ELISA (enzyme-linked immuno sorbent assay) kit for detecting prawn hemocyanin antibody

Abstract

The invention relates to the technical field of ELISA kits, in particular to an ELISA kit for detecting a prawn hemocyanin antibody, which comprises a pre-coated antigen plate, a standard substance solution, a 10 * PBS buffer solution, an enzyme-labeled secondary antibody, a TMB buffer solution and a stop solution, wherein the pre-coated antigen plate is formed by coating a PBS (Phosphate Buffer Solution) of hemocyanin as a coating solution, and the standard substance solution is a monoclonal antibody anti-SHC-2B3 solution. The ELISA kit for detecting the anti-haemocyanin antibody has the technical effects that the anti-haemocyanin antibody in a to-be-detected sample can be specifically recognized, whether animal immune haemocyanin reaction is successful or not can be quickly, accurately and qualitatively judged, and the content of the anti-haemocyanin antibody can be quantitatively detected.

Description

Technical Field

[0001] The present application relates to the technical field of ELISA kits, and in particular to an ELISA kit for detecting anti-prawn hemocyanin antibodies. Background Art

[0002] Shrimp Hemocyanin (SHC) is a giant blue protein molecule that plays a key role in transporting oxygen in the blood of shrimp. Shrimp Hemocyanin belongs to the hemocyanin family and is a very special class of proteins. Unlike mammalian hemoglobin, shrimp hemocyanin does not contain heme, but transports oxygen by directly binding to oxygen. This binding is reversible, allowing shrimp hemocyanin to effectively transport oxygen from the respiratory organs to various cells in the shrimp body. The structure of shrimp hemocyanin is very complex. It is composed of multiple protein subunits to form a large polymer structure. Each subunit contains two copper ions (Cu 2+ ), these Cu 2+ It is the key to the binding of shrimp hemocyanin with oxygen. When oxygen enters the shrimp's respiratory organs and enters the blood through the respiratory system, it will bind to Cu in the shrimp's hemocyanin. 2+ Combined with oxygen, the oxygenated hemocyanin is formed. The oxygenated hemocyanin is transported to various tissues and organs through the shrimp's blood circulation system.

[0003] Shrimp hemocyanin not only plays an important role in oxygen transport, but also has the functions of immunomodulation and antibacterial activity. Studies have shown that shrimp hemocyanin can activate immune cells in the shrimp immune system and enhance the disease resistance of shrimp. In addition, shrimp hemocyanin also has antibacterial activity and can resist pathogenic microorganisms inside and outside the shrimp. Due to the important role of shrimp hemocyanin in shrimp, it has become one of the hot spots in the field of biology and biomedical research. Scientists have conducted in-depth research on the structure and function of shrimp hemocyanin in order to better understand its biological role and explore its application potential in the fields of medicine and biotechnology. For example, shrimp hemocyanin is used as a vaccine carrier to improve shrimp immunity and disease resistance. As a hapten carrier, shrimp hemocyanin can combine with polypeptide antigens to form a complex, stimulating the body to produce specific antibodies through immune activation mechanisms.

[0004] In the preparation of antibodies against polypeptide antigens, the application of shrimp hemocyanin has the following advantages: 1. High immunogenicity: Shrimp hemocyanin, as a hapten carrier, can effectively activate the body's immune system and produce highly immunogenic antibodies; 2. Specificity and affinity: Through specific binding with polypeptide antigens, shrimp hemocyanin can promote the production of specific antibodies and improve the affinity and stability of antibodies; 3. Flexibility and customizability: Shrimp hemocyanin can be combined with different polypeptide antigens and customized according to needs, suitable for various immunological research and application fields.

[0005] By using shrimp hemocyanin as an antigen carrier, researchers can prepare antibodies against specific polypeptide antigens to achieve efficient and specific recognition and detection of polypeptides, which is of great significance for vaccine development, drug screening, disease diagnosis and treatment.

[0006] The development of this kit aims to provide a fast, accurate and reliable method for detecting whether animals have produced specific antibodies against shrimp hemocyanin and verifying whether the animal's immune response is successful. Through this kit, researchers can evaluate the immune response of animals to shrimp hemocyanin and further understand the application potential of shrimp hemocyanin as an antigen carrier in peptide antigen research. Summary of the invention

[0007] In order to provide an ELISA kit method for detecting whether an animal has produced specific antibodies against shrimp hemocyanin and verifying whether the animal's immune response is successful, the present application provides an ELISA kit for detecting anti-shrimp hemocyanin antibodies. The ELISA kit for detecting anti-shrimp hemocyanin antibodies has the technical effect of being able to quickly qualitatively determine whether the animal's immune hemocyanin reaction is successful and quantitatively detect the content of anti-shrimp hemocyanin antibodies.

[0008] The technical solution of this application In a first aspect, the present application provides an ELISA kit for detecting anti-shrimp hemocyanin antibodies, comprising a pre-coated antigen plate and a standard solution; The pre-coated antigen plate is a 96-well plate coated with a coating solution; the coating solution is a PBS buffer solution of hemocyanin with a concentration of 5 μg / mL; The standard solution is a monoclonal antibody anti-SHC-2B3 solution with a concentration of 5 μg / mL, wherein the solute is the monoclonal antibody anti-SHC-2B3 and the solvent is the sample diluent; The sample diluent is a porclin300 antibacterial agent solution with a volume percentage concentration of 0.1%, wherein the solute is the porclin300 antibacterial agent and the solvent is PBS buffer; The monoclonal antibody anti-SHC-2B3 is obtained by the following steps: (1) Synthesis of monoclonal antibody anti-SHC-2B3 gene fragment Synthesizing a monoclonal antibody anti-SHC-2B3 gene fragment, wherein the monoclonal antibody anti-SHC-2B3 gene fragment is composed of a light chain and a heavy chain of the monoclonal antibody anti-SHC-2B3; The nucleotide sequence of the light chain variable region VL and the light chain constant region CL contained in the light chain of the monoclonal antibody anti-SHC-2B3 is shown in SEQ ID NO.1; the nucleotide sequence of the heavy chain variable region VH and the heavy chain constant region CH1 contained in the heavy chain of the monoclonal antibody anti-SHC-2B3 is shown in SEQ ID NO.2; (2) Preparation of a recombinant plasmid containing the monoclonal antibody anti-SHC-2B3 gene ①, double-digest the vector plasmid pcDNA3.1 with BamH I and EcoR1 to obtain the linearized plasmid pcDNA3.1; ②, PCR amplify the monoclonal antibody anti-SHC-2B3 gene fragment, and obtain the monoclonal antibody anti-SHC-2B3 gene fragment after PCR amplification; The upstream primer sequence used in PCR amplification is AGCTCGGATCC + 20 bp sequence of the 5' of the monoclonal antibody anti-SHC-2B3 gene fragment, and the downstream primer sequence is GAATTCGG + 20 bp sequence of the 3' of the monoclonal antibody anti-SHC-2B3 gene fragment; ③. Ligation reaction The monoclonal antibody anti-SHC-2B3 gene fragment amplified by PCR was connected to the linearized plasmid pcDNA3.1 using T4 DNA ligase, and then transformed and extracted in sequence to obtain the recombinant plasmid pcDNA3.1; The transformation strain used is competent Escherichia coli DH5α; (3) transfecting the obtained recombinant plasmid pcDNA3.1 into Expi293 cells, and culturing and purifying the obtained recombinant Expi293 cells to obtain the monoclonal antibody anti-SHC-2B3; The purification steps are as follows: ①. Equilibrate the Protein A affinity chromatography column with 3 column volumes of PBS buffer; The Protein A affinity chromatography column is obtained by loading Protein A affinity chromatography resin into a chromatography column; ②, the supernatant collected by centrifugation of the recombinant Expi293 cell suspension obtained by culture was added to the Protein A affinity chromatography column equilibrated with PBS buffer at a flow rate of 2 mL / min; ③. Use PBS buffer to elute and remove non-specifically bound proteins and impurities; ④. Elute the recombinant antibody anti-SHC-2B3 using 0.5 mM glycine elution buffer at pH 3.5; ⑤. The eluted recombinant antibody anti-SHC-2B3 solution was adjusted to pH = 7.2 using a 0.1 mM Tris-HCl aqueous solution at pH = 10, and then concentrated by ultrafiltration to obtain a monoclonal antibody anti-SHC-2B3 concentrate.

[0009] By adopting the above technical solution, the hemocyanin in the pre-coated antigen plate specifically binds to the monoclonal antibody anti-SHC-2B3 in the standard solution, and can accurately identify whether the sample to be tested contains anti-shrimp hemocyanin antibodies, and calculate the content of the monoclonal antibody anti-SHC-2B3 in the sample to be tested through the standard curve equation prepared by the standard, which is used to determine whether the animal immune response is successful. In addition, the pre-coated antigen plate uses a 96-well plate, which can detect multiple samples at the same time, effectively improving the experimental efficiency.

