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Competitive ELISA using conjugated antibody can also be used to quantify levels of

circulating antibody in test serum. The solid phase has the antigen to which the

antibody will attach directly coated on to it. The test serum and the conjugated form

of antibody are mixed together and added to the reaction wells. The conjugated and

test antibody then compete to bind to the antigen. The level of antibody can again be

determined by the reduction in signal observed by addition of the substrate.

7.3.6 Dissociation enhanced lanthanide fluorescence immunoassay (DELFIA)

DELFIAis a time-resolved fluorometric assay which relies on the unique properties of

lanthanide chelate antibody labels. The lanthanides will generate a fluorescent signal

when stimulated with light of a specific wavelength. The light signal generated has a

long decay which enhances the negative to positive ratio of the assay. DELFIA offers a

signal enhancement greater than that possible from conventional enzyme-linked

assays. The lanthanide chelates are conjugated onto the secondary antibody and as

there are a number of lanthanides each with a unique signal which can be used,

multiplexing(more than one test carried out in the same reaction vessel) is possible.

The assay is carried out similarly to standard ELISA and may be competitive or non-

competitive. The assay is concluded by adding an enhancement solution which causes

dissociation of the lanthanide from the antibody molecules. The signal is generated

by stimulating the lanthanide with light of a specific wavelength and measuring the

Plate coated

with antibody

Native and conjugated

antigen compete for

coating antibody

Substrate added

to generate

coloured product

Sample and

conjugated

antigen added

Concentration

0

Absorbance

Standard curve

used to interpret

results

Fig. 7.15Competitive ELISA.

290 Immunochemical techniques