ELISA Plate Reader Research Articles

Lectin affinity bioassay: an assay method for glycoprotein enzyme

A simple and sensitive chromogenic microtitre plate assay for glycoprotein enzymes is described, using melanoma tissue plasminogen activator (t-PA) as a model enzyme. The assay is based on the binding of t-PA to immobilised lectin and quantitating the bound enzyme with plasminogen, fibrinogen fragments and chromogenic substrate S-2251 on an ELISA plate reader. Seven different lectins were examined for the binding of t-PA, and of these, concanavalin A was chosen for subsequent studies. The specificity of this binding can be inhibited dose-dependently in the presence of D-mannose and methyl alpha-D-mannoside, but not by D-glucose and D-lactose. The lower limit of the sensitivity of this assay is about 0.5 IU/ml. Comparison of the dose-response curves indicates that the sensitivity of this assay method is very similar to that of bioimmunoassay using anti-t-PA IgG to capture the antigen. The applicability of this method to other glycoprotein enzymes was also evaluated using alkaline phosphatase from bovine mucosa. The specificity of this method was related to the choice of substrate and this was shown by analysis of a mixture of t-PA and alkaline phosphatase. It is suggested that this assay can be adapted for the analysis of complex glycoprotein mixtures with the appropriate choice of lectin and substrate.

Use of a scanning densitometer or an ELISA plate reader for measurement of nanogram amounts of protein in crude extracts from biological tissues

Protein contents of crude extracts from plant and animal tissues can be rapidly assayed using a Coomassie blue dye-binding procedure combined with scanning densitometry. Total protein is extracted from 100 mg of fresh-frozen or dried-ground tissue using 1 ml of extraction buffer. One-microliter aliquots of standard solutions or crude extracts are spotted in rows on a suitably sized sheet of Whatman 3MM chromatography paper. The dried samples are stained with Coomassie brilliant blue R-250 (0.2%, w v , in acidified 50% MeOH) for 20 min and rinsed twice with acidified 20% MeOH. After drying, protein concentrations are read as reflectance using a scanning densitometer and peak heights or peak areas recorded using a digital integrator. In an alternative procedure, each spot is cut from the sample sheet and the dye-protein complex eluted in 1% sodium dodecyl sulfate (SDS) using an ultrasonic cleaner. Absorbance is subsequently read in a microwell sample holder at 590 nm with an enzyme-linked immunosorbent assay plate reader. Both procedures offer distinct advantages over previously reported methods. They are significantly faster when large numbers of samples are processed, they avoid interference by chlorophyll, dithiothreitol, SDS, 2-mercaptoethanol, Nonidet P-40, and phenylmethylsulfonyl fluoride (and other protease inhibitors) and they yield marked improvements in sensitivity, providing measurements of protein concentration below 100 and 200 ng·μl −1, respectively.

A highly sensitive chromogenic microtiter plate assay for plasminogen activators which quantitatively discriminates between the urokinase and tissue-type activators

A simple and highly sensitive chromogenic microtiter plate assay for plasminogen activators is described. The assay is based on plasmin cleavage of the synthetic tripeptide plasmin substrate H-D-norleucyl-hexahydrotyrosyllysine p-nitroaniline, which yields the yellow chromophore p-nitroanilide. Production of the latter compound is then quantitated spectrophotometrically at 405 nm on an ELISA plate reader. Linearity of the assay can be achieved over at least four orders of magnitude in a single experiment (0.01–100 milliPloug units) with appropriate incubation times. Capitalizing on tissue-type plasminogen activator's dependence on fibrin for enzymatic activity, the selective use of soluble fibrin products allows discrimination between urokinase and tissue-type activator. The utility of this aspect of the assay for the analysis of complex samples containing both types of plasminogen activitors is demonstrated.

An enzyme-linked immunosorbent assay (ELISA) for plasma cortisol

A direct ELISA for plasma cortisol is described which is carried out in a standard 96 well microtitre plate. In this ELISA cortisol-thyroglobulin conjugate is immobilised to the microtitre plate and competes with cortisol in the standard or plasma sample for antibody binding sites. Following washing the rabbit cortisol antibody bound to immobilised cortisol is incubated with peroxidase labelled goat antirabbit IgG. Following further washing o-phenylenediamine is added, colour developed, and the plate read at 492 nm on a standard ELISA plate reader. This ELISA shows good agreement with RIA and its sensitivity, specificity and precision allow its use in the routine steroid laboratory.

Rapid colormetric assay for cell viability: Application to the quantitation of cytotoxic and growth inhibitory lymphokines

A rapid colormetric microtiter assay has been developed to detect cytotoxic lymphokines produced by human lymphocytes activated with lectins or tumor cells. The viability of lymphotoxin-treated target cells was detected using a tetrazolium dye that is reduced to a blue formazan by living but not dead cells. The amount of dye formed was quantitated using a microplate spectrophotometer (ELISA plate reader) and visual observations confirmed the amount of formazan dye produced was directly proportional to the number of viable target cells. The advantages of using this colormetric method are that it requires no washing steps or radioisotopes and its precision and rapidity. Optimal conditions were established using the murine L929 and human ESH-5L cell lines as target cells for detecting lymphotoxins produced by human lymphocytes. The data indicate that the L929 cell line was 10–50-fold more sensitive than the ESH-5L line to the lytic activity of cytotoxins produced by human phytohemagglutinin-P-activated T lymphocytes, or the cytotoxins produced by peripheral blood lymphocytes stimulated with various tumor cell lines. This assay system was also useful in detecting antibodies capable of neutralizing lymphotoxin activity and thus should be a suitable method to aid in the molecular characterization of these lymphokines.