Anti-rabbit IgG goat recombinant monoclonal antibody as well as preparation and application thereof

Abstract

The invention discloses an anti-rabbit IgG goat recombinant monoclonal antibody as well as preparation and application thereof, and belongs to the technical field of biology. The recombinant monoclonal antibody is obtained by taking purified rabbit serum IgG as an immunogen, immunizing a goat, carrying out specific B cell screening and RT-PCR (Reverse Transcription-Polymerase Chain Reaction) to obtain a heavy chain variable region and a light chain variable region of the antibody, respectively inserting sequences of the variable regions into vector plasmids with constant region (heavy chain and light chain) genes, transfecting, expressing and purifying. The recombinant monoclonal antibody provided by the invention has high affinity with rabbit IgG, can recognize and detect rabbit IgG with high specificity and high sensitivity, can be used as a secondary antibody after HRP crosslinking, is applied to detection of rabbit IgG by methods such as western blot, immunohistochemistry and ELISA, is beneficial to obtaining more accurate detection and evaluation results, and has good application prospects. And the detection cost and the interference of background signals are reduced.

Description

Technical Field

[0001] The present application belongs to the field of biotechnology, and specifically relates to a goat recombinant monoclonal antibody against rabbit IgG and its preparation and application. Background Art

[0002] When humans and animals become sick, they need to rely on their own immune response, and specific immunity plays an important role. Humoral immunity occupies a certain position in specific immunity. Humoral immunity mainly relies on various immunoglobulins, of which IgG accounts for about 75% to 80%. It is the main antibody in the secondary immune response and the main target antibody for serological diagnosis and post-vaccination detection.

[0003] Rabbit antibodies are known for their high affinity, specificity, and diversity. In addition, rabbits and mice are evolutionarily different, so rabbit antibodies can recognize human antigen epitopes that are not immunogenic in mice, increase the total number of targetable epitopes, and help produce antibodies that cross-react with human antigens. Anti-rabbit IgG antibodies are used as secondary antibodies in immunology to detect rabbit primary antibodies. They can bind to markers to help detect and quantify the binding of rabbit antibodies to specific antigens. Therefore, the preparation of highly sensitive and accurate anti-rabbit IgG antibodies is of great significance for scientific research, diagnostic testing, and therapeutic applications.

[0004] Sheep antibodies have unique advantages as secondary antibodies. Sheep have 5 immunoglobulin isotypes, consisting of 5 heavy chain classes (IgM, IgG, IgA, IgD and IgE), and the IgG class is further subdivided into 3 subtypes (IgG1, IgG2, IgG3). As in other species, the heavy chain is combined with 2 light chains (Lambda and kappa). IgG-λ accounts for 95% of IgG in sheep. The affinity of sheep antibodies is usually 10 to 100 times higher than that of rodent antibodies; secondly, sheep monoclonal antibodies only bind to one specific area and can accurately distinguish closely related molecules (such as derivatives), which may produce highly specific antibodies against the desired target; in addition, sheep have a unique immune system, sheep antibodies can recognize "difficult" targets where other antibody technologies fail, and natural sheep antibodies can recognize a variety of heterologous epitopes.

[0005] In the current technical field, the goat anti-rabbit IgG antibody (DyLight488) provided by GeneTex is a polyclonal antibody with low specificity; the biotin-labeled goat anti-rabbit IgG (H+L) provided by Biotech is prepared by immunizing goats with rabbit IgG, then purifying the obtained antiserum with an affinity purification column, and then purifying it after adsorption and purification with human IgG, mouse IgG and rat IgG. It is still not a monoclonal antibody, so its specificity still has room for improvement. Summary of the invention

[0006] 1. Purpose of the Invention

[0007] The purpose of the present application is to provide a goat recombinant monoclonal antibody against rabbit IgG and its preparation and application. A new goat recombinant monoclonal antibody against rabbit IgG is successfully prepared by single B cell PCR technology. The recombinant monoclonal antibody can specifically recognize rabbit IgG protein in tissues, and can be used alone or after being labeled with horseradish peroxidase (HRP) to detect rabbit IgG protein in immunohistochemistry, (indirect / direct) ELISA, protein immunoblotting and the like. The recombinant monoclonal antibody has a higher binding affinity with rabbit IgG, which significantly improves the ability to detect rabbit IgG, providing a more reliable tool for accurate rabbit antibody detection.

[0008] 2. Technical solution

[0009] In order to achieve the above purpose, the technical solution adopted in this application is as follows:

[0010] The present application provides a goat recombinant monoclonal antibody against rabbit IgG, which can specifically bind to rabbit IgG protein, and the amino acid sequence of its heavy chain variable region is shown in SEQ ID NO.1, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO.2.

[0011] Furthermore, the amino acid sequence of the heavy chain constant region of the above-mentioned anti-rabbit IgG goat recombinant monoclonal antibody is shown in SEQ ID NO.5, and the amino acid sequence of the light chain constant region is shown in SEQ ID NO.6.

[0012] The present application also provides a nucleic acid encoding the heavy chain variable region and / or light chain variable region of the above-mentioned goat recombinant monoclonal antibody against rabbit IgG.

