Abstract
ObjectiveEnzyme-linked immunosorbent assays (ELISAs) are widely used to quantify immunoglobulin levels induced by infection or vaccination. Compared to conventional manual assays, automated ELISA systems offer more accurate and reproducible results, faster turnaround times and cost effectiveness due to the use of multianalyte reagents.DesignThe VaccZyme™ Human Anti-Haemophilus influenzae type B (Hib) kit (MK016) from The Binding Site Company was optimised to be used on an automated BioRad PhDTM system in the Immunology Laboratory (National Health Laboratory Service) in Tygerberg, South Africa.MethodsAn automated ELISA system that uses individual well incubation was compared to a manual method that uses whole-plate incubation.ResultsResults were calculated from calibration curves constructed with each assay. Marked differences in calibration curves were observed for the two methods. The automated method produced lower-than-recommended optical density values and resulted in invalid calibration curves and diagnostic results. A comparison of the individual steps of the two methods showed a difference of 10 minutes per incubation cycle. All incubation steps of the automated method were subsequently increased from 30 minutes to 40 minutes. Several comparative assays were performed according to the amended protocol and all calibration curves obtained were valid. Calibrators and controls were also included as samples in different positions and orders on the plate and all results were valid.ConclusionProper validation is vital before converting manual ELISA assays to automated or semi-automated methods.
Highlights
Quantitative analytical methods have advanced significantly since the development of the enzyme immunoassay (EIA) and enzyme-linked immunosorbent assay (ELISA) in 1971 by the groups of Perlmann and Engvall, and Schuurs and Van Weemen, respectively.[1,2,3] Before this, the only method for performing immunoassays was the radioimmunoassay (RIA), which was first described by Yalow and Berson in 1960.4the RIA had several shortcomings, for example the potential health threat of radioactivity, short half-lives of radioisotopes, cumbersome radioactive waste disposal, expensive counting equipment, etc.[3,5,6] An important shift from radioisotope-labelled liquid-phase assays to solidphase assays occurred in 1968
Modern commercial ELISA/EIA kits use 96-well microtitre plates, where either an antigen or an antibody is noncovalently bound to a solid-phase support
The 96-well microtitre plate included in the VaccZymeTM Human Anti-Haemophilus influenzae type B (Hib) kit (MK016) from The Binding Site Company (Birmingham, United Kingdom) is precoated with the Hib capsular polysaccharide antigen conjugated to human serum albumin
Summary
Quantitative analytical methods have advanced significantly since the development of the enzyme immunoassay (EIA) and enzyme-linked immunosorbent assay (ELISA) in 1971 by the groups of Perlmann and Engvall, and Schuurs and Van Weemen, respectively.[1,2,3] Before this, the only method for performing immunoassays was the radioimmunoassay (RIA), which was first described by Yalow and Berson in 1960.4the RIA had several shortcomings, for example the potential health threat of radioactivity, short half-lives of radioisotopes, cumbersome radioactive waste disposal, expensive counting equipment, etc.[3,5,6] An important shift from radioisotope-labelled liquid-phase assays to solidphase assays occurred in 1968. Miles and Hales[7] developed an immuno-radiometric technique, which used radioactively labelled antibodies instead of labelled antigens for measuring insulin in human plasma. Plastic tubes were subsequently coated with the antigen or antibody to create a solid-phase or immunosorbent platform.[8]. Modern commercial ELISA/EIA kits use 96-well microtitre plates, where either an antigen or an antibody is noncovalently bound to a solid-phase support. These methods are widely employed by laboratories and manufacturing companies for microbiological, virological and other serological diagnostic tests, validation of assays and general quality control. Automated pipetting devices have been used for more than two decades, the high cost associated with the technique remains a major limiting factor in developing countries and smaller laboratories.[3]
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