Immunoassay

Antibodies as Specific Reagents

Antibody = immunoglobulin protein with specific affinity for target molecule (antigen).
Can be polyclonal if produced by antigen injection into experimental animal, or monoclonal if produced by cell fusion and cell culture techniques. Monoclonal have better specificity and more reproducible properties from batch to batch.

In immunoassays the antibody serves as a specific reagent for the analyte antigen.
Broad classification into...

1. Competitive binding immunoassay - analyte competes with a labelled antigen for a limited pool of antibody molecules. (eg radioimmunoassay, EMIT)
   
2. Non-competitive - antibody present in excess and labelled. As analyte antigen is increased, amount of labelled antibody-antigen complex also increases. (eg ELISA)
   

Both types normally require separation of Ag-Ab complex from free Ag and/or Ab.

Labelling

Many alternatives to radioisotopes now available.

To achieve comparable sensitivity to radioactive label need..

or, more commonly, enzyme labelling, so that a single labelled molecule acting as a catalyst provides strong amplification (convert millions of substrate ---> product molecules)

EMIT

Enzyme Multiplied Immunoassay Technique

A competitive binding immunoassay that avoids the usual separation step.

As [Test antigen] is increased it occupies more of the antibody molecules, so fewer of the enzyme labels are blocked. Substrate ---> product response increases.

ELISA

Enzyme Linked Immunosorbent Assay
Wide application - diagnostic & research.

Non-competitive "sandwich" form below (cf Holme & Peck) requires two antibody-recognition sites (epitopes) on test antigen
- one of many ELISA variants .