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7.3.2 Triple antibody sandwich ELISA (TAS ELISA)

Triple antibody sandwich(TAS) ELISA, also known as indirect ELISA, is a widely used

method (Fig. 7.13). It is often used to identify antibodies in patient blood which may

be there as the result of infection. As with other immunoassays, layers of reagents are

built up, each dependent on the binding of the previous one. The system is used to test

patient blood for the presence of hepatitis B virus (HBV) antibodies as a diagnostic test

for this disease. In this test HBV coating antibody is bound to the wells of a microtitre

plate and HBV coat protein added to them. The live virus is not used as antigen as this

would be too dangerous to use in the laboratory. HBV coat protein is made synthetic-

ally specifically for use as antigen in this type of test. After incubation and washing,

patient serum is added which if it contains antibodies reacts to the antigen.

Anti-human antibody conjugated to an enzyme marker is then added which will bind

to the patient antibodies. Substrate is then added to identify samples which were

positive. The test works well for the diagnosis of HBV infection and is also used to

ensure that blood donations given for transfusion are free from this virus.

7.3.3 Double antibody sandwich ELISA (DAS ELISA)

Double antibody sandwich(DAS) ELISA is probably the most widely used immuno-

chemical technique in diagnostics (Fig. 7.14). It is rapid, robust, and reliable and can

be performed and the results interpreted with minimal training. The principle is the

same as other ELISA techniques in that the antigen is immobilised to a solid phase by

a primary antibody and detected with a second antibody which has been labelled with

a marker enzyme. The antigen creates a bridge between the two antibodies and the

ELISA plate coated
with antibody

Antigen trapped

by antibody

Secondary antibody

incubated on plate

Substrate added

to plate causing

colour change in

positive wells

Sample incubated

on plate

Anti-species

antibody

conjugate

incubated

on plate

Fig. 7.13TAS ELISA.

287 7.3 Immunoassay formats