Anti-rat IgG antibody, antibody composition and kit

Abstract

The present invention relates to the field of antibody technology, and specifically to an antibody, an antibody composition and a kit against rat IgG. The anti-rat IgG antibody provided by the present invention can specifically bind to rat IgG, but not to other similar proteins such as mouse IgG, and has a high affinity for rat IgG, and has high specificity, sensitivity and accuracy when used for rat IgG detection. The rat IgG detection kit provided by the present invention has the characteristics of high sensitivity, strong specificity, high accuracy, wide linear range, strong stability, etc., and has good application value in the detection of rat IgG content.

Description

Technical Field

[0001] The present invention relates to the field of antibody technology, in particular to an anti-rat IgG antibody, an antibody composition and a kit. Background Art

[0002] Rats are widely used as one of the most commonly used experimental animals in the evaluation of new drugs. Immunoglobulin G (IgG), as the most concentrated type of immunoglobulin in animal serum, plays a major role in antibody-mediated reactions. IgG levels are also a commonly used indicator in clinical immunoassays and humoral immune response assessments, and are of great significance for the comprehensive assessment of rats' immune response to drug effects. In addition, the level of IgG can indirectly reflect the strength of the body's immunity, and detecting the level of IgG in the blood of animal groups can be used to evaluate the health of the group.

[0003] The determination of IgG levels in rat serum has become a common indicator for clinical immunoassays and drug evaluation. Enzyme-linked immunosorbent assay (ELISA) is the simplest method for detecting rat IgG. An ideal rat IgG detection kit and method should have the following properties: high specificity, good sensitivity, good repeatability, high recovery rate, and wide linear range. However, since rat IgG and mouse IgG have a high homology, it is difficult to obtain anti-rat IgG antibodies with good specificity using mice as hosts. Therefore, it is urgent to reduce the cross-reaction of the kit for detecting rat IgG and improve its specificity. Summary of the invention

[0004] The present invention provides an anti-rat IgG antibody, an antibody composition and a kit.

[0005] Specifically, the present invention provides the technical solution described below.

[0006] In the first aspect, the present invention provides an antibody or an antigen-binding fragment thereof of rat IgG, wherein the amino acid sequences of the complementary determining regions CDR1, CDR2, and CDR3 of the heavy chain variable region of the antibody or the antigen-binding fragment thereof are shown in SEQ ID NO.6, 7, and 8, respectively, the amino acid sequence of the complementary determining region CDR1 of the light chain variable region is shown in SEQ ID NO.9, the amino acid sequence of CDR2 is SAS, and the amino acid sequence of CDR3 is shown in SEQ ID NO.10.

[0007] The rat IgG antibody or its antigen-binding fragment having the above-mentioned heavy chain CDR region and light chain CDR region sequences can specifically bind to rat IgG but not to mouse IgG, has a high affinity for rat IgG, can achieve specific, sensitive and accurate detection of rat IgG, and performs excellently as a detection reagent for rat IgG.

[0008] Preferably, the amino acid sequence of the heavy chain variable region of the antibody or antigen-binding fragment thereof is as shown in SEQ ID NO.13 or has at least 80% similarity to the sequence shown in SEQ ID NO.13, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.14 or has at least 80% similarity to the sequence shown in SEQ ID NO.14.

[0009] The above sequence similarity is preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 98%, more preferably at least 98.5%, more preferably at least 99%, more preferably at least 99.5%, more preferably at least 99.8%, more preferably at least 99.9%.

[0010] In some specific embodiments of the present invention, the amino acid sequence of the heavy chain variable region of the antibody or antigen-binding fragment thereof is shown in SEQ ID NO.13, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.14.

[0011] The above-mentioned antibody or antigen-binding fragment thereof may be a monoclonal antibody, Fab, Fab', F(ab')2, Fd, Fv or a single-chain antibody.

[0012] In some embodiments of the present invention, the antibody is a monoclonal antibody.

[0013] In a second aspect, the present invention provides a nucleic acid molecule encoding the above-mentioned antibody or antigen-binding fragment thereof.

