Labeled Immunoassays Labeled Immunoassays Some antigenantibody reactions not

Labeled Immunoassays

Labeled Immunoassays • Some antigen/antibody reactions not detected by precipitation or agglutination. • Looking for very small amounts. • Measured indirectly using a labeled reactant. • Referred to as receptor-ligand assays.

Terminology • Ligand is the substance to be measured. ▫ Defined as a molecule that binds to another molecule of a complementary configuration. ▫ Usually binds to the substance the test is trying to detect. • The receptor is what binds the specific target molecule.

Terminology Reactions may be competitive or non-competitive • Competitive – labeled known and patient unknown are added to reaction and “compete” for the target. ▫ For example, looking for an antibody. ▫ Add labeled reagent antibody of known specificity, patient sample and known antigen. ▫ Patient antibody competes with reagent antibody for the target antigen. ▫ Concentration is inversely proportional to results.

Terminology • Non-competitive ▫ Add patient sample, for example looking for antibody, to known reagent antigen. ▫ Reaction occurs and the concentration is directly related to the amount of antibody in patient sample.

Terminology Heterogeneous or homogeneous • Heterogeneous assays called separation assays ▫ Require multiple steps ▫ Careful washing of surface to remove unbound reagents and samples. • Homogeneous assays do NOT require a separation step. ▫ Mix reagents and patient sample. ▫ Measure the labeled product.

Homogeneous VS Heterogeneous Methods

Standards or Calibrators • • Substance of exact known concentration. Usually run for each new lot number Based on results create standard curve. Standard curve used to “read” results or built into machine to provide results.

Labels • Used to detect reaction which has occurred. • Most common are: ▫ ▫ Radioactive Enzymes Fluorescent Chemiluminescent

Radioimmunassay (RIA) • Competitive binding • Sensitive technique used to measure small concentrations of • • • antigens. Known quantity of antigen is made radioactive, usually with Iodine 125. Known labeled antigen and patient sample added to the reagent antibody Known antigen will compete with the unknown patient antigen for sites on the antibody. Radioactive label competes with patient for sites High radioactivity, small amount of patient substance Low radioactivity high amount of patient substance.

Radioimmunoassay • Thus, the results are inversely related to the label detected. • Standards are run and results read off of standard curve. • The bound antigens are separated from the unbound ones. ▫ Can measure the radioactivity of labeled free antigen in the supernatant solution. ▫ Can measure radioactivity of fixed labeled antigen to the well.

Radioimmunoassay

Radioimmunassay

Immunoradiometric Assay (IRMA) • Labeled antibody plus patient antigen • Solid phase antigen added to bind excess antibody. • Labeled antibody binds to both patient antigen, if present, and bound antigen. • Spin down to separate • Labeled antibody/antigen remain in solution. • Measure radioactivity.

IRMA • In IRMA, the antibodies are labeled with radioisotopes which are used to bind antigens present in the specimen. • When a positive sample is added to the tubes, radioactively labeled (labeled with I 125 or I 131 radioisotopes) antibodies bind to the free epitopes of antigens and form an antigenantibody complex. • Unbound labeled antibodies are removed by a second reaction with a solid phase antigen. • The amount of radioactive remaining in the solution is directly proportional to the antigen concentration.

Advantages/Disadvantages • Advantages ▫ Extremely sensitive and precise ▫ Detects trace amounts of analytes small in size. • Disadvantages ▫ ▫ Health hazard Disposal problems Short shelf life Expensive equipment necessary • Enzyme immunoassays have largely replaced radioimmunoassay.

Enzyme Immunoassay • Enzymes occur naturally and catalyze biochemical reactions. • Enzymes are ▫ ▫ ▫ Cheap Readily available Have a long shelf life Easily adaptable to automation. Automation relatively inexpensive. • Techniques pose no health hazards. • Little reagent enzyme necessary. • Can be used for qualitative or quantitative assays.