[0013] Furthermore, the nucleic acid also encodes the heavy chain constant region and / or light chain constant region of the above-mentioned goat recombinant monoclonal antibody against rabbit IgG.

[0014] Furthermore, the nucleotide sequence encoding the goat recombinant monoclonal antibody against rabbit IgG comprises:

[0015] The nucleotide sequence shown in SEQ ID NO.3 is used to encode the heavy chain variable region of the amino acid sequence shown in SEQ ID NO.1 of the above-mentioned goat recombinant monoclonal antibody against rabbit IgG;

[0016] The nucleotide sequence shown in SEQ ID NO.4 is used to encode the light chain variable region of the amino acid sequence shown in SEQ ID NO.2 of the above-mentioned goat recombinant monoclonal antibody against rabbit IgG.

[0017] Furthermore, the above nucleic acid also includes:

[0018] The nucleotide sequence shown in SEQ ID NO.7 is used to encode the heavy chain constant region of the amino acid sequence shown in SEQ ID NO.5 of the above-mentioned goat recombinant monoclonal antibody against rabbit IgG;

[0019] The nucleotide sequence shown in SEQ ID NO.8 is used to encode the light chain constant region of the amino acid sequence shown in SEQ ID NO.6 of the above-mentioned goat recombinant monoclonal antibody against rabbit IgG.

[0020] The present application also provides a recombinant expression vector, which contains the above-mentioned nucleic acid encoding the goat recombinant monoclonal antibody against rabbit IgG and can express the goat recombinant monoclonal antibody against rabbit IgG.

[0021] Furthermore, the above-mentioned recombinant expression vector includes: a pcDNA3.4 plasmid containing the above-mentioned nucleic acid encoding the goat recombinant monoclonal antibody against rabbit IgG.

[0022] The present application also provides a recombinant expression cell, which includes: the above-mentioned recombinant expression vector, or the above-mentioned nucleic acid encoding goat recombinant monoclonal antibody against rabbit IgG, which can be used to express goat recombinant monoclonal antibody against rabbit IgG.

[0023] Furthermore, the above-mentioned recombinant expression cells include: 293F cell line.

[0024] The present application also provides the use of the above nucleic acid, recombinant expression vector, and recombinant expression cell in the preparation of goat recombinant monoclonal antibodies against rabbit IgG.

[0025] The present application also provides a method for preparing the above-mentioned goat recombinant monoclonal antibody against rabbit IgG, comprising: using the above-mentioned recombinant expression vector to transfect cells to obtain recombinant expression cells; culturing the transfected recombinant expression cells; collecting and purifying the supernatant to obtain the goat recombinant monoclonal antibody against rabbit IgG.

[0026] The present application also provides a method for preparing the above-mentioned goat recombinant monoclonal antibody against rabbit IgG, comprising: culturing recombinant expression cells; collecting and purifying the supernatant to obtain the goat recombinant monoclonal antibody against rabbit IgG.

[0027] The present application also provides an HRP-labeled goat recombinant monoclonal antibody against rabbit IgG.

[0028] The present application also provides the above-mentioned goat recombinant monoclonal antibody against rabbit IgG, nucleic acid, recombinant expression vector, recombinant expression cell, method for preparing the goat recombinant monoclonal antibody against rabbit IgG, or use of the above-mentioned goat recombinant monoclonal antibody against rabbit IgG labeled with HRP in detecting rabbit IgG or preparing products for detecting rabbit IgG.

[0029] Furthermore, the above detection of rabbit IgG includes any one or more detection methods such as immunohistochemistry, ELISA, and protein immunoblotting.

[0030] 3. Beneficial effects

[0031] Compared with the prior art, the present application has the following beneficial effects:

[0032] The present application provides a goat recombinant monoclonal antibody against rabbit IgG, and its preparation and application. Comparison with commercially available antibodies shows that the goat recombinant monoclonal antibody against rabbit IgG provided by the present application binds to rabbit IgG protein molecules with high specificity and high sensitivity, can specifically identify and detect rabbit IgG protein, and is positive and highly expressed when detecting rabbit IgG protein. Therefore, the recombinant monoclonal antibody can be used in experiments such as immunohistochemistry, protein immunoblotting and indirect ELISA. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is the result of antibody SDT-GAR-1WB test.

[0034] Figure 2 This is the result of IHC detection of antibody SDT-GAR-1.

[0035] Figure 3 It is the indirect ELISA test result of HRP-labeled monoclonal antibody SDT-GAR-1.

[0036] Figure 4 This is the WB detection result of HRP-labeled monoclonal antibody SDT-GAR-1.

[0037] Figure 5 This is the IHC detection result of HRP-labeled monoclonal antibody SDT-GAR-1. DETAILED DESCRIPTION

[0038] The present application is further described below in conjunction with specific embodiments.

[0039] It should be noted that the terms such as "upper", "lower", "left", "right", "middle", etc. cited in this specification are only for the convenience of description and are not used to limit the scope of implementation. Changes or adjustments to their relative relationships should be regarded as the scope of implementation of this application without substantially changing the technical content.

[0040] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this application belongs; the term "and / or" used herein includes any and all combinations of one or more of the associated listed items.

[0041] If the specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. If the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be purchased commercially.

[0042] As used herein, the term "about" is used to provide flexibility and imprecision associated with a given term, measurement or value. The degree of flexibility for a particular variable can be easily determined by one skilled in the art.

