Goat recombinant monoclonal antibody against rabbit IgG and preparation and application thereof

Abstract

The application discloses a goat recombinant monoclonal antibody against rabbit IgG and a preparation and application thereof, and belongs to the technical field of biotechnology. The recombinant monoclonal antibody is obtained by taking purified rabbit serum IgG as an immunogen, immunizing a goat, screening specific B cells, and obtaining a heavy chain variable region and a light chain variable region of the antibody through RT-PCR. The variable region sequences are respectively inserted into a vector plasmid with constant region (heavy chain and light chain) genes, and the recombinant monoclonal antibody is obtained through transfection expression and purification. The recombinant monoclonal antibody provided in the application has high affinity with rabbit IgG, can recognize and detect rabbit IgG with high specificity and high sensitivity, and can be used as a secondary antibody after HRP cross-linking, applied to methods such as protein immunoblotting, immunohistochemistry and ELISA for detecting rabbit IgG, is conducive to obtaining more accurate detection and evaluation results, and reduces the interference of detection cost and background signal.

Description

Technical Field

[0001] This application belongs to the field of biotechnology, specifically relating to a goat recombinant monoclonal antibody against rabbit IgG and its preparation and application. Background Technology

[0002] When humans and animals develop diseases, they rely on their own immune response. Specific immunity plays an important role, and 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 also the main target antibody for serological diagnosis and post-vaccination detection.

[0003] Rabbit-derived antibodies are renowned for their high affinity, specificity, and diversity. Furthermore, rabbits and mice / greater mice differ in evolution, allowing rabbit-derived antibodies to recognize human antigenic epitopes that are not immunogenic in mice, increasing the total number of target epitopes and facilitating the production of antibodies that cross-react with human antigens. Anti-rabbit IgG antibodies are used as secondary antibodies in immunology to detect rabbit-derived primary antibodies. They can bind to markers, helping to detect and quantify the binding of rabbit-derived antibodies to specific antigens. Therefore, the preparation of highly sensitive and accurate anti-rabbit IgG antibodies is of significant importance for scientific research, diagnostic testing, and therapeutic applications.

[0004] Sheep-derived antibodies offer unique advantages as secondary antibodies. Sheep possess five immunoglobulin isotypes, composed of five heavy chain classes (IgM, IgG, IgA, IgD, and IgE), with IgG further subdivided into three subtypes (IgG1, IgG2, and IgG3). Like other species, the heavy chain is bound to two light chains (Lambda and Kappa). IgG-λ accounts for 95% of IgG in sheep. Sheep antibodies typically have 10 to 100 times higher affinity than rodent antibodies. Furthermore, sheep monoclonal antibodies bind to only a specific region and can precisely distinguish closely related molecules (e.g., derivatives), potentially producing highly specific antibodies against desired targets. In addition, sheep possess a unique immune system, enabling sheep antibodies to recognize "difficult" targets where other antibody technologies have failed, and natural sheep antibodies can recognize a variety of heterotopes.

[0005] In the current technological 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 Beyotime is prepared by immunizing goats with rabbit IgG, then purifying the obtained antiserum with an affinity purification column, and then purifying it by adsorption with human IgG, mouse IgG and rat IgG. It is still not a monoclonal antibody, so there is still room for improvement in its specificity. Summary of the Invention

[0006] 1. Purpose of the invention

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

[0008] 2. Technical Solution

[0009] To achieve the above objectives, the technical solution adopted in this application is as follows:

[0010] This application provides a goat recombinant monoclonal antibody against rabbit IgG, which can specifically bind to rabbit IgG protein. 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 above-mentioned goat recombinant monoclonal antibody against rabbit IgG has the heavy chain constant region amino acid sequence shown in SEQ ID NO.5 and the light chain constant region amino acid sequence shown in SEQ ID NO.6.

[0012] This application also provides a nucleic acid that encodes the heavy chain variable region and / or light chain variable region of the aforementioned anti-rabbit IgG goat recombinant monoclonal antibody.

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

[0014] Furthermore, the nucleotide sequence of the aforementioned goat recombinant monoclonal antibody encoding anti-rabbit IgG includes:

[0015] The heavy chain variable region of the amino acid sequence shown in SEQ ID NO.1, which encodes the above-mentioned anti-rabbit IgG, is the nucleotide sequence shown in SEQ ID NO.3.

