Ch 11: labeled immunoassays

to detect antigens and antibodies that may be mall in size or present in very low concentrations using a labeled reactant to monitor the specific antigen-antibody binding

• all reactants are mixed together simultaneously
• labeled antigen competes with unlabeled patient antigen for a limited number of antibody binding sites
• the amount of bound label is inversely proportional to the concentration of the labeled antigen

competitive immunoassays

• antibody is anchored and absorbed into a solid phase
• unknown patient antigen is reacted an captured by antibody
• Solid is washed to remove unbound antigen and an anti antibody with label is added to the reaction

the amount of label measured is directy proportional to the amount of patient antigen

non competitive immunoassay

using enzymes as labels which react with substrates to produce breakdown product that may be fluorogenic, luminescent, or chromogenic

• alkaline phosphatase
• horseradish peroxidase
• both have high sensitivity and easy to detect

highest conversion of substrate rates (turnover)

• HIV
• hepatitis B

• antigens that have multiple determinants such as 
• antibodies
• cytokines
• tumor markers

heterogeneous

heterogenous assay

• do not require a washing step
• enzyme activity is proportional to concentration of patient antigen present

qualitative observations using a fluorescent microscope

• identification of microorgansism in cell culture or infected tissue, neoplastic tissue
• used to determine concentration of therapeutic drugs

• involves the emission of light caused by oxidation reaction producing an excited molecule as it decays back to its original ground state
• can be used for heterogeneous and homogeneous assays because labels can be attached to either antigen/ antibody.