ELISA for antibody measurement: aspects related to data expression

Abstract

In an ELISA for antitoxoplasma IgG (antigen-coated polystyrene beads, horseradish peroxidase-coupled IgG or staphylococcal protein A), 3 modes of expressing the analytical results were considered, i.e. end-point antibody titre, untransformed absorbance reading at a single sample dilution, and antibody-activity unit from a calibration response curve (reference sera as calibrators). Criteria of merit for evaluation were defined as (a) stability of data under various conditions relating to both changes in assay design and minor variability of experimental conditions, and (b) linearity of response with dilutions of positive sera in negative serum, i.e., with positive sera sequentially defined as to antibody activity. Conclusions emerging were: single absorbance readings have some validity as indicators of trends but are very prone to systematic and random variability and inconsistent in response-antibody activity parallelism; parallelism of response proved to be an advantage of titration, but disadvantages are lack of practicability (manipulation and reagent costs involved) and lack of reliability (high levels of systematic and statistical error) The introduction of a reference scale allowing data to be expressed in activity units eliminates systematic components of error and gives analytical consistency. Use of the latter appears mandatory for between-run, between-laboratory and between-method normalization.