Enzyme Immunoassay • Enzymes selected according to ▫ ▫ Substrate molecules converted per molecule of enzyme. Ease and speed of detection. Stability. Availability and cost • Enzymes used include: ▫ ▫ Horseradish peroxidase Glucose-6 -phosphate dehydrogenase Alkaline phosphatase Β-D-galactosidase • Horseradish peroxidase and alkaline phosphatase are the most popular. ▫ Highest turnover ▫ High sensitivity ▫ Easy to detect

Types of Enzyme Immunoassay 1. Heterogeneous enzyme immunoassay ▫ Competitive ELISA ▫ Non competitive ELISA A. Immunoenzymometric assay (Indirect) B. Sandwich or capture ELISA 2. Homogenous enzyme immunoassay

Heterogenous EIA • Competitive ▫ Enzyme labeled antigen competes with unlabeled patient antigen for antibody sites. ▫ Wash to remove unbound reactants. ▫ Add substrate which causes color change. ▫ Results are inversely proportional to concentration. �More patient antigen bound, less color. �If little or no patient antigen bound, dark color. ▫ Used to measure small antigens such as insulin and estrogen. ▫ Enzyme produces color reaction

Heterogenous EIA Noncompetitive ▫ Noncompetitive are very popular. ▫ Often referred to as enzyme linked immunosorbent assay – ELISA ▫ Enzyme labeled reagent DOES NOT participate in the initial antigen-antibody reaction.

Noncompetitive EIA • Variety of solid support ▫ Microtiter plates ▫ Nitrocellulose membranes ▫ Magnetic beads • Advantages ▫ High sensitivity and specificity. ▫ Relatively simple to perform. ▫ Low cost.

Immunoenzymometric assay (Indirect) ▫ ▫ ▫ ▫ Antigen bound to solid phase Add patient sample, antibody will bind if present Wash Add known enzyme labeled antibody (anti Fc portion) Wash Add substrate Measure enzyme label

Sandwich or Capture Assays • Antibody bound to solid phase. • If looking for antigen must have multiple epitopes, bound antibody specific for one epitope, second labeled antibody added specific for a different epitope. • Antigens detected can be ▫ ▫ ▫ Antibodies Hormones Proteins Tumor markers Microorganisms especially viruses • Enzyme label used to detect reaction

Sandwich or Capture Assays • Add patient sample with antigen. • Antigen will bind to antibody bound to solid phase. • Add enzyme labeled antibody directed against a different epitope on the antigen. • Add substrate, measure intensity of color.

Homogenous immunoassay • Immunoassays those that do not require separation step. • An antigen is labelled by an enzyme. • When antibody reacts with the labelled antigen the enzyme is rendered inactive. • Free antigen completes labelled antigen for the antibody binding site preventing inhibition of enzyme activity. • Enzyme activity is thus directly proportional to the concentration of the free antigen

Homogenous immunoassay • Less sensitive BUT rapid, easy to perform and automate. • Chief use is to detect low molecular weight analytes such as: ▫ Hormones ▫ Therapeutic drugs ▫ Drugs of abuse • Can use serum or urine.

Chemiluminescent Immunoassays The process of chemiluminescence occurs when energy in the form of light is released from matter during a chemical reaction.

Chemiluminescent Immunoassays • Large number of molecules capable of chemiluminescence ▫ ▫ Luminol Acridium esters Ruthenium derivatives Nitrophenyl oxalates • Use sodium hydroxide as a catalyst • Light emission ranges from quick burst or flash to light which remains for a longer time. • Different types of instruments are required based on emission.

Chemiluminescent Immunoassays • Can be used for heterogeneous or homogeneous assays. • Can attach label to antigen or antibody. • Heterogeneous assays use competitive and sandwich assay. • Competitive assays used to measure smaller analytes. • Sandwich assays are used to measure larger analytes. • Many applications. • Can measure antigen or antibody. • Add chemiluminescently tagged analyte. • Measure light which is emitted which is directly related to concentration although competitive binding assays are available.

Chemiluminescent Immunoassay

Fluorescent Immunoassay • will be discussed in the following lecture