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

[0017] Furthermore, the aforementioned nucleic acids also include:

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

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

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

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

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

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

[0024] This application also provides the application of the above-mentioned nucleic acid, recombinant expression vector, and recombinant expression cell in the preparation of goat recombinant monoclonal antibody against rabbit IgG.

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

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

[0027] This application also provides an HRP-labeled goat recombinant monoclonal antibody against the above-mentioned anti-rabbit IgG.

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

[0029] Furthermore, the above-mentioned detection of rabbit IgG includes any one or more of the detection methods such as immunohistochemistry, ELISA, and Western blotting.

[0030] 3. Beneficial effects

[0031] Compared with the prior art, the advantages of this application are as follows:

[0032] This application provides a goat recombinant monoclonal antibody against rabbit IgG, its preparation, and its application. Comparison with commercially available antibodies shows that the goat recombinant monoclonal antibody against rabbit IgG provided in this application has high specificity and high sensitivity in binding to rabbit IgG protein molecules. It can specifically recognize and detect rabbit IgG protein, and shows positive high expression when detecting rabbit IgG protein. Therefore, this recombinant monoclonal antibody can be applied to experiments such as immunohistochemistry, Western blotting, and indirect ELISA. Attached Figure Description

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

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

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

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

[0037] Figure 5 This is the IHC test result of the HRP-labeled monoclonal antibody SDT-GAR-1. Detailed Implementation

[0038] The present application will be further described below with reference to specific embodiments.

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

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

[0041] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0042] As used herein, the term “about” is used to provide for the flexibility and imprecision associated with a given term, measure, or value. Those skilled in the art can readily determine the degree of flexibility for a particular variable.

[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” explicitly includes only A, only B, only C, and combinations thereof.

[0044] Concentration, amount, and other numerical data may be presented in range format herein. It should be understood that such range format is used solely for convenience and brevity and should be flexibly interpreted to include not only the values ​​explicitly stated as the limits of the range, but also all individual values ​​or subranges encompassed within the range, as if each value and subrange were explicitly stated. For example, a range of values ​​from about 1 to about 4.5 should be interpreted to include not only the explicitly stated limits of 1 to 4.5, but also individual numbers (such as 2, 3, 4) and subranges (such as 1 to 3, 2 to 4, etc.). The same principle applies to ranges that describe only a single value, such as “less than about 4.5,” which should be interpreted to include all the aforementioned values ​​and ranges. Furthermore, this interpretation should apply regardless of the breadth of the range or characteristic described.

[0045] Example 1

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

[0047] The preparation method includes:

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

[0049] Equilibration of the chromatography column: phosphate buffered saline (PBS) was passed through the Protein A affinity chromatography column at a flow rate of 1 mL / min until the pH of the effluent stabilized at 7.4.

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

[0051] Elution: IgG was eluted from Protein A using glycine-hydrochloric acid buffer, and then neutralized and stabilized with Tris-HCl;

[0052] Ultrafiltration: The collected purified IgG was concentrated using ultrafiltration centrifuge tubes to 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] Goats were immunized with purified rabbit serum IgG from Example 1. After the immunization procedure, peripheral blood mononuclear cells (PBMCs) were obtained by collecting blood from the jugular vein. A population of B cells capable of efficiently secreting antibodies was obtained through antigen capture, cell sorting, and limiting dilution. Specific antibody-secreting B cells secreting anti-rabbit IgG were screened using indirect ELISA. The heavy and light chain nucleic acid sequences of the antibody-secreting B cells were amplified by RT-PCR. The obtained nucleic acid sequences were constructed into the pcDNA3.4 vector using homologous recombination, transformed into TOP10 competent cells (DF1010M), and cultured overnight at 37°C. Single colonies were picked and sequenced to obtain the heavy and light chain nucleotide sequences. Plasmids containing different heavy and light chain nucleic acid sequences were paired and transfected into the Expi 293F cell line using a transfection reagent. Cell supernatant was collected. The supernatant of the transfected 293F cells was verified by ELISA and Western blotting to confirm that the nucleic acid sequence encoding the heavy chain variable region shown in SEQ ID NO.3 was consistent with SEQ ID NO.3. The plasmid encoding the nucleic acid sequence of the light chain variable region shown in NO.4 expressed an antibody that showed a good immune response to the antigen. This clone was named SDT-GAR-1.