[0014] According to the amino acid sequence of the antibody or its antigen-binding fragment provided above, those skilled in the art can obtain the nucleotide sequence of the nucleic acid molecule encoding the above antibody or its antigen-binding fragment. Due to the degeneracy of codons, the nucleotide sequence of the nucleic acid molecule encoding the above antibody or its antigen-binding fragment is not unique, and all nucleic acid molecules capable of encoding and producing the above antibody or its antigen-binding fragment are within the scope of protection of the present invention.

[0015] In some specific embodiments of the present invention, the nucleotide sequence of the nucleic acid molecule encoding the heavy chain variable region of the antibody or its antigen-binding fragment is shown as SEQ ID NO.17, and the nucleotide sequence of the nucleic acid molecule encoding the light chain variable region of the antibody or its antigen-binding fragment is shown as SEQ ID NO.18.

[0016] In a third aspect, the present invention provides a biological material, wherein the biological material contains the nucleic acid molecule described above; the biological material is an expression cassette, a vector or a host cell.

[0017] In the present invention, the expression cassette can be obtained by connecting a transcription or translation regulatory element such as a promoter upstream of the nucleic acid molecule and / or connecting a transcription or translation regulatory element such as a terminator downstream thereof.

[0018] In the present invention, the vector includes but is not limited to a plasmid vector, a phage vector, a virus vector, an artificial chromosome vector, and the like.

[0019] In the present invention, the host cells include microbial cells, insect cells or other animal cells.

[0020] In a fourth aspect, the present invention provides an antibody conjugate, wherein the antibody conjugate is obtained by conjugating the above-mentioned antibody or antigen-binding fragment thereof 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.

[0021] In a fifth aspect, the present invention provides a rat IgG antibody composition, wherein the antibody composition comprises the antibodies of (1) and (2):

[0022] (1) The amino acid sequences of the complementary determining regions CDR1, CDR2 and CDR3 of the heavy chain variable region are shown in SEQ ID NO.1, 2 and 3 respectively, the amino acid sequence of the complementary determining region CDR1 of the light chain variable region is shown in SEQ ID NO.4, the amino acid sequence of CDR2 is AAS, and the amino acid sequence of CDR3 is shown in SEQ ID NO.5;

[0023] (2) The amino acid sequences of the complementary determining regions CDR1, CDR2 and CDR3 of the heavy chain variable region are shown in SEQ ID NOs. 6, 7 and 8, respectively. The amino acid sequence of the complementary determining region CDR1 of the light chain variable region is shown in SEQ ID NO. 9, the amino acid sequence of CDR2 is SAS, and the amino acid sequence of CDR3 is shown in SEQ ID NO. 10.

[0024] Preferably, the amino acid sequence of the heavy chain variable region of the antibody described in (1) above is as shown in SEQ ID NO.11 or has at least 80% similarity to the sequence shown in SEQ ID NO.11, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.12 or has at least 80% similarity to the sequence shown in SEQ ID NO.12. The amino acid sequence of the heavy chain variable region of the antibody described in (2) above is as shown in SEQ ID NO.13 or has at least 80% similarity to the sequence shown in SEQ ID NO.13, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.14 or has at least 80% similarity to the sequence shown in SEQ ID NO.14.

[0025] In some specific embodiments of the present invention, the amino acid sequence of the heavy chain variable region of the antibody described in (1) above is shown as SEQ ID NO.11, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO.12. The amino acid sequence of the heavy chain variable region of the antibody described in (2) above is shown as SEQ ID NO.13, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO.14.

[0026] The antibody composition described above can be used as a paired antibody for detecting rat IgG by double antibody sandwich ELISA method, wherein the two antibodies in the antibody composition serve as a coating antibody and a labeled antibody, respectively (preferably, the antibody described in (1) serves as a labeled antibody, and the antibody described in (2) serves as a coating antibody). The double antibody sandwich ELISA method using the antibody composition to detect rat IgG has high specificity, sensitivity and accuracy.

