梅花鹿γ干扰素双单抗夹心ELISA方法的初步建立

Development of sandwich ELISA for the detection of cervine interferon-gamma

为利用抗牛γ干扰素(BoIFN-γ)特异性单克隆抗体(MAb)建立检测梅花鹿γ干扰素(CerIFN-γ)的双抗体夹心ELISA检测方法,本研究通过RT-PCR方法从双阳型梅花鹿外周血淋巴细胞总RNA中扩增出CerIFN-γcDNA,通过原核表达系统表达重组CerIFN-γ蛋白(rHis-CerIFN-γ);通过间接ELISA方法评价其与抗BoIFN-γMAb的反应性,筛选出与之反应最佳的两株MAb建立双抗体夹心ELISA检测方法。基因序列比对显示CerIFN-γ(JQ408441)与BoIFN-γ的同源性为95.6%,氨基酸序列的同源性为91.6%;SDS-PAGE检测结果显示CerIFN-γ在大肠杆菌BL21(DE3)中获得高效可溶性表达,大小为23 ku;间接ELISA结果显示42株MAb中有15株与rHis-CerIFN-γ反应性较好,其中1C12和5E11两株抗体结合活性最高;采用MAb 1C12为捕获抗体,以生物素标记的MAb 5E11为检测抗体建立的用于检测CerIFN-γ的双抗体夹心ELISA方法可检出3.05 ng/100μL的rHis-CerIFN-γ。本研究所建立的检测CerIFN-γ双抗体夹心ELISA方法,为进一步研究CerIFN-γ及相关疾病提供了实验手段。

To establish sandwich ELISA for the detection of cervus interferon-gamma （CerlFN-Y） using bovine IFN-v （BolFN-Y） monoclonal antibodies （MAb） that cross react with CerlFN-% the CerlFN-~/ gene was amplified by RT-PCR from the total RNA of cervus peripheral blood lymphocytes stimulated with ConA and cloned into expression vector pET-30a for expression in E.coli BL21 （DE3）. The recombinant protein （rHis-CerlFN-~/） was expressed and purified using as a coating antigen to scan the BolFN-3, MAb which cross-reacted with CerlFN-＇,/. Two BolFN-~ MAbs that cross-reacted with rHis-CerlFN-3, were selected for the development of sandwich ELISA. Alignment analysis indicated that the homology of cDNAs and deduced amino acid sequences were 95.6% and 91.6% between CerlFN-Y and BolFN--y, respectively. SDS-PAGE analysis showed that the rHis-CerlFN--Y（23 ku） was solubly expressed. The indirect ELISA indicated that 15 of 42 BolFN-YMAbs had relatively intensive reaction with CerlFN-~. Moreover, two of MAbs （1C12 and 5El 1） were used to develope the sandwich ELISA with MAb 1C 12 as the capture antibody and the biotin labelled MAb 5El 1 as the detection antibody, which had a detection limit of 3.05 ng/100 IxL of rHis-CerIFN-Y. The established sandwich ELISA for the detection of CerlFN--Yprovides a basis for the further studies of CerlFN--Y and CerlFN-3, related diseases.