[0043] As used herein, the term "at least one of" is intended to be synonymous with "one or more of." For example, "at least one of A, B, and C" expressly includes only A, only B, only C, and combinations of each thereof.

[0044] Concentration, amount and other numerical data can be presented in range format herein.It should be understood that such range format is only used for convenience and simplicity, and should be flexibly interpreted as not only including the numerical value clearly described as range limit, but also including all single numerical values ​​or sub-ranges contained in the range, just as each numerical value and sub-range are clearly described.For example, the numerical range of about 1 to about 4.5 should be interpreted as not only including the limit value of 1 to about 4.5 clearly described, but also including single numerals (such as 2,3,4) and sub-ranges (such as 1 to 3, 2 to 4, etc.).The same principle is applicable to the scope of only narrating a numerical value, such as "less than about 4.5," which should be interpreted as including all the above-mentioned values ​​and ranges.In addition, no matter how the breadth of the described range or feature is, this explanation should be applicable.

[0045] Example 1

[0046] This embodiment provides a method for preparing rabbit serum immunoglobulin G (IgG).

[0047] The preparation method comprises:

[0048] Sample pretreatment: The rabbit serum samples were appropriately diluted and filtered using a 0.45 μm filter membrane to remove particulate matter that might clog the chromatography column;

[0049] Equilibrate the column: Use phosphate buffered saline (PBS) to pass through the Protein A affinity chromatography column at a flow rate of 1 mL / min until the pH value of the effluent stabilizes at 7.4;

[0050] Sample loading: Load the pretreated sample onto the chromatography column, maintain a flow rate of 1 mL / min, and continue to wash with WashBuffer and PBS to completely elute the impurities;

[0051] Elution: Use glycine-HCl buffer to elute IgG from Protein A, and then use Tris-HCl to neutralize and stabilize IgG;

[0052] Ultrafiltration: Use ultrafiltration centrifuge tubes to concentrate the collected purified IgG, remove excess buffer, and adjust the IgG solution to the desired final volume and concentration.

[0053] Example 2

[0054] This embodiment provides a goat recombinant monoclonal antibody against rabbit IgG and its screening and preparation.

[0055] The rabbit serum IgG purified in Example 1 was used as an antigen to immunize goats; after the immunization procedure was completed, blood was collected from the jugular vein to obtain peripheral blood mononuclear cells (PBMC) of the goats; a B cell population that can efficiently secrete antibodies was obtained by antigen capture, cell sorting and limiting dilution, and a specific antibody B cell population secreting anti-rabbit IgG was screened out by indirect ELISA detection; the heavy and light chain nucleic acid sequences of B cells secreting specific antibodies were amplified by RT-PCR; the obtained nucleic acid sequence was constructed into a pcDNA3.4 vector by homologous recombination, TOP10 competent cells (DF1010M) were transformed and cultured at 37°C overnight, and single colonies were picked for sequencing to obtain heavy and light chain nucleotide sequences; plasmids connected with different heavy and light chain nucleic acid sequences were paired and combined, and transfected into Expi 293F cell lines by transfection reagents, and cell supernatants were collected; the supernatant of 293F cells after transfection was collected, and verified by ELISA and WB to confirm that the nucleic acid sequence encoding the heavy chain variable region shown in SEQ ID NO.3 was consistent with SEQ ID The antibody expressed by the plasmid of the nucleic acid sequence encoding the light chain variable region shown in NO.4 had a good immune response to the antigen, and this clone was named SDT-GAR-1.

[0056] The nucleic acid sequence encoding the heavy chain variable region (VH-DNA) is:

[0057] CTGTCGCAGGTTCAGCTGCAGGAGTCGGGACCCAGCCTGGTGAAGCCCTCACAGACCCTCTCCCTCACCTGCACGGTCTCTGGAGTCTCATTAAGTGACTCTGCTGTGGGCTGGGTCCCGCCAGGCTCCAGGAAAGGGGCTGGAGTGGCTTGGGATTATATTTAGTAATGGAAAAGCATTCTATCTGC CGGCCCTGAAATCCCGGCTCAAGCGTCACCAGGGACACCTCCAAGAGCCAAGTCTCCCTGTCACTGAGCAGCGTGACAACTGAAGACACAGCCGTGTACTATTGTGCAATCGGGAAGAATTATGTGGGCGCTATCCGCGGTGTAGATATTTGGGGCCGAGGACTCCTCGTCACCGTCTCCTCA(SEQID NO.3);

[0058] The nucleic acid sequence encoding the light chain variable region (VL-DNA) is:

[0059] TCCTATGAATTGACCCAGCCAACTTCTGTGTCGGTGGCCTTGGGACAGACGGCCGAGGTCACCTGCTCGGGAGACCTATTAAACGCGAATGTTGCTCACTGGTACCAGCAGAAGCCGGGCCAAGCCCCTGTGTTGCTCATTTATATGAATAATCAGTTGGCC TCGGAGATTCCTGCCCGGTTCTCCGGCTCCAGCTCAGGGAACACAGCCACCCTGACCATCAGCGGGGTCCAGGCTGAGGACGAGGCCGACTATTTCTGTCAATCTCTCACAGCACCGATCATATTTTCGGCAGCAAGACCAGGCTGACCGTCCTGGGT(SEQ ID NO.4).