[0027] In a sixth aspect, the present invention provides any of the following uses of the above-mentioned antibody or antigen-binding fragment thereof, the nucleic acid molecule, the biomaterial, the antibody conjugate, or the antibody composition:

[0028] (1) Use in the preparation of a product for detecting the presence or level of rat IgG in a sample;

[0029] (2) Application in the preparation of products for testing vaccine immunogenicity or immune efficacy;

[0030] (3) Use in the preparation of products for evaluating the efficacy and / or prognosis of immunotherapy drugs.

[0031] In the above (1), the sample may be a biological sample (such as blood, serum, etc.) derived from rats, or may be a product containing rat IgG prepared in vitro.

[0032] It is well known in the art that the IgG level in animals can reflect the effect of vaccine immunity. In the above (2), the immunogenicity or immune effect of the vaccine is evaluated by detecting the IgG content in vaccine-immunized rats.

[0033] It is well known in the art that the IgG level in an animal can reflect the body's response to immunotherapy drugs. In the above (3), the efficacy and / or prognosis of the immunotherapy drug is evaluated by monitoring the IgG content in the rats to which the drug is administered.

[0034] The immunotherapy includes but is not limited to tumor immunotherapy, autoimmune disease treatment, etc.

[0035] In a seventh aspect, the present invention provides use of the above-mentioned antibody or antigen-binding fragment thereof, the antibody conjugate or the antibody composition in detecting the presence or level of rat IgG in a sample for purposes other than disease diagnosis and treatment.

[0036] In the present invention, the method for detecting rat IgG can be an immunological detection method such as ELISA, chemiluminescent immunoassay, radioimmunoassay, fluorescent immunoassay, immunochromatography, etc.

[0037] In an eighth aspect, the present invention provides a kit comprising the above-described antibody or antigen-binding fragment thereof, or the antibody conjugate, or the antibody composition.

[0038] Preferably, the kit is a double antibody sandwich ELISA detection kit. The kit is used to detect rat IgG.

[0039] Preferably, the kit comprises a coated antibody and a labeled antibody, the amino acid sequences of the complementary determining regions CDR1, CDR2, and CDR3 of the heavy chain variable region of the labeled antibody are shown as SEQ ID NO.1, 2, and 3, respectively, the amino acid sequence of the complementary determining region CDR1 of the light chain variable region is shown as SEQ ID NO.4, the amino acid sequence of CDR2 is AAS, and the amino acid sequence of CDR3 is shown as SEQ ID NO.5; the amino acid sequences of the complementary determining regions CDR1, CDR2, and CDR3 of the heavy chain variable region of the coated antibody are shown as SEQ ID NO.6, 7, and 8, respectively, the amino acid sequence of the complementary determining region CDR1 of the light chain variable region is shown as SEQ ID NO.9, the amino acid sequence of CDR2 is SAS, and the amino acid sequence of CDR3 is shown as SEQ ID NO.10.

[0040] To facilitate detection, the labeled antibody may further include a detectable label (eg, HRP label); the kit may further include a second antibody carrying a detectable label to detect the antibody or antigen-binding fragment thereof.

[0041] The kit may also contain other reagents for ELISA detection, including but not limited to one or more selected from ELISA plates, coating buffer, diluent, rat IgG standard, PBST washing solution, blocking solution, color development solution, and stop solution.

[0042] In some specific embodiments of the present invention, the kit comprises an ELISA plate coated with the antibody in (2) of the fifth aspect, an HRP-labeled antibody in (1) of the fifth aspect, an HRP colorimetric substrate, a stop solution and a rat IgG standard.

[0043] The method for using the above-mentioned kit comprises: placing a sample to be tested containing rat IgG into the ELISA plate coated with the antibody in (2) of the above-mentioned fifth aspect, adding the antibody in (1) of the above-mentioned fifth aspect labeled with HRP for reaction, then adding an HRP color development substrate for color development reaction, adding a stop solution to stop the reaction after the color development is completed, and calculating the content of rat IgG in the sample to be tested according to the color development OD value.

[0044] In a ninth aspect, the present invention provides a method for detecting rat IgG, the method comprising: using the above-mentioned antibody or its antigen-binding fragment or the antibody conjugate or the antibody composition or the kit to detect the content of rat IgG in a sample to be tested.

