Method of detecting PCV2 antibody
An antibody and detection probe technology, applied in the fields of immunology and serology, can solve the problems of early diagnosis of difficult diseases, low antibody content, and limited signal amplification methods.
Pending Publication Date: 2021-04-23
HENAN INST OF SCI & TECH
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Problems solved by technology
Comparing commercially available ELISA kits with chemiluminescence kits, it is found that most commercial immunoassay kits have low sensitivity. The main reason is that the signal amplification method is limited.
In the early stage of viral infection, the antibody content is extremely low, so it is difficult to achieve early diagnosis of the disease with ordinary ELISA kits
Method used
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Embodiment １
[0028] 1. To detect PCV2 antibody, the steps are as follows:
[0029] Step 1, synthesis of gold nanoparticles:
[0030] Put 250ml of 0.01% chloroauric acid solution in a double-necked bottle, heat to boiling, add 10mL of 1% trisodium citrate solution under stirring, the color of the solution gradually changes from colorless to blue and finally to wine red, Continue heating to boiling for 10 minutes, remove the heat source, continue to stir until the solution cools to room temperature, filter through a 0.22 μm filter membrane, collect the filtrate, and store it in the dark at 4°C for later use;
[0031] Step 2, synthesis of detection probes
[0032] Take 200 µL of the above-prepared nano-gold filtrate, adjust its pH to 9.0, mix with 12 µL of HRP-goat anti-pig IgG with a concentration of 0.02 mg / mL, and incubate at room temperature for 30 min; add the same volume of 0.2 g / mL BSA solution to block for 30 min, Centrifuge for 15 minutes to remove excess antibody and BSA, resuspen...
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Abstract
The invention discloses a method for detecting a PCV2 antibody, which comprises the steps of 1, synthesizing nanogold; 2, synthesizing a detection probe, specifically, taking 200[mu]L of the prepared nanogold filtrate, regulating the pH value to 9.0, uniformly mixing with 12L[mu]L of HRP-goat anti-pig IgG with the concentration of 0.02 mg / mL, and incubating at room temperature for 30 minutes; adding a 0.2 g / mL BSA solution with the same volume to seal for 30 minutes, centrifuging for 15 minutes to remove redundant antibodies and BSA, re-suspending and washing precipitates once by using 220[mu]L of antibody diluent, and diluting the precipitates by 10 times by using the antibody diluent for later use; and 3, carrying out PCV2 antibody detection, specifically, (1) coating; (2) sealing; (3) adding a sample; (4), adding a detection probe; (5), developing; (6), terminating the reaction; and (7) conducting detection, specifically, detecting the absorbance value at 450 nm by a microplate reader. After an ELISA plate is coated with the PCV2 antigen, AuNPs are modified and coated with HRP-labeled goat anti-pig IgG to prepare the signal amplification probe, the loading rate is high, and the specificity is high. The prepared probe can be used for detecting the PCV2 antibody level in porcine serum with high sensitivity and high specificity.
Description
technical field [0001] The invention belongs to the technical field of immunology and serology, in particular to a method for detecting PCV2 antibody. Background technique [0002] With the expansion of breeding scale, the number of diseases infected in pigs is increasing day by day, which has brought huge economic losses to the production of pig farming. Porcine circovirus type 2 (PCV2) has strong pathogenicity and can infect pigs of different ages. After pregnant sows are infected, they are vertically infected to piglets through the placenta, and primiparous sows and new herds produce reproductive disorders. In addition to causing reproductive disorders, it can also lead to severe immunosuppression in pigs, which is prone to secondary or concurrent other infectious diseases. Clinically, the case fatality rate of single circovirus infection is very low, and more than 80% of cases belong to secondary infection. Therefore, it is particularly meaningful to develop accurate,...
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IPC IPC(8): G01N33/569G01N33/543
CPCG01N33/56983G01N33/54346G01N2333/01
Inventor 张守平胡建和王磊夏小静徐彦召
Owner HENAN INST OF SCI & TECH