CN112759629A - ELISA kit for detecting iridovirus antibody of micropterus salmoides and detection method thereof - Google Patents

Abstract

The invention relates to the technical field of immunodetection, in particular to an ELISA kit for detecting iridovirus antibodies of micropterus salmoides and a detection method thereof. The kit comprises an antibody capture agent, a solid phase support, a first antibody, an enzyme-labeled second antibody, a confining liquid, a developing liquid and a stop solution; wherein the antibody capture agent is recombinant Lateolabrax micropterus iridovirus LVMCPn protein, and the amino acid sequence of the antibody capture agent is shown as SEQ ID NO. 1. The kit has good sensitivity and specificity to LMBV antibodies, and has good repeatability and stability.

Description

ELISA kit for detecting iridovirus antibody of micropterus salmoides and detection method thereof

Technical Field

The invention relates to the field of immunodetection, in particular to an ELISA kit for detecting an antibody aiming at micropterus salmoides iridovirus and a detection method thereof.

Background

Largemouth bass (Micropterus salmoides) is a fish of the genus Largemouth bass, the family Sunglass, Largetus, commonly known as Micropterus salmoides, and is an important fish species for special fish culture in the south China. The largemouth black bass meat is delicious, has high nutritive value and the consumption is increased year by year. With the change of growth habits, the largemouth bass breeding gradually forms a large-scale breeding mode, and the largemouth bass yield is ten thousand jin under the condition of no diseases, so that the profit is considerable. However, under the condition of high-density culture, the disease of the largemouth bass is frequent, and the disease of the largemouth bass caused by frog iridovirus (Ranavirus) is the most serious, and the virus is combined with bacterial infection to cause the largemouth bass to be seriously rotten, the production performance to be reduced and the death rate to be high. The frog virus infecting micropterus salmoides is known as micropterus salmoides iridovirus (LMBV).

At present, a plurality of methods for detecting antibodies exist, and besides the traditional precipitation reaction, agglutination test and complement fixation test, labeled immunoassay such as enzyme-linked immunoassay, radioimmunoassay, fluorescent immunoassay, luminescent immunoassay and the like become main immunoassay technologies. However, the harm of the frog virus to the micropterus salmoides is not taken into consideration before, and the frog virus becomes the maximum pain point of the high-density culture of the micropterus salmoides along with the continuous improvement of the culture level of the micropterus salmoides; the frog virus has larger genome (80-120Kbp) and more complex pathogenicity, the preparation of immunological raw materials for detecting the LMBV specific antibody has certain technical difficulty, and no detection means which is accurate, simple and convenient and has high sensitivity to the LMBV specific antibody is available.

Disclosure of Invention

The present invention is directed to solving at least one of the above problems in the prior art. The invention discloses a detection method and a kit for specific iridovirus antibodies of micropterus salmoides based on LMBV virus proteins, fish IgM polyclonal antibodies and the like. The kit can analyze and detect LMBV specific antibodies generated by LMBV wild virus infection or artificial immunity micropterus salmoides, has good sensitivity and specificity, and can meet the requirement of LMBV specific antibody analysis and detection of micropterus salmoides.

The technical scheme of the invention is shown as follows.

The invention provides an antibody capture agent, which is a polypeptide with an amino acid sequence shown as SEQ ID NO. 1.

In yet another aspect, the present invention provides an ELISA kit for detecting iridovirus antibodies of micropterus salmoides, comprising an antibody capture agent as described above.

The invention takes the N-terminal structure domain (LVMCPn) sequence of LMBV main capsid protein (LMBV MCP) as reference, synthesizes and expresses recombinant sequences, and establishes an LMBV antibody indirect ELISA diagnostic kit by taking the expressed recombinant protein LVMCPn as an antibody capture agent. The kit has good specificity and high sensitivity.

The inventor researches that the complete sequence of LMBV capsid protein or the C-terminal domain synthetic protein of LMBV MCP is used as antigen, and finds that the sensitivity of different antigens for recognizing LMBV specific antibody in ELISA reaction is obviously different.

According to some embodiments of the invention, the concentration of the LVMCPn protein of the recombinant Perch micropterus iridovirus is greater than or equal to 8 μ g/mL, preferably 8 μ g/mL.

In the ELISA method detection process, the antigen coating concentration has a great influence on the test result, if the antigen coating concentration is high, the protein molecules are multilayered (stacking effect) due to the large acting force between the antigen protein molecules, the protein molecules are easy to wash, the non-specificity is increased, if the concentration is too low, an active surface which is not adsorbed with the antigen but is completely sealed is possibly left on the surface of the ELISA plate, the non-specificity is also increased, and therefore, the concentration of the antigen-coated protein must be screened.

In addition, the purity of the antibody is directly related to the specificity and sensitivity of an ELISA test, and the antibody with low purity often has excessive host cell high molecular compounds combined with specific antigens and can compete with the antigens for limited carrier surface positions to reduce effective adsorption rate, so that the high-purity antibody protein can improve reaction specificity.

According to the invention, through a large number of scientific experiments, the optimal dilution multiple of serum (a sample to be detected) is determined to be 20-40 times, preferably 20 times; the optimal antigen coating concentration is 4-16 mug/mL, and 8 mug/mL is preferable.

According to some embodiments of the invention, the kit further comprises a solid support, a first antibody, an enzyme-labeled second antibody, a blocking solution, a developing solution, and a stop solution.