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

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

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

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

[0060] Based on the above nucleic acid sequences encoding the heavy chain variable region and the light chain variable region, the amino acid sequences of the corresponding antibody's heavy chain variable region (VH) and light chain variable region (VL) 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 as follows:

[0066] GCCTCAACAACACCCCCGAAAGTCTACCCTCTGACTTCTTGCTGCGGGGACACGTCCAGCTCCATCGTGACCCTGGGCTGCCTGGTCTCCAGCTATATGCCCGAGCCGGTGACCGTGACCTGGAACTCTGGTGCCCTGACCAGCGGCGTGCACACCTTCCCGGCCATCCTGCAGTCCTCCGGGCTCTACTCTCTCAGCAGCGTGGTGACCGTGCCGGCCAGCACCTCAGGAGCCCAGACCTTCATCTGCAACGTAGCCCACCCGGCCAGCAGCACCAAGGTGGACAAGCGTGTTGAGCCCGGATGCCCGGACCCATGCAAACATTGCCGATGCCCACCCCCTGAGCTCCCCGGAGGACCGTCTGTCTTCATCTTCCCACCGAAACCCAAGGACACCCTTACAATCTCTGGAACGCCCGAGGTCACGTGTGTGGTGGTGGACGTGGGCCAGGATGACCCCGAGGTGCAGTTCTCCTGGTTCGTGGACAACGTGGAGGTGCGCACGGCCAGGACAAAGCCGAGAGAGGAGCAGTTCAACAGCACCTTCCGCGTGGTCAGCGCCCTGCCCATCCAGCACCAAGACTGGACTGGAGGAAAGGAGTTCAAGTGCAAGGTCCACAACGAAGCCCTCCCGGCCCCCATCGTGAGGACCATCTCCAGGACCAAAGGGCAGGCCCGGGAGCCGCAGGTGTACGTCCTGGCCCCACCCCAGGAAGAGCTCAGCAAAAGCACGCTCAGCGTCACCTGCCTGGTCACCGGCTTCTACCCAGACTACATCGCCGTGGAGTGGCAGAAAAATGGGCAGCCTGAGTCGGAGGACAAGTACGGCACGACCACATCCCAGCTGGACGCCGACGGCTCCTACTTCCTGTACAGCAGGCTCAGGGTGGACAAGAACAGCTGGCAAGAAGGAGACACCTACGCGTGTGTGGTGATGCACGAGGCTCTGCACAACCACTACACACAGAAGTCCATCTCTAAGCCTCCGGGTAAATGA(SEQID NO.7),

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

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

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

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

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

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

[0073] Example 3

[0074] This embodiment demonstrates the expression and specificity verification of the antibody SDT-GAR-1.

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

[0076] Expression vectors containing the genes for 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 1:1 and co-transfected into cells at a density of 3 × 10⁶ cells / year. 6 Cells were cultured in 100 mL of Expi 293F cells for 5 days. The cell supernatant was collected and purified by affinity chromatography using Protein A resin to obtain the antibody SDT-GAR-1.

[0077] Example 4

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

[0079] (1) Enzyme-linked immunosorbent assay

[0080] The specificity of antibody SDT-GAR-1 against rabbit IgG was assessed using 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 (50mM, pH 9.6), and added to the microplate at a rate of 100μL / well, and incubated overnight at 4°C.

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

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

[0085] Add 1:5000 secondary antibody (bovine anti-sheep) (manufacturer: Jackson, product number: 805-035-180), incubate at 37℃ for 1 hour;

[0086] Add TMB chromogenic reagent (manufacturer: Solarbio, product number: PR-1200) for color development, and read the absorbance value at OD450 using an ELISA reader.

[0087] Results analysis:

[0088] With an OD450 ≥ 0.3 as a positive result, the ELISA results showed that the antibody SDT-GAR-1 could specifically recognize IgG in rabbit serum (as shown in Table 1). It is easy to see from the OD450 that although the OD450 value of SDT-GAR-1 decreased with the increase of antibody dilution, it still remained above the positive judgment standard, showing higher sensitivity than commercially available antibodies. Moreover, the cross-reactivity with IgG from other species serum was also less than that of commercially available antibodies. The above results indicate that SDT-GAR-1 can specifically detect rabbit IgG.

[0089] Table 1. Results of indirect ELISA for cross-reactivity between antibody SDT-GAR-1 and commercially available antibodies.

[0090]

[0091] (2) Protein immunoblotting

[0092] The specificity of antibody SDT-GAR-1 against rabbit IgG was assessed using Western blot (WB).