[0045] The above detection method can be selected from immunological detection methods such as ELISA, chemiluminescent immunoassay, radioimmunoassay, fluorescent immunoassay, and immunochromatography.

[0046] The beneficial effects of the present invention include at least: the antibody provided by the present invention can specifically bind to rat IgG, but does not bind to other similar proteins such as mouse IgG, has high affinity for rat IgG, and has high specificity, sensitivity and accuracy when used for rat IgG detection. The kit for rat IgG detection developed based on the antibody has the characteristics of strong specificity, high sensitivity, high accuracy, wide linear range, strong stability, etc., and has good application value in the detection of rat IgG content. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0048] Figure 1 The SDS-PAGE test results of monoclonal antibodies 3E11 and 6F9 in Example 1 of the present invention are shown.

[0049] Figure 2 This is the standard curve prepared for the rat IgG standard in Example 2 of the present invention. DETAILED DESCRIPTION

[0050] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the drawings of the present invention. 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.

[0051] Example 1 Preparation of anti-rat IgG monoclonal antibody

[0052] 1. Animal immunization

[0053] Six-week-old female Balb / c mice were selected and immunized with naturally extracted rat IgG antigen mixed with an equal volume of water-soluble adjuvant. A second immunization was performed 14 days after immunization, and blood was collected 21 days after immunization to measure the titer. The immunization was boosted again three days before fusion.

[0054] 2. Cell fusion

[0055] The mice were killed by cervical dislocation, and the spleen was removed aseptically. The spleen was ground with a 200-mesh screen in a flat dish 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 described as follows.

[0056] (1) Myeloma (SP2 / 0) cell preparation

[0057] Resuscitate commercial SP2 / 0 cells, then suspend in complete culture medium (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 culture medium. After blowing down the cells, place them in a 50mL centrifuge tube, add serum-free culture medium 1640 to 50mL, count, and then centrifuge for use.

[0058] (2) Preparation of immune spleen cells

[0059] 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 min 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 again with a sieve, add 1640 to 50 mL of serum-free culture medium, count it, and set it aside after centrifugation.

[0060] (3) Preparation of feeder cells

[0061] Take an unimmunized Balb / c mouse, bleed it from its orbit, and collect the negative serum. Inject 10 mL of RPMI-1640 basal solution (without serum) into the mouse's peritoneal cavity, then aspirate and place it in another centrifuge tube for later use. The solution contains peritoneal macrophages.

[0062] (4) Integration

[0063] 1-2×10 7 SP2 / 0 and 10 8 Mix the immune cells in a 50mL centrifuge tube and centrifuge at 1000 r / min for 8min. After discarding the supernatant, place the centrifuge tube containing the cell mixture in a 37℃ water bath, then add 1 mL (sigma) of 50% PEG preheated to 37℃, stir and let stand for 30s. After standing, add 10mL of RPMI-1640 basal solution (without serum) preheated at 37℃. After mixing, centrifuge at 1000 r / min for 5 min, discard the supernatant and let stand at 37℃ for 5-8min. Then mix with the feeder cell suspension, add to the prepared semi-solid culture medium, mix well, pour into the plate, 10mL per plate, and culture in a 37℃, 5% CO2 incubator. When the cell clusters in the fusion plate grow to white spots visible to the naked eye, use a 10µL pipette tip to pick out the white spots into a 96-well culture plate. When the cell colonies grow to 1 / 4 of the culture well and the culture medium turns slightly yellow, perform antibody detection.

[0064] 3. Screening of hybridoma positive cells

[0065] Positive hybridoma cells were screened by indirect ELISA, and the steps are as follows.

[0066] (1) Antigen coating: Dilute the antigen to 1 μg / mL with coating buffer; add 100 μL to each microwell, shake gently, and store in a refrigerator at 4°C overnight; wash three times with a plate washer and pat dry.

[0067] (2) Block the ELISA plate: Add 200 μL of blocking solution (5% skimmed milk powder) to each well of the microwell, shake gently, and incubate at room temperature for 2 hours; then discard the liquid in the well.

