CN103267846A - An ELISA kit for detecting type 2 porcine circovirus IgM antibody - Google Patents

Abstract

The invention discloses an ELISA (Enzyme Linked Immunosorbent Assay) kit for detecting a porcine circovirus 2 (PCV2) IgM antibody. The kit comprises a porcine IgM antibody coated ELISA batten, an enzyme-labeled antigen of the PCV2 antibody, a cleaning solution, serum, an enzyme-labeled antigen diluent, a color-substrate solution, a stop solution, and PCV2 negative and positive control serum. The kit has the characteristics of good specificity and high sensitivity, is easy to operate, needs short detection time, and can be used for IgM (Immunoglobulin M) antibody detection of PCV2 and diagnosis of early infection.

Description

An ELISA kit for detecting type 2 porcine circovirus IgM antibody

technical field

The invention belongs to the technical field of veterinary medicine, and in particular relates to an ELISA kit for detecting type 2 porcine circovirus IgM antibody, which can be used for the detection of type 2 porcine circovirus IgM antibody and the diagnosis of early infection. Background technique

Clinically, type 2 circovirus (PCV2) infection is very common in pigs. Enzyme-linked immunosorbent assay (ELISA) is a commonly used detection method. The existing methods are mainly only for IgG antibody detection, and cannot distinguish maternal antibodies. Antibodies after PCV2 infection and vaccine immunization cannot effectively diagnose the disease and help formulate reasonable prevention and control measures.

At present, the method for detecting PCV2-IgM reported in China is the indirect ELISA method, but the sensitivity and specificity of this method are not ideal enough, and it cannot be used in clinical practice; the method for detecting PCV2-IgM antibody in foreign countries is mainly Spanish INGENASA company The capture method antibody detection kit produced, but the kit needs to prepare monoclonal antibodies against PCV2, the production cost is high, the detection principle is relatively complicated, and the detection time is too long, which are not conducive to clinical promotion and use.

Contents of the invention

The technical problem to be solved by the present invention is to provide an ELISA kit for detecting PCV2-IgM antibody for the deficiencies of the above-mentioned prior art. It has the advantages of less time, etc., and can be used for the detection of IgM antibody of PCV2 and the diagnosis of early infection.

In order to solve the above-mentioned technical problems, the technical solution adopted in the present invention is: an ELISA kit for detecting PCV2-IgM antibodies, which is characterized in that the kit includes ELISA strips coated with anti-pig IgM antibodies, and anti-pig Enzyme-labeled antigen for porcine circovirus type 2 antibody.

The above kit also includes washing solution, serum and enzyme-labeled antigen diluent, substrate chromogenic solution, stop solution, negative control and positive control. in:

The ELISA strip: each kit contains 5 ELISA plates, each plate contains the detachable ELISA strip, and the specification is 8 wells × 12 strips;

The washing solution: 200ml of 10 times concentrated 1M phosphate solution (PBS) with a pH of 7.4 containing 0.5% by volume of Tween-20;

The enzyme-labeled antigen of the anti-type 2 porcine circovirus antibody: 0.5 ml of 100-fold concentrated horseradish peroxidase-labeled Cap protein recombinant antigen of type 2 circovirus;

Described serum and enzyme-labeled antigen diluent: pH is 100ml of 1M PBS solution of 7.4;

The substrate chromogenic solution: TMB chromogenic solution 50ml;

The stop solution: 50ml of 2M sulfuric acid solution;

The negative and positive controls: 0.2 ml each of standard PCV2 negative and positive serum.

The kit of the present invention is based on a principle different from the indirect method and the antibody capture method. The basic principle is: firstly, the ELISA plate is coated with anti-pig IgM antibody to capture the total IgM antibody in the test sample, and then a horseradish peroxidase (HRP)-labeled enzyme label that can bind to the PCV2-specific antibody is added Antigen, finally for display and termination reaction. If the sample contains specific PCV2-IgM antibody, it will be captured by the capture antibody first, and then bound to the HRP-labeled antigen, showing a positive reaction; otherwise, it will show a negative reaction.

For PCV2, IgM antibody theoretically only appears after infection or immunization, so the detection of IgM can distinguish maternal antibodies from antibodies produced after infection, and the combined detection of IgG and IgM can more accurately determine the timing of infection and The level of infection rate, analysis of antibody indicators produced after vaccine immunization to evaluate the effect of the vaccine.

Use this kit to detect standard positive serum for porcine reproductive and respiratory syndrome virus, standard positive serum for classical swine fever virus, standard positive serum for pseudorabies virus, standard positive serum for foot-and-mouth disease virus, standard positive serum for porcine parvovirus, and standard positive serum for Escherichia coli , the results were all negative. It shows that there is no cross-reaction between the diagnostic antigen used in this kit and other pathogenic antibodies.