[0093] Cell samples were HeLa, NIH / 3T3, and C6 cells in good culture condition. The primary antibody was a rabbit monoclonal antibody against Histone H3 protein (manufacturer: Startec, catalog number: S0B2239P, concentration: 0.5 mg / mL) diluted 1:1000. The secondary antibody was purified SDT-GAR-1 (1:500). A commercially available antibody was used as a positive control. The third antibody was HRP-crosslinked donkey anti-goat antibody (manufacturer: Jackson, catalog number: 705-005-003) (1:10000). Specific experimental procedures included:

[0094] Dissolve RIPA lysis buffer at room temperature (Radio Immunoprecipitation Assay Lysis) Prepare a 1mM buffer (radioimmunoprecipitation lysis buffer) and add an appropriate amount of protease inhibitor (PMSF). Add 200μL of RIPA to each well to fully wet the cell surface. Lyse the cells on ice for 15 min, centrifuge at 1000rpm for 10 min at 4℃, and collect the supernatant. Set up the electrophoresis tank and use a 4%–20% gradient gel for sample loading. Add 10μL of protein sample to each well. Set the voltage to 100V and run for 60 min. After observing clear band separation, stop electrophoresis, peel off and cut the gel for transfer. Cut a PVDF membrane slightly larger than the gel and special filter paper. Activate the PVDF membrane with methanol. Wet the filter paper and gel with transfer buffer. Install the filter paper, gel, membrane, and filter paper in the order of negative to positive on the sponge pad of the transfer apparatus, and remove air bubbles with a plate. Set up the transfer apparatus and transfer the membrane under ice bath conditions. Set the voltage and transfer for 60 min.

[0095] After transfer, the membrane was shaken at room temperature for 2 hours with 15 mL of milk powder blocking buffer. The corresponding antibody was diluted with 1×PBS to prepare the primary antibody dilution buffer, and incubated overnight at 4°C. The PVDF membrane was then washed three times with TBST for 10 minutes each time. The corresponding secondary antibody was then prepared with TSBT and incubated on a shaker at room temperature for 2 hours. The corresponding triantibody was then prepared with TSBT and incubated on a shaker at room temperature for 2 hours. Finally, the membrane was washed three times with TBST for 10 minutes each time. The developing solution was prepared at a 1:1 ratio and incubated on the PVDF membrane for 5 minutes before developing and photographing.

[0096] Results analysis:

[0097] In Western blot assays, the SDT-GAR-1 monoclonal antibody demonstrated superior performance. For example... 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, with no obvious impurities observed. This result not only confirms that SDT-GAR-1 antibody can specifically recognize and bind to rabbit IgG, but also indicates that it does not cross-react with IgG from other species, demonstrating high specificity. Therefore, when used as a secondary antibody in Western blot assays, SDT-GAR-1 antibody can provide detection results comparable to commercially available antibodies, with higher specificity and affinity.

[0098] (3) Immunohistochemistry

[0099] The specificity and affinity of antibody SDT-GAR-1 against rabbit IgG were assessed using immunohistochemistry (IHC).

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

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

[0102] Dewaxing and hydration: (All operations are performed in a fume hood) The sections are placed in the following order: (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 the operation, the sections are removed and rinsed with distilled water three times, 5 minutes each time.

[0103] Antigen retrieval: Place the tissue sections in a staining chamber containing sodium citrate buffer or EDTA retrieval solution. Add 2L of distilled water to the antigen retrieval pot and place the staining chamber in a pressure cooker for high-temperature and high-pressure retrieval for 20 minutes. Then, stop heating, open the lid, remove the container containing the tissue sections, allow it to cool naturally to room temperature, rinse with distilled water, and wash three times with 1×PBS for 3 minutes each time.

[0104] After antigen retrieval, the slides were placed in 3% H2O2 solution for 10 minutes (operated in a fume hood), then washed three times with 1×PBS for 5 minutes each time to block endogenous peroxidase. Non-specific antigens were blocked with 5% BSA + 10% sheep serum (prepared using PBST) at room temperature for 30 minutes. After blocking, the blocking solution on the tissue slides was knocked off, and diluted primary antibody was added at 80-100 μL per well, adjusting the amount according to the size of the immunohistochemistry pen circle. The slides were then placed in a humidified chamber and slowly placed in a refrigerator at 4°C overnight.