[0068] (3) Sample addition: Take 50 μL of supernatant from each well of the hybridoma to be tested and add it to the wells of the ELISA plate in order. Shake gently. Incubate at 37°C for 1 hour, wash 4 times with a plate washer, and pat dry the liquid in the wells.

[0069] (4) Add enzyme-labeled antibody: First, dilute the enzyme-labeled secondary antibody to the appropriate working concentration using diluent according to the instructions, add 100 μL to each well, shake gently, and incubate at 37°C for 30 min; then wash 4 times and pat dry the washing solution.

[0070] (5) Add color developing solution: Add 100 μL of single-component color developing solution to each well, shake gently, and incubate at 37°C for 10 min.

[0071] (6) Termination reaction: Add 50 μL of stop solution to each well to terminate the reaction.

[0072] (7) Determination result: OD of the microplate reader 450 The reading was performed at 2.1 times that of the negative well, which was considered positive.

[0073] The hybridoma positive cells obtained by screening are expanded and cultured as follows: a single cluster of positive cells can be moved to a 24-well culture plate. When the cells in the 24-well plate grow well, they are expanded to culture dishes for culture. Finally, after the cells are frozen, they are inoculated intraperitoneally into mice to collect ascites.

[0074] Two monoclonal antibodies that specifically bind to rat IgG but not mouse IgG were obtained through the above hybridoma cell screening and were named monoclonal antibodies 3E11 and 6F9, respectively.

[0075] 4. Cryopreservation of hybridoma cells of monoclonal antibodies 3E11 and 6F9

[0076] When hybridoma cells have grown all over the culture dish, use a pipette to blow down the cells and collect them in a 50 mL centrifuge tube for counting. After centrifugation, count the cells according to the number of cells: 5 × 10 6 / mL of 90% calf serum freezing solution containing DMSO was added to resuspend the cells, and then placed in a cryovial, placed in a -80℃ refrigerator overnight, and then frozen in liquid nitrogen the next day.

[0077] 5. Large-scale preparation of monoclonal antibodies 3E11 and 6F9

[0078] The hybridoma cells were injected into the peritoneal cavity of mice. The abdomen of the mice swelled after about 7 days. The ascites was collected and the antibodies were purified by Protein G affinity chromatography and detected by SDS-PAGE. The results are as follows: Figure 1 shown.

[0079] 6. Variable region sequence determination of monoclonal antibodies 3E11 and 6F9

[0080] The number of hybridoma cells collected was greater than 10 6, the total RNA extraction kit (Beijing Solaibao 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 according to the instructions of the reverse transcription kit (Beijing Solaibao Technology Co., Ltd.), and the amplified DNA was constructed into a T vector and sent to Qingke Bio for sequencing. The gene sequencing results are as follows: the heavy chain variable region sequence of monoclonal antibody 3E11 is 360bp long, encoding 120 amino acids, the DNA sequence is shown in SEQ ID NO.15, and the protein sequence is shown in SEQ ID NO.11, wherein the amino acid sequences of the complementary determining regions CDR1, CDR2, and CDR3 of the heavy chain variable region are shown in SEQ ID NO.1, 2, and 3, respectively; the light chain variable region sequence is 333bp long, encoding 111 amino acids, the DNA sequence is shown in SEQ ID NO.16, and the protein sequence is shown in SEQ ID NO.12, wherein the amino acid sequence of the complementary determining region CDR1 of the light chain variable region is shown in SEQ ID NO.4, the amino acid sequence of CDR2 is AAS, and the amino acid sequence of CDR3 is shown in SEQ ID NO.5. The heavy chain variable region sequence of monoclonal antibody 6F9 is 360 bp long, encoding 120 amino acids, the DNA sequence is shown in SEQ ID NO.17, and the protein sequence is shown in SEQ ID NO.13, wherein the amino acid sequences of the complementary determining regions CDR1, CDR2, and CDR3 of the heavy chain variable region are shown in SEQ ID NO.6, 7, and 8, respectively; the light chain variable region sequence is 321 bp long, encoding 107 amino acids, the DNA sequence is shown in SEQ ID NO.18, and the protein sequence is shown in SEQ ID NO.14, wherein the amino acid sequence of the complementary determining region CDR1 of the light chain variable region is shown in SEQ ID NO.9, the amino acid sequence of CDR2 is SAS, and the amino acid sequence of CDR3 is shown in SEQ ID NO.10.

