody of the pig IgG provided by the invention can be specifically combined with the pig IgG, has no cross reaction with other similar proteins, has higher affinity with the pig IgG, and has higher stability; the detection of the pig IgG by the double-antibody sandwich ELISA method based on the antibody has higher sensitivity, specificity and accuracy, can realize accurate and high-flux detection of the IgG content in pig serum or other serum analogues or products containing the pig IgG, and has better application prospect in pig IgG detection.

Drawings

FIG. 1 is a diagram showing the result of SDS-PAGE electrophoresis of monoclonal antibodies 4H1 and 9G5 purified according to the preferred embodiment of the present invention; wherein M is a protein molecular weight marker, and the unit is kDa.

FIG. 2 is a standard curve established by the detection of pig IgG by the double-antibody sandwich ELISA method in the preferred embodiment of the invention.

Detailed Description

The invention aims to provide a kit capable of accurately, rapidly and massively detecting the content of pig IgG in a sample.

The invention adopts the following technical scheme:

the invention provides an ELISA plate for coating an anti-pig IgG monoclonal antibody 9G 5; the light and heavy chain variable region DNA sequences of antibody 9G5 were: SEQ ID NO. 5 and SEQ ID NO. 6. The amino acid sequences of the light chain and heavy chain variable regions are respectively as follows: SEQ ID NO. 7 and SEQ ID NO. 8.

The invention provides an anti-pig IgG monoclonal antibody 4H1 marked by HRP; the light chain and heavy chain variable region DNA sequences are respectively as follows: SEQ ID NO. 1 and SEQ ID NO. 2. The amino acid sequences of the light chain and heavy chain variable regions are respectively as follows: SEQ ID NO. 3 and SEQ ID NO. 4.

The invention also provides a kit for detecting the pig IgG, wherein a pig IgG detection reagent is arranged in the kit. Preferably, the kit comprises an ELISA plate coated with a monoclonal antibody of anti-pig IgG, namely pig IgG-9G5, an HRP-labeled monoclonal antibody of anti-pig IgG, namely pig IgG-4H1 solution, an HRP chromogenic substrate and a standard substance.

The invention also provides a using method of the kit for detecting the pig IgG, a sample to be detected containing the pig IgG is added to an ELISA plate coated with the monoclonal antibody pig IgG-9G5 of the anti-pig IgG, an HRP-labeled monoclonal antibody pig IgG-4H1 solution of the anti-pig IgG is added for reaction, then an HRP chromogenic substrate is added, and the content of the pig IgG in the sample to be detected is calculated according to a chromogenic OD value.

The following examples are illustrative of the invention and are not intended to limit the scope of the invention. Unless otherwise indicated, the technical means used in the examples are conventional means well known to those skilled in the art, and all raw materials used are commercially available.

EXAMPLE 1 preparation of anti-porcine IgG monoclonal antibodies

1. Immunization of animals

Female Balb/c mice with the age of 6-8 weeks are selected, and are immunized after being emulsified by using a natural extracted pig IgG antigen and an equal volume of Freund's adjuvant, wherein the immunization period is two weeks, blood is taken for measuring titer after 3 times of immunization, and the immunization is boosted again three days before fusion.

2. Cell fusion

Mice were sacrificed by cervical scission, spleens were removed by aseptic manipulation, and spleen cell suspensions were prepared by squeeze milling in a plate. The prepared syngeneic myeloma cells and the spleen cells of the mice are mixed according to a certain proportion, and a fusogenic agent polyethylene glycol (PEG) is added. Under the action of polyethylene glycol, various lymphocytes can be fused with myeloma cells to form hybridoma cells. The specific operation is as follows:

myeloma (SP 2/0) cell activation:

thawing and resuscitating commercial SP2/0 cells, suspending in nutrient solution (RPMI-1640, adding 10% calf serum), and culturing in incubator at 37deg.C under 5% CO ₂; passaging is performed 3-5 days later, and the culture medium is replaced one day before the fusion. The culture medium in the culture dish is completely discarded on the same day as the fusion day, the culture medium is replaced by a serum-free culture medium, the cells are blown down, then the cells are placed in a 50mL centrifuge tube, and the serum-free culture medium 1640 to 50mL are added for counting and then are centrifuged for standby.

Preparation of immune spleen cells:

One of the Balb/c mice with enhanced immunity is taken, the orbit is bloodletting and sacrificed (serum is collected, namely positive serum), soaked in 75% alcohol for 5-10min for sterilization, then the mice are fixed on an dissecting plate for dissection, spleen is taken out and sheared, the mice are placed in a sterilized screen for grinding, the ground spleen cells are filtered once by the screen, counted after being added with 1640-50 mL of serum-free culture medium, and the mice are centrifuged for standby.