[0060] According to the above nucleic acid sequences encoding the heavy chain variable region and the light chain variable region, the amino acid sequences of the heavy chain variable region (VH) and the light chain variable region (VL) of the corresponding antibody are obtained:

[0061] Amino acid sequence of the heavy chain variable region (VH):

[0062] LSQVQLQESGPSLVKPSQTLSLTCTVSGVSLSDSAVGWVRQAPGKGLEWLGIIFSNGKA FYLPALKSRLSVTRDTSKSQVSLSLSSVTTEDTAVYYCAIGKNYVGAIRGVDIWGRGLLVTV SS(SEQ ID NO.1);

[0063] Amino acid sequence of the light chain variable region (VL):

[0064] SYELTQPTSVSVALGQTAEVTCSGDLLNANVAHWYQQKPGQAPVLLIYMNNQLASEIP ARFSGSSSGNTATLTISGVQAEDEADYFCQSSHSTDHIFGSKTRLTVLG (SEQ ID NO. 2).

[0065] The nucleic acid (CH-DNA) sequence encoding the heavy chain constant region of antibody SDT-GAR-1 is:

[0066] GCCTCAACAACACCCCCGAAAGTCTACCCTCTGACTTCTTGCTGCGGGGACACGTCCAGCTCCATCGTGACCCTGGGCTGCCTGGTCTCCAGCTATATGCCCGAGCCGGTGACCGTGACCTGGAACTCTGGTGCCCTGACCAGCGGCGTGCACACCTTCCCGGCCATCCTGCAGTCCTCCGGGCTCTACTCTCTCAGCAGCGTGGTGACCGTGCCGGCCAGCACCTCAGGAGCCCAGACCTTCATCTGCAACGTAGCCCACCCGGCCAGCAGCACCAAGGTGGACAAGCGTGTTGAGCCCGGATGCCCGGACCCATGCAAACATTGCCGATGCCCACCCCCTGAGCTCCCCGGAGGACCGTCTGTCTTCATCTTCCCACCGAAACCCAAGGACACCCTTACAATCTCTGGAACGCCCGAGGTCACGTGTGTGGTGGTGGACGTGGGCCAGGATGACCCCGAGGTGCAGTTCTCCTGGTTCGTGGACAACGTGGAGGTGCGCACGGCCAGGACAAAGCCGAGAGAGGAGCAGTTCAACAGCACCTTCCGCGTGGTCAGCGCCCTGCCCATCCAGCACCAAGACTGGACTGGAGGAAAGGAGTTCAAGTGCAAGGTCCACAACGAAGCCCTCCCGGCCCCCATCGTGAGGACCATCTCCAGGACCAAAGGGCAGGCCCGGGAGCCGCAGGTGTACGTCCTGGCCCCACCCCAGGAAGAGCTCAGCAAAAGCACGCTCAGCGTCACCTGCCTGGTCACCGGCTTCTACCCAGACTACATCGCCGTGGAGTGGCAGAAAAATGGGCAGCCTGAGTCGGAGGACAAGTACGGCACGACCACATCCCAGCTGGACGCCGACGGCTCCTACTTCCTGTACAGCAGGCTCAGGGTGGACAAGAACAGCTGGCAAGAAGGAGACACCTACGCGTGTGTGGTGATGCACGAGGCTCTGCACAACCACTACACACAGAAGTCCATCTCTAAGCCTCCGGGTAAATGA(SEQID NO.7),

[0067] The amino acid sequence of the heavy chain constant region (CH) is:

[0068] ASTTPPKVYPLTSCCGDTSSSIVTLGCLVSSYMPEPVTVTWNSGALTSGVHTFPAILQSSGLYSLSSVVTVPASTSGAQTFICNVAHPASSTKVDKRVEPGCPDPCKHCRCPPPELPGGPSVFIFPPKPKDTLTISGTPEVTCVVVDVGQDDPEVQFSWFVDNVEVR TARTKPREEQFNSTFRVVSALPIQHQDWTGGKEFKCKVHNEALPAPIVRTISRTKGQAREPQVYVLAPPQEELSKSTLSVTCLVTGFYPDYIAVEWQKNGQPESEDKYGTTTSQLDADGSYFLYSRLRVDKNSWQEGDTYACVVMHEALHNHYTQKSISKPPGK(SEQ ID NO.5);

[0069] The nucleic acid sequence (CL-DNA) encoding the light chain constant region of antibody SDT-GAR-1 is:

[0070] CAGCCCAAGTCCGCACCCTCAGTCACCCTGTTCCCGCCCTCCACGGAGGAGCTCAATGCCAACAAGGCCACCGTGGTGTGTCTCATCAGCGACTTCTACCCGGGTAGCGTGACCGTGGTCTGGAAGGCAGATGGCAGCACCATCAATCAGAACGTGAAGAC CACCCAGGCCTCCAAACAGAGCAACAGCAAGTACGCGGCCAGCAGCTACCTGACCCTGACGGGCAGCGAGTGGAAGTCTAAGAGCAGTTACAGCTGCGAGGTCACGCACGAGGGGAGCACCGTGAAGAAGACAGTGAAGCCCTCAGAGTGTTCTTAG(SEQ ID NO.8),

[0071] The amino acid sequence of the light chain constant region (CL) is:

[0072] QPKSAPSVTLFPPSTEELNANKATVVCLISDFYPGSVTVVWKADGSTINQNVKTTQAS KQSNSKYAASSYLTLTGSEWKSKSSYSCEVTHEGSTVKKTVKPSECS (SEQ ID NO. 6).

