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

Abstract

A standardized enzyme-linked immunosorbent assay (ELISA) was used to examine the capacity of immunoassay plates to prevent non-specific protein binding under blocking conditions. Data from 16 types of 96-well microtitre plate from seven commercial sources, are described. Plates were evaluated with respect 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 ≥0.05, in not more than one well, were defined as within acceptable limits. Major problems were seen in high binding γ-irradiated polystyrene plates, from all sources, where only ≤30% of plates were acceptable. These showed high, randomly distributed, non-specific binding, with some wells showing absorbance values >2.0. Similar results were obtained when high binding plates were repeatedly γ-irradiated, and after γ-irradiation of low binding polystyrene plates. For high binding, non-γ-irradiated polystyrene plates, approximately 70% of plates were acceptable. Better results (86–100% acceptability) were observed for all low binding polystyrene plates. Only one source in three provided acceptable, low binding, polyvinylchloride plates. This paper confirms a widely held view that non-specific binding to certain plates could be a serious factor in both the development and application of ELISAs. Therefore, the test protocol described is proposed as an additional quality control method for certifying ELISA plates by commercial companies.