1. Immunoassays are tests that take advantage of the specificity of antibodies to their protein epitotes.
Qualitative example: pregnancy test kits, HIV test strips
Quantitative example: test kit for GMO detection
Much like the Bradford test where a series of controls are used as a quantitative comparison
ELISA… performed in microplates
Ouchterlony…performed using agarose gels
Western blots … performed using SDS-PAGE
Immunoassays

2. Labeling of Antibodies
Labeling consists of adding a marker to detect a union of the epitote to the antibody
Most often an oxidation reaction emitting a color when the antibody has attached to the substrate
Can be by direct antibody binding or through the use of a secondary antibody (indirect detection)
The secondary antibody binds the antigen and the primary antibody binds the secondary antibody
Allows for use of polyclonal secondary antibodies
Labeling of Antibodies

3. Enzyme-Linked Immunosorbent Assay (ELISA)
Types of ELISA Methods:  Direct ELISA: Antigens are immobilized and enzyme-conjugated primary antibodies are used to detect or quantify antigen concentration. The specificity of the primary antibody is very important.
PROS: minimum procedure; avoids cross-reactivity from secondary antibody.
CONS: requires labeling of all primary antibodies - high cost; not every antibody is suitable for labeling.
Indirect ELISA: Primary antibodies are not labeled, but detected instead with enzyme-conjugated secondary antibodies that recognize the primary antibodies.
PROS: secondary antibodies are capable of signal amplification; many available secondary antibodies can be used for different assays; unlabeled primary antibodies retain maximum immunoreactivity.
CONS: cross-reactivity may occur.
Enzyme-Linked Immunosorbent Assay (ELISA)

4. Enzyme-Linked Immunosorbent Assay (ELISA)
Sandwich ELISA: The antigen to be measured is bound between a layer of capture antibodies and a layer of detection antibodies. The two antibodies must be very critically chosen to prevent cross-reactivity or competition of binding sites.
PROS: sensitive, high specificity, antigen does not need to be purified prior to use.
CONS: antigens must contain at least two antibody binding sites.
Competitive ELISA: The antigen of interest from the sample and purified immobilized antigen compete for binding to the capture antibody. A decrease in signal when compared to assay wells with purified antigen alone indicates the presence of antigens in the sample.
PROS: crude or impure samples may be used, high reproducibility.
CONS: lower overall sensitivity and specificity.
Enzyme-Linked Immunosorbent Assay (ELISA)

5. Reading Elisa Elisa results are quantified using a microplate reader
Uses the principles of spectrophotometry
And is able to quantify reading in all wells providing high throughput efficiency