[0010] Preferably, it also includes 10×PBS buffer, enzyme-labeled secondary antibody, TMB buffer and stop solution; The enzyme-labeled secondary antibody is a solution of goat anti-mouse IgG labeled with horseradish peroxidase; The horseradish peroxidase-labeled goat anti-mouse IgG solution is prepared by mixing horseradish peroxidase-labeled goat anti-mouse IgG and PBS buffer in a volume ratio of 1:1000-5000; preferably, the horseradish peroxidase-labeled goat anti-mouse IgG:PBS buffer is 1:5000; The stop solution is 0.1 mol / L HCl aqueous solution.

[0011] By adopting the above technical scheme, the ELISA kit for detecting anti-prawn hemocyanin antibodies is configured with 10×PBS buffer, enzyme-labeled secondary antibody, TMB buffer and stop solution. During the ELISA experiment, the experimenter does not need to match other reagents separately, which is convenient for use and also reduces the possible influence between different reagents, so that the detection result of the ELISA kit for detecting anti-prawn hemocyanin antibodies is more accurate and stable.

[0012] Preferably, the coating process of the pre-coated antigen plate is as follows: (1) Dissolve hemocyanin in PBS buffer to prepare a coating solution with a concentration of 5 μg / mL; (2) Coating: Add 50 μL of 5 μg / mL coating solution to each well of a 96-well plate, coat at 4°C for 16 h, then discard the liquid in the wells and wash 3-5 times with PBS buffer to obtain a coated 96-well plate; (3) Blocking: After the 96-well plate is coated in step (2), 100 μL of 10% BSA blocking solution is added to each well, and then the coated 96-well plate is incubated at room temperature of 25° C. for 1 h, and then the liquid in the wells is discarded and washed 3-5 times with PBS buffer to obtain a blocked 96-well plate; (4) The 96-well plate blocked in step (3) was placed in a 37°C environment to stabilize for 30 minutes to obtain a pre-coated antigen plate.

[0013] By adopting the above technical solution, the process of coating the antigen plate is simple, the operation is convenient, the preparation efficiency is high, and it is easy to prepare on a large scale.

[0014] Preferably, the 96-well plate is a 96-well plate with detachable strips, with a specification of 8 wells×12 strips.

[0015] By adopting the above technical solution, the experimenter can flexibly use the number of detachable strips according to the specific experimental design, which is convenient for use, and can also save costs and avoid the waste of the entire 96-well plate.

[0016] Preferably, an ELISA kit for detecting anti-shrimp hemocyanin antibodies consists of a pre-coated antigen plate, 100 mL of 10×PBS buffer, 100 mL of sample diluent, 0.1 mL of standard solution, 100 μL of enzyme-labeled secondary antibody, 100 mL of TMB buffer and 100 mL of stop solution; The number of pre-coated antigen plates is 1, 2, 3, 4 or 5.

[0017] In a second aspect, a method for using an ELISA kit for detecting anti-prawn hemocyanin antibodies adopts the following technical solution: A method for using an ELISA kit for detecting anti-prawn hemocyanin antibodies, comprising the following steps: (1) Take out the ELISA kit for detecting anti-shrimp hemocyanin antibodies stored at 2-8°C and equilibrate it to room temperature 25°C; (2) Preparation of standard solution for standard curve preparation: dilute the standard solution with a concentration of 5 μg / mL with sample diluent to obtain standard solutions with concentrations of 0.5000 μg / mL, 0.2500 μg / mL, 0.1250 μg / mL, 0.0625 μg / mL, and 0.0313 μg / mL, respectively; (3) Preparation method of the sample to be tested ① Take the sample to be tested, centrifuge it at 12000r / min for 5min, and take the supernatant; The sample to be tested is animal serum immunized with hemocyanin; The animal serum is mouse or rabbit serum; ② Use sample diluent to dilute the supernatant 1000-2000 times to obtain the diluted sample to be tested; (4) Adding samples to the pre-coated antigen plate: Add the standard solutions with the above concentrations of 0.5000 μg / mL, 0.2500 μg / mL, 0.1250 μg / mL, 0.0625 μg / mL, and 0.0313 μg / mL, the diluted test samples, and the negative control to the pre-coated antigen plate, respectively. The sample volume is 200 μL per well. Two parallel samples are set for each concentration of the standard solution, the diluted test sample, and the negative control. After incubation at 37°C for 1 hour, wash with PBS buffer for 3-5 times; The negative control uses PBS buffer; (5) Enzyme-labeled secondary antibody labeling: 200 μL of enzyme-labeled secondary antibody was added to the pre-coated antigen plate after sample addition, the standard solution of different concentrations, the diluted test sample and the negative control wells, incubated at room temperature at 25°C for 1-2 hours, and then washed with PBS buffer for 3-5 times; (6) Color reaction: Add 50 μL of TMB buffer to each well of the pre-coated antigen plate after enzyme-labeled secondary antibody labeling, and perform color reaction at room temperature 25°C for 15 min; (7) Termination reaction: Add 200 μL of stop solution to each well of the pre-coated antigen plate after the color development reaction to terminate the reaction and obtain a stop reaction solution; (8) At a working wavelength of 450 nm, measure the OD of the termination reaction solution in each well of the pre-coated antigen plate after the termination reaction. 450 Calculate the average OD of two parallel samples of different concentrations of standard solution, diluted test sample and negative control 450 value; (9) The concentration of the standard solution in step (2) is used as the horizontal axis, and the average OD of the standard solution with different concentrations in step (8) is used as the horizontal axis. 450 The values ​​are plotted as the ordinate, and the curve is fitted to obtain the standard curve equation; (10) The average OD of the sample to be tested 450 The value is substituted into the standard curve equation and combined with the dilution multiple of the sample to be tested to obtain the concentration of anti-shrimp hemocyanin antibodies in the sample to be tested.

[0018] By adopting the above technical solution, the experimental steps are simple and easy for the experimenter to operate, and the entire experimental process takes a short time and can be completed in about 4 hours.

[0019] In a third aspect, an ELISA kit for detecting anti-shrimp hemocyanin antibodies is used in detecting animals immunized with hemocyanin; The animal is a mouse or a rabbit.

[0020] By adopting the above technical scheme, the ELISA kit for detecting anti-prawn hemocyanin antibodies is used to detect the animal serum immunized with hemocyanin, so that the content of anti-prawn hemocyanin antibodies in the animal serum immunized with hemocyanin can be quantitatively detected, and then whether the animal immune response is successful can be quickly and accurately determined.

[0021] Beneficial technical effects of this application The ELISA kit for detecting anti-shrimp hemocyanin antibodies in the present application has simple experimental steps, is easy for experimenters to operate, and the entire experimental process takes a short time and can be completed in about 4 hours; Furthermore, the ELISA kit for detecting anti-shrimp hemocyanin antibodies in the present application has a linear range of 0.0313-0.5000 μg / mL, R 2 ≥0.9800, good specificity, no cross-reaction with non-anti-shrimp hemocyanin antibodies, can accurately determine whether the animal immune response is successful, and can be stably stored for 12 months. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 , the electrophoresis diagram of the purity determination of the solution of the monoclonal antibody anti-SHC-2B3 using the SDS-PAGE gel electrophoresis method in Example 1; Figure 2 , a blot image of the solution of monoclonal antibody anti-SHC-2B3 detected by Western blot method in Example 1; Figure 3 , the standard curve equation obtained in Example 5, wherein the abscissa is the concentration of the monoclonal antibody anti-SHC-2B3 solution, and the ordinate is the average OD 450 value. DETAILED DESCRIPTION

[0023] The present application is further described in detail below through embodiments and in conjunction with the accompanying drawings, but the present application is not limited thereto.

[0024] The reagents used in the examples of this application, except for the special reagents listed in the following table, are all commercially available; In each embodiment of the present application, the 10% BSA blocking solution used is a PBS buffer solution of BSA, and the amount of BSA and PBS buffer is such that the mass volume ratio of BSA:PBS buffer is 10 g:100 mL; The PBS buffer used was the dilution obtained by diluting 10× PBS buffer 10 times with deionized water; The PBST buffer used was a dilution obtained by diluting 10×PBS / Tween buffer 10 times with deionized water.

