Introduction

The Enzyme-Linked Immunosorbent Assay [ELISA] is a highly sensitive procedure to quantify the concentration of an antibody or antigen in a sample. The estimation of the analyte concentration depends upon the construction of a standard curve. The standard curve is prepared by making serial dilutions of one known concentration of the analyte across a range of concentrations near the expected unknown concentration. The concentration of unknown samples is determined by interpolation which relies on a properly generated standard curve.

It is imperative to understand that the standard curve run at different times will not yield the exact OD values for every run. The reason for this variation includes inter-operator and assay such as the incubation conditions and pipettors used. Thus, the OD@450 nm value for a point on the standard curve on one given day may not be the same on the next day. To obtain accurate results in calculating concentrations of unknown samples, the interpolation of data must not be done using a standard curve from a previous run. A separate standard curve should be generated for every run along with unknown samples to yield reliable data.

These findings demonstrate inter-operator variability by comparing separate assays performed by two operators using the same lot of an ELISA kit. Additionally, to assess inter-assay variability, we compare the results of separate runs of ELISA by the same operator using the same lot from the ELISA kit.

For research use only, not for diagnostic or therapeutic use.