Enzyme Linked  Immuno sorbent  Assay    Antigen of interest is adsorbed on to plastic surface sorbent  This antibody is recognized by second antibody (Immuno) This antibody is recognized by second antibody (immuno) which has enzyme attached (enzyme-linked).  Substrate reacts with enzyme to produce product, usually colored.

Use an enzyme to detect the binding of antigen (Ag)antibody (Ab). The enzyme converts a colorless substrate (chromogen) to a colored product, indicating the presence of Ag : Ab binding.  An ELISA can be used to detect either the presence of Antigens or antibodies in a sample depending how the test is designed. ELISA was developed in 1970 and became rapidly accepted.

ELISA Qualitative/Quantitative determines antigen or antibody is present or absent Quantitative determines the quantity of the antibody  Titer The highest dilution of the specimen usually serum which gives a positive reaction in the test

TYPES OF ELISA Solid phase immunoassay  Enzyme Label Competitive assay  Non-competitive assay 

Competitive Elisa Used to determine small molecule antigens. (T3,T4,progesterone etc.) antibody coated microwell serum antigen and labelled antigen added together--competition. antibody-antigen-enzyme complex bound is inversely related to the concentration of antigen present in the sample. The bound enzyme conjugate reacts with the chromogenic substrate added to produce a color reaction (blue to yellow color). . Increased serum antigen results in reduced binding of the antigen-enzyme conjugate with the capture antibody producing less enzyme activity and color (yellow) formation

Noncompetitive Sandwich Assay  Direct Assay  Antigen capture ELISA Antigen adsorbed directly detected by labeled enzyme Antibody capture ELISA Antibody adsorbed directly by labeled enzyme. Indirect Assay  Antigen directly adsorbed on to the solid phase is first incubated with patient serum, and then with a labeled antibody specific for human immunoglobulin

Sandwich Assay  Antigens such as tumor markers, hormones and serum proteins may be determined. Antigen in the sample binds with the capture antibody on the microwell and becomes immobilized. The antibody of the enzyme conjugate binds with the immobilized antigen to form a sandwich of antibody-antigen-antibody/enzyme bound to the microwell.

The design described above using two monoclonal anti-hormone-antibodies in combination with coated tube technique results in an assay which is rapid, sensitive, precise and easy to perform [113]. Standard curve of LH.

Enzyme-lined Immunosorbent Assay (ELISA) Adsorb antibody (or antigen) onto a plastic surface (e.g., an ELISA plate) using chemical coupling or a high pH buffer. Add samples or standards to different wells. Wash Add enzyme-labelled antibody

Enzyme-lined Immunosorbent Assay (ELISA) Wash Add enzyme substrate. Wells containing enzyme go coloured. Coloured wells mean antgen present in sample. Amount of colour is proportional to the amount of antigen in the sample (or standard).

Radioimmunoassay RIAs

Radioimmunoassays Radioimmunoassays are competitive immunoassays. In competitive immunoassays labelled antigen competes with unlabelled antigen for the antigen binding sites of a finite amount of antibody.

Radioimmunoassays In radioimmunoassays the antigen is often labelled with 125I (iodine 125 – a radioactive isotope of iodine which emits gamma rays). The gamma rays emitted by 125I can be detected using a (gamma) scintillation counter.

Procedure Add samples (or standards) to the RIA tubes and incubate. Add 125I-labelled antigen to the tubes and incubate. Remove bound from free antigen (both labelled and unlabelled antigen). Count the amount of bound radioactively-labelled antigen.

Radioimmunoassays The more (unlabelled) Ag in the sample the less chance the labelled antigen has of binding to the Ag-binding sites of the Ab. The less (unlabelled) Ag in the sample the greater the chance of labelled Ag binding to the Ab.

Radioimmunoassays Like other assays standard amount of antigen are added to some assay tubes and a standard curve is determined. The amount of radioactivity in the tubes containing sample (unknown Ag concentration) is compared to the standard curve and the antigen concentration read from the graph.

Used to measure hormones in serum Insulin (first RIA) Growth hormone (GH) Prolactin (PRL) Thyroid simulating hormone (TSH)

Immunoradiometric Assay (IRMA) Other types of RIA Immunoradiometric Assay (IRMA)

Immunoradiometric Assay (IRMA) Immunoradiometric assay is an assay for the quantitative determination of hormones in human serum. Two monoclonal antibodies recognizing different binding sites on the antigen (Hormone) are used in excess. One of them is labeled with iodine-125 (tracer); the other is immobilized on the inner surface of the tube (coated tube system)

During the incubation both antibodies react with hormone molecules of the sample to form a sandwich-type complex bound to the tube. Afterwards, the remaining excess of tracer is completely removed. After washing, the radioactivity of the tubes is measured. The radioactivity is directly proportional to the hormone concentration of the respective sample. Using samples with known hormone concentrations (standards), a radioactivity concentration profile (standard curve) is constructed by which hormone values of unknown samples can be determined.

The design described above using two monoclonal anti-hormone-antibodies in combination with coated tube technique results in an assay which is rapid, sensitive, precise and easy to perform [113]. Standard curve of LH.

Assays using enzyme-labelled antibodies Radioisotopes are unsafe and there are disposal problems. Enzymes much safer.

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