[0025] A validation method for an ELISA kit for detecting anti-shrimp hemocyanin antibodies is as follows: In each example of the present application, P / N value = OD corresponding to the standard 450 Average value / negative control OD 450 Corresponding average value; 1. Specificity verification The mouse sera that were immunized and not immunized with hemocyanin were selected as the test samples for detection, and PBS buffer was used as the negative control. Two parallel samples were made for each test sample and negative control, and 200 μL was added to each well. The OD values ​​of the test samples and negative controls were read. 450 Calculate the average OD of two replicates of each sample or negative control. 450 Value, to determine the specificity of the ELISA kit used to detect anti-shrimp hemocyanin antibodies; Judgment criteria: average OD 450 When the value is <0.20, it indicates that the specificity of the ELISA kit for detecting anti-shrimp hemocyanin antibodies is good; 2. Linear verification Select different batches of ELISA kits for detecting anti-shrimp hemocyanin antibodies, and detect the standard curves in different batches of ELISA kits for detecting anti-shrimp hemocyanin antibodies respectively. Make 2 parallel samples for each concentration of standard, add 200 μL to each well, and calculate the average OD of 2 parallel samples of each concentration of standard 450 The value was calculated and the corresponding standard curve was fitted for linear verification of the standard curve in the ELISA kit for detecting anti-shrimp hemocyanin antibodies; Judgment standard: R 2When ≥0.9800, it indicates that the linearity of the ELISA kit for detecting anti-shrimp hemocyanin antibodies is good; 3. Storage stability verification Six ELISA kits from the same batch for detecting anti-shrimp hemocyanin antibodies were selected and stored at 4°C for 30 min, 1 month, 3 months, 6 months, 9 months, and 12 months, respectively. ELISA tests were performed on the same sample to be tested, and 2 parallel samples were made for each sample to be tested. The average OD of the 2 parallel samples of each sample to be tested was calculated. 450 The average OD values ​​were calculated after 1 month, 3 months, 6 months, 9 months, and 12 months of storage. 450 The value and the average OD value of the sample were stored for 30 min. 450 The change rate of the value (%) was used to verify the storage stability of the ELISA kit for detecting anti-shrimp hemocyanin antibodies; the average OD 450 Change rate of value (%) = | (average OD after storage for 1 month, 3 months, 6 months, 9 months or 12 months 450 Value - average OD after 30 minutes of storage 450 value) / average OD after 30 min storage 450 value|×100%; Judgment standard: when the change rate (%) is ≤5%, it indicates that the storage stability of the ELISA kit for detecting anti-prawn hemocyanin antibodies is good. Example

[0026] Example 1 An ELISA kit for detecting anti-shrimp hemocyanin antibodies, comprising a pre-coated antigen plate, 100 mL 10×PBS buffer, 100 mL sample diluent, 0.1 mL standard solution, 100 μL enzyme-labeled secondary antibody (dilution multiple 1:5000), 100 mL TMB buffer and 100 mL stop solution; 1. The pre-coated antigen plate: the pre-coated antigen plate is a 96-well plate coated with a coating solution; The coating process of the above pre-coated antigen plate is as follows: (1) Dissolve hemocyanin in PBS buffer to prepare coating solutions with concentrations of 15 μg / mL, 5 μg / mL, 1.6 μg / mL, 0.5 μg / mL, 0.18 μg / mL, and 0.06 μg / mL, respectively; (2) Coating: Add 50 μL of coating solution to each well of a 96-well plate, coat at 4°C for 16 h, then discard the liquid in the wells, wash with PBS buffer, gently shake the 96-well plate, discard the liquid in the wells, and repeat the PBS buffer wash twice to obtain a coated 96-well plate; For the above washing, the amount of PBS buffer used each time was 200 μL / well; In the above 96-well plate, the distribution of coating solutions of different concentrations is as follows: The coating solution added in row 1 has a concentration of 15 μg / mL; The coating solution added in row 2 has a concentration of 5 μg / mL; The coating solution added in row 3 has a concentration of 1.6 μg / mL; The coating solution added in row 4 has a concentration of 0.5 μg / mL; The coating solution added in row 5 has a concentration of 0.18 μg / mL; The coating solution added in row 6 has a concentration of 0.06 μg / mL; Coating solutions with concentrations of 15 μg / mL, 5 μg / mL, 1.6 μg / mL, 0.5 μg / mL, 0.18 μg / mL, and 0.06 μg / mL were added to columns 1, 2, 3, 4, 5, and 6 of rows 7 and 8, respectively; (3) Blocking: After the 96-well plate was coated in step (2), 100 μL of 10% BSA blocking solution was added to each well, and then the coated 96-well plate was blocked at room temperature (25° C.) for 1 h, and then the liquid in the wells was discarded, and the wells were washed with PBS buffer. The 96-well plate was gently shaken, and the liquid in the wells was discarded. The PBS buffer washing was repeated twice to obtain a blocked 96-well plate; For the above washing, 200 μL / well of PBS buffer was used each time; (4) placing the 96-well plate blocked in step (3) in a 37° C. environment for 30 minutes to ensure the stability and activity of the coated antigen, thereby obtaining a pre-coated antigen plate; 2. The sample diluent is a solution of porclin300 antibacterial agent with a volume percentage concentration of 0.1%, wherein the solute is porclin300 antibacterial agent and the solvent is PBS buffer solution; 3. The standard solution is a monoclonal antibody anti-SHC-2B3 solution, in which the solute is the monoclonal antibody anti-SHC-2B3 and the solvent is the sample diluent; The monoclonal antibody anti-SHC-2B3 is obtained by the following steps: (1) Synthesis of monoclonal antibody anti-SHC-2B3 gene fragment Synthesizing a monoclonal antibody anti-SHC-2B3 gene fragment, wherein the monoclonal antibody anti-SHC-2B3 gene fragment is composed of a light chain and a heavy chain of the monoclonal antibody anti-SHC-2B3; The nucleotide sequences of the light chain variable region VL and the light chain constant region CL contained in the light chain of the monoclonal antibody anti-SHC-2B3 are shown in SEQ ID NO.1; The nucleotide sequences of the heavy chain variable region VH and the heavy chain constant region CH1 contained in the heavy chain of the monoclonal antibody anti-SHC-2B3 are shown in SEQ ID NO.2; The synthesis of the above gene fragments was completed by GenScript Biotech Co., Ltd.; (2) Preparation of a recombinant plasmid containing the monoclonal antibody anti-SHC-2B3 gene ①. Double-digest the vector plasmid pcDNA3.1 with BamH I and EcoR1 to obtain linearized plasmid pcDNA3.1; the enzyme digestion system is shown in the following table: Enzyme digestion system components Volume / Mass Plasmid pcDNA3.1 1 μg BamHI (10U / μL) 1μL EcoR1(10U / μL) 1μL 10× Buffer 1μL <![CDATA[H2O]]> Make up to 10 μL Incubate at 37°C for 30 min. ② PCR amplification of monoclonal antibody anti-SHC-2B3 gene fragment amplifying the monoclonal antibody anti-SHC-2B3 gene fragment by PCR to obtain a monoclonal antibody anti-SHC-2B3 gene fragment solution after PCR amplification; The specific components and dosage of the PCR amplification system are shown in the following table: The above 2×Phanta Max Buffer, dNTP Mix (10 mM each), Phanta Max Super-Fidelity DNA Polymerase and ddH2O were all from Phanta Max Super-Fidelity DNA Polymerase; The upstream primer sequence is AGCTCGGATCC + 20 bp sequence of the 5' of the monoclonal antibody anti-SHC-2B3 gene fragment; The above downstream primer sequence is GAATTCGG + 20 bp sequence of the 3' of the monoclonal antibody anti-SHC-2B3 gene fragment; the program of the PCR amplification system is set as: pre-denaturation (95°C, 5 min), cyclic amplification (30 cycles in total, each cycle includes denaturation (95°C, 30 s), annealing (65°C, 30 s), extension (72°C, 10 s)) and final extension (72°C, 5 min); ③. Ligation reaction The monoclonal antibody anti-SHC-2B3 gene fragment amplified by PCR was connected to the linearized plasmid pcDNA3.1 using T4 DNA ligase to obtain the corresponding solution