Preparation of feeder cells:

One non-immunized Balb/c mouse was taken, the orbit was exsanguinated, and serum was collected as negative serum. The mouse was intraperitoneally injected with HAT-configured medium 1640 ml, and after blowing, aspirated into another centrifuge tube for further use, the solution contained macrophages in the abdominal cavity.

Fusion:

1X 10 ⁷-2×10⁷ SP2/0 and 10 ⁸ immunocytes were mixed in a 50mL centrifuge tube, centrifuged at 1000r/min for 8min. After discarding the supernatant, the centrifuge tube containing the cell mixture was placed in a 37℃water bath, followed by adding 1mL (sigma) of 50% PEG pre-warmed to 37℃and allowed to stand for 30s after stirring. After standing, a serum-free medium 1640 was added at 37℃pre-warmed. Centrifuging at 1000r/min for 5min, discarding supernatant, and standing at 37deg.C for 5-8min. Subsequently mixed with feeder cell suspension, seeded in 96-well plates at 200. Mu.L/well and incubated in a 5% CO ₂ incubator at 37 ℃. The HAT medium was changed to continue culturing on day 4 after the fusion. And (4) when the colony of the fused cells grows to 1/4 of the culture hole and the culture medium turns yellow slightly, detecting the antibody.

3. Selection of hybridoma positive clones and cloning of cells

The purpose of the selective culture is to screen the fused hybridoma cells using HAT selective medium. In HAT medium, unfused myeloma cells lack hypoxanthine-guanine-phosphoribosyl transferase and cannot synthesize DNA by salvage pathways to die. Unfused spleen cells have hypoxanthine-guanine-phosphoribosyl transferase, but do not survive in vitro for long periods and die. Only fused hybridoma cells survive and proliferate in HAT medium due to the hypoxanthine guanine phosphoribosyl transferase obtained from spleen cells and the unlimited proliferation of myeloma cells. The specific operation is as follows:

positive hybridoma cells were screened by indirect ELISA as follows:

coating antigen: diluting the antigen to 1-10 mug/mL with coating buffer; adding 100 μl of the solution into each of the microwells, shaking gently, and cooling overnight at 4deg.C or 37 deg.C for 2 hr; throwing away the liquid in the hole (taking the liquid in the hole as dry as possible); washing for 2-3 min for 3 times.

Blocking the positions of the enzyme-labeled wells not coated with antigen: 200. Mu.L of blocking solution (5% skimmed milk powder or 2% BSA) was added to each well, gently shaken well, and allowed to stand at 37℃for 1h; throwing away the liquid in the hole; washing buffer solution by Kong Jiaman times, standing for 2-3 min, throwing away liquid in the hole, beating to dry, and washing 3 times by using the washing buffer solution. And (3) sample adding, namely taking 50 mu L of supernatant from each hole of the hybridoma to be detected, sequentially adding the supernatant into the enzyme-labeled holes, and gently shaking the mixture. Washing at 37 ℃ for 1h, and drying.

Adding enzyme-labeled anti-antibody: diluting the enzyme-labeled secondary antibody to a proper working concentration according to instructions by using a diluent, adding 100 mu L of the enzyme-labeled secondary antibody into each hole, gently shaking the mixture, and standing the mixture at 37 ℃ for 30min; then washing and beating to dry. Adding a color development liquid: each well was added with 100. Mu.L of freshly prepared color development solution, gently shaken well, at 37℃for 10min. Terminating the reaction: 50. Mu.L of stop solution was added to each well.

Determination result: the enzyme label instrument OD ₄₅₀ is used for reading, and the detection result is 3 times larger than that of the negative hole, so that the detection result can be judged positive.

Cloning of hybridoma cells (limiting dilution method):

preparing a mouse feeder cell layer before cloning; gently blowing the hybridoma cells to be cloned from the culture well, and counting the number of living cells by using a blood cell counting plate; diluting cells to 5, 10, 30 cells/mL with complete medium;

the three concentrations of cell suspension were added to the prepared 96-well culture plates of feeder cells at 100. Mu.L/well, so that each well contained 0.5, 1 and 3 cells, respectively. Culturing until 4 days of fluid infusion is one drop, carefully observing the growth condition of cells in each hole on 5-6 days, and recording;

Detecting the specific antibody, namely detecting when the cell clone grows 1/3-1/2 field of view on the 7 th to 9 th days after cloning; after cloning by the same method for three times, the cells which are positive by selecting single-hole single-group for the last time can be transferred to a 24-hole culture plate, and when the cells in the 24-hole plate grow well, the mice can be inoculated in the abdominal cavity to collect ascites.