The kit of the present invention is compared with the kit of Ingenasa Company of Spain: the ELISA kit for detecting PCV2-IgM antibody of Ingenasa Company is widely used in developed countries such as Europe and the United States, and is currently internationally recognized as a high-quality diagnosis for detecting PCV2 antibody Reagent test kit. The kit and the ELISA kit for detecting PCV2-IgM antibody from INGENASA Company were used to detect 146 clinically submitted sera, and the results showed that the detection results of the two methods were in good agreement. The results are shown in Table 1 below.

Table 1 kit of the present invention and INGENASA company kit comparison test

Note: Kappa coefficient > 0.75 means that the two methods have good detection consistency, and the larger the value, the better the consistency.

Moreover, compared with the kit for detecting PCV2-IgM antibody from INGENASA, this kit saves the enzyme-labeled antibody incubation process and 6 times of washing process, thus saving nearly 1 hour of detection time.

The kit of the invention has the characteristics of good specificity and high sensitivity, simple operation process and less detection time, and can be used for the detection of IgM antibody of type 2 porcine circovirus and the diagnosis of early infection.

Detailed ways

Preparation of antibody capture microtiter plate (ELISA plate):

Dilute the commercially available anti-pig IgM antibody with 0.05M carbonate buffer (pH9.6), determine the optimal coating concentration of the antibody as 0.5 μg/mL by square array method, take 8 detachable 96-well microtiter plates, Add 100 μl to each well, coat at 4°C for 24 hours, wash twice with phosphate solution containing 0.05% Tween-20 (PBST; pH 7.4), block with 5% skimmed milk powder at 37°C for 1 hour, and wash fully with PBST , and air-dry at room temperature to prepare an antibody capture microtiter plate.

Preparation of enzyme-labeled antigen (enzyme-labeled antigen against porcine circovirus type 2 antibody):

According to the DNA sequence of PCV2, two primers were designed, the upstream primer was: GCGAATTCCGTCTGCAAACGGCAGGCAATGT; the downstream primer was: GCGTCGACTTACGGATTCAGCGGCGGGTC. The gene corresponding to the 186-233 amino acids of the Cap protein was amplified from the porcine PCV2 genome by PCR, and cloned into the pET32-a(+) prokaryotic expression vector (the cloning site was between EcoR I and Sal I), Obtain the pET32dCap recombinant expression plasmid. Transformed into Escherichia coli BL21(DE3) pLySs, and expressed the recombinant TdCap fusion protein with reference to Novagen pET Syetem Manual, the protein was expressed in soluble form. The expressed fusion protein TdCap was purified with His-Bind protein purification kit (product of Novagen).

Horseradish peroxidase (HRP) was coupled to fusion protein TdCap by modified sodium periodate method. The specific process is as follows: add 0.2ml of freshly prepared 0.1M _NaIO4 solution to 5mg/ml HRP solution, stir at room temperature in the dark for 20 minutes, dialyze against 1mM sodium acetate buffer (PH4.4) at 4°C overnight, add 20μl of 0 .2MPH9.5 carbonate buffer solution to raise the pH of the above hydroformylated HRP to 9.0~9.5, then immediately add 2ml of TdCap protein to be labeled to 1ml0.01M carbonate buffer solution, gently Stir for 2 hours, add 0.1ml of newly prepared 4mg/ml NaBH4 solution, mix well, place at 4°C for 2 hours, dialyze against 0.15M PBS (pH7.4) at 4°C overnight, add an equal volume of saturated ammonium sulfate drop by drop under stirring , placed at 4°C for 1 hour, centrifuged at 3000 rpm for half an hour, and discarded the supernatant. The precipitate was washed twice with half-saturated ammonium sulfate, and finally the precipitate was dissolved in a small amount of 0.15M phosphate buffer (pH7.4), and dialyzed against 0.15M phosphate buffer (pH7.4) buffered saline to remove ammonium After ionization (detected with Naphthalene's reagent), centrifuge at 10,000 rpm for 30 minutes to remove the precipitate, and the supernatant is the enzyme-labeled antigen. After determining the ELISA titer of the enzyme-labeled antigen by a single titration method, it was diluted to 100 times the concentration used.

Preparation of buffer solution: Prepare 0.1M PBS (PH7.4) as serum and enzyme-labeled antigen diluent of PCV2 antibody detection kit, 1M PBS (PH7.4) containing 0.5% Tween-20 as 10× concentrated washing solution .