[0081] 7. Antibody affinity constant detection

[0082] Prepare the ELISA plate according to the indirect ELISA method for screening positive hybridoma cells in 3 above, 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 (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. The results are shown in Table 1.

[0083] Table 1

[0084]

[0085] Example 2 Establishment of a double antibody sandwich ELISA kit and method for detecting rat IgG content

[0086] 1. Antibody titer detection

[0087] Prepare the ELISA plate according to the method (see indirect ELISA screening of positive hybridoma cells in 3 of Example 1), dilute the purified monoclonal antibodies 3E11 and 6F9 to the same concentration, dilute the first well at a ratio of 1:1000, and dilute the subsequent 96 wells in turn at a ratio of 1:500, 1000, 2000, 4000, 8000, 16000, 32000, 64000, 128000, 256000, 512000, 100 μL / well, and incubate at 37°C for 1 hour. After washing the plate with PBST, add HRP-goat anti-mouse IgG (1:5000) and detect the color. Result determination: OD 450 The dilution factor greater than 2.1 times of the negative well can be determined as the antibody titer.

[0088] OD corresponding to each dilution factor 450 The readings are shown in Table 2.

[0089] Table 2

[0090]

[0091] 2. HRP labeling of monoclonal antibody 3E11

[0092] Add antibody 3E11 to a dialysis bag and immerse in 0.01M CB buffer for overnight dialysis at 4°C. Take 10mg HRP and dissolve in 2mL water. Prepare a fresh NaIO4 solution, 0.1M, add 0.4mL 0.1M NaIO4 to 2mL HRP solution, mix well, and dialyze at room temperature in the dark for 45min. Immerse in 10mM NaAc buffer for overnight dialysis at 4°C. Take out the antibody and HRP solution that have been dialyzed overnight and add 0.1mL 0.2M pH 9.5 carbonate buffer, then immediately add the antibody that has been taken out to the HRP solution, and stir gently for 2 hours at room temperature in the dark. Weigh 0.04g NaBH4 and dissolve it in 10mL water, take 0.4mL and add it to the reaction solution, mix well, and place at 4°C for 2h. Take it out and put it in a dialysis bag, dialyze it in 0.01M PBS, change the solution once after 2h, and dialyze it overnight at 4°C. Take out the labeling solution (i.e., containing the enzyme-labeled antibody HRP-3E11) that has been dialyzed overnight, add an equal volume of glycerol, and store at -20°C.

[0093] 3. Preparation of monoclonal antibody 6F9 coated ELISA plate

[0094] Dilute the monoclonal antibody 6F9 to 2 μg / mL with 0.05M pH 9.6 carbonate coating buffer. Add 0.1 mL to the reaction wells of the 96-well polystyrene reagent plate and incubate at 4°C overnight. The next day, discard the solution in the wells and wash 3 times with washing buffer for 3 minutes each time. After the above steps, block each well of the reaction plate with 2% BSA solution, 0.3 mL per well, and place at room temperature for 2 hours. Discard the solution in the wells, dry in a dry room, evacuate, and store at 4°C.

[0095] 4. Establishment of double antibody sandwich ELISA method and determination of sensitivity

[0096] 30 minutes before the experiment, take out the ELISA plate coated with monoclonal antibody 6F9, return to room temperature, wash the plate 3 times and spin dry. Add 100 μL of rat IgG standard (Solarbio, catalog number SP032) of different dilution concentrations, the rat IgG dilution concentrations are 80 ng / mL, 40 ng / mL, 20 ng / mL, 10 ng / mL, 5 ng / mL, 2.5 ng / mL, 1.25 ng / mL, and set up a blank control. 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-3E11 working solution to the reaction well, incubate in a room temperature incubator for 30 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 450 nm using an ELISA reader. The standard curve was drawn with different concentrations of rat IgG standard as the horizontal axis and the corresponding OD value as the vertical axis to establish a regression equation. The results showed that the detection range of this method was 1.25-80 ng / mL, R 2 is 0.9995 ( 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 detecting rat IgG using the double antibody sandwich ELISA method was 0.013509 ng / mL (see Table 3).