4. Monoclonal antibodies 4H1 and 9G5 variable region sequencing

The number of hybridoma cells collected was greater than 10 ⁶: extracting total RNA of two cell lines by using a Beijing Soxhlet technology limited company total RNA extraction kit according to the operation of a specification; the first strand of cDNA is synthesized and specifically amplified by using a reverse transcription kit of Beijing Soxhlet technology Co., ltd., and the amplified DNA is constructed on a T vector and sent to a Optimago for sequencing. Obtaining a gene sequencing result: the variable region sequence of the 4H1 cell strain light chain is 315bp in length, 107 amino acids are encoded, the DNA sequence is shown as SEQ ID NO. 1, and the protein sequence is shown as SEQ ID NO. 3; the heavy chain variable region has a sequence length of 366bp, codes 122 amino acids, the DNA sequence is shown as SEQ ID NO. 2, and the protein sequence is shown as SEQ ID NO. 4. The variable region sequence of the 9G5 cell strain light chain is 324bp in length, 108 amino acids are encoded, the DNA sequence is shown as SEQ ID NO. 5, and the protein sequence is shown as SEQ ID NO. 7; the heavy chain variable region has a length of 351bp, codes 117 amino acids, the DNA sequence is shown as SEQ ID NO. 6, and the protein sequence is shown as SEQ ID NO. 8.

5. Mass production of monoclonal antibodies 4H1 and 9G5

The hybridoma cells after the strain establishment were injected into the abdominal cavity of the mice, and ascites were collected for about 7 days, and the antibody was purified by Protein G affinity chromatography (FIG. 1).

Example 2 construction of double antibody sandwich ELISA kit for detecting pig IgG content

1. Antibody affinity constant detection

The ELISA plate was prepared according to the method of (indirect ELISA screening positive hybridoma cells), and the purified antibody was diluted to the same concentration, 100. Mu.L/well, and incubated at 37℃for 1 hour. After PBST washing the plates, eluting with sodium thiocyanate with different concentrations, sequentially adding 0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5 and 5.0mol/L of solution 60 mu L/hole, standing at room temperature for 15min, washing the plates by PBST, adding secondary antibodies (goat anti-mouse IgG-HRP, 1:5000), and performing color development detection. And (3) result judgment: the relative affinity constant of the antibody was determined as the concentration of sodium thiocyanate corresponding to a decrease in OD ₄₅₀ after elution to 50% without elution, expressed in mol/L (Table 1).

TABLE 1

2. HRP-tag of monoclonal antibody derived from cell line 4H1 (anti-pig IgG antibody 2)

Anti-pig IgG antibody 2 was added to the dialysis bag and dialyzed overnight at 4 ℃ in 0.01M CB buffer. 10mg of HRP was dissolved in 2mL of water. Fresh NaIO ₄ solution (0.1 mol/L) was prepared, 0.4mL of 0.1mol/L NaIO ₄ was added to 2mL of HRP solution, mixed well, and immersed in 10mM NaAc buffer solution at 4℃overnight for dialysis at room temperature in the absence of light for 45 min. The overnight dialyzed antibody and HRP solution were removed and 0.1mL of 0.2mol/L carbonate buffer pH 9.5 was added. The antibody that had been removed was then immediately added to the HRP solution and gently stirred at room temperature for 2h in the dark. 0.04g of NaBH ₄ is weighed and dissolved in 10mL of water, 0.4mL is added to the reaction solution, and the mixture is placed at 4 ℃ for 2 hours. Taking out, placing in a dialysis bag, dialyzing in 0.01mol/L PBS, changing the solution once after two hours, and dialyzing at 4 ℃ overnight. Taking out the overnight dialyzed labeling solution, adding equal volume of glycerol, and storing at-20deg.C.

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

Anti-porcine IgG antibody 1 was diluted to 2 μg/mL with 0.05M pH9.6 carbonate coating buffer. 100 μl was added to 96-well polystyrene ELISA plate wells at 4deg.C overnight. The next day, the solution in the wells was discarded, and washed 3 times with wash buffer for 3min each. After the above procedure, each well of the reaction plate was blocked with 250. Mu.L per well with 2% BSA solution for 2h at room temperature. The solution in the hole is discarded, and the drying room is dried and vacuumized and stored at 4 ℃.