According to some embodiments of the invention, the solid support is selected from microplate, beads, microparticles, paddles, chips, plastics, membranes, and the like, made of polystyrene, polyethylene, cellulose, nitrocellulose, cellulose acetate, silicide, and the like. Preferably a polystyrene microplate.

The solid phase support is an adsorbent and a container in the ELISA determination process, does not participate in chemical reaction, and still retains the original immunological activity after the antibody or protein antigen is adsorbed thereon.

According to some embodiments of the invention, the first antibody is a polyclonal antibody against micropterus salmoides IgM, preferably a polyclonal antibody against rabbit micropterus salmoides IgM. The first antibody is used for identifying the LMBV-resistant IgM antibody (namely LMBV-specific IgM antibody of the micropterus salmoides) adsorbed by the solid-phase antigen in the micropterus salmoides serum sample.

According to some embodiments of the invention, the first antibody is diluted 2000 to 6000 times, preferably 4000 times, by volume.

According to some embodiments of the invention, the enzyme-labeled secondary antibody is selected from HRP (horseradish peroxidase) -labeled goat anti-rabbit IgG.

According to some embodiments of the invention, the enzyme-labeled second antibody is diluted 4000-fold or 8000-fold by volume.

The concentration of the enzyme-labeled second antibody has great influence on the test result, the concentration is too high, the chance of nonspecific binding is increased, false positive may occur, and the concentration is too low, and false negative may occur if no effective binding exists. Through scientific experiments, the optimal dilution multiple of the enzyme-labeled second antibody is determined to be 1:4000 or 8000.

According to some embodiments of the invention, the blocking solution is selected from a protein-free blocking solution.

According to some embodiments of the invention, the blocking solution is selected from a proteinaceous blocking solution containing 0.1% BSA or horse serum, or a protein-free blocking solution containing a concentration of sugar molecules.

Blocking is a process of recoating with a high concentration of an irrelevant protein solution following coating, the concentration used for antigen coating is low, unoccupied voids remain on the surface of the solid support after adsorption, and blocking is a process of allowing a large amount of irrelevant protein to fill the voids, thereby excluding the reabsorption of interfering substances in the subsequent step of ELISA.

According to some embodiments of the invention, the color developing solution is selected from Tetramethylbenzidine (TMB), o-phenylenediamine, 2, 2-diazanyl-bis- (3-ethylbenzthiazoline-6-sulfonic acid) diamine salt (ABTS) or other HRP colored conjugate.

The developing solution is used for combining with the label on the enzyme-labeled second antibody for developing color, and can be selected according to actual needs.

According to some embodiments of the invention, the stop solution is sulfuric acid.

The purpose of the stop solution is to stop the enzymatic reaction (or immunological reaction), and an appropriate stop solution may be selected depending on the actual enzyme label.

The invention also provides a method for detecting the iridovirus antibody of the micropterus salmoides by using the kit, which comprises the following steps:

1) solid-phase coating: coating the solid phase support with an antibody capture agent, and then sealing with a sealing solution;

2) sample adding: adding a diluted sample to be detected;

3) adding a first antibody: adding the diluted primary antibody;

4) enzyme-labeled secondary antibody: adding a diluted enzyme-labeled secondary antibody;

5) color development: adding a color development liquid;

6) and (4) terminating: adding a stop solution;

7) and (3) detection and judgment: measuring OD of the final sample liquid obtained in the step 6) by using an enzyme-labeling instrument_450nmAnd (4) judging the result.

According to some embodiments of the invention, the test sample is a test serum.

According to some embodiments of the invention, the method further comprises a positive control test and a negative control test; when a positive control test is carried out, replacing the sample to be detected in the step 2) with a positive control; when the negative control test is carried out, the sample to be tested in the step 2) is replaced by a negative control.

According to some embodiments of the invention, the P/N>When 3, the test result is true; when S is more than or equal to 2.1 XN, the sample is judged to be positive; wherein P is a positive control OD_450nmValue, N is the negative control OD_450nmThe value S is the OD of the sample to be measured_450nmA value; when N is less than 0.05, the amount is 0.05.

According to some embodiments of the invention, the positive control is an antibody prepared after immunization of micropterus salmoides with LBMV inactivated virus; the negative control is micropterus salmoides serum without LBMV antibody.

According to some embodiments of the invention, the antibody capture agent, the first antibody and the enzyme-labeled second antibody are added in equal volumes.

The invention has the beneficial effects that:

the invention optimizes the LMBV MCPN end structure domain (LVMCPn), synthesizes and expresses the recombinant protein rLVMCPn, coats the antigen through the rLVMCPn, utilizes the prepared positive serum and negative serum and the Micropterus salmoides IgM polyclonal antibody, determines the antigen coating concentration and the serum dilution, determines the kit assembly and other steps, establishes the LMBV antibody indirect ELISA diagnostic kit, and is used for detecting the LMBV antibody level in the Micropterus salmoides serum. The kit can analyze and detect LMBV specific antibodies generated by LMBV wild virus infection or artificial immunity micropterus salmoides, has good sensitivity and specificity, and can meet the requirement of LMBV specific antibody analysis and detection of micropterus salmoides.

Drawings

FIG. 1 is an analysis diagram of the sequence structure of LMBV MCP;

FIG. 2 is a Western Blot result chart of three expression proteins of LMBV MCP.

