Enzyme-linked fluorescence assay: Ultrasensitive solid-phase assay for detection of human rotavirus

Abstract

Enzyme-linked immunosorbent assay (ELISA) has proven to be a useful assay system for the direct detection of infectious agents. However, when the usual color-producing substrates are employed, relatively large amounts of substrate must be hydrolyzed by the bound enzyme before detection can be achieved. We attempted to improve the sensitivity of ELISA by utilizing a substrate that yields a fluorescent product on enzyme action. The enzyme-linked fluorescence assay (ELFA) based on this principle was approximately 100 times more sensitive than the corresponding ELISA or radioimmunoassay for the detection of human rotavirus in a standard stool suspension. In addition, the ELFA for human rotavirus was capable of detecting antigen in six specimens that were negative by ELISA. Five of these specimens were obtained late in the course of confirmed rotavirus infections. ELFA provides a simple, reliable, ultrasensitive method for the rapid detection of viral antigen.

Chemiluminescent assay of enzymes using proenhancers and pro‐anti‐enhancers

“…These features combine to produce a sensitive assay with a limited dynamic range. The detection limit for the proenhancer-based assay for alkaline phosphatase is 100 attomoles and this is comparable to the published detection limit for the colorimetric assay (50 attomoles) (Yolken and Stopa, 1979) but not as sensitive as the chemiluminescent assay using an adamantyl 1,2-dioxetane phenyl phosphate (detection limit 0.001 attomoles) or the bioluminescent assay using firefly luciferin-0-phosphate (detection limit 0.01 attomoles) (Miska and Geiger, 1989).…”

Rapid detection of rotavirus in stool by latex agglutination: Comparison with radioimmunoassay and electron microscopy and clinical evaluation of the test

“…Rapid diagnosis of viral infections by antigen detection is based on the fact that viral antigens are excreted in body fluids and are detectable by various methods. Latex agglutination is but one of the techniques now available [Birch et al, 1979;Grauballe et al, 1977;Harris et al, 1979;Matsuno and Nagayoshi, 1978;Provonost et al, 1981;Sanekata et al, 1979;Sarkkinen et al, 1979;Sarkkinen, 1981;Zissis et al, 1978;Yolken and Stopa, 1979a;Yolken et al, 19801 but has some advantages. It is extremely rapid, does not require any complicated machinery, and can be performed outside a microbiological laboratory.…”

Viral Diseases of the Digestive System

“…The references which follow are cited as examples and are not intended to be all-inclusive. Furthermore, the tech niques have been applied mainly to rotaviruses other than EDIM virus: neutrahzation (Kraft, 1961;Blackwell et al, 1966), complement fixation (Wilsnack et al, 1969;Kapikian et al, 1976;Thouless et al, 1977b), direct immunofluorescent staining or precipitin (Wilsnack et al, 1969;Spence et al, 1975;Foster α/., 1975;Peterson α/., 1976), immune electron microscopy (Kapikian et al, 1974;Bridger and Woode, 1975), immunoelectroosmophoresis (Tufvesson and Johnsson, 1976;Middleton et al, 1976), enzyme-linked im munosorbent assay (ELISA) (Scherrer and Bernard, 1977;El lens etal., 1978;Yolken etal., 1978a,b,c), radioimmunoas say (Acres and Babiuk, 1978;Kalica et al, 1977;Middleton et al, 1977), immunodiffusion (Woode et al, 1976), hemagglutination inhibition (Fauvel et al, 1978), enzymelinked fluorescence assay (ELISA) (Yolken and Stopa, 1979), an unlabeled soluble enzyme peroxidase-antiperoxidase method , plaque reduction test (Estes and Graham, 1980), serologic trapping on antibody-coated electron microscope grids (Nicolaieff et al, 1980), a solid phase system (SPACE, solid phase aggregation of coupled erythrocytes) for detection of rotaviruses in feces (Bradbume et al, 1979), and immune electron microscopy with serum in agar diffusion (Lamontagne et al, 1980). More recently, Sheridan and Aurelian (1981) have described an ELISA test for EDIM virus which should prove beneficial for both practical (serologic) purposes and for investigations of the antigenic structure of the virion.…”