Estimation of the protein-binding capacity of microplate wells using sequential ELISAs

A non-linear mathematical method for estimating the protein binding capacity of a microwell is developed by applying Langmuir-type binding principles to data obtained from sequential ELISAs (Underwood and Steele (1991) J. Immunol. Methods 142, 83). Experimental data from sequential ELISAs measuring the binding of rabbit and mouse immunoglobulin G and of BSA to Immulon 2 microwells agree well with the binding curves predicted by our simple mathematical model. Estimates of Immulon 2 microwell capacity for these proteins when coated in a 100 microliters/well volume are between 250 and 325 ng/well. The method provides a convenient, quantitative method for estimating microwell capacity without the use of radioisotopes. A spreadsheet form of the method is also presented, so that the mathematical calculations for any set of experimental data may be performed readily using the spreadsheet's SOLVER function. The method is not applicable to data from experiments where significant desorption occurs or where binding to the solid phase deviates significantly from the simple Langmuir model.