Detailed Description

The technical solutions and effects of the present invention will be further described and illustrated with reference to the following specific examples, but the present invention is not limited to these specific embodiments. The test methods used in the examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are commercially available reagents and materials unless otherwise specified.

Wherein, the rabbit anti-micropterus salmoides IgM polyclonal antibody is provided by Guangdong sea Daorhike veterinary research institute, Inc. The confining liquid used in the experiment is a protein-free confining liquid containing sugar molecules with a certain concentration, and is purchased from livestock veterinary research institute, ltd, of the great east China sea. The stop solution used in the experiment was 2M sulfuric acid.

Nocardia seriolae, Aeromonas schubertii and Aeromonas hydrophila are respectively obtained by separating from a certain largemouth black bass culture field in Jiangmen of Guangdong, and are identified as Nocardia seriolea, Aeromonas schubertii and Aeromonas hydrophilum through morphological characteristics, physiological and biochemical characteristics and molecular biological characteristics.

Iridovirus (LMBV) of largemouth bass, which is separated from a certain largemouth bass culture field in the Guangdong river; siniperca chuatsi Infectious Spleen and Kidney Necrosis Virus (ISKNV) and Mandarin fish rhabdovirus (SCRV) were isolated from Siniperca chuatsi farm in Guangdong. All three viruses were identified using whole genome sequencing.

EXAMPLE 1 expression purification of recombinant protein

Constructing expression plasmids of LMBV MCP complete sequence (LVMCP), N-terminal domain (LVMCPn) of LMBV MCP protein and C-terminal domain (LVMCPc) of LMBV MCP, and transforming the expression plasmids into Escherichia coli expression strain Rosetta (DE3) pLysS for expression to obtain recombinant proteins LVMCP, LVMCPn and LVMCPc.

Wherein, the amino acid sequences of LVMCP, LVMCPn and LVMCPc are respectively as follows: 2, 1 and 3; the structural analysis of the LMBV MCP protein sequence is shown in figure 1.

The expression of 3 engineering bacteria was identified by Western Blot, and the results are shown in FIG. 2, wherein M is protein Ladder, and lanes 1-3 are Western blots of LVMCP, LVMCPn and LVMCPc, respectively.

The 3 recombinant proteins were purified by affinity chromatography and the protein concentrations were calibrated using a commercial BCA protein quantification kit.

Example 2LMBV antibody positive, negative sera and quality control

1. Preparation of LMBV antibody-positive serum

Culturing LMBV virus on carp epithelial cell (EPC), adding formaldehyde inactivated virus with final concentration of 0.2%, emulsifying virus stock solution with VSA201 adjuvant after quality inspection to prepare vaccine, and storing at 4 deg.C after quality inspection. 200-300 g of 50 healthy largemouth bass is selected, the prepared LMBV inactivated vaccine is used for abdominal immunity, the immunization dose is 200 mu L/mouse, 2 times of immunity are carried out, and the interval of each immunization is 15 days. All tail fin root veins are collected and the antibody titer is evaluated by combining an ELISA test. Selecting a serum sample with relatively high titer (the serum sample is diluted by 40 times and the OD450nm value is about 1.0) as LMBV antibody positive serum, and storing at-20 ℃ for later use.

2. Preparation of LMBV antibody negative serum

200-300 g of healthy largemouth bass 50 tails are selected, PBS emulsified by VSA201 adjuvant is used as a control vaccine for intraperitoneal immunization, and the immunization and the test method are prepared by the positive serum. Selecting a serum sample without immune reaction as LMBV antibody negative serum, and storing at-20 ℃ for later use.

3. LMBV antibody detection quality control product

The specificity of the LMBV antibody ELISA detection method is evaluated by taking antibody positive serum of 5 common pathogens for micropterus salmoides culture as a quality control product.

The quality control product preparation and inspection method comprises the following steps: respectively culturing (1) rhabdovirus, (2) siniperca chuatsi infectious spleen and kidney necrosis virus and (3) nocardia seriolae, (4) schubert aeromonas and (5) aeromonas hydrophila, inactivating virus or bacteria, and emulsifying the inactivated matter with VSA201 adjuvant to prepare the vaccine. 200 and 300 g of healthy largemouth bass 50 tails are selected, the abdominal cavity of the prepared inactivated vaccine is used for immunization, the immunization dose is 200 mu L/mouse, the immunization is carried out for 3-4 times, and the interval of each immunization is 15 days. All immunized tail fin roots were bled intravenously and antibody titers were assessed in conjunction with either the neutralization assay, or the latex agglutination assay. Selecting serum samples with relatively high bacterial or toxic antibody titer (neutralization test (rhabdovirus, mandarin infectious spleen and kidney necrosis virus) or latex agglutination test (Nocardia seriolae, Schubert aeromonas, hydrophila) and serum samples with antibody titer more than 8.) as quality control products, and storing at-20 deg.C.

