A double antibody sandwich enzyme-linked immunosorbent assay for detection of soft-shelled turtle iridovirus antigens

Abstract

A double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) for detection of the soft-shelled turtle iridovirus (STIV) was developed using a specific monoclonal antibody (mAb) against STIV and anti-STIV rabbit serum. Using DAS-ELISA, the detection limit of STIV was found to be 10(3)PFU/ml. The positive rate of 15 STIV samples was 100%, while the positive rate of 100 other aquatic virus samples was 0%. These data show that DAS-ELISA is highly specific and sensitive for the detection of STIV. In clinical tests, 128 samples isolated from pond-reared turtles were subjected to DAS-ELISA and PCR. The overall agreement between the results obtained by DAS-ELISA and PCR was 98.4%. The results indicate that the DAS-ELISA method could be used for diagnosing diseases caused by STIV.

Fig. 1

Immunofluorescence staining of the CO cells infected with STIV with different mAbs. Con: uninfected CO cells. Photographs were taken at 24 h post-infection.

Fig. 2

Detection limit of double antibodies sandwich ELISA coated by mAb 2F9. CON was negative control of uninfected CO cell debris. The samples infected with STIV were diluted 10-fold to 10⁶–10¹ PFU/ml serially. The values of 10⁶–10³ PFU/ml were above critical value 0.228, and two times higher than negative control (0.155). It showed the detection limit of DAS-ELISA was 10³ PFU/ml.

Fig. 3

Specificity of double antibodies sandwich ELISA coated by mAb 2F9. CON was negative control of uninfected CO cell debris. The average value of STIV samples was 0.975, above critical value and four times higher than the control's. The value of others samples were similar to control's. It indicated that the mAb 2F9 react with STIV specifically.