Miniaturization of β-galactosidase immunoassays using chromogenic and fluorogenic substrates

Abstract

• Enzyme immunoassay techniques are widely used to quantify various antigens and antibodies. The final step of these techniques (i.e. enzyme reaction) may be carried out in several ways (e.g. chromogenic, fluorogenic, or radioactive substrate and thermometric measurement). • This paper compares the effectiveness of the chromogenic and the fluorogenic substrates in the β-galactosidase immunoassay. Using microtitration plates (150 υl samples) coated with anti-human IgE, and anti-human IgE labeled with E. coli β-galactosidase, the lowest concentrations of IgE that one could detect employing either the chromogenic ( o-nitrophenyl- β-galactopyranoside) or the fluorogenic (4-methyl-umbelliferyl-β- D-galactopyranoside) substrate were determined. It was found that both substrates were almost equally effective in measuring the lowest concentration of IgE (0.075–0.13 IU/ml) under the optimal conditions. But, using fluorogenic substrate and suitable apparatuses the enzymes immunoassay can be miniaturized. Thus by using decreasing volumes of reagents, progressively smaller amounts of antigen were quantified: as the sample volumes were reduced from 150 to 10 μl and finally to 0.3 μl a progressive decrease from 7 × 10 7 molecules of IgE to 2.9 × 10 7 molecules and to 1.5 × 10 6 molecules was observed. The corresponding lowest detection limits were 0.075 IU/ml, 0.46 IU/ml and 0.8 IU/ml.