Example 3 ELISA reactivity of three recombinant proteins

The immunological reaction of the micropterus salmoides serum antibody with 3 recombinant proteins (prepared in example 1) was detected using an indirect ELISA method. ELISA detection solid phase coating materials are respectively selected: LVMCP, LVMCPn and LVMCPc. The specific method comprises the following steps:

(1)3 recombinant proteins were diluted with 0.05mol/L carbonate (PH 9.6) to 2 μ g/mL, 4 μ g/mL, and 8 μ g/mL coated elisa plates, 100 μ L/well, incubated at 37 ℃ for 1 hour, and at 4 ℃ overnight, respectively;

(2) washing the microplate 3 times, 5 min/time, with 0.05% Tween-20 (PBST); adding 100 mu L of sealing liquid of 5% skimmed milk powder into each hole, and sealing at 37 ℃ for 1 hour;

(3) wash 3 times with PBST, 3 min/time; diluting the largemouth bass serum (LMBV antibody positive serum or LMBV antibody negative serum) prepared in example 2 by 1:20 times, adding into the well, and incubating for 30 minutes at 37 ℃;

(4) wash 3 times with PBST, 3 min/time; adding rabbit anti-micropterus salmoides IgM polyclonal antibody (first antibody), diluting at 1:4000, and incubating at 37 ℃ for 1 hour;

(5) HRP-labeled goat anti-rabbit IgG was added according to the commercial reagent instructions (Shanghai Biyuntian Biotech Co., Ltd.), diluted at 1:8000, and incubated at 37 ℃ for 45 minutes. Wash 5 times with PBST, 3 min/time;

(6) adding 100 mu L/hole of color development liquid (TMB), developing for 5-15 minutes in a dark place at room temperature, and finally adding 100 mu L of 2mol/L sulfuric acid to terminate the reaction; OD determination with microplate reader_450nmThe value is obtained.

Performing ELISA analysis according to the optimization method, evaluating the reactivity of the three recombinant proteins and LMBV antibody strong positive (serum samples with 40-fold serum dilution and OD450nm value of about 1.0) and weak positive (serum samples with 40-fold serum dilution and OD450nm value of 0.3-0.4) two types of sera, and screening the optimal solid phase coating. During the test, a blank control well without serum sample was set.

The results are shown in table 1, and analysis gave: (1) of the 3 recombinant proteins, LVMCPn showed the best ELISA reactivity (OD) with both strong positive and weak positive sera to LMBV antibody_450nmHighest value), the sensitivity is higher;

(2) among the 3 recombinant proteins, when the coating concentration of LVMCPn is increased to 8 mug/mL, the negative control change is small, and the P/N value is calculated, so that the P/N value of the LVMCPn hole is high, and the specificity of the antigen is good.

In conclusion, LVMCPn was selected as the LMBV antibody for detection of the solid phase coating.

TABLE 1 ELISA reactivity (OD) of LMBV antibodies with different antigens_450nmMean value)

Example 4 construction of LMBV specific antibody Indirect ELISA method

1. Optimal antigen and antibody concentrations used (checkerboard titration)

The positive serum diluted in a gradient way is taken as a sample, a rabbit anti-micropterus salmoides IgM polyclonal antibody and HRP marked goat anti-rabbit IgG dilution are fixed, antigens with different concentrations are coated by an ELISA solid phase, and the optimal antigen use concentration and the sample dilution are known by a chessboard titration method. The specific operation is carried out according to the following steps:

(1) solid phase coating of LMBV recombinant protein (LVMCPn): diluting LVMCPn to 16, 8, 4, 2, 1, 0.5 μ g/mL, respectively coating polystyrene reaction plate, incubating at 37 deg.C for 1 hr, incubating at 4 deg.C overnight, adding washing solution at 250 μ L/well the next day, washing continuously for 3 times, and drying;

(2) and (3) sealing: adding 150 μ L/well of sealing liquid, sealing at 37 deg.C for 1 hr, adding 250 μ L/well of washing liquid, continuously washing for 3 times, drying, and storing at 4 deg.C;

(3) sample adding: placing the ELISA plate at room temperature for 30 minutes, diluting positive and negative serum according to the ratio of 1:20, 1:40, 1:80, 1:160 and 1:320, loading sample according to 100 mu L/hole, incubating for 30 minutes at 37 ℃, loading washing solution according to 250 mu L/hole, continuously washing for 3 times, and drying;

(4) adding a first antibody: adding rabbit anti-micropterus salmoides IgM polyclonal antibody diluted by 1:4000 as a first antibody, incubating for 1 hour at 37 ℃ in 100 mu L/hole;

(5) addition of a second antibody: adding HRP-labeled goat anti-rabbit IgG diluted at the ratio of 1:8000 into the mixture, incubating the mixture at the temperature of 100 mu L/hole for 45 minutes, adding a washing solution into the mixture at the temperature of 250 mu L/hole, continuously washing the mixture for 3 times, and patting the mixture dry;

(6) adding TMB color development liquid, developing at 50 μ L/hole in dark at room temperature for 5-15 min;

(7) adding 50 mu L of stop solution into each hole to stop the reaction, reading within 10 minutes after the reaction is stopped, and determining the OD of an enzyme-linked immunosorbent assay (OD)_450nmAnd (6) reading.

(8) Calculation of Positive serum OD_450nmValue/negative serum OD_450nmThe value is the P/N value, and the optimal dilution of the antigen serum is determined according to the ratio. During the test, a test control without the sample is set as a blank control. The results are shown in tables 2 and 3.

TABLE 2 serum ELISA test results for different concentrations of antigen and different dilution times

TABLE 3P/N values of ELISA reactions

According to the analysis of the P/N value of the test result, when the P/N value is maximum, the coating concentration of the recombinant protein LVMCPn is 8 mug/mL, and the serum dilution multiple is 20 times.