[0097] Table 3

[0098]

[0099] 5. Specificity detection of double antibody sandwich ELISA method

[0100] 30 minutes before the experiment, take out the ELISA plate coated with monoclonal antibody 6F9, return to room temperature, wash the plate 3 times and spin dry. Add 100 μL of other similar protein samples with different dilution multiples, including bovine IgG, rabbit IgG, chicken IgG, chicken IgM, duck IgG, mouse IgG, chicken IgY and human IgG, and all samples are diluted to high concentration (4 gradients), 320 ng / mL, 160 ng / mL, 80 ng / mL, 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-3E11 working solution to the reaction well, seal the plate, incubate in a room temperature incubator for 45 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 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 with an ELISA reader (within 5 min). The results showed that monoclonal antibodies 3E11 and 6F9 did not react with other similar proteins (Table 4).

[0101] Table 4

[0102]

[0103] 6. Stability test of double antibody sandwich ELISA kit

[0104] The ELISA plate coated with monoclonal antibody 6F9, HRP-labeled monoclonal antibody 3E11, and rat IgG standard were placed at 37°C for accelerated stability test for 15 days (equivalent to 24 months at 4°C). Then the plate was taken out for testing. The testing method was as follows: the ELISA plate was taken out, washed 3 times and dried, 100 μL of rat IgG standard with different dilution concentrations was added, and the rat IgG dilution concentrations were 80 ng / mL, 40 ng / mL, 20 ng / mL, 10 ng / mL, 5 ng / mL, 2.5 ng / mL, 1.25 ng / mL and a blank control was set. After sealing the plate, it was incubated in a room temperature incubator for 2 hours, washed 4 times and dried. 100 μL of enzyme-labeled antibody HRP-3E11 working solution was added to the reaction well, and after sealing the plate, it was incubated in a room temperature incubator for 45 min, washed 4 times and dried. Add 100 μL of chromogenic substrate TMB to the reaction wells, seal the plate and color develop in the dark at room temperature for 15 min, add 50 μL of stop solution (2M sulfuric acid solution), and immediately measure the OD value at 450 nm with an enzyme reader (within 5 minutes). Use rat IgG standards of different concentrations 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 kit has good stability. Among them, the OD values ​​corresponding to each concentration of rat IgG standard are as follows: 450 As shown in Table 5.

[0105] Table 5

[0106]

[0107] 7. Spike recovery rate

[0108] 30 min before the experiment, take out the ELISA plate coated with monoclonal antibody 6F9, return to room temperature, wash the plate 3 times and spin dry. Select 3 rat sera (serum 1, 2, 3) for a certain dilution and 1 SP2 / 0 cell culture supernatant, add rat IgG at different concentrations and set up a blank control. After sealing the plate, incubate at room temperature for 2h, wash the plate 4 times and spin dry. Add 100 μL enzyme-labeled antibody HRP-3E11 working solution to the reaction well, seal the plate and incubate at room temperature for 45 min, wash the plate 4 times and spin dry. Add 100 μL color substrate TMB to the reaction well, seal the plate and color develop at room temperature in the dark for 15 min, add 50 μL stop solution (2M sulfuric acid solution), and immediately measure the OD value at 450nm wavelength with an ELISA reader (within 5 minutes). The test results show that the spike recovery rate is within the normal range of 70%-130% (Table 6).

[0109] Table 6

[0110]

[0111] 8. Linear Dilution

[0112] 30 min before the experiment, take out the ELISA plate coated with monoclonal antibody 6F9, return to room temperature, wash the plate 3 times and spin dry. After selecting 4 rat sera for a certain dilution, add high concentration rat IgG, dilute within the dynamic range of the standard curve, and evaluate linearity. After sealing the plate, incubate at room temperature for 2h, wash the plate 4 times and spin dry. Add 100 μL of enzyme-labeled antibody HRP-3E11 working solution to the reaction well, seal the plate and incubate at room temperature for 45 min, wash the plate 4 times and spin dry. Add 100 μL of color substrate TMB to the reaction well, seal the plate and color 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 a wavelength of 450nm with an ELISA reader (within 5 minutes). The recovery rate at each dilution ratio is shown in Table 7. The test results show that this method can accurately determine the content of rat serum IgG and has a good linear relationship in a wide dilution range.

