Development of a ultramicro assay method for high-molecular- weight substances-- " Immunoenzymatic cycling "

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A new ultramicro assay mthod for macromolecules, named as "immunoenzymatic assay",were developed by combining the sandwich enzyme immunoassay and the NAD enzymatic cycling. Glass beads. 100 <micrn>m in diameter. were coated with antibody to horse ferritin (mol. wt. 500.000), which was used as a model of high-molecular- weight sabstance. (1) Mass of 240 glass beads were incubated for 5 min at 38 C in 54 no of solution containing 1-15 x10-19 mol of ferritin. Ferritin was bound to Its antibody on the surface of glass beads. The glass beads was incubated in 290 nl of fab -<beta> -D-galactosidase conjigate solution. and then the glass beads were incubated in 3.2 <micrn> 1 of gatactosidase reaction mixture containing methylumberllifery1-<beta>-D- galactoside as substrate. Galactose released was fluorometrically determined. This enzyme immunoassay using glass beads shoritens the period for assay from 2 days (for the previous routine assay) to 2.5 h. The small volumes of solutions. which were … More 1/50-1/500 of those in the routine assay. save the amount of conjugate and substrate. (2) The same.procedures were carried out as in (1) except for 1-5 x10-20 mol of ferritin and the glass beads binding Fab-conjugate were incubated with 2-nitropheny1-<beta> -D-galactoside as substrtate. Galactose released were converted by galactose dehydrogense to NADH in the presence of excess NAD^+. This NADH was amplified 2.000-fold by NAD cycling and deterimed fluorometrically. This immunoenzymatic cycling assay with 240 galss beads has 30-fold higher sensitivitgy than the assay (1) with the highest sensitiviry for enzyme immunoassay. (3) A single glass bead with 30-100 <micrn>m diameter was incubated with 1-10 x10^<-22> mol of ferritin in 2.l nl of its solution. For Fab'-conjugate and galactosidase reactions. 15 nl of these solutions were used. NADH formed was amplified 109,000-fold using double NAD cycling. The single bead assay measures 60-600 molecules of ferritin and its sensitivity is the highest ever amoung assayes currently in use. Less