2. Optimal use concentration of the first antibody and the second antibody

2.1 Rabbit anti-IgM polyclonal antibody (first antibody) optimization Using concentration

Referring to the optimization result of chessboard titration, the enzyme-labeled reaction plate is coated with LVMCPn of 8 mug/mL, 20 times diluted positive serum and negative serum are added, and rabbit anti-micropterus salmoides IgM polyclonal antibody (primary antibody) is diluted in a gradient way to determine the optimal using concentration of the antibody. The specific operation is carried out according to the following steps:

(1) solid phase coating of LMBV recombinant protein (LVMCPn): diluting LVMCPn to 8 μ g/mL, coating polystyrene reaction plate, incubating at 37 deg.C for 1 hr, incubating at 4 deg.C overnight, washing at 250 μ L/well for 3 times, and drying;

(2) and (3) sealing: adding 150 μ L/well of sealing liquid, sealing at 37 deg.C for 1 hr, adding 250 μ L/well of washing liquid, continuously washing for 3 times, drying, and storing at 4 deg.C;

(3) sample adding: placing the ELISA plate at room temperature for 30 minutes, diluting positive and negative serum according to a ratio of 1:20, adding sample according to 100 mu L/hole, incubating for 30 minutes at 37 ℃, adding washing solution according to 250 mu L/hole, continuously washing for 3 times, and patting to dry;

(4) adding a first antibody: adding 1000, 2000, 4000, 8000 and 16000 times diluted rabbit anti-micropterus salmoides IgM polyclonal antibody (primary antibody) respectively, 100 μ L/well, incubating at 37 deg.C for 1 hr;

(5) addition of a second antibody: adding HRP-labeled goat anti-rabbit IgG diluted 8000 times, incubating at 100 μ L/well for 45 min at 37 deg.C, adding washing solution at 250 μ L/well, washing continuously for 3 times, and patting to dry;

(6) adding TMB color development liquid, developing at 50 μ L/hole in dark at room temperature for 5-15 min;

(7) adding 50 mu L of stop solution into each hole to stop the reaction, reading within 10 minutes after the reaction is stopped, and determining the OD of an enzyme-linked immunosorbent assay (OD)₄₅₀And reading in nm.

(8) Calculation of Positive serum OD_450nmValue/negative serum OD_450nmThe value is the P/N value, and the optimal dilution of the antigen serum is determined according to the ratio. During the test, a test control without the sample is set as a blank control. The results are shown in tables 4 and 5 below.

TABLE 4 different dilutions of the first antibody ELISA assay results

TABLE 5P/N values of ELISA reactions

According to the analysis of the P/N value of the test result, when the P/N value is maximum, the dilution ratio of the rabbit anti-micropterus salmoides IgM polyclonal antibody (primary antibody) is 4000 times.

2.2 HRP-labeled goat anti-rabbit IgG concentration optimization

Referring to the optimization result, the enzyme-labeled reaction plate is coated with LVMCPn of 8 mug/mL, 20-fold diluted positive serum and negative serum are added, the rabbit anti-micropterus salmoides IgM polyclonal antibody (primary antibody) is diluted 4000-fold, and the commercial HRP-labeled goat anti-rabbit IgG (secondary antibody) is subjected to gradient dilution to determine the optimal using concentration of the secondary antibody. The specific operation is carried out according to the following steps:

(1) solid phase coating of LMBV recombinant protein (LVMCPn): diluting LVMCPn to 8 mu g/mL, coating a polystyrene enzyme label plate, incubating at the temperature of 100 mu L/well for 1 hour at 37 ℃, incubating overnight at the temperature of 4 ℃, continuously washing for 3 times on the next day by adding a washing solution into each well at the temperature of 250 mu L, and patting dry;

(2) and (3) sealing: adding 150 μ L/well of sealing liquid, sealing at 37 deg.C for 1 hr, adding 250 μ L/well of washing liquid, continuously washing for 3 times, drying, and storing at 4 deg.C;

(3) sample adding: placing the ELISA plate at room temperature for 30 minutes, diluting positive and negative serum according to a ratio of 1:20, adding sample according to 100 mu L/hole, incubating for 30 minutes at 37 ℃, adding washing solution according to 250 mu L/hole, continuously washing for 3 times, and patting to dry;

(4) adding a first antibody: adding a 4000-fold diluted rabbit anti-micropterus salmoides IgM polyclonal antibody (primary antibody), 100 mu L/well, and incubating at 37 ℃ for 1 hour;

(5) addition of a second antibody: adding commercial HRP-labeled goat anti-rabbit IgG diluted by 2000, 4000, 8000 and 16000 times respectively, incubating at 37 deg.C for 45 min, adding washing solution at 250 μ L/well, washing continuously for 3 times, and patting to dry;

(6) adding TMB color development liquid, developing at 50 μ L/hole in dark at room temperature for 5-15 min;

(7) adding 50 mu L of stop solution into each hole to stop the reaction, reading within 10 minutes after the reaction is stopped, and determining the OD of an enzyme-linked immunosorbent assay (OD)₄₅₀And reading in nm.

(8) Calculation of Positive serum OD_450nmValue/negative serum OD_450nmThe value is the P/N value, and the optimal dilution of the antigen serum is determined according to the ratio. During the test, a test control without the sample is set as a blank control.

The results are shown in tables 6 and 7 below.

