Evaluation of ELISA with ABTS, 2-2'-azino-di-(3-ethylbenzthiazoline sulfonic acid), as the substrate of peroxidase and its application to the diagnosis of schistosomiasis

A micro-technique of enzyme-linked immunosorbent assay (ELISA) using ABTS, 2,2'-azino-di-(3-ethylbenzthiazoline sulfonic acid), as a substrate for horseradish peroxidase (HRP) conjugate was studied. In a comparative study among 4 substrates, namely; 5-aminosalicylic acid (5AS), O-phenylenediamine (OPD), O-tolidine (OT) and ABTS, for HRP in terms of sensitivity, it was found that ABTS was the most sensitive, stable and the best in visuality by its bluish-green color. The precise reproducibility of ELISA readings was attained by making correction of observed absorbance with positive reference serum. Sera from 50 patients positive for S. japonicum (Sj) egg and from 50 healthy residents in non-infested areas were diluted at 1:200 and examined by this procedure of ELISA against crude egg antigen. The upper limit of 99% critical range of the absorbance among negative sera was 0.080. The reactions of egg positive cases were beyond this value, being 0.505 at the minimal absorbance (1.259 +/- 0.278), and were easily distinguished from negatives even by naked eyes. Cross-reactivity to Sj egg antigen was studied using sera from 75 cases with 22 kinds of varied parasitic infections. Among them, S. mansoni and S. haematobium infections showed higher absorbance while the Others did a little higher than the normal range. The highest absorbance shown by a Trichobilharzia brevis case was 0.177. The results indicated that micro-ELISA technique with ABTS developed in the present study is applicable to the field survey because of its high sensitivity, specificity and reproducibility.