4. Establishment of double-antibody sandwich ELISA method and determination of sensitivity

And taking out the ELISA plate coated with the anti-pig IgG antibody 1 30min before the experiment, recovering to room temperature, washing the plate for 3 times, and spin-drying. 100. Mu.L of pig IgG standard with different dilution concentration was added, pig IgG dilution concentration was 400ng/mL, 200ng/mL, 100ng/mL, 50ng/mL, 25ng/mL, 12.5ng/mL, 6.25ng/mL and blank control was added, while 100. Mu.L of blank dilution (PBST solution, 0.1mol/L phosphate buffer containing 0.05% Tween 20) was added to 20 blank wells. And (3) incubating the plates for 2 hours in a room temperature incubator, washing the plates for 4 times and spin-drying. Adding 100 mu L enzyme-labeled antibody HRP-4H1 working solution into the reaction holes, sealing the plates, incubating for 30min in a room temperature incubator, washing the plates for 4 times and spin-drying. 100 μl of chromogenic substrate TMB was added to the wells, the plates were sealed and developed at room temperature in the absence of light for 15min, 50 μl of stop solution 2M sulfuric acid solution was added, and the OD was measured immediately (within 5 min) at 450nm using an ELISA reader. And drawing a standard curve by taking pig IgG standards with different concentrations as an abscissa and corresponding OD values as an ordinate, and establishing a regression equation. The results showed that the detection range was 0.4-25ng/mL and R ² was 0.9998 (FIG. 2). The average value of the concentration OD of 20 zero standard substances is detected, the two standard deviations are added, and the corresponding detectable concentration is calculated, so that the result shows that the detection sensitivity of the double-antibody sandwich ELISA method is 1.373586ng/ml (table 2).

TABLE 2

5. Double-antibody sandwich ELISA specific detection

5.1 Cross detection with other species standards

And taking out the ELISA plate coated with the anti-pig IgG antibody 130 min before the experiment, recovering to room temperature, washing the plate for 3 times, and spin-drying. 100. Mu.L of a standard of pig IgG (from Solarbio under the trade name SP 037) or a high concentration of a structurally similar protein of pig IgG was added, wherein all samples of pig IgM, pig IgE, bovine IgG, rabbit IgG, chicken IgM, duck IgG, mouse IgG, chicken IgY, human IgG were diluted at high concentrations of 320ng/mL, 160ng/mL, 80ng/mL, 40ng/mL, respectively. And (3) incubating the plates for 2 hours in a room temperature incubator, washing the plates for 4 times and spin-drying. Adding 100 mu L enzyme-labeled antibody HRP-4H1 working solution into the reaction holes, sealing the plates, incubating for 45min in a room temperature incubator, washing the plates for 4 times and spin-drying. 100 μl of chromogenic substrate TMB was added to the wells, the plates were sealed and developed at room temperature in the absence of light for 15min, 50 μl of stop solution 2M sulfuric acid solution was added, and the OD was measured immediately (within 5 min) at 450nm using an ELISA reader. The results showed that the antibody pair did not react with other similar proteins (table 3).

TABLE 3 Table 3

5.2 Cross detection with IgG in meats of other species

And taking out the ELISA plate coated with the anti-pig IgG antibody 130 min before the experiment, recovering to room temperature, washing the plate for 3 times, and spin-drying. And adding 100 mu L of samples with the same dilution ratio, homogenizing all the samples of pork, beef, mutton, chicken, duck and goose, wherein the dilution ratio is 1:100. And (3) incubating the plates for 2 hours in a room temperature incubator, washing the plates for 4 times and spin-drying. Adding 100 mu L enzyme-labeled antibody HRP-4H1 working solution into the reaction holes, sealing the plates, incubating for 45min in a room temperature incubator, washing the plates for 4 times and spin-drying. 100 μl of chromogenic substrate TMB was added to the wells, the plates were sealed and developed at room temperature in the absence of light for 15min, 50 μl of stop solution 2M sulfuric acid solution was added, and the OD was measured immediately (within 5 min) at 450nm using an ELISA reader. The results showed that the antibody pair did not react with other meats and that the IgG content of the pork homogenate was 13.66935. Mu.g/mL calculated from the standard curve (Table 4).