TABLE 6 different dilutions of the second antibody ELISA test results

TABLE 7P/N values of ELISA reactions

When the P/N value is maximum, the dilution factor of the commercial HRP-labeled goat anti-rabbit IgG (secondary antibody) is 8000 or 4000 times, according to the P/N value analysis of the test results.

3. Optimum incubation conditions

Referring to the optimization result, in the detection of the LMBV antibody, an enzyme-labeled reaction plate is coated with LVMCPn of 8 mug/mL, positive serum and negative serum which are diluted by 20 times are added, a rabbit anti-micropterus salmoides IgM polyclonal antibody (primary antibody) is diluted by 4000 times, and a commercial HRP-labeled goat anti-rabbit IgG (secondary antibody) is diluted by 8000. The test will further optimize the optimal incubation conditions-temperature and time-of the serum to be tested and the primary antibody.

3.1 optimization of incubation conditions for serum to be tested

The specific operation is carried out according to the following steps:

(1) solid phase coating of LMBV recombinant protein (LVMCPn): diluting LVMCPn to 8 μ g/mL, coating polystyrene reaction plate, incubating at 37 deg.C for 1 hr, incubating at 4 deg.C overnight, washing at 250 μ L/well for 3 times, and drying;

(2) and (3) sealing: adding 150 μ L/well of sealing liquid, sealing at 37 deg.C for 1 hr, adding 250 μ L/well of washing liquid, continuously washing for 3 times, drying, and storing at 4 deg.C;

(3) sample adding of serum to be detected: placing the ELISA plate at room temperature for 30 minutes, diluting positive serum and negative serum according to a ratio of 1:20, adding sample according to 100 mu L/hole, setting incubation conditions of (first) 37 ℃ for 1 hour, second 37 ℃ for 30 minutes and (third) room temperature for 1 hour; adding a washing solution into a hole with the volume of 250 mu L, continuously washing for 3 times, and patting to dry;

(4) adding a first antibody: adding a 4000-fold diluted rabbit anti-micropterus salmoides IgM polyclonal antibody (primary antibody), 100 mu L/well, and incubating at 37 ℃ for 1 hour;

(5) addition of a second antibody: adding HRP-labeled goat anti-rabbit IgG diluted 8000 times, incubating at 100 μ L/well for 45 min at 37 deg.C, adding washing solution at 250 μ L/well, washing continuously for 3 times, and patting to dry;

(6) adding TMB color development liquid, developing at 50 μ L/hole in dark at room temperature for 5-15 min;

(7) adding 50 mu L of stop solution into each hole to stop the reaction, reading within 10 minutes after the reaction is stopped, and determining the OD of an enzyme-linked immunosorbent assay (OD)₄₅₀And reading in nm.

(8) Calculation of Positive serum OD_450nmValue/negative serum OD_450nmThe value is the P/N value, and the optimal dilution of the antigen serum is determined according to the ratio. During the test, a test control without the sample is set as a blank control. The results are shown in tables 8 and 9 below.

TABLE 8 ELISA test results of samples to be tested under different incubation conditions

TABLE 9P/N values of ELISA reactions

According to the analysis of the P/N value of the test result, when the P/N value is maximum, the optimal incubation condition of the serum to be detected is 37 ℃ for 30 minutes or room temperature for 1 hour, and in order to save the detection time, the optimal incubation condition of the serum to be detected is preferably 37 ℃ for 30 minutes.

3.2 Rabbit anti-Lateolabrax micropterus IgM polyclonal antibody incubation condition optimization

The specific operation is carried out according to the following steps:

(1) solid phase coating of LMBV recombinant protein (LVMCPn): diluting LVMCPn to 8 μ g/mL, coating polystyrene reaction plate, incubating at 37 deg.C for 1 hr, incubating at 4 deg.C overnight, washing at 250 μ L/well for 3 times, and drying;

(2) and (3) sealing: adding 150 μ L/well of sealing liquid, sealing at 37 deg.C for 1 hr, adding 250 μ L/well of washing liquid, continuously washing for 3 times, drying, and storing at 4 deg.C;

(3) sample adding of serum to be detected: placing the ELISA plate at room temperature for 30 minutes, diluting positive and negative serum at a ratio of 1:20, loading the sample at a concentration of 100 μ L/well at 37 ℃ for 30 minutes; adding a washing solution into a hole with the volume of 250 mu L, continuously washing for 3 times, and patting to dry;

(4) adding a first antibody: adding a 4000-fold diluted rabbit anti-micropterus salmoides IgM polyclonal antibody (a first antibody) with the concentration of 100 mu L/hole, setting incubation conditions of firstly 37 ℃ for 1 hour, secondly 37 ℃ for 30 minutes and thirdly room temperature for 1 hour;

(5) addition of a second antibody: adding HRP-labeled goat anti-rabbit IgG diluted 8000 times, incubating at 100 μ L/well for 45 min at 37 deg.C, adding washing solution at 250 μ L/well, washing continuously for 3 times, and patting to dry;

(6) adding TMB color development liquid, developing at 50 μ L/hole in dark at room temperature for 5-15 min;

(7) adding 50 mu L of stop solution into each hole to stop the reaction, reading within 10 minutes after the reaction is stopped, and determining the OD of an enzyme-linked immunosorbent assay (OD)_450nmAnd (6) reading.