containing the recombinant plasmid pcDNA3.1; The composition and dosage of the reactants, related reagents, etc. used in the above connection process are shown in the following table: Reactants, related reagents, etc. Volume / Mass T4 DNA ligase (10U / μL) 1μL Monoclonal antibody anti-SHC-2B3 gene fragment after PCR amplification 200ng Linearized plasmid pcDNA3.1 500ng 10×T4 DNA ligase reaction buffer 2μL <![CDATA[H2O]]> Add to 20 μL The reactants and related reagents used in the above connection process were mixed evenly and reacted at 16°C for 12 hours; ④. Transformation of recombinant plasmid Take 10 μL of the ligation solution containing the recombinant plasmid pcDNA3.1 and add it to 50 μL of competent E. coli DH5α, mix well, let it stand on ice for 30 min, transfer it to a 42°C environment and let it stand for 90 s, then let it stand on ice for another 2 min, then transfer it to 5 mL of LB liquid culture medium and incubate it at 37°C for 1 h to obtain a bacterial solution of E. coli DH5α containing the recombinant plasmid pcDNA3.1; The bacterial solution of Escherichia coli DH5α containing the recombinant plasmid pcDNA3.1 was evenly spread on an LB agar plate containing 10 μg / mL ampicillin antibiotics, and inverted cultured at 37°C for 16 hours to obtain recombinant Escherichia coli DH5α colonies corresponding to the recombinant plasmid pcDNA3.1; A single colony of the Escherichia coli DH5α containing the recombinant plasmid pcDNA3.1 was picked and inoculated into 5 mL of LB liquid culture medium, and cultured for 16 hours to obtain a fermentation broth of the recombinant Escherichia coli DH5α corresponding to the recombinant plasmid pcDNA3.1; The results of the sanger sequencing (completed by Shanghai Qingke Biotechnology Co., Ltd.) showed that the recombinant Escherichia coli DH5α was successfully obtained; ⑤. Recombinant plasmid extraction The recombinant plasmid was extracted from the fermentation liquid of the recombinant Escherichia coli DH5α obtained above using a plasmid extraction kit to obtain the recombinant plasmid pcDNA3.1 fermented by the recombinant Escherichia coli DH5α; The results of the sanger sequencing (completed by Shanghai Qingke Biotechnology Co., Ltd.) showed that the recombinant plasmid pcDNA3.1 fermented by recombinant Escherichia coli DH5α was successfully obtained; (3) Expression of a monoclonal antibody anti-SHC-2B3 gene in recombinant Expi293 cells ①, Expi293 cell transfection The PEI transfection reagent and the recombinant plasmid pcDNA3.1 fermented by recombinant Escherichia coli DH5α were mixed at a ratio of 0.1 mL of PEI transfection reagent: 10 μg of recombinant plasmid pcDNA3.1 fermented by recombinant Escherichia coli DH5α to obtain a mixture of transfection reagent / recombinant plasmid pcDNA3.1 fermented by recombinant Escherichia coli DH5α, and the mixture was allowed to stand at 30°C for 15 min; the mixture of transfection reagent / recombinant plasmid pcDNA3.1 fermented by recombinant Escherichia coli DH5α was transfected into Expi293 cells, and 10 μg of the recombinant plasmid pcDNA3.1 fermented by recombinant Escherichia coli DH5α was used for 10 6 The ratio of Expi293 cells was used to obtain recombinant Expi293 cells. ②, Recombinant Expi293 cell culture The recombinant Expi293 cells were cultured in a cell culture incubator at 37° C. and 5% CO2 volume percentage concentration for 1 week to obtain a recombinant Expi293 cell culture fluid containing the recombinant antibody anti-SHC-2B3; The supernatant of the recombinant Expi293 cell culture medium containing the recombinant antibody anti-SHC-2B3 was collected by centrifugation; (4) Purification of a monoclonal antibody anti-SHC-2B3 ①. Purification of monoclonal antibody anti-SHC-2B3 loading the Protein A affinity chromatography resin into a chromatography column to form a Protein A affinity chromatography column; The Protein A affinity chromatography column was equilibrated with 3 column volumes of PBS buffer; 1L of the collected supernatant of the recombinant Expi293 cell culture fluid containing the recombinant antibody anti-SHC-2B3 was loaded onto a Protein A affinity chromatography column equilibrated with a PBS buffer, the flow rate was controlled at 2mL / min, and then the non-specifically bound proteins and impurities were eluted in sequence with a PBS buffer to retain the recombinant antibody anti-SHC-2B3 bound to the Protein A affinity chromatography resin; The recombinant antibody anti-SHC-2B3 bound to Protein A was eluted from the Protein A affinity chromatography column using a 0.5 mM glycine elution buffer at pH 3.5; The eluted recombinant antibody anti-SHC-2B3 solution was adjusted to pH = 7.2 with a 0.1 mM Tris-HCl aqueous solution at pH = 10, and then ultrafiltration and concentration were performed to obtain a monoclonal antibody anti-SHC-2B3 concentrated solution; ② SDS-PAGE gel electrophoresis The purity of the monoclonal antibody snti-SHC-2B3 concentrate was tested by SDS-PAGE. The electrophoresis diagram was as follows: Figure 1 As shown; Grayscale analysis showed that the purity of monoclonal antibody anti-SHC-2B3 in the monoclonal antibody snti-SHC-2B3 concentrate was ≥90%; ③、Western blot The monoclonal antibody anti-SHC-2B3 concentrate was analyzed by Western blot. The specific steps are as follows: a. Dilute hemocyanin with WB loading buffer to a concentration of 1 mg / mL and boil at 95°C for 5 min to obtain a pretreated hemocyanin sample; The WB loading buffer mentioned above is 100 mM DTT, 1% SDS, 20% glycerol and 1% methyl blue; b. Add the pretreated hemocyanin sample to the sample wells of the precast gel, add 100 ng of the pretreated hemocyanin sample to each well, perform electrophoresis for 1 h at a constant voltage of 120 V, and remove the gel; c. Cut a piece of nitrocellulose membrane of the same size as the gel, soak the nitrocellulose membrane and filter paper with rapid transfer buffer, and then place them in a wet transfer apparatus in a "sandwich" manner, in the following order: electrode (-) - sponge - filter paper - gel - nitrocellulose membrane - filter paper - sponge - electrode (+), constant current 300mA, power on for 1h; The pore size of the above-mentioned nitrocellulose membrane is 0.22 μm; d. After the transfer, take out the nitrocellulose membrane, wash it with water once, and then place it in PBST buffer containing 5% skim milk powder and block it at 30℃ for 10 minutes to prevent nonspecific binding; The above-mentioned PBST buffer containing 5% skimmed milk powder has a mass volume ratio of skimmed milk powder to PBST buffer of 5 g:100 mL; e. Wash the nitrocellulose membrane three times with PBST buffer, 5 min each time; f. The monoclonal antibody anti-SHC-2B3 obtained above was added as a specific primary antibody into PBST buffer containing 5% skim milk powder to obtain a primary antibody solution with a concentration of 1 μg / mL. The nitrocellulose membrane was placed in the primary antibody solution and incubated at room temperature (25°C) for 1 hour. g. Wash the nitrocellulose membrane three times with PBST buffer, 5 min each time; h. Add horseradish peroxidase (HRP)-labeled goat anti-mouse IgG as the secondary antibody into PBST buffer containing 5% skim milk powder to obtain a secondary antibody solution with a concentration of 1 μg / mL, and incubate at room temperature (25°C) for 1 h; i. Wash the nitrocellulose membrane three times with PBST buffer, 5 min each time, to remove non-specifically bound secondary antibodies and other impurities; j. Use ECL color developing solution to develop the final band until the color is clear, and then perform image analysis; k. Result: Figure 2 As shown, the results showed that the monoclonal antibody anti-SHC-2B3 can specifically bind to shrimp hemocyanin, and the antigenic determinants of specific binding are located on the two subunits of shrimp hemocyanin with molecular weights of 73kDa and 75kDa; 4. The enzyme-labeled secondary antibody is a solution of goat anti-mouse IgG labeled with horseradish peroxidase (HRP); The horseradish peroxidase (HRP)-labeled goat anti-mouse IgG solution has a volume ratio of horseradish peroxidase (HRP)-labeled goat anti-mouse IgG to PBS buffer of 1:5000; 5. The stop solution is 0.1 mol / L HCl aqueous solution.