[0113] Table 7

[0114]

[0115] Example 3 Determination of IgG content in rat serum

[0116] 30 min before the experiment, take out the ELISA plate coated with monoclonal antibody 6F9, return to room temperature, wash the plate 3 times and spin dry. Select 8 rat sera (numbered 1, 2, 3, 4, 5, 6, 7, and 8), the sample dilution multiples are 10,000 times and 20,000 times, and add 100L of rat serum samples with different dilution multiples to each well. The dilution concentrations of rat IgG standard are 80ng / mL, 40ng / mL, 20ng / mL, 10ng / mL, 5ng / mL, 2.5ng / mL, 1.25ng / mL and a blank control is set. After sealing the plate, incubate it in a room temperature incubator for 2h, wash the plate 4 times and spin dry. Add 100 μL of enzyme-labeled antibody HRP-3E11 working solution to the reaction wells, seal the plate and incubate in a room temperature incubator for 45 minutes, wash the plate 4 times and spin dry, add 100L of color substrate TMB to the reaction wells, seal the plate and color develop at room temperature in the dark for 15 minutes, add 50L of stop solution (2M sulfuric acid solution), and immediately measure the OD value at a wavelength of 450nm with an enzyme reader (within 5 minutes). The test results show that this method can accurately determine the content of rat serum IgG and has a good linear relationship within a certain range (Table 8).

[0117] Table 8

[0118]

[0119] 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. An anti-rat 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 shown in SEQ ID NO.6, 7 and 8, respectively; the amino acid sequence of the complementary determining region CDR1 of the light chain variable region is shown in SEQ ID NO.9, the amino acid sequence of CDR2 is SAS, and the amino acid sequence of CDR3 is shown in SEQ ID NO.

10.

2. The 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 antibody or antigen-binding fragment thereof is shown in SEQ ID NO.13, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.

14.

3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, 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.

4. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3.

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

6. 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 3 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.

7. A rat IgG antibody composition, characterized in that The antibody composition comprises the following antibodies (1) and (2): (1) the amino acid sequences of the complementary determining regions CDR1, CDR2 and CDR3 of the heavy chain variable region are shown in SEQ ID NO.1, 2 and 3 respectively, the amino acid sequence of the complementary determining region CDR1 of the light chain variable region is shown in SEQ ID NO.4, the amino acid sequence of CDR2 is AAS, and the amino acid sequence of CDR3 is shown in SEQ ID NO.5; (2) The amino acid sequences of the complementary determining regions CDR1, CDR2 and CDR3 of the heavy chain variable region are shown in SEQ ID NO.6, 7 and 8, respectively; the amino acid sequence of the complementary determining region CDR1 of the light chain variable region is shown in SEQ ID NO.9, the amino acid sequence of CDR2 is SAS, and the amino acid sequence of CDR3 is shown in SEQ ID NO.

10.

8. Any of the following uses of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, the nucleic acid molecule according to claim 4, the biomaterial according to claim 5, the antibody conjugate according to claim 6, or the antibody composition according to claim 7: (1) Use in the preparation of a reagent for detecting the presence or level of rat IgG in a sample; (2) Application in the preparation of reagents for testing the immunogenicity or immune effect of vaccines based on IgG detection; (3) Use in the preparation of reagents for evaluating the efficacy and / or prognosis of immunotherapeutic drugs based on IgG detection.

9. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, the antibody conjugate according to claim 6, or the antibody composition according to claim 7 for detecting the presence or level of rat IgG in a sample for non-disease diagnosis and treatment purposes.

10. A kit, characterized in that: It comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, or the antibody conjugate according to claim 6, or the antibody composition according to claim 7.

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