3.3.6. Antibodies in Assay

Monoclonal Antibody. When antibodies are produced from B cells, some of them are polyclonal - i.e., binds with more than one type of antigen, whereas others are monoclonal - i.e., binds with only one type of antigen. Usually, antibodies are produced by immunizing an animal (such as a mouse). The antibodies produced are collected and the monoclonal antibodies are separated. The monoclonal antibodies (Mab) is the key for immunoassays.

ELISA. Although the antibody-antigen binding is highly specific, the direct detection of this measurement has been difficult. Therefore, labels are used for measuring the complexation product. Enzymes, fluorescent molecules, and radioactive labels are used for the labeling of the complexation. An example is ELISA (Enzyme-Linked Immuno Sorbent Assay). In this method, a known amount of enzyme-labeled antigen is added to a sample with unknown antigen concentration. When this mixture reacts with antibody, wnzyme-labeled and non-labeled antigens compete for the binding sites of the antibody. The more antigen there is in the sample, the lower is the fraction of enzyme-labeled antigen in the antigen-antibody complex. After removal of unbound antigen the amount of bound enzyme-labeled antigen is determined via the enzyme-catalyzed reaction (Fig. 3. 18). Widely used indicator enzymes are horseradish peroxidase and alkaline phosphatase. Urease and deaminases have also been used in immunosensors.

antibody

Fab

IgG

IgG

IgM

effective antibody valence

1

1

2

up to 10

antigen valence

1

1

n

n

equilibrium constant

104

104

107

1011

advantage of multivalence

-

103 fold

107 fold

definition of bining

affinity

affinity

avidity

avidity

Fig. 3.17. Antibody avidity.

Fig. 3.18. Competitive binding immunoassay (ELISA).