[0073] Example 3

[0074] In this example, the expression and specificity verification of the antibody SDT-GAR-1 were performed.

[0075] (1) Expression of antibody SDT-GAR-1

[0076] The expression vectors containing the genes encoding the light chain (SEQ ID NO.4 and SEQ ID NO.8) and the heavy chain (SEQ ID NO.3 and SEQ ID NO.7) of the goat monoclonal antibody SDT-GAR-1 were paired in a 1:1 ratio and co-transfected into cells at a density of 3×10 6 / mL 100mL Expi 293F cells, the cell supernatant was collected after 5 days of culture, and the antibody SDT-GAR-1 was purified by affinity chromatography using Protein A resin.

[0077] Example 4

[0078] This example provides specificity and affinity verification of the antibody SDT-GAR-1.

[0079] (1) Enzyme-linked immunosorbent assay

[0080] The specificity of the antibody SDT-GAR-1 anti-rabbit IgG was evaluated by indirect enzyme-linked immunosorbent assay (ELISA).

[0081] The specific experimental steps include:

[0082] Rabbit, human, mouse, rat and bovine serum were used as antigens, coated with carbonate buffer (50 mM, pH 9.6), added to the ELISA plate at a volume of 100 μL / well, and incubated at 4°C overnight;

[0083] The next day, the cells were blocked with 1% bovine serum albumin (BSA) at 37°C for 1 h.

[0084] The purified antibody SDT-GAR-1 was used as the primary antibody and the commercially available antibody as the positive control, and was diluted in a gradient ratio of 1:4 and incubated at 37°C for 1 hour;

[0085] Add 1:5000 secondary antibody (bovine anti-sheep) (manufacturer: Jackson, catalog number: 805-035-180) and incubate at 37°C for 1 h;

[0086] TMB color developing solution (manufacturer: Solebow, catalog number: PR-1200) was added for color development, and the absorbance value of OD450 was read by an enzyme reader.

[0087] Result analysis:

[0088] Taking OD450 ≥ 0.3 as positive, the ELISA test results showed that the antibody SDT-GAR-1 can specifically recognize IgG in rabbit serum (as shown in Table 1). It is not difficult to find from OD450 that with the increase of the antibody dilution multiple, although the OD450 value of SDT-GAR-1 decreases, it still remains above the positive judgment standard, showing a higher sensitivity than commercially available antibodies, and the cross-reaction with serum IgG of other species is also less than that of commercially available antibodies. The above results show that SDT-GAR-1 can specifically detect rabbit IgG.

[0089] Table 1 Indirect ELISA results of cross-reactivity between antibody SDT-GAR-1 and commercially available antibodies

[0090]

[0091] (2) Protein immunoblotting

[0092] The specificity of the SDT-GAR-1 anti-rabbit IgG antibody was evaluated by Western blot (WB).

[0093] The cell samples were Hela, NIH / 3T3, and C6 cells in good culture. Histone H3 protein rabbit monoclonal antibody (manufacturer: Startech, catalog number: S0B2239P, concentration: 0.5 mg / mL) was used as the primary antibody at a dilution of 1:1000, the purified antibody SDT-GAR-1 was used as the secondary antibody (1:500), the commercially available antibody was used as the positive control, and HRP-crosslinked donkey anti-sheep (manufacturer: Jackson, catalog number: 705-005-003) (1:10000) was used as the tertiary antibody. The specific experimental steps included:

[0094] Dissolve RIPA lysis buffer (Radio Immunoprecipitation Assay Lysis buffer, radioimmunoprecipitation lysis buffer), and add an appropriate amount of protease inhibitor (PMSF) at a concentration of 1mM; add 200μL of RIPA to each well of cells to fully wet the cell surface, lyse the cells on ice for 15min, centrifuge at 1000rpm at 4℃ for 10min, and take the supernatant; install the electrophoresis tank, use 4% to 20% gradient gel to load the sample, add 10μL of protein sample to the gel well tank per well, voltage 100V, 60min, after observing obvious separation of the bands, end the electrophoresis, peel and cut the gel for transfer; cut the PVDF membrane slightly larger than the gel, and the special filter paper, activate the PVDF membrane with methanol, wet the filter paper and gel with transfer buffer, install them on the sponge pad of the transfer instrument in the order of filter paper, gel, membrane, and filter paper from negative to positive, and use a plate to remove bubbles; install the transfer instrument, the transfer process is under ice bath conditions, set the voltage, and transfer for 60min;

[0095] After the transfer, shake with 15mL of milk powder blocking solution at room temperature for 2h, dilute the corresponding antibody with 1×PBS, prepare the primary antibody dilution solution, incubate at 4℃ overnight, and then wash the PVDF membrane with TBST three times, 10 minutes each time; then prepare the corresponding secondary antibody with TSBT, incubate the PVDF membrane, and incubate on a shaker at room temperature for 2 hours; then prepare the corresponding tertiary antibody with TSBT, incubate the PVDF membrane, and incubate on a shaker at room temperature for 2 hours; finally, wash the membrane three times with TBST, 10 minutes each time; finally, prepare the developer in a 1:1 ratio, incubate the PVDF membrane for 5 minutes, and take pictures.

