Abstract: This study was designed to develop an indirect ELISA for detection of sheep antibodies against Echinococcosis granulosa, which will provide a fast and simple means for detection Echinococcosis granulosa of sheep. The amino acid sequences of EG95 published in GenBank were analyzed using DNAStar software, and the hydrophilic epitopes were selected, named EG95s. Then the gene of EG95s were cloned and expressed. As a result, an indirect ELISA for detection of sheep antibodies against Echinococcosis granulosa were developed using the purified recombinant fusion protein as the coating antigen, therefore a variety of conditions were optimized. The results of SDS-PAGE showed that the recombinant fusion protein, GST-1EG95s and HIS1EG95s, were efficiently expressed, which were soluble and purified easily Then the results of Western blot showed that the expression products were reacted with positive serum of sheep with Echinococcosis granulosa, which indicated they were good immunogenicity. The optimized conditions of indirect ELISA showed that: HIS-1EG95s was better than GST-1EG95s as the coating antigen. The determine criteria of the indirect ELISA were: Samples with an OD450 nm value ≥ 0.235 were judged as positive, and with an OD450 nm value≤ 0.191 were judged as negative, while with an OD450 nm value ranging between them was suspicious. 70 positive and negative serum of sheep collected in Xinjiang with or without Echinococcosis granulosa were detected by the indirect ELISA respectively, and the results showed that this indirect ELISA method was completely consistent with the indirect ELISA provided by Wallaceville Animal Research Centre of New Zealand. There were no cross reactivity detected with other proteins. The variant coefficient in batch and between batches varied from 3.8% to 5.6% and from 5.7% to 8.5%, respectively. All these results showed that the indirect ELISA possessed specificity, sensitivity and good reproducibility. The indirect ELISA for detection of sheep antibodies against Echinococcosis granulosa were successfully developed, which provide a fast and simple means, and can be used for clinical detection.

Key words: Echinococcosis granulosa of sheep, EG95s, recombinant fusion protein, indirect ELISA, detection