A2_1 Principles of competitive

Nearly all immunoassays are competitive or immunometric in format. Because they have some shared characteristics they are both explained in this chapter. ELISA (Enzyme-Linked Immunosorbent Assay) is a subgroup of immunometric assays although the term is sometimes used loosely (but incorrectly) to describe any immunoassay. Immunometric assays are commonly known as sandwich assays because the analyte becomes sandwiched between two antibodies (the capture and labeled antibodies).

This chapter explains the basic principles of competitive and immunometric assays, starting with the kinetics of antibody-antigen binding reactions, based on the Law of Mass Action. The determinants of assay sensitivity are examined in detail, for both competitive and immunometric assays, with simulations of assays to show how sensitivity can best be optimized during the process of immunoassay design. Three-dimensional graphs are used to explain the relationships between antibody concentration, equilibrium constant, specific activity, non-specific binding and assay sensitivity. There are sections on the impact of solid-phases on assay kinetics and considerations for the determination of antibody levels in samples.

The editor is grateful to Xiyang Mi and Rui Liu, who discovered these errors in this chapter.

In equation (1), [Ag] = [Ab] should be [Ag] + [Ab]

In equation (3), the first two expressions in the denominator should be enclosed in brackets. The correct denominator is ([Abt]-[Ag-Ab])[Ag].

In equation (25), there should be a minus sign after [Abt]. The correct expression is [B]/[F] = Keq {[Abt] – [Agt] X [B]/[F] ÷ (1 + [B]/[F])}

On pages 39 and 40 there are a number of incorrect references to “NSM”. This should be “non-specific binding” or “NSB”.

This chapter is based on material in earlier editions of The Immunoassay Handbook written by Chris Davies, formerly Scientific Advisor at Ortho Clinical Diagnostics, and more recently, International Project Manager for Biotechnology at the Welsh Development Agency. He is currently the Business Development Manager for Life Sciences for the Welsh Government. Chris has achieved global recognition for his ability to explain immunoassay principles through his chapters in The Immunoassay Handbook.

Immunoassay, competitive, reagent-limited, immunometric, sandwich assay, reagent-excess, ELISA, capture bridge assay, specificity, sensitivity, antibodies, antigens, kinetics, Law of Mass Action, equilibrium constant, affinity constant, Scatchard Plot, non-specific binding, Sips plot, dose-response curve, bridge recognition, matrix effects, signal-to-noise ratio, antibody occupancy, immunoassay design, assay optimization, solid-phase, sticking coefficient, boundary layer.