[0096] Result analysis:

[0097] In immunoblotting, SDT-GAR-1 monoclonal antibody showed excellent performance. Figure 1 As shown, compared with commercially available antibodies, the detection results of SDT-GAR-1 antibody in Hela (human), NIH / 3T3 (mouse) and C6 (rat) cell lines showed that its bands were single and clear, and no obvious mixed bands were observed. This result not only confirmed that the SDT-GAR-1 antibody can specifically recognize and bind to rabbit IgG, but also showed that it does not cross-react with IgG from other species, showing a high degree of specificity. Therefore, when used as a secondary antibody in Western blot experiments, the SDT-GAR-1 antibody can provide detection effects comparable to commercially available antibodies, and has higher specificity and affinity.

[0098] (3) Immunohistochemistry

[0099] Immunohistochemistry (IHC) was used to evaluate the specificity and affinity of the antibody SDT-GAR-1 anti-rabbit IgG.

[0100] The applicant's self-developed rabbit anti-Vimentin (manufacturer: Startech, catalog number: S0B2254P) was used as the primary antibody, the monoclonal antibody SDT-GAR-1 was used as the secondary antibody, the commercially available antibody was used as the positive control, and the HRP-crosslinked donkey anti-sheep (manufacturer: Jackson, catalog number: 705-005-003) was used as the tertiary antibody. The specific experimental steps included:

[0101] Place the cancer tissue sections in a drying oven at 63℃~65℃ for about 1 hour;

[0102] Dewaxing and hydration: (All operations were completed in a fume hood) The sections were placed in (1) xylene twice, 7 minutes each time; (2) anhydrous ethanol twice, 5 minutes each time; (3) 90% ethanol twice, 5 minutes each time; (4) 75% ethanol once, 3 minutes; (5) 50% ethanol once, 3 minutes; after finishing, remove the sections and rinse with distilled water three times, 5 minutes each time;

[0103] Antigen repair: Place the slices in a staining box containing sodium citrate buffer or EDTA repair solution, add 2L of distilled water to the antigen repair pot, place the staining box in a pressure cooker for high temperature and high pressure repair for 20 minutes; then end the heating, open the lid, take out the container containing the tissue slices, cool naturally to room temperature, rinse with distilled water, and wash 3 times with 1×PBS, 3 minutes each time;

[0104] Place the antigen-recovered sections in a 3% H2O2 solution for 10 minutes (operate in a fume hood), then wash three times with 1×PBS for 5 minutes each time to block endogenous peroxidase; block nonspecific antigens with 5% BSA + 10% sheep serum (prepared with PBST) at room temperature for 30 minutes. After blocking, knock off the blocking solution on the tissue sections, add the diluted primary antibody, 80-100 μL per well, adjust appropriately according to the size of the immunohistochemistry pen circle, put the slides into a wet box, and then slowly put them into a refrigerator at 4°C and let them stand overnight;

[0105] After incubation, take out the wet box and let it stand at room temperature for 10 minutes; rinse 3 times with 1×PBST, 5 minutes each time, add secondary antibody, and incubate at room temperature for 30 minutes; wash 3 times with 1×PBST, 5 minutes each time, and add tertiary antibody;

[0106] Use DAB colorimetric solution for color development, 100 μL per slice, add freshly prepared colorimetric solution to each slice, color development for 1 to 2 minutes, observe under a microscope, and stop color development when brown-yellow color appears, then rinse with distilled water;

[0107] Restain with hematoxylin for 2 to 5 minutes, rinse with distilled water for 5 minutes, decolorize in 1×PBS for 30 seconds, and rinse with distilled water for 5 minutes; dehydrate and dry with gradient ethanol, (1), 50% ethanol, dehydrate for 5 minutes; (2), 75% ethanol, dehydrate for 5 minutes; (3), 90% ethanol, dehydrate for 5 minutes; (4), dehydrate with anhydrous ethanol 3 times, 5 minutes each time; (5), dehydrate with xylene 2 times, 5 minutes each time;

[0108] Add 50-100 μL of neutral gum to each tissue section, and then slowly add a coverslip;

[0109] The sealed slides were dried overnight and observed under a microscope the next day and scanned with a tissue slicer to obtain the immunohistochemical results.

[0110] Result analysis:

[0111] The anti-rabbit IgG recombinant monoclonal antibody SDT-GAR-1 applied in this study showed clear staining effect and accurate localization in immunohistochemistry (IHC) staining results in human breast cancer tissue and human gastric tissue. Compared with commercially available antibodies, SDT-GAR-1 showed no significant difference in staining effect, such as Figure 2 As shown. This shows that SDT-GAR-1 can specifically recognize rabbit IgG, thus confirming that the antibody has a high degree of specificity and affinity for rabbit IgG protein. These results not only demonstrate the potential application of SDT-GAR-1 in IHC experiments, but also highlight its reliability and effectiveness in detecting rabbit antibodies.

