Immunoassays and ELISA Flashcards

Flashcards about Immunoassays and ELISA

Immunoassay

Tests that take advantage of the specificity of antibodies to their protein epitopes.

Qualitative Immunoassay Example

Pregnancy, COVID, HIV test kits.

Quantitative Immunoassay Example

Test kit for GMO detection.

ELISA

Immunoassay performed in microplates.

Ouchterlony

Immunoassay performed using agarose gels.

Western blots

Immunoassay performed using SDS-PAGE.

Labeling of Antibodies

Adding a marker to detect a union of the epitope to the antibody.

Direct Antibody Binding

Antibody directly binds to the antigen.

Indirect Detection

Uses a secondary antibody; the secondary antibody binds the antigen and the primary antibody binds the secondary antibody.

Direct ELISA

Antigens are immobilized and enzyme-conjugated primary antibodies are used to detect or quantify antigen concentration.

Direct ELISA - PRO

Minimum procedure; avoids cross-reactivity from secondary antibody.

Direct ELISA - CON

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.

Indirect ELISA - PRO

Secondary antibodies are capable of signal amplification; many available secondary antibodies can be used for different assays; unlabeled primary antibodies retain maximum immunoreactivity.

Indirect ELISA - CON

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.

Sandwich ELISA - PRO

Sensitive, high specificity, antigen does not need to be purified prior to use.

Sandwich ELISA - CON

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.

Competitive ELISA - PRO

Crude or impure samples may be used, high reproducibility.

Competitive ELISA - CON

Lower overall sensitivity and specificity.

Microplate reader

used to quantify Elisa results.

Spectrophotometry

Principle used in Elisa quantification.