Enzyme-Linked Immunosorbent Assay (ELISA) is used mainly to detect and quantify proteins, antibodies, peptides, or hormones in a sample. In ELISA, antigens are immobilized on a solid support, either coated directly or through specific capture antibodies. Primary detection antibodies are then applied, forming a complex with the antigen. If conjugated with an enzyme, the detection antibody can directly be used to quantify the antigen, or can itself be quantified by another enzyme-conjugated secondary antibody. The method chosen depends on which target you are investigating.
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.
- 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.
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[Brochure] Antibody Pairs for ELISA, IP-WB, Protein Phosphorylation, Protein-Protein Interaction.