[0112] Example 5

[0113] This example provides HRP-labeled monoclonal antibody SDT-GAR-1 and a preparation method thereof.

[0114] The preparation method thereof comprises:

[0115] Take 5 mg of the antibody to be labeled, add 1455 μL 1× PBS, 45 μL 1 M sodium carbonate solution, and adjust the pH of the antibody to 9.6;

[0116] Weigh 2 mg of horseradish peroxidase (HRP) and dissolve it in 0.4 mL of distilled water. Add 0.6 mg of HRP to 1 mg of the purified antibody SDT-GAR-1.

[0117] Weigh an appropriate amount of NaIO4 powder, add an appropriate amount of pure water according to the weighed amount, so that the final concentration is 20 mg / mL; add 45 μL of freshly prepared 20 mg / ml NaIO4 solution to the upper liquid, and react at room temperature in the dark for 20 minutes (add 22.5 μL of 20 mg / mL NaIO4 solution for every mg of HRP, the actual amount added is determined by the mass of HRP, and is scaled up in equal amounts);

[0118] Add 40 μL of ethylene glycol and react at room temperature in the dark for 30 min (add 20 μL of ethylene glycol for every mg of HRP. The actual amount added is determined by the mass of HRP and is amplified in equal amounts);

[0119] Add the activated HRP to the dialyzed antibody and mix well; dialyze in 2L of 0.05M pH 9.6 carbonate buffer and stir at 4°C overnight;

[0120] Weigh an appropriate amount of NaBH solution, add an appropriate amount of pure water to dissolve it, so that the final concentration is 10 mg / mL; add 80 μL of freshly prepared 10 mg / ml NaBH solution, mix well, react at 4°C in the dark for 2 hours, replace with 0.01M pH7.4 PBS for dialysis, and incubate at 4°C overnight (change PBS once in the middle);

[0121] Pipette the dialysate into a 2 mL centrifuge tube, add 50% saturated ammonium sulfate solution, let stand at 4°C for 30 min, centrifuge at 12000 rpm, 30 min, 4°C, discard the supernatant, and drain; if the volume is 500 μL, add 250 μL of saturated ammonium sulfate solution, and so on;

[0122] The precipitate was dissolved in a small amount of 0.01M pH7.4 PBS (500 μL), and half the volume of saturated ammonium sulfate solution was added. After standing at 4°C for 30 min, the mixture was centrifuged at 12000 rpm for 30 min at 4°C.

[0123] The supernatant was discarded, the precipitate was drained, and the precipitate was dissolved in a small amount of PBS. The volume of PBS was calculated according to the amount of cross-linked antibody (multiply the cross-linked antibody (mg) by 0.8 and then divided by 20, which is the volume of PBS (mL)). After cross-linking, the HRP-labeled monoclonal antibody SDT-GAR-1 was obtained and named Goat Anti-Rabbit IgG (H+L) (HRP Conjugate).

[0124] Example 6

[0125] This example provides the application of the HRP-labeled monoclonal antibody SDT-GAR-1.

[0126] In this example, Goat Anti-Rabbit IgG (H + L) (HRP Conjugate) was used as the secondary antibody for indirect ELISA detection, and a commercially available antibody was used as a positive control. The specific steps were referred to the enzyme-linked immunosorbent assay in Implementation 4, except that: the primary antibody was the applicant's self-developed product Keratin 5 Recombinant Rabbit mAb (SDT-014-67) (manufacturer: Start, item number: S0B2033), with an initial concentration of 1000 ng / μL; the secondary antibody used was the Goat Anti-Rabbit IgG (H + L) (HRP Conjugate) provided in Example 5, diluted 1:10000 and incubated.

[0127] Result analysis:

[0128] The results are as follows Figure 3 As shown, as the concentration of the primary antibody decreases, the OD450 also gradually decreases. This trend is consistent with the antibody products sold on the market, showing no significant difference. These findings indicate that there is a high affinity between Goat Anti-Rabbit IgG (H + L) (HRPConjugate) and the rabbit primary antibody, and its performance is comparable to that of antibody products on the market. Therefore, it can be inferred that Goat Anti-Rabbit IgG (H + L) (HRP Conjugate) has potential application value as an enzyme-labeled secondary antibody in ELISA experiments, and can provide detection effects comparable to those of commercially available antibodies.

[0129] Example 7

[0130] This example provides the application of the HRP-labeled monoclonal antibody SDT-GAR-1.

[0131] In this example, Goat Anti-Rabbit IgG (H+L) (HRP Conjugate) was used as a secondary antibody, and a commercially available antibody was used as a positive control for WB detection. The specific steps were referred to the protein immunoblotting in Implementation 4, except that: HeG2 cells were added to this example, the primary antibody used was the applicant's self-developed product β-actin Recombinant Rabbit mAb (manufacturer: Start, item number: S0B0074), and the secondary antibody used was the Goat Anti-Rabbit IgG (H+L) (HRPConjugate) provided in Example 5.

