Identification of unacceptable background caused by non-specific protein adsorption to the plastic surface of 96-well immunoassay plates using a standardized enzyme-linked immunosorbent assay procedure

A standardized enzyme-linked immunosorbent assay (ELISA) was used to examin e the capacity of immunoassay plates to prevent non-specific protein bindin g under blocking conditions. Data from 16 types of 96-well microtitre plate from seven commercial sources, are described. Plates were evaluated with r espect to their capacity to adsorb a conjugated antibody in diluent buffer containing non-ionic detergent Tween 20 (0.05%) and skimmed milk proteins ( 5%). Plates with an absorbance value of greater than or equal to 0.05, in n ot more than one well, were defined as within acceptable limits. Major prob lems were seen in high binding gamma-irradiated polystyrene plates, from al l sources, where only less than or equal to 30% of plates were acceptable. These showed high, randomly distributed, non-specific binding, with some we lls showing absorbance values > 2.0. Similar results were obtained when hig h binding plates were repeatedly gamma-irradiated, and after gamma-irradiat ion of low binding polystyrene plates. For high binding, non-gamma-irradiat ed polystyrene plates, approximately 70% of plates were acceptable. Better results (86-100% acceptability) were observed for all low binding polystyre ne plates. Only one source in three provided acceptable, low binding, polyv inylchloride plates. This paper confirms a widely held view that non-specif ic binding to certain plates could be a serious factor in both the developm ent and application of ELISAs. Therefore, the test protocol described is pr oposed as an additional quality control method for certifying ELISA plates by commercial companies. (C) 1999 Elsevier Science B.V. All rights reserved .