Homogeneous Enzyme-linked Binding Assay Research Articles

Homogeneous Enzyme-Linked Binding Assay Mediated by the Interaction of Avidin with Biotin: Mistletoe Lectin I Assay

We have examined the feasibility of using the specific interaction between mistletoe lectin I (ML I) and <TEX>${\beta}$</TEX>-Dgalactose instead of the anti-ML I antibody in developing a homogeneous type competitive binding assay for ML I. We also have examined the feasibility of adapting the biotin/avidin mediated homogeneous assay for this system. Alkaline phosphatase (AKP) was employed as a single substrate enzyme label. The dose-response curve shows a detection range of 1-25 <TEX>${\mu}$</TEX>g/mL and a linear response with a correlation coefficient of 0.99. To demonstrate the analytical utility of this method, 10 <TEX>${\mu}$</TEX>g/mL of ML I was spiked into distilled water. The results show that the mean recovery was 10.03 <TEX>${\mu}$</TEX>g/mL with an SD of 0.18. The difference between the spiked value and the mean recovery was 0.03 <TEX>${\mu}$</TEX>g/mL, with a relative error of 0.3 and 1.6 % of RSD.

Biotin-Avidin Mediated Homogeneous Enzyme-Linked Binding Assays for Riboflavin and Riboflavin Binding Protein Using Alkaline Phosphatase as the Labelt

The feasibility of using alkaline phosphatase (ALP) as a generic label in a homogeneous type assay is examined. In the proposed ALP-based biotin-avidin mediated assay, the activity of the ALP-biotin conjugate is inhibited in the presence of the avidin-riboflavin conjugate, and the observed inhibition is reversed in an amount proportional to the concentration of riboflavin binding protein (RBP) added. The ALP activity of the mixture is re-inhibited upon additions of free analyte riboflavin, to an extent proportional to the concentration of riboflavin added to the assay mixture. An avidin-riboflavin conjugate with a desired inhibitory effect is prepared by protecting the binding-site of avidin with 2-iminobiotin during the conjugation reaction at pH 6.5. The catalytic activity of ALP, conjugated with biotin, is inhibited to a significant degree (up to 86%) by the binding of the avidin-riboflavin conjugate, and is fully recovered upon additions of RBP.

Adapting Homogeneous Enzyme-Linked Competitive Binding Assays to Microtiter Plates

Recently devised homogeneous enzyme-linked binding assays useful for the rapid detection of carbohydrate structure/content of intact glycoproteins (via use of lectins as binders) and for quantitating given vitamins (e.g., biotin; using soluble binding proteins) are adapted successfully to a microtiter plate reader format. The problem of nonspecific adsorption of the binders and enzyme-saccharide/vitamin conjugates is solved via the addition of Tween 20 to the assay buffer. More convenient and reliable photometric detection of the preferred labeling enzyme, glucose-6-phosphate dehydrogenase (G6PDH), is accomplished by monitoring the rate of generation of reduced thio-NAD (from thio-NAD) at 405 nm instead of NADH (from NAD) at 340 nm. By employing these modifications it is shown that homogeneous enzyme-linked binding assays can be readily adapted to microtiter plates without loss in analytical assay performance. Results further suggest that other homogeneous assays based on G6PDH, including commercial EMIT assays used routinely in clinical chemistry laboratories for detecting drugs of abuse, could, in principle, be run on microtiter plates to significantly enhance sample throughput.

Lectin-based homogeneous enzyme-linked binding assay for estimating the type and relative amount of carbohydrate within intact glycoproteins

The feasibility of using a new lectin-based homogeneous enzyme-linked binding assay for estimating the type and relative amount of specific carbohydrate structures within intact glycoproteins is examined. Malate dehydrogenase-galactose, -mannose, and - N-acetylglucosamine conjugates are utilized in conjunction with Jacalin, concanavalin A, and wheat germ agglutinin, respectively. The catalytic activity of the glyco-enzyme conjugates is inhibited significantly (>60%) in solution in the presence of the respective lectins. The observed inhibition for each reagent set is reversed in proportion to the type and relative amount of specific carbohydrates present within test glycoproteins added to the assay mixture. Competitive binding ED 50 values for a number of synthetic and native model glycoproteins correlate well with the known carbohydrate content of these species. The proposed method is much faster than previous solid-phase lectin-based enzymelinked methods used to probe carbohydrate content/structure (<15 min) and has the potential to be fully automated.

Homogeneous enzyme-linked binding assay for studying the interaction of lectins with carbohydrates and glycoproteins

A simple and rapid homogeneous enzyme-linked binding assay method for studying lectin-carbohydrate interactions is described. The method is based on the homogeneous inhibition of appropriate enzyme-saccharide conjugates by specific carbohydrate-binding lectins. In the presence of carbohydrate structures recognized by the lectins, enzyme activity is regained in an amount of proportional to the concentration of carbohydrate. The new method can be used to rapidly assess the relative carbohydrate specificity of the various lectins and for the selective analytical detection of simple saccharides and complex glycoproteins. Indeed, when Jacalin lectin is used in conjunction with a malate dehydrogenase-galactose conjugate, selective measurement of human IgA (immunoglobulin A) at microgram per milliliter levels in less than 10 min is possible. The potential for using this analytical methodology for determining changes in the carbohydrate structure of intact recombinant glycoproteins is also discussed.