An ELISA for detection of antibodies against porcine epidemic diarrhoea virus (PEDV) based on the specific solubility of the viral surface glycoprotein.
An ELISA for detection of antibodies against porcine epidemic diarrhoea virus (PEDV) based on the specific solubility of the viral surface glycoprotein.
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DOI：
10.1016/0378-1135(92)90100-8
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发表时间：
1992-09
影响因子：
3.3
通讯作者：
Kihm U
中科院分区：
文献类型：
--
作者：
Knuchel M;Ackermann M;Müller HK;Kihm U
用低渗条件和非离子洗涤剂从感染的Vero细胞胞浆中提取猪流行性腹泻病毒(PEDV)病毒蛋白。改变提取缓冲液的pH和NaC l浓度，以增加病毒粒子刺突糖蛋白(S蛋白)和核衣壳蛋白(N蛋白)的溶解度。用单抗、高免血清和恢复期猪血清通过免疫沉淀和免疫印迹法对这些蛋白进行鉴定和监测。在pH为4时，S蛋白的溶解度最高，而在pH为9时，N蛋白的溶解度最好。因此，无论是S蛋白还是N蛋白都有可能被浓缩，增加缓冲液中的氯化钠浓度对这方面没有好处。用浓缩的S蛋白和N蛋白分别作为S-EL ISA和N-EL ISA的抗原。S-ELISA法是两种免疫检测方法中最有效的一种。在感染PEDV的猪血清中，抗S蛋白抗体的检测时间比抗N蛋白抗体的检测时间更长。使用这种灵敏度更高的ELISA法，可以观察到瑞士只有少量农场感染了PEDV。
Viral proteins of porcine epidemic diarrhoea virus (PEDV) were extracted from the cytoplasm of infected Vero cells using hypotonic conditions and a non-ionic detergent. Both the pH and the NaCl concentration of the extraction buffer were varied in attempts to increase the solubility of the virion spike glycoproteins (S-protein) and of the nucleocapsid proteins (N-protein). Monoclonal antibodies, hyperimmune sera and convalescent pig sera were used to identify and monitor these proteins by immunoprecipitation and Western blots. The solubility of the S-protein was optimal at pH 4, whereas that of the N-protein was optimal at pH 9. Consequently, it was possible to enrich for either S-protein or N-protein; increases in the NaCl concentration of the buffer were of no advantage in this respect. Enriched preparations of the S-protein and N-protein were used as ELISA antigen for the S-ELISA and N-ELISA, respectively. The S-ELISA proved to be the more effective of the two immunoassays. Antibodies against S-protein remained detectable for longer periods of time than anti-N-protein antibodies in the sera of PEDV-infected pigs. Using this ELISA of increased sensitivity, it was observed that only a small number of farms in Switzerland had been infected with PEDV.
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影响因子：
5
作者：
Correa I;Jiménez G;Suñé C;Bullido MJ;Enjuanes L
通讯作者：
Enjuanes L
影响因子：
3.3
作者：
CALLEBAUT, P;DEBOUCK, P;PENSAERT, M
通讯作者：
PENSAERT, M
影响因子：
3.7
作者：
STURMAN, LS
通讯作者：
STURMAN, LS
影响因子：
3.3
作者：
Hofmann M;Wyler R
通讯作者：
Wyler R
影响因子：
5
作者：
TALBOT, PJ;BUCHMEIER, MJ
通讯作者：
BUCHMEIER, MJ