[0132] Result analysis:

[0133] like Figure 4As shown, Goat Anti-Rabbit IgG (H + L) (HRP Conjugate) and commercially available antibodies both showed a clear single band in the Western blot experiment, and no other non-specific miscellaneous bands were observed. Under the same experimental conditions, compared with commercially available antibodies, the band intensity of Goat Anti-Rabbit IgG (H + L) (HRP Conjugate) was more significant, indicating that the antibodies of the present application have higher specificity, affinity and sensitivity. These characteristics make Goat Anti-Rabbit IgG (H + L) (HRP Conjugate) have obvious advantages as an enzyme-labeled secondary antibody in Western blot experiments, and can provide more reliable detection results.

[0134] Example 8

[0135] This example provides the application of the HRP-labeled monoclonal antibody SDT-GAR-1.

[0136] In this embodiment, Goat Anti-Rabbit IgG (H + L) (HRP Conjugate) was used as a secondary antibody for immunohistochemistry detection. The specific steps refer to the immunohistochemistry in Implementation 4, except that: the detection materials used in this embodiment are gastric cancer tissue, thyroid cancer tissue and ovarian cancer tissue, wherein the primary antibody for the first tissue is the applicant's self-developed rabbit anti-Claudin 18.2 (manufacturer: Start, item number: S0B2070P), and the primary antibodies for the latter two tissues are rabbit anti-PAX8 (manufacturer: Start, item number: S0B2071P), and PBS is used as a negative control for the primary antibody, and the secondary antibody is Goat Anti-Rabbit IgG (H + L) (HRP Conjugate).

[0137] Result analysis:

[0138] like Figure 5As shown, the experimental group showed accurate localization compared with the PBS control group, which confirmed that Goat Anti-Rabbit IgG(H+L)(HRP Conjugate) could specifically bind to rabbit IgG. This result indicates that Goat Anti-Rabbit IgG(H+L)(HRP Conjugate) can effectively be used as a secondary antibody in immunohistochemistry (IHC) experiments to play its detection role. In addition, despite the differences in the rabbit primary antibodies used, Goat Anti-Rabbit IgG(H+L)(HRPConjugate) can still recognize rabbit IgG proteins, further demonstrating its wide applicability and high recognition ability for rabbit antibodies. These findings emphasize the potential and reliability of Goat Anti-Rabbit IgG(H+L)(HRP Conjugate) as a secondary antibody in IHC experiments.

[0139] It is understandable that those skilled in the art can make equivalent substitutions or changes based on the technical solution and concept of the present invention, and all these changes or substitutions should fall within the protection scope of the claims attached to the present invention.

Claims

1. A goat recombinant monoclonal antibody against rabbit IgG, characterized in that: The amino acid sequence of the heavy chain variable region of the recombinant monoclonal antibody is shown in SEQ ID NO.1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.

2.

2. The goat recombinant monoclonal antibody against rabbit IgG according to claim 1, characterized in that: The amino acid sequence of the heavy chain constant region of the recombinant monoclonal antibody is shown in SEQ ID NO.5, and the amino acid sequence of the light chain constant region is shown in SEQ ID NO.

6.

3. A nucleic acid, characterized in that The nucleic acid encodes the heavy chain variable region and / or light chain variable region of the anti-rabbit IgG goat recombinant monoclonal antibody according to claim 1 or 2.

4. A nucleic acid according to claim 3, characterized in that The nucleic acid comprises: The nucleotide sequence shown in SEQ ID NO.3 is used to encode the heavy chain variable region of the amino acid sequence shown in SEQ ID NO.1; The nucleotide sequence shown in SEQ ID NO.4 is used to encode the light chain variable region of the amino acid sequence shown in SEQ ID NO.

2.

5. A recombinant expression vector, characterized in that: The recombinant expression vector contains the nucleic acid according to claim 3 or 4.

6. A recombinant expression cell, characterized in that: The recombinant expression cell comprises the recombinant expression vector according to claim 5, or the nucleic acid according to claim 3 or 4.

7. Use of the nucleic acid according to claim 3 or 4, or the recombinant expression vector according to claim 5, or the recombinant expression cell according to claim 6 in preparing goat recombinant monoclonal antibodies against rabbit IgG.

8. The method for preparing the anti-rabbit IgG goat recombinant monoclonal antibody according to claim 1, characterized in that: The method comprises: Transfecting cells with the recombinant expression vector of claim 5 to obtain recombinant expression cells; culturing the transfected recombinant expression cells; collecting and purifying the supernatant to obtain anti-rabbit IgG recombinant monoclonal antibodies; Or culturing the recombinant expression cell according to claim 6; collecting and purifying the supernatant to obtain the anti-rabbit IgG recombinant monoclonal antibody.

9. A HRP-labeled goat recombinant monoclonal antibody against rabbit IgG, characterized in that: The recombinant monoclonal antibody is the anti-rabbit IgG goat recombinant monoclonal antibody according to claim 1 or 2.

10. Use of the recombinant monoclonal antibody according to claim 1, 2 or 9, or the nucleic acid according to claim 3 or 4, or the recombinant expression vector according to claim 5, or the recombinant expression cell according to claim 6, or the preparation method according to claim 8 in detecting rabbit IgG or preparing a product for detecting rabbit IgG.

11. The use according to claim 10, characterized in that: The detection of rabbit IgG includes any one or more of immunohistochemistry, protein immunoblotting or ELISA.

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