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

[0106] Use DAB chromogenic solution for chromogenic development. Add 100 μL of freshly prepared chromogenic solution to each slice and develop for 1-2 minutes. Observe under a microscope until a brownish-yellow color appears. Then rinse with distilled water to stop the chromogenic process.

[0107] Counterstain with hematoxylin for 2-5 minutes, rinse with distilled water for 5 minutes, destain and return to blue in 1×PBS for 30 seconds, rinse with distilled water for 5 minutes; then dehydrate and dry with a gradient of ethanol: (1) 50% ethanol, dehydrate for 5 minutes; (2) 75% ethanol, dehydrate for 5 minutes; (3) 90% ethanol, dehydrate for 5 minutes; (4) anhydrous ethanol, dehydrate 3 times, 5 minutes each time; (5) xylene, dehydrate 2 times, 5 minutes each time.

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

[0109] After mounting, the slides were left to air dry overnight. The next day, they were observed under a microscope and the immunohistochemical results were obtained by scanning the slides with a tissue sectioning instrument.

[0110] Results analysis:

[0111] The anti-rabbit IgG recombinant monoclonal antibody SDT-GAR-1, which was applied for in this study, showed clear staining effects and accurate localization in immunohistochemical (IHC) staining of 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 in the figure, this indicates that SDT-GAR-1 can specifically recognize rabbit IgG, thus confirming that the antibody has high specificity and affinity for rabbit IgG protein. These results not only demonstrate the application potential of SDT-GAR-1 in IHC experiments, but also highlight its reliability and effectiveness in detecting rabbit-derived antibodies.

[0112] Example 5

[0113] This embodiment provides the HRP-labeled monoclonal antibody SDT-GAR-1 and its preparation method.

[0114] Its preparation methods include:

[0115] Take 5 mg of the antibody to be labeled, add 1455 μL of 1×PBS and 45 μL of 1M sodium carbonate solution to 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 purified antibody SDT-GAR-1.

[0117] Weigh an appropriate amount of NaIO4 powder and add an appropriate amount of pure water according to the weighed amount to make the final concentration 20 mg / mL; add 45 μL of freshly prepared 20 mg / mL NaIO4 solution to the above solution and react at room temperature in the dark for 20 min (add 22.5 μL of 20 mg / mL NaIO4 solution per mg HRP, the actual amount added depends on the mass of HRP, and scale up the scale by equal volume);

[0118] Add 40 μL of ethylene glycol and react at room temperature in the dark for 30 min (add 20 μL of ethylene glycol per mg HRP; the actual amount added depends on the mass of HRP, and scale up the reaction accordingly).

[0119] Add the activated HRP to the dialyzed antibody and mix well; dialyze in 2L of 0.05M pH9.6 carbonate buffer and stir overnight at 4°C.

[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, then replace with 0.01 M pH 7.4 PBS for dialyzing, and incubate at 4°C overnight (replace PBS once during the process).

[0121] Transfer the dialysate to a 2 mL centrifuge tube, add 50% saturated ammonium sulfate solution, let stand at 4°C for 30 min, then centrifuge at 12000 rpm for 30 min at 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] Dissolve the precipitate in a small amount of 0.01M pH7.4 PBS (500μL), then add half a volume of saturated ammonium sulfate solution, let stand at 4℃ for 30 min, then centrifuge at 12000 rpm for 30 min at 4℃.

[0123] Discard the supernatant, drain, dissolve the precipitate in a small amount of PBS, and calculate the volume of PBS based on the amount of cross-linked antibody (multiply the cross-linked antibody (mg) by 0.8 and then divide by 20 to get the volume of PBS (mL)). After cross-linking is completed, the HRP-labeled monoclonal antibody SDT-GAR-1 is obtained and named Goat Anti-Rabbit IgG(H+L) (HRP Conjugate).

[0124] Example 6

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

[0126] In this embodiment, 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 are the same as those in Example 4, except that: the primary antibody was the applicant's self-developed product Keratin 5 Recombinant Rabbit mAb (SDT-014-67) (manufacturer: Startech, catalog number: S0B2033), with an initial concentration of 1000 ng / μL; the secondary antibody was Goat Anti-Rabbit IgG(H+L)(HRP Conjugate) provided in Example 5, diluted at 1:10000 and incubated.

