Further studies on the ELISA-spot technique. Its application to particulate antigens and a potential improvement in...

Applications of the ELISA-spot technique to particulate antigens (sheep erythrocytes and E. coli) are reported; sheep erythrocytes were used to ensure a rigorous comparison of the spot assay and the haemolytic plaque assay. The spot technique was also applied to soluble antigens (dextran and trinitrophenyl-lipopolysaccharide) to assess the putative occurrence of non-haemolytic antibodies. In most instances, the spot assay disclosed higher numbers of antibody-secreting cells than the plaque assay. An examination of the kinetics of spot formation demonstrated that spot development was most rapid during the first and second hours of enzyme activity and slowed thereafter, although the numbers of spots at 16 h were higher than those at 2 h. To shorten the assay time a redox reaction (which yields an insoluble formazan) was coupled to the enzymatic reaction. In duplicate assays, this improved technique gave larger numbers of spots in a shorter time, than the conventional assay.