[0027] The above-mentioned ELISA kit for detecting anti-prawn hemocyanin antibodies is stored at 2-8°C.

[0028] The method for using the ELISA kit for detecting anti-prawn hemocyanin antibodies comprises the following specific steps: 1. Take out the ELISA kit for detecting anti-shrimp hemocyanin antibodies stored at 2-8°C and equilibrate to room temperature (25°C); 2. Dilute the monoclonal antibody anti-SHC-2B3 solution with a sample diluent to obtain monoclonal antibody anti-SHC-2B3 solutions with concentrations of 15 μg / mL, 5 μg / mL, 1.6 μg / mL, 0.5 μg / mL, 0.18 μg / mL, and 0.06 μg / mL, respectively; 3. Adding samples to the pre-coated antigen plate: Add the monoclonal antibody anti-SHC-2B3 solution and negative control (the negative control uses PBS buffer) with the above concentrations of 15 μg / mL, 5 μg / mL, 1.6 μg / mL, 0.5 μg / mL, 0.18 μg / mL, and 0.06 μg / mL to the pre-coated antigen plate, respectively, with a sample volume of 200 μL per well. Set up 2 parallel samples for each concentration of monoclonal antibody anti-SHC-2B3 solution and negative control, and incubate at 37°C for 1 hour; After the above incubation, the liquid in each well of the pre-coated antigen plate was discarded, and then washed with PBS buffer, gently shaken, and then the liquid in each well of the pre-coated antigen plate after incubation was discarded, and then washed with PBS buffer twice; In the above washing process, the amount of PBS buffer used for each washing was 200 μL / well; The distribution of the above-mentioned monoclonal antibody anti-SHC-2B3 solutions of different concentrations and negative controls in each well of the pre-coated antigen plate is as follows: A monoclonal antibody anti-SHC-2B3 solution with a concentration of 15 μg / mL was added to each well of rows 1 to 6 in columns 1 and 2; A monoclonal antibody anti-SHC-2B3 solution with a concentration of 5 μg / mL was added to each well of rows 1 to 6 in columns 3 and 4; A monoclonal antibody anti-SHC-2B3 solution with a concentration of 1.6 μg / mL was added to each well of rows 1 to 6 in columns 5 and 6; A monoclonal antibody anti-SHC-2B3 solution with a concentration of 0.5 μg / mL was added to each well of the 1st to 6th rows of the 7th and 8th columns; a monoclonal antibody anti-SHC-2B3 solution with a concentration of 0.18 μg / mL was added to each well of the 1st to 6th rows of the 9th and 10th columns; a monoclonal antibody anti-SHC-2B3 solution with a concentration of 0.06 μg / mL was added to each well of the 1st to 6th rows of the 11th and 12th columns; a negative control was added to each well of the 7th and 8th rows of the 1st to 6th columns; 4. Enzyme-labeled secondary antibody labeling: After loading the sample, add 200 μL of enzyme-labeled secondary antibody to each well of the pre-coated antigen plate, the monoclonal antibody anti-SHC-2B3 solution with different concentrations, and the negative control, and incubate at room temperature (25°C) for 1 hour; Then discard the liquid in each well of the pre-coated antigen plate after incubation, wash with PBS buffer, shake gently, discard the liquid in each well of the pre-coated antigen plate after incubation, and repeat washing with PBS buffer twice; In the above washing process, the amount of PBS buffer used for each washing was 200 μL / well; 5. Color reaction: Add 50 μL TMB buffer to each well of the pre-coated antigen plate after enzyme-labeled secondary antibody labeling, and perform color reaction at room temperature (25°C) for 15 minutes; 6. Stop reaction: add 200 μL of stop solution to each well of the pre-coated antigen plate after the color development reaction to stop the reaction and obtain the stop reaction solution; 7. Use an ELISA reader to measure the OD of the termination reaction solution in each well of the pre-coated antigen plate after the termination reaction at a working wavelength of 450 nm. 450 value; Judgment criteria: average OD of negative control 450 When the value is less than 0.20, the experiment is established, otherwise the experiment is invalid; the average OD of the negative control 450 The OD of the stop reaction solution for all negative control wells 450 The average value of .

[0029] According to the detection OD 450 The results of the changes in the average values ​​show that the optimal concentration of the coating solution is 5μg / mL, and the optimal concentration of the monoclonal antibody anti-SHC-2B3 solution is 0.5μg / mL.

[0030] Example 2 An ELISA kit for detecting anti-prawn hemocyanin antibodies, comprising 4 pre-coated antigen plates coated with a coating solution with a concentration of 5 μg / mL, 100 mL of 10×PBS buffer, 100 mL of sample diluent, 0.1 mL of standard solution, 100 μL of enzyme-labeled secondary antibody (dilution multiple 1:5000), 100 mL of TMB buffer and 100 mL of stop solution; 1. The pre-coated antigen plate: the pre-coated antigen plate is a 96-well plate coated with antigen; The coating process of the above pre-coated antigen plate is as follows: (1) Dissolve hemocyanin in PBS buffer to prepare a coating solution with a concentration of 5 μg / mL; (2) Coating: Add 50 μL of 5 μg / mL coating solution to each well of four 96-well plates, coat at 4°C for 16 h, then discard the liquid in the wells, wash with PBS buffer, gently shake the 96-well plates, discard the liquid in the wells, and repeat the PBS buffer wash twice to obtain four coated 96-well plates; For the above washing, the amount of PBS buffer used each time was 200 μL / well; (3) Blocking: Add 100 μL of 10% BSA blocking solution to each well of the four 96-well plates coated in step (2), and then incubate the four 96-well plates at room temperature (25°C) for 1 h, room temperature (25°C) for 2 h, 37°C for 1 h, and 37°C for 2 h, respectively. Then, discard the liquid in the wells, wash with PBS buffer, gently shake the 96-well plates, discard the liquid in the wells, and repeat the PBS buffer washing twice to obtain four blocked 96-well plates; For the above washing, 200 μL / well of PBS buffer was used each time; (4) placing the four blocked 96-well plates in step (3) in a 37° C. environment for 30 minutes to ensure the stability and activity of the coated antigen, thereby obtaining a pre-coated antigen plate; 2. The sample diluent is the same as the sample diluent in Example 1; 3. The standard solution is the same as the standard solution in Example 1; 4. The enzyme-labeled secondary antibody is the same as the enzyme-labeled secondary antibody in Example 1; 5. The stop solution is the same as that in Example 1.

[0031] The above-mentioned ELISA kit for detecting anti-prawn hemocyanin antibodies is stored at 2-8°C.

[0032] The method for using the ELISA kit for detecting anti-prawn hemocyanin antibodies comprises the following specific steps: 1. The same as step 1 of the method for using an ELISA kit for detecting anti-prawn hemocyanin antibodies in Example 1; 2. Dissolve the monoclonal antibody anti-SHC-2B3 in the sample diluent to obtain a monoclonal antibody anti-SHC-2B3 solution with a concentration of 0.5 μg / mL; 3. Adding samples to the pre-coated antigen plates: Add the above-mentioned monoclonal antibody anti-SHC-2B3 solution with a concentration of 0.5 μg / mL and the negative control (the negative control uses PBS buffer) to 4 pre-coated antigen plates, respectively, with a sample volume of 200 μL per well, and incubate at 37°C for 1 hour; The distribution of the above monoclonal antibody anti-SHC-2B3 solution with a concentration of 0.5 μg / mL and the negative control in each pre-coated antigen plate is as follows: In row 1, add monoclonal antibody anti-SHC-2B3 solution at a concentration of 0.5 μg / mL; Add negative control in row 2; After the above incubation, the liquid in each well of the 4 pre-coated antigen plates was discarded, and then washed with PBS buffer, gently shaken, and then the liquid in each well of the pre-coated antigen plates after incubation was discarded, and then washed with PBS buffer twice; In the above washing process, the amount of PBS buffer used for each washing was 200 μL / well; 4. Enzyme-labeled secondary antibody labeling: After loading the sample, add 200 μL of enzyme-labeled secondary antibody to each well of the pre-coated antigen plate, the monoclonal antibody anti-SHC-2B3 solution and the negative control, and incubate at room temperature (25°C) for 1 hour; Then discard the liquid in each well of the pre-coated antigen plate after incubation, wash with PBS buffer, shake gently, discard the liquid in each well of the pre-coated antigen plate after incubation, and repeat washing with PBS buffer twice; In the above washing process, the amount of PBS buffer used for each washing was 200 μL / well; 5. Color development reaction: the same as step 5 of the method for using an ELISA kit for detecting anti-prawn hemocyanin antibodies in Example 1; 6. Termination of reaction: the same as step 6 of the method for using an ELISA kit for detecting anti-shrimp hemocyanin antibodies in Example 1; 7. Use an ELISA reader to measure the OD of the termination reaction solution in each well of the pre-coated antigen plate after the termination reaction at a working wavelength of 450 nm. 450 value; According to the measured OD 450 The corresponding P / N value is calculated to determine the blocking temperature and time of the coated antigen. The results are shown in the following table: It can be seen from the data analysis in the above table that the blocking temperature and time of the coated antigen are incubated at room temperature (25°C) for 1-2 hours; considering energy consumption, preferably, the blocking temperature and time of the coated antigen are incubated at room temperature (25°C) for 1 hour.

[0033] Example 3 An ELISA kit for detecting anti-prawn hemocyanin antibodies, comprising a pre-coated antigen plate coated with a coating solution with a concentration of 5 μg / mL, 100 mL of 10×PBS buffer, 100 mL of sample diluent, 0.1 mL of standard solution, 100 μL of enzyme-labeled secondary antibody, 100 mL of TMB buffer and 100 mL of stop solution; 1. The pre-coated antigen plate: the pre-coated antigen plate is a 96-well plate coated with antigen; The coating process of the above pre-coated antigen plate is as follows: (1) Dissolve hemocyanin in PBS buffer to prepare a coating solution with a concentration of 5 μg / mL; (2) Coating: Add 50 μL of 5 μg / mL coating solution to each well of a 96-well plate, coat at 4°C for 16 h, then discard the liquid in the wells, wash with PBS buffer, gently shake the 96-well plate, discard the liquid in the wells, and repeat the PBS buffer wash twice to obtain a coated 96-well plate; For the above washing, the amount of PBS buffer used each time was 200 μL / well; (3) Blocking: After the 96-well plate was coated in step (2), 100 μL of 10% BSA blocking solution was added to each well of the plate, and then the coated 96-well plate was incubated at room temperature (25° C.) for 1 h, and then the liquid in the wells was discarded, and the plate was washed with PBS buffer. The 96-well plate was gently shaken, and the liquid in the wells was discarded. The plate was washed with PBS buffer twice to obtain a blocked 96-well plate. For the above washing, 200 μL / well of PBS buffer was used each time; (4) placing the 96-well plate blocked in step (3) in a 37° C. environment for 30 minutes to ensure the stability and activity of the coated antigen, thereby obtaining a pre-coated antigen plate; 2. The sample diluent is the same as the sample diluent in Example 1; 3. The standard solution is the same as the standard solution in Example 1; 4. The enzyme-labeled secondary antibody is a solution of goat anti-mouse IgG labeled with horseradish peroxidase (HRP); The solutions of the goat anti-mouse IgG labeled with horseradish peroxidase (HRP) are calculated according to the volume ratio of the goat anti-mouse IgG labeled with horseradish peroxidase (HRP) to the PBS buffer solution, which are 1:1000, 1:2500, 1:5000, 1:7500, and 1:10000 respectively; 5. The stop solution is the same as that in Example 1.