TABLE 4 Table 4

6. Double-antibody sandwich ELISA stability detection

6.1 Accelerated stability test at 37 ℃

The ELISA plate coated with the monoclonal antibody 9G5, the HRP-labeled monoclonal antibody 4H1 and the standard pig IgG were subjected to an accelerated stability test at 37℃for 15 days (about 24 months at 4 ℃). And then taking out for detection, wherein the detection method is that the ELISA plate is taken out for washing the plate for 3 times and spin-drying. Pig IgG standards were added at 100. Mu.L of varying dilution concentration, with pig IgG dilution concentrations of 400ng/mL, 200ng/mL, 100ng/mL, 50ng/mL, 25ng/mL, 12.5ng/mL, 6.25ng/mL and blank. And (3) incubating the plates for 2 hours in a room temperature incubator, washing the plates for 4 times and spin-drying. Adding 100 mu L enzyme-labeled antibody HRP-4H1 working solution into the reaction holes, sealing the plates, incubating for 45min in a room temperature incubator, washing the plates for 4 times and spin-drying. 100. Mu.L of chromogenic substrate TMB was added to the wells, the plates were sealed and developed at room temperature in the absence of light for 15min, 50. Mu.L of stop solution 2M sulfuric acid solution was added, and the OD value was measured immediately (within 5 min) at a wavelength of 450nm using an ELISA reader. And drawing a standard curve by taking pig IgG standards with different concentrations as an abscissa and corresponding OD values as an ordinate, and establishing a regression equation. The results showed good stability of the kit (Table 5).

TABLE 5

6.2 Stability test at 4 DEG C

The ELISA plate coated with the monoclonal antibody 9G5, the HRP-labeled monoclonal antibody 4H1 and the standard pig IgG are placed at 4 ℃ for stability experiments, taken out when being placed for 0 day, 6 months, 12 months, 18 months and 24 months respectively, and then taken out for detection. The detection method comprises the steps of taking out an ELISA plate, washing the plate for 3 times, spin-drying, adding 100 mu L of pig IgG standard substances with different dilution concentrations, wherein the dilution concentrations of pig IgG are 400ng/mL, 200ng/mL, 100ng/mL, 50ng/mL, 25ng/mL, 12.5ng/mL and 6.25ng/mL, and setting blank control. And (3) incubating the plates for 2 hours in a room temperature incubator, washing the plates for 4 times and spin-drying. Adding 100 mu L enzyme-labeled antibody HRP-4H1 working solution into the reaction holes, sealing the plates, incubating for 45min in a room temperature incubator, washing the plates for 4 times and spin-drying. 100. Mu.L of chromogenic substrate TMB was added to the wells, the plates were sealed and developed at room temperature in the absence of light for 15min, 50. Mu.L of stop solution 2M sulfuric acid solution was added, and the OD value was measured immediately (within 5 min) at a wavelength of 450nm using an ELISA reader. And drawing a standard curve by taking pig IgG standards with different concentrations as an abscissa and corresponding OD values as an ordinate, and establishing a regression equation. The results showed that the stability of the kit was still good after long-term storage at 4 ℃ (Table 6).

TABLE 6

7. Recovery rate of adding mark

Pig IgG is added into selected healthy pig plasma at different concentration levels of high, medium and low 3, and if the content of the sample is high, the pig IgG can be used for proper dilution. Table 7 shows the standard recovery rate of pig IgG, and the detection result shows that the standard recovery rate is in the normal range of 70% -130%.

TABLE 7

8. Linear dilution

Adding known high-concentration pig IgG into 4 parts of healthy pig plasma, performing 1:2,1:4,1:8 and 1:16 times dilution, performing recovery experiment, and performing linear evaluation. The linear recovery of pig IgG is shown in table 8.

TABLE 8

Example 3 comparison of test results with other brands of the same type of kit abroad

The foreign minodro (ab 291065) brand was selected for analogy testing, and experiments were performed in the manner best suited for each brand (operating fully according to its instructions). The performance of the kit was compared by the following two aspects.

A. Standard curve comparison: the results showed that the fitted curve of the kit (R ² = 0.99996) was superior to the comparative branded kit (R ² = 0.99757), and the background performance of the kit was excellent (zero pore value 0.02) and lower than the branded kit (zero pore value 0.043) (table 9).

TABLE 9

B. Sample measurement result comparison: 30 pig serum samples are randomly selected and measured simultaneously (unit mg/mL), and the content of IgG in normal pig serum ranges from 3 mg/mL to 20mg/mL (different contents according to ages and individuals) according to literature reports. The measurement result shows that the measurement content of the kit is consistent with foreign brands and ranges from 3 mg/mL to 20mg/mL (Table 10).