[0127] Results analysis:

[0128] The results are as follows Figure 3 As shown, OD450 gradually decreased with decreasing primary antibody concentration, a trend consistent with commercially available antibody products, indicating no significant difference. These findings suggest that Goat Anti-Rabbit IgG(H+L) (HRPConjugate) exhibits high affinity for rabbit-derived primary antibodies, and its performance is comparable to commercially available antibody products. 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, providing detection effects comparable to commercially available antibodies.

[0129] Example 7

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

[0131] In this embodiment, Goat Anti-Rabbit IgG(H+L)(HRP Conjugate) was used as the secondary antibody, and a commercially available antibody was used as a positive control for Western blotting. The specific steps are the same as in Example 4, except that HeG2 cells were added in this embodiment, the primary antibody was the applicant's self-developed product β-actin Recombinant Rabbit mAb (manufacturer: Startech, catalog number: S0B0074), and the secondary antibody was Goat Anti-Rabbit IgG(H+L)(HRPConjugate) provided in Example 5.

[0132] Results analysis:

[0133] like Figure 4As shown, both Goat Anti-Rabbit IgG(H+L)(HRP Conjugate) and commercially available antibodies exhibited clear single bands in Western blot experiments, with no other non-specific bands observed. Under the same experimental conditions, compared to the commercially available antibody, the band intensity of Goat Anti-Rabbit IgG(H+L)(HRP Conjugate) was significantly higher, indicating that the antibody of this application possesses higher specificity, affinity, and sensitivity. These characteristics give Goat Anti-Rabbit IgG(H+L)(HRP Conjugate) a significant advantage as an enzyme-labeled secondary antibody in Western blot experiments, providing more reliable detection results.

[0134] Example 8

[0135] This embodiment 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 the secondary antibody for immunohistochemical detection. The specific steps are the same as those in Embodiment 4, except that the detection materials used in this embodiment are gastric cancer tissue, thyroid cancer tissue, and ovarian cancer tissue. The primary antibody for the first tissue is the applicant's self-developed rabbit anti-Claudin 18.2 (manufacturer: Startech, catalog number: S0B2070P), and the primary antibodies for the latter two tissues are rabbit anti-PAX8 (manufacturer: Startech, catalog number: S0B2071P). 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] Results analysis:

[0138] like Figure 5As shown, the experimental group exhibited more accurate localization compared to the PBS control group, confirming that Goat Anti-Rabbit IgG(H+L)(HRP Conjugate) specifically binds to rabbit IgG. This result demonstrates that Goat Anti-Rabbit IgG(H+L)(HRP Conjugate) can effectively serve as a secondary antibody in immunohistochemical (IHC) assays. Furthermore, despite differences in the rabbit-derived primary antibody used, Goat Anti-Rabbit IgG(H+L)(HRPConjugate) still recognized rabbit IgG protein, further demonstrating its broad applicability and high recognition ability for rabbit-derived antibodies. These findings highlight the potential and reliability of Goat Anti-Rabbit IgG(H+L)(HRP Conjugate) as a secondary antibody in IHC experiments.

[0139] It is understood that those skilled in the art can make equivalent substitutions or modifications to the technical solutions and concepts of this invention, and all such substitutions or modifications should fall within the protection scope of the appended claims.

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 the light chain variable region of the goat recombinant monoclonal antibody against rabbit IgG as described in claim 1 or 2.

4. A nucleic acid according to claim 3, characterized in that, The nucleic acids include: 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 as described in claim 3 or 4.

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

7. The use of the nucleic acid of claim 3 or 4, or the recombinant expression vector of claim 5, or the recombinant expression cell of claim 6 in the preparation of a goat recombinant monoclonal antibody against rabbit IgG.

8. The method for preparing the goat recombinant monoclonal antibody against rabbit IgG according to claim 1, characterized in that, The method includes: Recombinant expression cells were obtained by transfecting cells with the recombinant expression vector described in claim 5; the transfected recombinant expression cells were cultured; the supernatant was collected and purified to obtain a recombinant monoclonal antibody against rabbit IgG. Alternatively, the recombinant expression cells as described in claim 6 can be cultured; the supernatant can be collected and purified to obtain the anti-rabbit IgG recombinant monoclonal antibody.

9. A goat recombinant monoclonal antibody against rabbit IgG labeled with HRP, characterized in that, The recombinant monoclonal antibody is the goat recombinant monoclonal antibody against rabbit IgG as described in claim 1 or 2.

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

11. The application according to claim 10, characterized in that, The detection of rabbit IgG includes any one or more of the following methods: immunohistochemistry, Western blotting, or ELISA.

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