[0034] The above-mentioned ELISA kit for detecting anti-prawn hemocyanin antibodies is stored at 2-8°C.

[0035] The method for using the ELISA kit for detecting anti-prawn hemocyanin antibodies comprises the following specific steps: 1. The same as step 1 of the method for using an ELISA kit for detecting anti-prawn hemocyanin antibodies in Example 1; 2. The same as step 2 of the method for using an ELISA kit for detecting anti-prawn hemocyanin antibodies in Example 2; 3. Adding samples to the pre-coated antigen plate: Add the above-mentioned monoclonal antibody anti-SHC-2B3 solution with a concentration of 0.5 μg / mL and the negative control (the negative control uses PBS buffer) to the pre-coated antigen plate, with a sample volume of 200 μL per well, and incubate at 37°C for 1 hour; The distribution of the monoclonal antibody anti-SHC-2B3 solution with a concentration of 0.5 μg / mL and the negative control in the pre-coated antigen plate is as follows: In row 1, add monoclonal antibody anti-SHC-2B3 solution at a concentration of 0.5 μg / mL; Add negative control in row 2; After the above incubation, the liquid in each well of the pre-coated antigen plate was discarded, and then washed with PBS buffer, gently shaken, and then the liquid in each well of the pre-coated antigen plate after incubation was discarded, and then washed with PBS buffer twice; In the above washing process, the amount of PBS buffer used for each washing was 200 μL / well; 4. Enzyme-labeled secondary antibody labeling: After loading the sample, add 200 μL of enzyme-labeled secondary antibody with a dilution of 1:1000, 1:2500, 1:5000, 1:7500 or 1:10000 to the pre-coated antigen plate, the wells containing the monoclonal antibody anti-SHC-2B3 solution and the negative control, and incubate at room temperature (25°C) for 1 hour; The distribution of the enzyme-labeled secondary antibodies at dilution multiples of 1:1000, 1:2500, 1:5000, 1:7500 and 1:10000 in each well of the pre-coated antigen plate is as follows: Add enzyme-labeled secondary antibody at a dilution of 1:1000 to columns 1 and 2; In columns 3 and 4, add enzyme-labeled secondary antibody at a dilution of 1:2500; In columns 5 and 6, add enzyme-labeled secondary antibody at a dilution of 1:5000; In columns 7 and 8, add enzyme-labeled secondary antibody at a dilution of 1:7500; In columns 9 and 10, add enzyme-labeled secondary antibody at a dilution of 1:10,000; Then discard the liquid in each well of the pre-coated antigen plate after incubation, wash with PBS buffer, shake gently, discard the liquid in each well of the pre-coated antigen plate after incubation, and repeat washing with PBS buffer twice; In the above washing process, the amount of PBS buffer used for each washing was 200 μL / well; 5. Color reaction: Add 50 μL TMB buffer to each well of the pre-coated antigen plate labeled with enzyme-labeled secondary antibodies at different dilution multiples, and perform color reaction at room temperature (25°C) for 15 minutes; 6. The same as step 6 of the method for using an ELISA kit for detecting anti-prawn hemocyanin antibodies in Example 1; 7. Use an ELISA reader to measure the OD of the termination reaction solution in each well of the pre-coated antigen plate after the termination reaction at a working wavelength of 450 nm. 450 value; According to the measured OD 450 The corresponding P / N value is calculated to determine the dilution multiple of the enzyme-labeled secondary antibody. The results are shown in the following table: It can be seen from the data analysis in the above table that the dilution multiple of the enzyme-labeled secondary antibody is 1:1000-5000; considering the preparation cost, preferably, the dilution multiple of the enzyme-labeled secondary antibody is 1:5000.

[0036] Example 4 An ELISA kit for detecting anti-prawn hemocyanin antibodies, comprising 3 pre-coated antigen plates coated with a coating solution with a concentration of 5 μg / mL, 100 mL of 10×PBS buffer, 100 mL of sample diluent, 0.1 mL of standard solution, 100 μL of enzyme-labeled secondary antibody (dilution multiple 1:5000), 100 mL of TMB buffer and 100 mL of stop solution; 1. Each of the pre-coated antigen plates is the same as the pre-coated antigen plate in Example 3; 2. The sample diluent is the same as the sample diluent in Example 1; 3. The standard solution is the same as the standard solution in Example 1; 4. The enzyme-labeled secondary antibody is the same as the enzyme-labeled secondary antibody in Example 1; 5. The stop solution is the same as that in Example 1.

[0037] The above-mentioned ELISA kit for detecting anti-prawn hemocyanin antibodies is stored at 2-8°C.

[0038] The method for using the ELISA kit for detecting anti-prawn hemocyanin antibodies comprises the following specific steps: 1. The same as step 1 of the method for using an ELISA kit for detecting anti-prawn hemocyanin antibodies in Example 1; 2. The same as step 2 of the method for using an ELISA kit for detecting anti-prawn hemocyanin antibodies in Example 2; 3. The loading of samples on each pre-coated antigen plate is the same as the loading of samples on the pre-coated antigen plate in step 3 of the method for using an ELISA kit for detecting anti-shrimp hemocyanin antibodies in Example 3; 4. Enzyme-labeled secondary antibody labeling: Add 200 μL of enzyme-labeled secondary antibody diluted 1:5000 to each well containing the sample and negative control in the three pre-coated antigen plates after sample loading, and incubate the three pre-coated antigen plates at room temperature (25°C) for 30 minutes, 1 hour, and 2 hours respectively; Then discard the liquid in each well of the three pre-coated antigen plates after incubation, wash with PBS buffer, shake gently, discard the liquid in each well of the pre-coated antigen plates after incubation, and repeat washing with PBS buffer twice; In the above washing process, the amount of PBS buffer used for each washing was 200 μL / well; 5. Add 50 μL TMB buffer to each well of the pre-coated antigen plate after enzyme-labeled secondary antibody labeling at different incubation times, and color development reaction at room temperature (25°C) for 15 minutes; 6. The same as step 6 of the method for using an ELISA kit for detecting anti-prawn hemocyanin antibodies in Example 1; 7. Use an ELISA reader to measure the OD of the termination reaction solution in each well of the pre-coated antigen plate after the termination reaction at a working wavelength of 450 nm. 450 value; According to the measured OD 450 The corresponding P / N value was calculated and the incubation time of the enzyme-labeled secondary antibody was determined. The results are shown in the following table: It can be seen from the data analysis in the above table that the incubation time of the enzyme-labeled secondary antibody is 1-2 hours at room temperature (25°C); considering energy consumption, preferably, the incubation time of the enzyme-labeled secondary antibody is 1 hour at room temperature (25°C).

[0039] Example 5 An ELISA kit for detecting anti-prawn hemocyanin antibodies, comprising a pre-coated antigen plate coated with a coating solution with a concentration of 5 μg / mL, 100 mL of 10×PBS buffer, 100 mL of sample diluent, 0.1 mL of standard solution, 100 μL of enzyme-labeled secondary antibody (dilution multiple 1:5000), 100 mL of TMB buffer and 100 mL of stop solution; 1. The pre-coated antigen plate is the same as the pre-coated antigen plate in Example 3; 2. The sample diluent is the same as the sample diluent in Example 1; 3. The standard solution is the same as the standard solution in Example 1; 4. The enzyme-labeled secondary antibody is the same as the enzyme-labeled secondary antibody in Example 1; 5. The stop solution is the same as that in Example 1.

[0040] The above-mentioned ELISA kit for detecting anti-prawn hemocyanin antibodies is stored at 2-8°C.