Preparation of background color developing solution: Weigh 200mg of tetramethylbenzidine (TMB), dissolve it in 100ml of absolute ethanol or DMSO, dilute to 1000ml with double distilled water, and configure developing solution A. Weigh 9.33g of citric acid, 14.6g of disodium hydrogen phosphate (Na ₂ HPO ₄ 12H ₂ O), and 6.4ml of 0.75% urea hydrogen peroxide, adjust the pH value to 5.0-5.4, and distilled water to 1000ml , Prepare chromogenic solution B. Chromogenic solution A and B are mixed in equal volumes to form a chromogenic solution.

Stop solution: Measure 111.2ml of concentrated sulfuric acid (18M) and dilute to 1000ml to prepare a 2M sulfuric acid solution, which is used as the stop solution for color development.

Negative and positive controls and judgment criteria:

During the ELISA process, there will be certain errors between different operators and different testing batches, such as errors in sample addition and reaction time, etc. These errors will lead to errors in the OD value of the test samples. However, the test sample OD value/standard sample OD value (S/P value) can eliminate errors and accurately reflect the test results. Add 0.2mg/ml NaN3 to the PCV2 standard negative and positive serum, respectively, so that it can be stored at 4°C for a long time without much change in antibody titer.

Judgment criteria: When the OD value of the standard positive serum is ≥0.8; the OD value of the standard negative serum is <0.2, indicating that the test is valid. When S/P≥0.2, the sample is positive for anti-PCV2-IgM antibody, and when S/P<0.2, the sample is negative for anti-PCV2-IgM antibody.

Assembly of the kit:

a. ELISA strips: each kit contains 5 ELISA plates, each plate contains detachable ELISA strips coated with anti-pig IgM antibody, and the specification is 8 wells x 12 strips.

b. Washing solution: 200ml of 10 times concentrated 1M PBS (pH7.4) containing 0.5% Tween-20.

c. 500 μl of 100-fold concentrated enzyme-labeled antigen.

d. Serum and enzyme-labeled antigen diluent 50ml.

e. Substrate chromogenic solution 50ml.

f. Stop solution: 50ml of 2M sulfuric acid solution.

g. Standard PCV2 negative and positive serum 0.2ml each.

The basic operation steps of sample detection are as follows:

a. Dilute the serum 100 times with serum diluent, add 100 μl to each well, seal the plate, and incubate at 37°C for 60 minutes.

b. Add 300 μl of washing solution to each well to wash the plate 5 times, blot the remaining liquid in the air, dilute the enzyme-labeled antigen 100 times with the enzyme-labeled antigen diluent, add 100 μl to each well, seal the plate, and incubate at 37°C for 30 minutes.

c. Add 300 μl of washing solution to each well to wash the plate 5 times, blot the remaining liquid in the air, add 50 μl of substrate TMB to each well, and develop color at 37°C in the dark for 15 minutes.

d. Add 50 μl of stop solution 2M sulfuric acid to each well to stop color development, and immediately read with a microplate reader at a wavelength of 450 nm.

Note: Except for the 100-fold concentrated enzyme-labeled antigen, other kit components and serum to be tested should be returned to room temperature before use.

Claims (3)

1. an ELISA kit for detecting type 2 porcine circovirus IgM antibodies, characterized in that, the kit includes the ELISA strip coated with anti-porcine IgM antibodies and the enzyme of anti-type 2 porcine circovirus antibodies labeled antigen. 2. a kind of ELISA kit that detects type 2 porcine circovirus IgM antibody as claimed in claim 1, is characterized in that, described kit also comprises washing liquid, serum and enzyme-labeled antigen diluent, substrate color development solution, stop solution, negative control and positive control serum. 3. a kind of ELISA kit that detects type 2 porcine circovirus IgM antibody as claimed in claim 2, is characterized in that, described ELISA strip: each test kit comprises 5 ELISA plates, and each plate contains The disassembled ELISA strip has a specification of 8 holes x 12 strips; Said washing solution: 200ml of 1M phosphate solution containing 0.5% volume percent Tween-20 containing 0.5% by volume of pH 7.4 concentrated by 10 times; The enzyme-labeled antigen of the anti-type 2 porcine circovirus antibody: 0.5 ml of 100-fold concentrated horseradish peroxidase-labeled Cap protein recombinant antigen of type 2 circovirus; Said serum and enzyme-labeled antigen diluent: 100ml of 1M phosphate solution with a pH of 7.4; The substrate chromogenic solution: TMB chromogenic solution 50ml; The stop solution: 50ml of 2M sulfuric acid solution; The negative and positive controls: 0.2 ml each of standard PCV2 negative and positive serum.