Ultrasensitive enzymatic radioimmunoassay: application to detection of cholera toxin and rotavirus.

Abstract

Rotavirus and enterotoxin-producing bacteria are major causes of diarrheal disease in humans. A method of rapid diagnosis, ultrasensitive enzymatic radioimmunoassay, has been developed to quantitatively detect cholera toxin and rotavirus. The method uses features of both enzyme-linked immunosorbent assay and radioimmunoassay; however, the sensitivity of the assay is 100-to 1000-fold more sensitive than the two parent assays. Ultrasensitive enzymatic radioimmunoassay should also be useful in measuring other biologically important agents such as drugs and hormones.In recent years, the capability to detect small quantities of biologically important molecules has been greatly expanded by the development of radioimmunoassays (RIA) (1, 2). These assays have been especially useful for the detection of circulating substances, such as hormones, drugs, and infectious agents. However, RIA have certain practical limitations. The short half-life of the isotopes used limits the shelf life of the reagents. In addition, the need for y-emitting isotopes subjects the users of RIA to a radiation hazard. Enzyme immunoassays (also known as enzyme-linked immunosorbent assays or ELISA) have been developed in an attempt to overcome those problems (3-5). ELISA is similar in design to solid-phase RIA except that an enzyme is used as the immunoglobulin marker instead of a 'y-emitting isotope. This enzyme-antibody conjugate is bound to the solid phase by a series of antibody-antigen reactions and essentially converts the substrate to products with a visible yellow color, which can be measured spectrophotometrically. The fact that a single molecule of enzyme is capable of reacting with a large number of substrate molecules provides for amplification and, thus, a high degree of sensitivity (3, 6). Because stable enzymes such as alkaline phosphatase [orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1] can be used, ELISA also has the advantage of using reagents with a long shelf life.In practice, however, the sensitivity of ELISA systems has not significantly exceeded that of RIA (7). In order to combine the advantages of both RIA and ELISA, we developed a practical ultrasensitive enzymatic radioimmunoassay (USERIA) for the detection of antigens such as human rotavinus (an important cause of infantile gastroenteritis) and cholera toxin. These assay systems are a 1000-fold more sensitive than both RIA and ELISA for the detection of these agents. MATERIALS AND METHODSReagents.[3H]AMP (generally 3H-labeled, 15 Ci/mmol, New England Nuclear; 1 Ci = 3.7 X 10O°becquerels) was purified by column chromatography with DEAE-Sephadex (A-25; Pharmacia) in a stepwise manner as follows: (i)

Development of a Highly Sensitive Bead‐ELISA to Detect Bacterial Protein Toxins

“…These sensitivities are much higher than those of previously reported methods for assay of TDH (14), Shiga toxin (19), and cholera enterotoxin (7), and are comparable to that for Shiga toxin reported by Donohue-Rolfe et al (4) and that for cholera enterotoxin reported by Harris et al (6). Variation in sensitivity with different toxins has been repeatedly observed with different immunized rabbits.…”

“…A good method should be simple, accurate, reproducible, cheap, and sensitive. In general, immunological methods are better than others, and of the various immunological methods so far reported (3,5,6,9,21,27,31), ELISA is the most widely used, because it is the most sensitive, accurate, and reproducible. The sensitivities of the ELISA's reported previously, however, varied significantly.…”

Antibodies

“…by liquid or solid extraction procedures) prior to assay in order to obtain reliable data. Alternatively, the assay can be made more sensitive to permit analyses of smaller quantities of a compound (13).…”

Single-Molecule ELISA

“…First it must be noted that similar sensitivity claims were made for 2 methods described in reports published some 30 years ago (4,5 ). Both methods relied on enzymes as signal amplifiers.…”