[0041] The method for using the ELISA kit for detecting anti-prawn hemocyanin antibodies comprises the following specific steps: 1. The same as step 1 of the method for using an ELISA kit for detecting anti-prawn hemocyanin antibodies in Example 1; 2. Dissolve the monoclonal antibody anti-SHC-2B3 in the sample diluent to obtain monoclonal antibody anti-SHC-2B3 solutions with concentrations of 0.5000 μg / mL, 0.2500 μg / mL, 0.1250 μg / mL, 0.0625 μg / mL, and 0.0313 μg / mL, respectively; 3. Preparation method of samples to be tested (1) Select the sera of two BALB / c mice not immunized with shrimp hemocyanin as test samples I and II, and select the sera of two BALB / c mice immunized with shrimp hemocyanin as test samples I' and II', centrifuge at 12000r / min for 5min, remove insoluble substances, and take the supernatant respectively; The above-mentioned process of immunizing BALB / c mice with shrimp hemocyanin is as follows: ①, dissolve hemocyanin in PBS buffer at a concentration of 1 mg / mL to obtain a hemocyanin solution as an immunogen; ②, select two healthy BALB / c mice weighing 20-25g for immunization, the immunization is divided into four times, the first immunization is the primary immunization, and the next three are booster immunizations, each immunization is separated by two weeks, and two immunized BALB / c mice are obtained; The above-mentioned BALB / c mice were purchased from Southern Model Organisms Corporation; Primary immunization: BALB / c mice were subcutaneously injected with hemocyanin solution supplemented with complete Freund's adjuvant at multiple points. The volume ratio of hemocyanin solution to complete Freund's adjuvant was 1:1, and the mixture was fully emulsified before injection. The amount of hemocyanin injected into BALB / c mice in the above primary immunization was 100 μg / mouse; Enhanced immunization: BALB / c mice were subcutaneously injected with hemocyanin solution supplemented with incomplete Freund's adjuvant at multiple points. Hemocyanin and incomplete Freund's adjuvant were mixed in a volume ratio of 1:1 and injected after being fully emulsified. The amount of hemocyanin injected into BALB / c mice in the above booster immunization was 100 μg / mouse; (2) using a sample diluent, diluting the four supernatants 1000 times to obtain diluted sample I, diluted sample I', diluted sample II, and diluted sample II', respectively; 4. Sample loading on the pre-coated antigen plate: add the above-mentioned monoclonal antibody anti-SHC-2B3 solution with a concentration of 0.5000 μg / mL, 0.2500 μg / mL, 0.1250 μg / mL, 0.0625 μg / mL, 0.0313 μg / mL, diluted sample I, diluted sample I', diluted sample II, diluted sample II' and negative control (PBS buffer) to the pre-coated antigen plate, with a sample loading volume of 200 μL per well. Set up 2 parallel samples for each concentration of monoclonal antibody anti-SHC-2B3 solution, diluted sample I, diluted sample I', diluted sample II, diluted sample II' and negative control, and incubate at 37°C for 1 hour. After the above incubation, the liquid in each well of the pre-coated antigen plate was discarded, and then washed with PBS buffer, gently shaken, and then the liquid in each well of the pre-coated antigen plate after incubation was discarded, and then washed with PBS buffer twice; In the above washing process, the amount of PBS buffer used for each washing was 200 μL / well; 5. Enzyme-labeled secondary antibody labeling: After adding the sample, add 200 μL of enzyme-labeled secondary antibody to each well of the pre-coated antigen plate, the monoclonal antibody anti-SHC-2B3 solution with different concentrations, the diluted sample I, the diluted sample I', the diluted sample II, the diluted sample II' and the negative control, and incubate at room temperature (25°C) for 1 hour; Then discard the liquid in each well of the pre-coated antigen plate after incubation, wash with PBS buffer, shake gently, discard the liquid in each well of the pre-coated antigen plate after incubation, and repeat washing with PBS buffer twice; In the above washing process, the amount of PBS buffer used for each washing was 200 μL / well; 6. The same as step 6 of the method for using an ELISA kit for detecting anti-prawn hemocyanin antibodies in Example 1; 7. The same as step 7 of the method for using an ELISA kit for detecting anti-prawn hemocyanin antibodies in Example 1; 8. Use an ELISA reader to measure the OD of the termination reaction solution in each well of the pre-coated antigen plate after the termination reaction at a working wavelength of 450 nm. 450Calculate the average OD of two parallel samples of monoclonal antibody anti-SHC-2B3 solution with different concentrations, diluted sample I, diluted sample I', diluted sample II, diluted sample II' and negative control. 450 value; Judgment criteria: average OD of negative control 450 When the value is less than 0.20, the experiment is established, otherwise the experiment is invalid; the average OD of the negative control 450 The value is the OD of the termination reaction solution in all negative control wells. 450 The average of the values.

[0042] The concentration of monoclonal antibody anti-SHC-2B3 solution was used as the horizontal axis, and the average OD 450 The value is plotted as the ordinate and the curve equation is fitted, such as Figure 3 As shown, the standard curve equation is y = 2.896x + 1.231, R 2 =0.9816; The average OD of the diluted sample I and the diluted sample II 450 The values ​​were all less than 0.20, while the average OD of diluted sample Ⅰ' and diluted sample Ⅱ' after immunization with shrimp hemocyanin was 450 The value was significantly higher than 0.20, indicating that the ELISA kit used to detect anti-shrimp hemocyanin antibodies did not cross-react with non-anti-shrimp hemocyanin antibodies and had good specificity.

[0043] The average OD of the diluted sample I' and the diluted sample II' 450 Substituting the values ​​into the standard curve equation, the concentrations of monoclonal antibody anti-SHC-2B3 in the diluted sample Ⅰ' and the diluted sample Ⅱ' were obtained to be 0.37μg / mL and 0.35μg / mL, respectively. Multiplying by the corresponding dilution factor 1000, the concentrations of monoclonal antibody anti-SHC-2B3 in the sample Ⅰ' and the sample Ⅱ' were 370μg / mL and 350μg / mL, respectively, indicating that the two BALB / c mice had a good immune response to shrimp hemocyanin.

[0044] Example 6 An ELISA kit for detecting anti-shrimp hemocyanin antibodies is validated, and the specific steps are as follows: 1. Specificity verification: According to the method for using an ELISA kit for detecting anti-prawn hemocyanin antibodies in Example 5, bovine serum, horse serum, and sheep serum were taken as samples to be tested for specificity verification. The results are shown in the following table: Samples to be tested <![CDATA[Average OD 450 Value]]> Bovine serum 0.15 Horse serum 0.12 Sheep serum 0.12 From the data analysis in the above table, we can see that the average OD of bovine serum, horse serum and sheep serum 450 The values ​​were all less than 0.20, which further indicated that the established ELISA kit for detecting anti-shrimp hemocyanin antibodies did not cross-react with non-anti-shrimp hemocyanin antibodies and had good specificity.

[0045] 2. Linear verification According to the method for using an ELISA kit for detecting anti-prawn hemocyanin antibodies in Example 5, three different batches of ELISA kits for detecting anti-prawn hemocyanin antibodies were taken for linearity verification. The results are shown in the following table: It can be seen from the data in the above table and the data analysis of Example 5 that four different batches of ELISA kits for detecting anti-shrimp hemocyanin antibodies have a concentration of 0.5000 μg / mL, 0.2500 μg / mL, 0.1250 μg / mL, 0.0625 μg / mL, 0.0313 μg / mL monoclonal antibody anti-SHC-2B3 solution and the corresponding average OD after ELISA determination 450 The linearity of the curve obtained by value fitting is good, R 2 ≥0.9816, all meet R 2 ≥0.9800. Therefore, monoclonal antibody anti-SHC-2B3 can be used as a positive internal reference in the ELISA kit for detecting anti-shrimp hemocyanin antibodies.

[0046] 3. Storage stability verification According to the method for using an ELISA kit for detecting anti-prawn hemocyanin antibodies in Example 5, storage stability verification was performed, and the results are shown in the following table: From the data analysis in the above table, it can be seen that the ELISA kit for detecting anti-shrimp hemocyanin antibodies was stored at 4°C for 1 month, 3 months, 6 months, 9 months, and 12 months, and the average OD of the sample was tested. 450The change rate (%) of the value is 0.4%-1.3%, which is consistent with the change rate (%) ≤ 5%. This shows that the established ELISA kit method for detecting anti-shrimp hemocyanin antibodies has good storage stability.

[0047] In summary, the ELISA kit for detecting anti-shrimp hemocyanin antibodies in the present application has simple experimental steps, is easy for experimental personnel to operate, and the entire practical process takes a short time; at the same time, the linear range is 0.0313-0.5000 μg / mL, R 2 ≥0.9800, good specificity, no cross-reaction with non-anti-shrimp hemocyanin antibodies, and can be stably stored for 12 months. Therefore, the ELISA kit for detecting anti-shrimp hemocyanin antibodies in the present application can quickly and accurately determine whether the animal immune response is successful.

[0048] The above are all preferred embodiments of the present application, and the protection scope of the present application is not limited thereto. Therefore, any equivalent changes made according to the structure, shape, and principle of the present application should be included in the protection scope of the present application.