(8) Calculation of Positive serum OD_450nmValue/negative serum OD_450nmThe value is the P/N value, and the optimal dilution of the antigen serum is determined according to the ratio. During the test, a test control without the sample is set as a blank control. The results are shown in tables 10 and 11 below.

TABLE 10 different incubation conditions of the first antibody ELISA test results

TABLE 11P/N values of ELISA reactions

According to the analysis of the P/N value of the test result, when the P/N value is maximum, the optimal incubation condition of the rabbit anti-micropterus salmoides IgM polyclonal antibody is 37 ℃ for 1 hour.

4. Specificity test

The LMBV antibody ELISA detection reagent is prepared by applying optimized test conditions, and antibody positive serum (prepared by using holobacteria/virus inactivator) of (1) Nocardia seriolia seriolae, (2) Aeromonas schubertii (Aeromonas schubertii), (3) Aeromonas hydrophila, (4) Siniperca chuatsi (ISKNV) and (5) rhabdovirus (SHRV) is respectively detected, and the specificity of the detection method is evaluated. During the test, a test control without the sample is set as a blank control. The results are given in Table 12 below.

TABLE 12 ELISA detection specificity assay

Except LMBV antibody positive serum, the serum has no reaction with other common pathogenic antibodies, which indicates that the specificity of the method is good.

5. Repeatability test

Assembly of 3 batches of reagents using optimized assay conditionsBox(laboratory preparations). Detecting 45 Perciformes micropterus serum samples, repeating each serum sample in the same plate for 5 times, performing statistical analysis on the detection results, and respectively evaluating the reagentsBoxCoefficient of variation for intra-and inter-batch repeats. The maximum Coefficient of Variation (CV) of the kit in batches and among batches is 5 percent and 6 percent respectively, and the kit has good repeatability and stability.

EXAMPLE 5 practical application case

500 micropterus salmoides (approximately 40 g/tail) were randomly transported from the pond to two cement ponds, labeled group 1 and group 2, 250/pond (groups). Preparing an oil emulsion inactivated vaccine by taking the inactivated LMBV whole virus as an antigen, wherein the group 1 largemouth black bass abdominal cavity vaccine is 100 microliters per tail; group 2, the largemouth black bass is normally fed and not immunized. After 21 days, blood is respectively collected from tail veins of two groups of fishes, and serum is separated; the method is applied to detecting the micropterus salmoides serum sample.

The ELISA detection method comprises the following steps:

(1) solid-phase coating: diluting LVMCPn to 8 μ g/ml, coating polystyrene reaction plate, incubating at 37 deg.C for 1 hr, incubating at 4 deg.C overnight, washing at 250 μ L/well for 3 times, and drying;

(2) and (3) sealing: adding 150 μ L/well of sealing liquid, sealing at 37 deg.C for 1 hr, adding 250 μ L/well of washing liquid, continuously washing for 3 times, drying, and storing at 4 deg.C;

(3) sample adding of serum to be detected: placing the ELISA plate at room temperature for 30 minutes, diluting the serum to be detected of group 1 (47 parts by random selection) and group 2 (47 parts by random selection) according to a ratio of 1:20, adding the sample according to 100 mu L/hole, and carrying out the sample addition at 37 ℃ for 30 minutes; adding a washing solution into a hole with the volume of 250 mu L, continuously washing for 3 times, and patting to dry;

(4) adding a 4000-fold diluted rabbit anti-micropterus salmoides IgM polyclonal antibody at the temperature of 37 ℃ for 1 hour at 100 mu L/hole;

(5) adding HRP-labeled goat anti-rabbit IgG diluted 8000 times, incubating at 100 μ L/well for 45 min at 37 deg.C, adding washing solution at 250 μ L/well, washing continuously for 3 times, and patting to dry;

(6) adding TMB color development liquid, developing at 50 μ L/hole in dark at room temperature for 5-15 min;

(7) adding 50 mu L of stop solution into each hole to stop the reaction, reading within 10 minutes after the reaction is stopped, and determining the OD of an enzyme-linked immunosorbent assay (OD)₄₅₀And reading in nm.

The microplate reader readings are shown in table 13 below.

The results were analyzed to yield:

(1) group 1 is the LMBV inactivated vaccine immunization group, wherein row a, row 1, is an LMBV antibody positive serum sample; the remaining 47 were randomly selected micropterus salmoides serum samples from the group 1 pool; ELISA antibody detection results show that 47 serum samples OD selected randomly_450nmThe average value is 0.229 +/-0.151; group 2 was the normal-fed, non-immunized group, in which row E1 is the LMBV antibody negative serum sample; the remaining 47 are randomly selected micropterus salmoides serum samples in the group 2 pool; 47 serum sample OD_450nmThe average value was 0.097. + -. 0.033. The statistical analysis (T test) shows that the difference between the two is extremely obvious, which indicates that the established ELISA detection method can evaluate the LMBV antibody change after different immune treatments;

(2) for OD in group 2_450nmTwo serum samples (0.264 and 0.222) with higher values were tested for serum by qPCRThe LMBV nucleic acids are all positive, which indicates that the LMBV antibody of the immunized micropterus salmoides is increased due to the influence of LMBV wild virus.

(3) By detecting a large amount of serum samples and combining the qPCR method for LMBV nucleic acid detection, the Cut-off value of the ELISA method is 2.1 XN according to the C.O (N is a negative control OD)_450nmAnd when N is less than 0.05, the amount is 0.05). According to this determination method, 14/47 serum samples were positive for LMBV antibody in group 1 and 2/47 was positive for LMBV antibody in group 2.

