Immunoassay and Primary Reactions Lecture Notes

Flashcards covering the definitions and principles of primary reactions, specifically focusing on Radioimmunoassay (RIA) and various Enzyme Immunoassay (EIA) techniques.

Primary Reactions

Reactions where the antigen and antibody binding is visible through labeling or tagging with substances that can be seen microscopically or macroscopically.

Secondary Reactions

Reactions where the actual binding of antigen and antibody is not seen directly, but the results, such as precipitation and agglutination, are visible.

Radioimmunoassay (RIA)

An in-vitro technique introduced by Berson and Yalow in 1960 used to measure concentrations of antigens, such as insulin or hormones, making use of radiation.

Competitive binding

A principle of RIA where a reagent and a patient analyte compete for available binding sites to ensure specificity.

Picogram range

The sensitivity level of RIA, capable of measuring down to $10^{-12}\,g$.

Half-life

The duration representing the decay of radioactive material; if it decays too far (e.g., from 90% to 45%), it becomes unsuited for laboratory detection.

Gamma and Beta Spectrometers

Special types of spectrophotometers used to measure the decay in radioimmunoassays.

$$^{131}I$$

A gamma-emitting isotope used in RIA with a half-life of only $8.05\,days$.

$$^{125}I$$

The most popular radioactive label in RIA; it emits gamma rays, can be easily incorporated into proteins, and has a half-life of $60\,days$.

$^3H$ (Tritiated Hydrogen)

A beta-emitting isotope used as a radioactive label in RIA.

Crystal Scintillation Counter

Instrument used to detect high energy gamma rays from labels like $^{125}I$ or $^{131}I$.

Liquid Scintillation Counter

Instrument used to detect beta-emitting isotopes, characterized by higher detection efficiency.

RIST (Radioimmunosorbent Test)

A non-competitive binding assay where Anti-IgE is covalently coupled to Sephadex particles to detect the concentration of IgE in patient samples.

RAST (RadioAllergoSorbent Test)

A quantitative blood test used to identify specific allergens by using paper discs coated with different antigens to measure increased IgE levels.

RIP (Radioimmunoprecipitation Assay)

A double antibody technique with a sensitivity of approximately $1\,IU\,IgE/ml$ ($2.4\,ng$), though susceptible to interference by serum factors.

IRMA (Immunoradiometric Assay)

A non-competitive immunoassay that uses labeled antibodies in excess to ensure all unknown analyte is involved in the reaction.

Immunoenzyme

An antibody labeled with an enzyme used as a reagent to detect a specific antigen.

Heterogeneous EIA

A type of enzyme immunoassay where the antigen-antibody reaction does not affect enzyme activity, requiring physical separation of bound and unbound antigens.

Homogeneous EIA

An enzyme immunoassay where the reaction modulates enzyme activity, meaning no separation of bound and free antigen is required.

Direct ELISA

An assay where an enzyme-labeled primary antibody binds directly to a target antigen immobilized on a plate.

Indirect ELISA

A technique where antigen is coated on a well, patient antibody is added, and then detected by an enzyme-conjugated secondary antibody.

Sandwich ELISA

A technique where an antibody is coated on a well to capture an antigen, which is then bound by a second enzyme-linked antibody specific for a different epitope.

Competitive ELISA

An assay used to measure antigen concentration where the higher the concentration of antigen in the sample, the lower the final absorbance.

EMIT (Enzyme Multiplied Immunoassay Technique)

An example of a homogeneous immunoassay used primarily for low molecular weight analytes such as haptens (e.g., Morphine or Digoxin).