Table 10

Example 4 pig serum IgG content assay

And taking out the ELISA plate coated with the monoclonal antibody 9G 530 min before the experiment, recovering to room temperature, washing the plate for 3 times, and spin-drying. Slaughterhouse procurement serum was selected in 4 parts (No. 1, 2,3, 4), 100 μl of pig serum samples/standard at different dilution factors were added, the sample dilution factors were 5-fold and 10-fold, pig IgG standard dilution concentrations were 400ng/mL, 200ng/mL, 100ng/mL, 50ng/mL, 25ng/mL, 12.5ng/mL, 6.25ng/mL and blank control was set. And (3) incubating the plates for 2 hours in a room temperature incubator, washing the plates for 4 times and spin-drying. Adding 100 mu L enzyme-labeled antibody HRP-4H1 working solution into the reaction well, sealing the plate, incubating 45 min in a room temperature incubator, washing the plate for 4 times and spin-drying. 100. Mu.L of chromogenic substrate TMB was added to the wells, the plates were sealed and developed at room temperature in the absence of light 15 min. Mu.L of stop solution 2M sulfuric acid solution was added and the OD was measured immediately (within 5 minutes) at 450nm using an ELISA reader. The detection result shows that the method can accurately measure the content of pig serum IgG and has good linear relation within a certain range (Table 11).

TABLE 11

While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.

Claims

1. An anti-pig IgG antibody or antigen-binding fragment thereof, wherein the amino acid sequences of complementarity determining regions CDR1, CDR2, CDR3 of the heavy chain variable region and the amino acid sequences of complementarity determining regions CDR1, CDR2, CDR3 of the light chain variable region are GYTFTSYV, INPYNDGT, ARYDGYGFPY, respectively, and QDVGTT, RAS, QQYTRYPYT, respectively.

2. The antibody or antigen-binding fragment thereof of claim 1, wherein 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. 7 or has at least 80% similarity to the sequence as shown in SEQ ID No. 7 and the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 8 or has at least 80% similarity to the sequence as shown in SEQ ID No. 8.

3. The antibody or antigen-binding fragment thereof of claim 1 or 2, wherein the antibody or antigen-binding fragment thereof is a monoclonal antibody, fab ', F (ab') 2, fv, or single chain antibody.

4. A nucleic acid molecule encoding the antibody or antigen-binding fragment thereof of any one of claims 1-3.

5. A biological material comprising the nucleic acid molecule of claim 4, wherein the biological material is a recombinant DNA, an expression cassette, a transposon, a plasmid vector, a viral vector, an engineering bacterium or a host cell.

6. An antibody conjugate, wherein the antibody conjugate is obtained by coupling the antibody or the antigen binding fragment thereof according to any one of claims 1 to 3 with a label selected from one or more of an enzyme label, a biotin label, a fluorescent dye label, a chemiluminescent dye label, and a radiolabel.

7. An anti-pig IgG antibody composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1-3, and anti-pig IgG antibody 4H1 or antigen-binding fragment thereof;

The amino acid sequences of complementarity determining regions CDR1, CDR2 and CDR3 of the heavy chain variable region of the antibody 4H1 or antigen binding fragment thereof are GFTFSNHP, ISTGGSYT, SRHGYDPERSWYFDV respectively, and the amino acid sequences of complementarity determining regions CDR1, CDR2 and CDR3 of the light chain variable region are SSVSY, STS, QQRSRYPYT respectively.

8. Use of an antibody or antigen binding fragment thereof according to any one of claims 1-3 or a nucleic acid molecule according to claim 4 or a biological material according to claim 5 or an antibody conjugate according to claim 6 or an antibody composition according to claim 7 for the preparation of a product for qualitative or quantitative detection of pig IgG.

9. The antibody or antigen binding fragment thereof of any one of claims 1-3 or the antibody conjugate of claim 6 or any one of the following uses of the antibody composition of claim 7:

(1) Qualitative or quantitative detection of pig IgG for non-disease diagnosis and treatment purposes;

(3) Is used for quality control of pig IgG-containing products.

10. A pig IgG double antibody sandwich enzyme-linked immunosorbent assay kit, characterized in that the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3 is used as a first antibody, and the anti-pig IgG antibody 4H1 or antigen-binding fragment thereof according to claim 7 is used as a second antibody; or alternatively

An anti-pig IgG antibody 4H1 or an antigen-binding fragment thereof according to claim 7 as a first antibody and an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 3 as a second antibody.