TABLE 13 serum sample LMBV antibody test results

Combining a large number of sample detection results, the ELISA detection method comprises the following steps:

1. solid-phase coating: diluting LVMCPn to 8 μ g/mL, coating polystyrene reaction plate, incubating at 37 deg.C for 1 hr, incubating at 4 deg.C overnight, washing at 250 μ L/well for 3 times, and drying;

2. and (3) sealing: adding 150 μ L/well of sealing liquid, sealing at 37 deg.C for 1 hr, adding 250 μ L/well of washing liquid, continuously washing for 3 times, drying, and storing at 4 deg.C;

3. sample adding of serum to be detected: the ELISA plate was placed at room temperature for 30 minutes, and positive (47 random aliquots) and negative (47 random aliquots) sera were diluted at a ratio of 1:20, loaded at 100. mu.L/well, and incubated at 37 ℃ for 30 minutes; adding a washing solution into a hole with the volume of 250 mu L, continuously washing for 3 times, and patting to dry;

4. adding a first antibody: adding a 4000-fold diluted rabbit anti-micropterus salmoides IgM polyclonal antibody (primary antibody), 100 mu L/well, 37 ℃, 1 hour;

5. addition of a second antibody: adding HRP-labeled goat anti-rabbit IgG diluted 8000 times, incubating at 100 μ L/well for 45 min at 37 deg.C, adding washing solution at 250 μ L/well, washing continuously for 3 times, and patting to dry;

6. adding TMB color development liquid, developing at 50 μ L/hole in dark at room temperature for 5-15 min;

7. stop the reaction by adding 50. mu.L of stop solution to each well, and reading the enzyme within 10 minutes after the reaction is stoppedStandard instrument OD₄₅₀And reading in nm.

8. And a result judgment method comprises the following steps: (1) the test result is true when P/N > 3 (P is positive control OD_450nmN is negative control OD_450nm) (ii) a (2) Positive when S is not less than 2.1 XN (S is OD of sample to be detected)_450nmN is negative control OD_450nmAnd when N is less than 0.05, the amount is 0.05).

While the invention has been disclosed with reference to specific embodiments, it will be apparent that other embodiments and variations of the invention may be devised by those skilled in the art without departing from the true spirit and scope of the invention, and it is intended that the following claims be interpreted to include all such embodiments and equivalent variations. In addition, the contents of all references cited herein are hereby incorporated by reference.

SEQUENCE LISTING

<110> Guangdong sea Daorhusbandry veterinary research institute Co., Ltd

<120> ELISA kit for detecting iridovirus antibody of micropterus salmoides and detection method thereof

<130> 111

<160> 3

<170> PatentIn version 3.5

<210> 1

<211> 222

<212> PRT

<213> Micropterus salmoides

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His Phe Ser Val Gly Val Pro Arg Ser Gly Asp Tyr Val Leu Asn Ser

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Claims (10)

1. An antibody capture agent, which is characterized in that the antibody capture agent is a polypeptide with an amino acid sequence shown as SEQ ID NO. 1.

2. An ELISA kit for detecting iridovirus antibodies of micropterus salmoides, comprising the antibody capture agent of claim 1.

3. The kit of claim 2, further comprising a solid support, a first antibody, an enzyme-labeled second antibody, a blocking solution, a developing solution, and a stop solution.

4. The kit of claim 3, wherein the first antibody is an anti-micropterus salmoides IgM polyclonal antibody.

5. The kit of claim 3, wherein the enzyme-labeled secondary antibody is selected from the group consisting of HRP-labeled goat anti-rabbit IgG.

6. The method for detecting iridovirus antibodies of micropterus salmoides by using the kit of any one of claims 2 to 5, which is characterized by comprising the following steps:

1) solid-phase coating: coating the solid phase support with an antibody capture agent, and then sealing with a sealing solution;

2) sample adding: adding a diluted sample to be detected;

3) adding a first antibody: adding the diluted primary antibody;

4) enzyme-labeled secondary antibody: adding a diluted enzyme-labeled secondary antibody;

5) color development: adding a color development liquid;

6) and (4) terminating: adding a stop solution;

7) and (3) detection and judgment: measuring OD of the final sample liquid obtained in the step 6) by using an enzyme-labeling instrument_450nmAnd (4) judging the result.

7. The method of claim 6, further comprising a positive control test and a negative control test; when a positive control test is carried out, replacing the sample to be detected in the step 2) with a positive control; when the negative control test is carried out, the sample to be tested in the step 2) is replaced by a negative control.

8. The method of claim 7, wherein P/N is>When 3, the test result is true; when S is more than or equal to 2.1 XN, the sample is judged to be positive; wherein P is a positive control OD_450nmValue, N is the negative control OD_450nmThe value S is the OD of the sample to be measured_450nmA value; when N is less than 0.05, the amount is 0.05.

9. The method according to claim 7 or 8, wherein the positive control is an antibody prepared after LBMV inactivated virus immunization of micropterus salmoides; the negative control is micropterus salmoides serum without LBMV antibody.

10. Use of the antibody capture agent of claim 1 or the kit of any one of claims 2 to 5 in the preparation of a reagent for detecting iridovirus antibodies of micropterus salmoides.

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