Chemiluminescent detection kit for bovine foot-and-mouth disease 3ABC antibody

A technology for chemiluminescent detection and bovine foot-and-mouth disease, applied in the field of immunological detection

Active Publication Date: 2017-04-26
LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

But so far, there is no report on the chemiluminescent immunoassay method for

Method used

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  • Chemiluminescent detection kit for bovine foot-and-mouth disease 3ABC antibody
  • Chemiluminescent detection kit for bovine foot-and-mouth disease 3ABC antibody
  • Chemiluminescent detection kit for bovine foot-and-mouth disease 3ABC antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1 Preparation of 3ABC fusion protein

[0034] Total RNA was extracted from the blister fluid of cattle infected with foot-and-mouth disease virus according to the operating instructions of the RNeasy® Mini Kit kit; using the extracted RNA as a template, cDNA was synthesized using SMART® MMLV reverse transcriptase and oligo dT primers (purchased from Takara); the design Six sets of primers, using the synthesized cDNA as a template, first use primers 3ABC-F and 3C-46-R to amplify the first gene, and then use 3C-46-F and 3C-163-R to amplify the second gene , and then use 3C-163-F and 3ABC-R to amplify the third gene. Finally, the three amplified genes were fused into a mutated full-length 3ABC gene by fusion PCR, and the gene sequence is shown in SEQ ID NO:1. The primer sequence is as follows (or SEQ ID NO: 3~8):

[0035] 3ABC-F: 5'-CGGGATCCATCTCAATTCCTTCCCAAAAGTCC-3'

[0036] 3ABC-R: 5'-CCGCTCGAGT CTCATGGTGTGGTTCGGGGT-3'

[0037] 3C-46-F: 5'-CGTACCTCGTTACCTTTT...

Embodiment 2

[0044] Example 2 Optimization and establishment of CLIA method for detecting bovine foot-and-mouth disease 3ABC antibody

[0045] The calf serum with high infection titer 30 days after challenge was used as the standard positive control serum, and the blocking rate of this serum was 98% detected by the FMDVPrioCHECK® NSP ELISA kit; the newborn fetal bovine serum (FBS) was used as the standard negative serum . Apply the checkerboard titration experiment to determine the optimal 3ABC antigen coating concentration and serum dilution concentration, in which the 3ABC antigen concentrations are respectively coated with 2ug, 1ug, 500ng and 250ng, and the standard negative and positive serums are respectively made 5 times, 10 times and 20 times , 40-fold and 80-fold dilutions. According to the comprehensive consideration of economic factors and signal-to-noise ratio, the optimal conditions were selected as antigen coating conditions of 250ng and serum dilution of 20 times. Under thi...

Embodiment 3

[0046] Example 3 Sensitivity and specificity evaluation

[0047] 1. Establishment of serum plate

[0048] a. A total of 149 serum samples came from clinically healthy cattle. These cattle had not been vaccinated. They were tested with the FMD liquid-phase blocking ELISA kit developed by Lanzhou Veterinary Research Institute. These sera had no corresponding O, A, and Asia1 antibodies . These sera were used to evaluate the diagnostic sensitivity of this CLIA kit.

[0049] b. A total of 168 serum samples were collected from clinically healthy cattle that had been immunized with inactivated foot-and-mouth disease vaccine, and these serum samples were collected 30-180 days after immunization. These sera were used to evaluate the diagnostic sensitivity of this CLIA kit.

[0050] c. A total of 143 serum samples came from experimentally challenged cattle. These serum samples are serum samples collected 10-200 days after the challenge. These sera were used to evaluate the diagnost...

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Abstract

The invention discloses a chemiluminescent detection kit for a bovine foot-and-mouth disease 3ABC antibody and belongs to the field of immunological detection. The chemiluminescent detection kit comprises a 96-well chemiluminescent immunoassay plate, an enzyme-labeled antibody, a serum diluent, a chemiluminescent substrate, a chemiluminescent enhancer and PBST washing liquid, wherein the 96-well chemiluminescent immunoassay plate is coated with 3ABC fusion protein, the enzyme-labeled antibody is a horseradish peroxidase labeled rabbit anti-bovine lgG antibody, the serum diluent comprises 0.01% of Tween-20, 1% of casein, 10% of horse serum and 1% of escherichia coli, the chemiluminescent substrate refers to a Luminol solution, and the chemiluminescent enhancer comprises IPP, H2O2 and Tween-20. Compared with commercially-available kit products, the chemiluminescent detection kit is high in sensitivity, higher in specificity and superior to other products in diagnosibility.

Description

technical field [0001] The invention belongs to the field of immunological detection, in particular to a chemiluminescent detection kit for bovine foot-and-mouth disease 3ABC antibody. Background technique [0002] Foot-and-mouth disease is an ancient political and economic disease, which seriously threatens the healthy development of animal husbandry and import and export trade. Early and accurate diagnosis of foot-and-mouth disease is undoubtedly crucial for the prevention and control of the epidemic of foot-and-mouth disease. Therefore, developing a fast, sensitive and accurate method for diagnosing foot-and-mouth disease is a current research hotspot. So far, the methods mentioned by the World Health Organization (OIE) for the diagnosis of foot-and-mouth disease mainly include: virus isolation test, virus neutralization test, complement fixation test, various enzyme-linked immunoassays (ELISA) and real-time quantitative PCR. However, these methods have many shortcoming...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N33/569G01N33/531G01N21/76C07K19/00C12N15/62C12N15/70C12N15/66
CPCC07K14/005C07K2319/00C12N15/62C12N15/66C12N15/70C12N2770/32122G01N21/763G01N33/531G01N33/56983G01N33/68G01N2333/09
Inventor 常惠芸刘泽众邵军军赵付荣李秀梅张永光
Owner LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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