An enzyme-linked lectin binding assay for quantitative determination of lectin receptors.
An enzyme-linked lectin binding assay (ELBA) has been developed for the detection of soluble lectin binding substances (receptors) and the determination of their relative affinity for the lectin. The assay is based on competitive binding to enzyme-labeled lectin of a known lectin receptor, bound to a solid phase, and unknown sample receptors. In this paper the assay is exemplified with the mannose/glucose-specific pea lectin, with the glycoprotein ovalbumin as its receptor, and with horseradish peroxidase (EC 1.11.1.7) as the enzyme used for labeling. Also a method was developed for the preparation of peroxidase-labeled lectin. Labeling was started by mixing equimolar amounts of lectin and periodate-oxidized enzyme at pH 4.5 at a final concentration of 10(-4)M, after which conjugation was started by raising the pH to 9.5. This resulted in complete conjugation, after which the product could be diluted 50-500 times for application in ELBA. For the ELBA ovalbumin was adsorbed onto polystyrene microtiter plates. Sample receptors, added together with the enzyme-labeled lectin, inhibited binding of the latter to ovalbumin. Bound enzyme activity was colorimetrically determined after addition of o-phenylenediamine. Relative lectin affinity (KL) was expressed as (formula; see text) in which [X]50% is the concentration of sample receptor necessary to inhibit 50% of the binding of a certain amount of lectin, and [M]50% is the concentration of D-mannose necessary to inhibit 50% binding of the same amount of lectin. With this technique lectin affinity of both monovalent and polyvalent lectin binding substances can be estimated: low KL values mean high lectin affinity.
D001667:Binding, Competitive; D055598:Chemical Phenomena; D002621:Chemistry; D007887:Fabaceae; D006735:Horseradish Peroxidase; D006863:Hydrogen-Ion Concentration; D010544:Peroxidases; D010946:Plants, Medicinal; D011975:Receptors, Mitogen / Q000032:analysis*; D012995:Solubility
D011975:Receptors, Mitogen; D006735:Horseradish Peroxidase; D010544:Peroxidases