Claims

1. An ELISA kit for detecting anti-prawn hemocyanin antibodies, characterized in that: Includes pre-coated antigen plates and standard solutions; The pre-coated antigen plate is a 96-well plate coated with a coating solution; the coating solution is a PBS buffer solution of hemocyanin with a concentration of 5 μg / mL; The standard solution is a monoclonal antibody anti-SHC-2B3 solution with a concentration of 5 μg / mL, wherein the solute is the monoclonal antibody anti-SHC-2B3 and the solvent is the sample diluent; The sample diluent is a porclin300 antibacterial agent solution with a volume percentage concentration of 0.1%, wherein the solute is the porclin300 antibacterial agent and the solvent is PBS buffer; The monoclonal antibody anti-SHC-2B3 is obtained by the following steps: (1) Synthesis of monoclonal antibody anti-SHC-2B3 gene fragment Synthesizing a monoclonal antibody anti-SHC-2B3 gene fragment, wherein the monoclonal antibody anti-SHC-2B3 gene fragment is composed of a light chain and a heavy chain of the monoclonal antibody anti-SHC-2B3; The nucleotide sequence of the light chain variable region VL and the light chain constant region CL contained in the light chain of the monoclonal antibody anti-SHC-2B3 is shown in SEQ ID NO.1; the nucleotide sequence of the heavy chain variable region VH and the heavy chain constant region CH1 contained in the heavy chain of the monoclonal antibody anti-SHC-2B3 is shown in SEQ ID NO.2; (2) Preparation of a recombinant plasmid containing the monoclonal antibody anti-SHC-2B3 gene ①, double-digest the vector plasmid pcDNA3.1 with BamH I and EcoR1 to obtain linearized plasmid pcDNA3.1; ② PCR amplification of monoclonal antibody anti-SHC-2B3 gene fragment amplifying the monoclonal antibody anti-SHC-2B3 gene fragment by PCR to obtain the monoclonal antibody anti-SHC-2B3 gene fragment after PCR amplification; The upstream primer sequence used in PCR amplification is AGCTCGGATCC + 20 bp sequence of the 5' of the monoclonal antibody anti-SHC-2B3 gene fragment, and the downstream primer sequence is GAATTCGG + 20 bp sequence of the 3' of the monoclonal antibody anti-SHC-2B3 gene fragment; ③. Ligation reaction The monoclonal antibody anti-SHC-2B3 gene fragment amplified by PCR was connected to the linearized plasmid pcDNA3.1 using T4 DNA ligase, and then transformed and extracted in sequence to obtain the recombinant plasmid pcDNA3.1; The transformation strain used is competent Escherichia coli DH5α; (3) Transfecting the obtained recombinant plasmid pcDNA3.1 into Expi293 cells, and culturing and purifying the obtained recombinant Expi293 cells to obtain the monoclonal antibody anti-SHC-2B3; The purification steps are as follows: ①. Equilibrate the Protein A affinity chromatography column with 3 column volumes of PBS buffer; The Protein A affinity chromatography column is obtained by loading Protein A affinity chromatography resin into a chromatography column; ②, the supernatant collected by centrifugation of the recombinant Expi293 cell suspension obtained by culture was added to the Protein A affinity chromatography column equilibrated with PBS buffer at a flow rate of 2 mL / min; ③. Use PBS buffer to elute and remove non-specifically bound proteins and impurities; ④. Elute the recombinant antibody anti-SHC-2B3 using 0.5 mM glycine elution buffer at pH 3.5; ⑤. The eluted recombinant antibody anti-SHC-2B3 solution was adjusted to pH = 7.2 using a 0.1 mM Tris-HCl aqueous solution at pH = 10, and then concentrated by ultrafiltration to obtain a monoclonal antibody anti-SHC-2B3 concentrate.

2. An ELISA kit for detecting anti-prawn hemocyanin antibodies according to claim 1, characterized in that: It also includes 10× PBS buffer, enzyme-labeled secondary antibody, TMB buffer, and stop solution; The enzyme-labeled secondary antibody is a solution of goat anti-mouse IgG labeled with horseradish peroxidase; The horseradish peroxidase-labeled goat anti-mouse IgG solution is prepared by mixing horseradish peroxidase-labeled goat anti-mouse IgG and PBS buffer in a volume ratio of 1:1000-5000. The stop solution is 0.1 mol / L HCl aqueous solution.

3. An ELISA kit for detecting anti-prawn hemocyanin antibodies according to claim 2, characterized in that: In the solution of goat anti-mouse IgG labeled with horseradish peroxidase, the volume ratio of goat anti-mouse IgG labeled with horseradish peroxidase to PBS buffer is 1:5000.

4. An ELISA kit for detecting anti-prawn hemocyanin antibodies according to claim 1, characterized in that: The coating process of the pre-coated antigen plate is as follows: (1) Dissolve hemocyanin in PBS buffer to prepare a coating solution with a concentration of 5 μg / mL; (2) Coating: Add 50 μL of 5 μg / mL coating solution to each well of a 96-well plate, coat at 4°C for 16 h, then discard the liquid in the wells and wash 3-5 times with PBS buffer to obtain a coated 96-well plate; (3) Blocking: After the 96-well plate was coated in step (2), 100 μL of 10% BSA blocking solution was added to each well, and then the coated 96-well plate was incubated at room temperature of 25°C for 1 h, and then the liquid in the wells was discarded and washed 3-5 times with PBS buffer to obtain a blocked 96-well plate; (4) The 96-well plate blocked in step (3) was placed in a 37°C environment to stabilize for 30 minutes to obtain a pre-coated antigen plate.

5. An ELISA kit for detecting anti-prawn hemocyanin antibodies according to claim 4, characterized in that: The 96-well plate is a 96-well plate with detachable strips, with a specification of 8 wells×12 strips.

6. An ELISA kit for detecting anti-prawn hemocyanin antibodies according to claim 5, characterized in that: It consists of a pre-coated antigen plate, 100 mL 10× PBS buffer, 100 mL sample diluent, 0.1 mL standard solution, 100 μL enzyme-labeled secondary antibody, 100 mL TMB buffer, and 100 mL stop solution; The number of pre-coated antigen plates is 1, 2, 3, 4 or 5.

7. A method for using an ELISA kit for detecting anti-prawn hemocyanin antibodies according to any one of claims 1 to 6, characterized in that: The steps include: (1) Take out the ELISA kit for detecting anti-shrimp hemocyanin antibodies stored at 2-8°C and equilibrate it to room temperature 25°C; (2) Preparation of standard solution for standard curve preparation: dilute the 5 μg / mL standard solution with sample diluent to obtain standard solutions with concentrations of 0.5000 μg / mL, 0.2500 μg / mL, 0.1250 μg / mL, 0.0625 μg / mL, and 0.0313 μg / mL, respectively; (3) Preparation method of the sample to be tested ① Take the sample to be tested, centrifuge it at 12000r / min for 5min, and take the supernatant; The sample to be tested is animal serum immunized with hemocyanin; The animal serum is mouse or rabbit serum; ② Use sample diluent to dilute the supernatant 1000-2000 times to obtain the diluted sample to be tested; (4) Loading samples on the pre-coated antigen plate: Add the above-mentioned standard solutions with concentrations of 0.5000 μg / mL, 0.2500 μg / mL, 0.1250 μg / mL, 0.0625 μg / mL, and 0.0313 μg / mL, the diluted test samples, and the negative control to the pre-coated antigen plate, respectively. The loading volume is 200 μL per well. Two parallel samples are set for each concentration of standard solution, diluted test sample, and negative control. After incubation at 37°C for 1 hour, wash with PBS buffer for 3-5 times. The negative control uses PBS buffer; (5) Enzyme-labeled secondary antibody labeling: Add 200 μL of enzyme-labeled secondary antibody to each well of the pre-coated antigen plate, the standard solution of different concentrations, the diluted test sample and the negative control, incubate at room temperature 25°C for 1-2 hours, and then wash with PBS buffer for 3-5 times; (6) Color reaction: Add 50 μL of TMB buffer to each well of the pre-coated antigen plate after enzyme-labeled secondary antibody labeling, and allow the color reaction to develop for 15 min at room temperature (25°C); (7) Termination reaction: Add 200 μL of stop solution to each well of the pre-coated antigen plate after the color development reaction to terminate the reaction and obtain a stop reaction solution; (8) At a working wavelength of 450 nm, measure the OD of the termination reaction solution in each well of the pre-coated antigen plate after the termination reaction. 450 Calculate the average OD of two parallel samples of standard solution with different concentrations, diluted test sample and negative control. 450 value; (9) Take the concentration of the standard solution in step (2) as the horizontal axis and the average OD of the standard solution of different concentrations in step (8) as the horizontal axis. 450 The values ​​are plotted as the ordinate, and the curve is fitted to obtain the standard curve equation; (10) The average OD of the sample to be tested 450 Substitute the value into the standard curve equation obtained in (9) and combine it with the dilution factor of the sample to be tested to obtain the concentration of anti-shrimp hemocyanin antibodies in the sample to be tested.

8. Use of an ELISA kit for detecting anti-shrimp hemocyanin antibodies according to any one of claims 1 to 6 in detecting animals immunized with hemocyanin; The animal is a mouse or a rabbit.

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