Principles of ELISA

Immunoassay

• Immunochemical reactions form the basis of a diverse range of sensitive and specific clinical assays.

• In a typical immunochemical analysis, an antibody is used as a reagent to detect an antigen of interest.

• The exquisite specificity and the high affinity of antibodies for specific antigens, coupled with the unique ability of antibodies to cross link antigens, allow the identification and quantitation of specific substances by a variety of methods.

Enzyme-immuno Assay(EIA)

• Labeled immunochemical assays• Nonlsotopic Immunoassay

Competitive Versus Noncompetitive Reaction Formats

Heterogeneous Versus Homogeneouslmmunochemical Assays

• Immunochemical assays that require a separation of the free from the bound label are termed heterogeneous; those that do not are called homogeneous.

Enzyme-Linked Immunosorbent Assay(ELISA)

• ELISA is a heterogeneous Enzyme Immunoassay (EIA).

• a solid phase antibody. • enzyme-labeled antibody• a "sandwich complex" of

solid phase Ab-Ag-Ab-enzyme is form

• Targets – Human, mouse, rat as well as veterinary

• Sample – Serum, plasma, cell supernatant and urine analysis

• Specificity against both native and recombinant antigens

Example of a typical ELISA kit protocol

High sensitivity

Types of ELISA formats

Troubleshooting tips

Sample preparation guide

• General guidelines – Protocol included with your kit for product-specific

details regarding sample preparation and compatible sample types.

• Optimum sample preparation procedures will vary depending on the target and assay of interest

• Consult the literature for experimental examples similar to your own when developing a new assay.

Sample preparation guide

Types of ELISA formats

Western blot

Colorimetric detection

Peroxidase

H2O2 + chromogen Color complex

(o-dianisidine) + H2O

(phenylamine) λ = 520 nm