A double-antibody sandwich ELISA was developed for the detection of antigenic differences between wild and vaccine-derived strains of poliovirus type 2 and poliovirus type 3. Antibodies were prepared in rabbits by immunization with purified antigens of vaccine strains (type 2: Sabin P712, and type 3: Sabin Leon) and wild type strains (type 2: MEF, and type 3: Pool 30). Immunoblotting analysis of all antisera demonstrated that the IgG antibodies raised in rabbits have specificity towards the main structural proteins (Vp1, Vp2 and Vp3) of poliovirus. IgG fractions were purified from antisera by affinity chromatography, on a protein A-activated Sepharose 4B column. Purified IgG antibodies were used for coating of microtest plates (catching antibodies). The same reagents labelled with horseradish peroxidase were used as conjugates, after cross-adsorption with antigens of the same type heterologous virus strains (strain-specific conjugates). 29 poliovirus type 2 strains and 73 poliovirus type 3 strains isolated from clinical samples, were differentiated intratypically, as vaccine-derived or wild types, no intermediate strains were found and all samples tested fell in two distinct (vaccine/wild) categories. As little as 40 ng of poliovirus antigens was detected in stool samples from healthy children or from polio patients cultivated in monkey kidney tissue cultures. Preparation of strain specific conjugates did not require large amounts of poliovirus antigens. The developed ELISA, which is economic and capable of (1) detection of low amounts of poliovirus antigens in cultivated clinical samples, and (2) intratypic differentiation of poliovirus antigens as either vaccine-derived or wild type, is therefore well suited for large scale screening of poliovirus isolates.