immunoassays

involve the binding of an antibody (Ab) with an antigen (Ag) to detect an analyte used to measure analytes in low concentrations (< 1 µmol/L) that are difficult to measure by traditional methods examples: proteins hormones metabolites therapeutic drugs drugs of abuse nucleic acids

either antigens or antibodies

On antibodies the functional domain us the F( ab) portion - this is the part that can bind to a site on the antigen

IgG is the most common immunochemical reagent

an antigenic determinant or epitope

a substance capable of inducing an immune response & the formation of an Ab

the strength of a single antibody - antigen interaction (IgG) affinity is measured using a Hapten ( Hp) ( low MW Ag with only one epitope )

strength of all interactions combined (IgM)

When an Ab reacts with an Ag that is structurally similar to the Ag that induced Ab production - the greater the similarity the greater the Rxn

labeled product is detected a label is attached to the Ag or Ab label can be radioactive element, fluorescent dye, or enzyme more sensitive technique

nothing is attached to the Ag or Ab ( no label used ) Turbidimetry or Nephelometry is used for direct measurement ( in solution) A precipitation pattern can be looked at in agar gel less anlytically sensitive technique

if binding reagent is an antibody = immunoassay ( majority)*** if binding reagent is a receptor ( ex. estrogen receptor) = receptor assay if binding reagent is a transport protein ( ex. TGB) = Competitive protein-binding assay

most common : horseradish peroxidase ( HRP) alkaline phosphatase (ALP) glucose- 6- phosphate dehydrogenase enzymes: biological catalysts that increase rate of conversion & arent consumed in reaction enzymes can catalyze many substrates molecules enzyme activity can be monitored by the amount of product formed or the effect of the product on a coupled reaction

compounds that absorb radiant energy of one wavelength & emit radiant energy of a longer wavelength emitted light is detected at an angle of 90 degrees from the path of excitation light by a fluorometer or a modified spectrophotometer ( 2 monochromators ) florescein ( a fluorescent label) can be substituted for an enzyme label measure fluorescence

Emit a photon of light as a result of electrical, biochemical, or chemical reactions Luminol • 1st chemiluminescent label used in immunoassays • emits light under alkaline conditions Acridinium Esters • triple-ringed organic molecule • linked by an ester bond to an organic chain

radioactive elements as the label measures radioactivity largely replaced by non-isotopic labels

labelled Ag* competes with an unlabelled patient Ag for a limited number of binding sites ( equal competition ) labelled Ag concentration id constant & limited as concentration of patient Ag increases, more binds to Ab resulting in less bind of labelled Ag* Ag* is inversely proportional to patient analyte 2 types: heterogenous - requires separation homogenous - no separation

• test sample ( quality control, calibrator or patient ) is added to test tube • labelled Ag & Ab reagents are added • incubation • separation of free labelled ( unbound Ag) • bound labeled Ag is measured

also known as immunometric assays labelled reagent Ab is used to detect Ag excess labelled Ab is required to ensure that the labeled Ab reagent does not limit the reaction concentration of Ag is directly proportional to the bound labeled Ab examples: IRMA - immunoradiometric assay IEMA- immunoezymatic assay

only one antigenic site involved ( epitope) labelled Ab ( in excess) is added to the patient sample resulting Ab - Ag complex is precipitated the amount of label in the precipitate is measured direct relationship *** as patient Ag increases radioactivity in ppt increases or enzyme activity in ppt increases

also called Ag capture assay • immobilized Ab captures patient Ag • washing removes unreacted molecules • labeled Ab is added • labeled Ab binds to captured Ag • washing removes free labeled Ab • signal from bound labeled AB is proportional to Ag captured

• immobilized Ag captures patient Ab • washing removes unreacted molecules • labeled Ab is added • labeled Ab binds to captured Ab • washing removes free labeled Ab • signal from bound labeled Ab is proportional to the Ab captured

free labeled reactants must be distinguished from bound labeled reactant Heterogeneous immunoassays: • physical separation needed ( bound labeled Ag from free labeled Ag) • methods; adsorption, precipitation, interaction with solid phase • ex: RIA, EIA, FIA, ELISA Homogenous immunoassays: - no physical separation step needed

uses particles to trap small antigens ( labeled or not) a mixture of charcoal & cross-linked dextran is mis commonly used charcoal: porous , combines with small molecules to remove them fom solution dextran: prevents nonspecific protein binding to charcoal other adsorbents: silica, ion exchange resin, Sephadex After adsorption, sample is centrifuged. Free labeled Ag is found in the precipitate