A rapid method for estimating the binding of ligands to ELISA microwells.
This report presents a rapid and simple assay for estimating to what extent the surface of ELISA microwells is coated by a ligand of choice such as, for example, proteins, peptides, hormones, polysaccharides and nucleic acids. The method also provides a practical approach for defining the conditions required for optimal coating, such as ligand concentration, coating buffer, temperature and duration of coating and also for evaluating the efficiency of the reagents used to saturate the ELISA microwells. The important advantage of this procedure is that, in contrast to conventional ELISA procedure, the detection of the microwell-adhered ligand is not achieved by using an antibody. It is therefore the solution of choice when, as is often the case, no primary specific antibody is available. The test consists of three steps: first the ligand is allowed to adsorb to the microwells. Second, alkaline phosphatase is added to bind to any residual microwell surface not occupied by the ligand. Finally, substrate is added and the resulting color reaction is measured. Light absorbancy is inversely correlated with the level of ligand adherence. The results obtained by this method match those of direct ligand quantitation, as evaluated by a regular ELISA procedure.
Steinitz
M
M
The Department of Pathology, The Hebrew University - Hadassah Medical School, POB 12272, Jerusalem, Israel. michaels@md2.huji.ac.il
Baraz
L
L
D000469:Alkaline Phosphatase / Q000378:metabolism; D000595:Amino Acid Sequence; D004797:Enzyme-Linked Immunosorbent Assay / Q000295:instrumentation / Q000379:methods*; D016346:Gene Products, vif / Q000378:metabolism*; D016333:HIV Protease / Q000378:metabolism*; D006801:Humans; D008024:Ligands; D008969:Molecular Sequence Data; D010455:Peptides / Q000378:metabolism; D013997:Time Factors
D016346:Gene Products, vif; D008024:Ligands; D010455:Peptides; D000469:Alkaline Phosphatase; D016333:HIV Protease