Kit for indirect ELISA (Enzyme-Linked Immunosorbent Assay) detection of IgG or IgA antibodies of PRRSV (Porcine reproductive and respiratory syndrome virus) of pigs

A kit and antibody technology, applied in the field of biological detection reagents, can solve the problem that saliva samples are not widely used

Inactive Publication Date: 2015-02-04
苏州市吴江区畜牧兽医站 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

But saliva samples have not be...

Method used

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  • Kit for indirect ELISA (Enzyme-Linked Immunosorbent Assay) detection of IgG or IgA antibodies of PRRSV (Porcine reproductive and respiratory syndrome virus) of pigs
  • Kit for indirect ELISA (Enzyme-Linked Immunosorbent Assay) detection of IgG or IgA antibodies of PRRSV (Porcine reproductive and respiratory syndrome virus) of pigs
  • Kit for indirect ELISA (Enzyme-Linked Immunosorbent Assay) detection of IgG or IgA antibodies of PRRSV (Porcine reproductive and respiratory syndrome virus) of pigs

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Expression and purification of embodiment 1 recombinant N protein

[0031] 1.1 Amplification of the N protein gene

[0032] According to the American strain PRRSV JXA1 strain (GenBank: KC422729), design a pair of specific primers including the whole gene sequence of N protein: P1:5'-CCG GAATTC ATGCCAAATAACAACGGCAAG-3' (SEQ ID NO. 1);

[0033] P2:5'-CCG CTCGAG TCATGCTGAGGGTGATGCTGT-3' (SEQ ID NO. 2),

[0034] EcoRI and XhoI restriction enzyme sites were inserted at the upstream and downstream 5' ends, respectively. The primers were synthesized by Yingjun Biotechnology Co., Ltd., and the size of the amplified fragment was 372bp. Total RNA was extracted from the viral cell culture medium, and the target gene was amplified by RT-PCR according to the kit instructions. The PCR amplification program was: pre-denaturation at 94°C for 3 min; 35 cycles of 94°C for 45 s, 55°C for 45 s, and 72°C for 1 min; extension at 72°C for 10 min. After the reaction, 5 μL of the PCR pr...

Embodiment 2

[0042] The establishment of embodiment 2 serum IgG ELISA detection method

[0043] 1.1 Determination of optimal antigen coating concentration and serum dilution concentration

[0044] The N protein purified in Example 1 was diluted into 6 dilutions with pH9.6, 0.05M carbonate buffer for coating 10μg / mL, 5μg / mL, 2.5μg / mL, 1.25μg / mL, 0.625μg / mL and 0.313μg / mL, two wells were replicated for each dilution, 100μL / well, and coated overnight at 4°C. PBST (PBS containing 0.5% Tween-20) was washed three times, and 100 μL of 3% BSA (diluted in PBST) was added to each well to block in a 37° C. incubator for 2 hours. PRRSV standard positive sera and negative sera were diluted 1:10, 1:20, 1:40, 1:80 in blocking solution respectively, 100 μL per well, and incubated in a 37°C incubator for 1 hour for ELISA square array experiment. The HRP-labeled anti-pig IgG antibody was diluted 1:5000 times in PBST (the dilution factor recommended by the instructions), 100 μL per well, and incubated in ...

Embodiment 3

[0070] The establishment of embodiment 3 serum, saliva IgA antibody ELISA detection method

[0071] Modify the indirect ELISA method for IgG in serum, optimize the reaction conditions, and establish an indirect ELISA method suitable for the detection of IgA antibody in serum. The operation steps and requirements are the same as the IgG detection method in serum, and the enzyme-labeled secondary antibody is replaced by HRP-labeled goat anti-pig IgA. The negative and positive sera used were the serum samples separated before immunization and 35 days after immunization, respectively. Blood was collected from the anterior vena cava of the animals. After the blood was collected and coagulated at room temperature, it was kept overnight at 4°C. After the serum was precipitated, the serum samples were separated by centrifugation at 1000×g for 10 minutes.

[0072]At the same time, on the basis of the serum IgG detection method, the reaction conditions were improved and optimized to de...

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Abstract

The invention belongs to the field of biological detection reagents and relates to a kit for indirect ELISA (Enzyme-Linked Immunosorbent Assay) detection of IgG or IgA antibodies of PRRSV (Porcine reproductive and respiratory syndrome virus) of pigs. The kit is used for indirect ELISA detection of the IgG or IgA antibodies of the PRRSV in serum or saliva of pigs and comprises (a) an antibody detection board, (b) an HRP-labeled goat anti-pig IgG antibody solution or HRP-labeled goat anti-pig IgA, (c) a positive control, namely PRRSV standard positive serum or saliva, (d) a negative control, namely PRRSV negative serum or saliva, (e) sample diluting liquid, (f) 10* concentrated washing liquid, (g) substrate developing liquid, and (h) stopping liquid. The kit is good in PRRSV specificity and sensitivity when being used for detection of PRRSV IgG or IgA antibodies in serum or saliva of pigs. A repeatability test also shows that the kit is good in repeatability.

Description

technical field [0001] The invention belongs to the field of biological detection reagents, and relates to a kit for indirect ELISA detection of pig PRRSV virus IgG or IgA antibodies. Background technique [0002] Porcine reproductive and respiratory syndrome (PRRS), also known as porcine highly pathogenic blue ear disease, is caused by porcine reproductive and respiratory syndrome virus (Porcine reproductive and respiratory syndrome virus, PRRSV) infection. The prevalence and distribution of the disease in China is very wide, and the harm is becoming more and more serious. It has become an important infectious disease restricting the development of pig industry. Clinically, the disease is mainly manifested as persistent high fever, dry feces, respiratory disorder and decreased reproductive performance. Abortion, stillbirth and mummified fetuses occur in sows in late pregnancy[1]. It has the characteristics of high incidence, long duration, difficult treatment and easy recu...

Claims

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Application Information

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IPC IPC(8): G01N33/68
CPCG01N33/56983
Inventor 樊彦红何成华赵秀美任喆夏兵兵汪袁
Owner 苏州市吴江区畜牧兽医站
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