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presence of the enzyme causes a colour change in thechromogenic(colour-producing)

substrate. The marker enzyme used is usually either horseradish peroxidase (HRP) or

alkaline phosphatase (AP). Other enzymes have been used and claims have been made

for increased sensitivity but this is at the expense of more complex substrates and

buffers. In some systems the enzyme is replaced with a radioactive label and this

format is known as the immunoradiometric assay(IRMA). DAS ELISA is used

extensively in horticulture and agriculture to ensure that plant material is free of

virus. Potato tubers that are to be used as seed for growing new crops have to be free

of potato viruses and screening for this is carried out by DAS ELISA. There are many

potato viruses but potato leafroll virus (PLRV) in particular causes considerable

problems. PLRV antibodies are coated onto the wells of ELISA plates and then the

sap to be tested is added. After incubation, the plates are washed and PLRV antibody

conjugated to alkaline phosphatase is added. The plates are incubated and after

washing, substrate is used to identify the positive wells. The system again requires

the presence of the antigen (PLRV) for the sandwich of antibodies to be built up.

7.3.4 Enhanced ELISA systems

The maximum sensitivity of ELISA is in the picomole range and there have been

many attempts to increase the detection threshold for assays beyond this. The

physical limitations are based on the dynamics of the double binding event and the

subsequent generation of signal above the background substrate value. Most workers

have concentrated their efforts on the amplification of signal. Antibody binding

cannot be improved as it is primarily a random event modified by the individual

avidities of the antibodies themselves. Some improvement in some assays can be

ELISA plate coated
with antibody

Antigen trapped

by antibody

Antibody/enzyme

conjugate

incubated on plate

Substrate added

to plate causing

colour change in

positive wells

Sample incubated

on plate

Fig. 7.14DAS ELISA.

288 Immunochemical techniques