為建立一種檢測(cè)非洲豬瘟病毒（ASFV）抗體的間接ELISA（iELISA）方法，對(duì)構(gòu)建的ASFV P30基因表達(dá)工程菌誘導(dǎo)表達(dá)后，將獲取的重組ASFV P30蛋白進(jìn)行純化和Western-blot檢測(cè)，然后以純化的重組蛋白為抗原，建立了ASFV抗體iELISA檢測(cè)方法，并進(jìn)行了特異性、靈敏性、重復(fù)性試驗(yàn)；同時(shí)與基于ASFV P30-2His6蛋白（N、C末端各融合1個(gè)His6標(biāo)簽）的iELISA方法進(jìn)行獸醫(yī)臨床樣本比較試驗(yàn)。結(jié)果顯示：重組ASFV P30蛋白和重組ASFV P30-2His6蛋白在Western-blot檢測(cè)中，均能與豬ASFV陽(yáng)性血清產(chǎn)生特異性雜交帶；基于重組ASFV P30蛋白iELISA的最佳反應(yīng)條件為，抗原蛋白包被質(zhì)量濃度20 μg/mL、血清樣品稀釋度1:1 000、酶標(biāo)二抗稀釋度1:40 000、血清樣品檢測(cè)OD450陽(yáng)性結(jié)果臨界值0.22。該方法僅對(duì)ASFV陽(yáng)性血清呈特異性反應(yīng)，1:3 200稀釋的陽(yáng)性血清仍可檢出，批內(nèi)試驗(yàn)和批間試驗(yàn)變異系數(shù)均小于10%，可以消除His標(biāo)簽所造成的假陽(yáng)性反應(yīng)。本研究建立的ASFV P30 iELISA檢測(cè)方法為ASFV抗體檢測(cè)提供了一種有效手段。
Establishment and Application of the iELISA for Detection of Antibodies against ASFV
In order to establish an indirect enzyme-linked immunosorbent assay（iELISA）for detecting antibodies against African swine fever virus（ASFV），the gene expression engineering strain of recombinant ASFV P30 was induced and expressed，followed by purification and Western-blot detection of the recombinant proteins，then the iELISA method was established by taking the purified recombinant proteins as antigens，its specificity，sensitivity and repeatability were subsequently evaluated. Meanwhile，it was compared with the iELISA based on ASFV P30-2His6 protein（one His6 tag was fused respectively at N and C terminals）for veterinary clinical samples. The results showed that，by Western-blot detection，both the recombinant proteins of ASFV P30 and ASFV P30-2His6 could produce specific hybrid band with ASFV positive serum；the optimal reaction conditions of iELISA based on the recombinant ASFV P30 proteins were 20 μg/mL mass concentration of antigen protein coating，1:1 000 dilution of serum samples，1:40 000 dilution of goat anti-swine IgG conjugate，and 0.22 critical value of positive serum samples OD450. The established method was specific only to ASFV positive serum，1:3 200 diluted positive serum could still be detected，and the coefficients of variation（CV）of intra assay and inter assay were both less than 10%，which could eliminate any false positive reaction caused by His tag. Therefore，an effective tool was provided for detecting antibodies against ASFV by the established ASFV P30 iELISA in the study.
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