Establishment of double-antigen sandwich chemiluminescent enzyme immunoassay for antibody detection against classical swine fever virus Erns antigen
10.16303/j.cnki.1005-4545.2025.09.03
- VernacularTitle:检测猪瘟病毒Erns蛋白抗体的双抗原夹心化学发光方法的建立
- Author:
Zihan YANG
1
;
Zhongdi LIU
;
Yixiao ZHANG
;
Qingshan ZUO
;
Qichao SONG
;
Zunbao WANG
;
Yidi GUO
;
Changchun TU
;
Wenjie GONG
Author Information
1. 吉林大学动物医学学院人兽共患病研究教育部重点实验室,吉林长春 130062;中国农业科学院长春兽医研究所,吉林长春 130012
- Publication Type:Journal Article
- Keywords:
classical swine fever;
Erns protein;
protein expression;
chemiluminescent detection meth-od
- From:
Chinese Journal of Veterinary Science
2025;45(9):1834-1842
- CountryChina
- Language:Chinese
-
Abstract:
To establish a highly specific,sensitive,and efficient method for detecting antibodies a-gainst the Erns protein of classical swine fever virus(CSFV),and to distinguish CSFV vaccine strains from wild strains infections in combination with the E2 subunit vaccine.The purified Erns protein of the CSFV expressed by baculovirus was conjugated to carboxylated magnetic beads as a solid-phase carrier and horseradish peroxidase(HRP),separately.A double-antigen sandwich chemiluminescent enzyme immunoassay(CLEIA)was developed by optimizing various reaction parameters using a fully automated chemiluminescence analyzer.This method was then applied to quantitatively detect Erns protein antibodies in sera from pigs infected with prevalent strains and those immunized with the CSFV E2 subunit vaccine and challenged with field strains.The results showed that the optimal conditions for coupling protein-to-magnetic bead were as follows:coupling buffer pH of 8.0,a protein coupling amount of 2.5 mg/g,blocking solution of 10％BSA,serum sample volume of 20 μL.The optimal dilution of enzyme-labeled antigen was at 1:500 with a one-step reaction time of 15 minutes.The cutoff value of the established CLEIA method for detecting CSFV Erns protein antibodies was 5.83 U/mL and a diagnostic sensitivity of 1:128.No cross-reac-tivity was observed with positive sera against African swine fever virus,pseudorabies virus,porcine circovirus type 2,porcine epidemic diarrhea virus,porcine reproductive and respiratory syndrome virus,or porcine gastroenteritis virus.Additionally,the method yielded negative results with sera from pigs immunized with the E2 subunit vaccine.In repeatability tests,the intra-assay coefficient of variation(CV)ranged from 0.77％to 11.56％,and the inter-assay CV ranged from 10.30％to 14.55％,both below 15％.The positive and negative concordance rates with a commercial CSFV Erns protein antibody detection kit were 95.24％and 92.71％,separately,with an overall concord-ance rate of 93.23％.The double-antigen sandwich chemiluminescence method established in this study exhibits high sensitivity,excellent repeatability,and suitability for automated detection,making it applicable for serological differentiation between CSFV E2 subunit vaccination and infec-tion with prevalent strains.
