Labeled Immunoassays Some antigen/antibody reactions not detectedby 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 moleculeof a complementary configuration. Usually binds to the substance the test is trying todetect. The receptor is what binds the specific targetmolecule. “Sandwich” technique is an example.
Terminology Reactions may be competitive or non-competitive Competitive – labeled known and patientunknown 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 forthe 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 directlyrelated to the amount of antibody in patient sample.
Terminology Heterogeneous or homogeneous Heterogeneous assays called separationassays Require multiple steps Careful washing of surface to remove unboundreagents and samples. Homogeneous assays do NOT require aseparation step. Mix reagents and patient sample. Measure the labeled product.
Competitive Binding Add known labeled antigen Add unknown antigen Will compete with each other for sites on boundantibody molecule. Must wash off unreacted substances. Type of label on known antigen will determinemethod of detection.
Noncompetitive Binding Patient sample added. Will react with its homologous antigen orantibody, depending upon what is being testedfor. The reaction is measured and the concentrationis directly related to the detected amount.
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 builtinto machine to provide results.
Labels Used to detect reaction which has occurred. Most common are: Radioactive Enzymes Fluorescent Chemiluminescent
Radioimmunassay (RIA) Competitive binding Uses Iodine 125 (I 125) as label Radioactive label competes with patient for sites High radioactivity, small amount of patientsubstance Low radioactivity high amount of patientsubstance. Refer to your textbook for diagrams.
Radioimmunoassay 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 onthe antibody. 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. High radioactivity indicates a low concentration of patient antigen wasbound to the reagent antibody. Low radioactivity indicates a high concentration of patient antigen wasbound to the reagent antibody. Thus, the results are inversely related to the label detected. Standards are run and results read off of standard curve.
Immunoradiometric Assay (IRMA) Labeled antibody plus patient antigen Solid phase antigen added to bind excessantibody. Labeled antibody binds to both patient antigen, ifpresent, and bound antigen. Spin down to separate Labeled antibody/antigen remain in solution. Measure radioactivity.
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 replacedradioimmunoassay.
Enzyme Immunoassay Enzymes occur naturally and catalyzebiochemical reactions. Enzymes are Cheap Readily available Have a long shelf life Easily adaptable to automation. Automation relatively inexpensive.
Enzyme Immunoassay Techniques pose no health hazards. Little reagent enzyme necessary. Can be used for qualitative or quantitativeassays. Enzymes selected according to Substrate molecules converted per molecule ofenzyme. Ease and speed of detection. Stability. Availability and cost
Enzyme Immunoassay Enzymes used include: Horseradish peroxidase Glucose-6-phosphate dehydrogenase Alkaline phosphatase Β-D-galactosidase Horseradish peroxidase and alkalinephosphatase are the most popular. Highest turnover High sensitivity Easy to detect
Heterogenous EIA Competitive Enzyme labeled antigen competes with unlabeledpatient 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 andestrogen.
Competitive ELISA Unknown antigen competes with labeled known antigen Enzyme produces color reaction
Heterogenous EIA Noncompetitive are very popular. Often referred to as enzyme linkedimmunosorbent assay – ELISA Enzyme labeled reagent DOES NOT participatein the initial antigen-antibody reaction. Sandwich technique Advantages High sensitivity and specificity. Relatively simple to perform. Low cost.
Noncompetitive EIA Variety of solid support Microtiter plates Nitrocellulose membranes Magnetic beads Procedure Antigen bound to solid phase Add patient sample, antibody will bind if present Wash Add known enzyme labeled antibody Wash Add substrate Measure enzyme label
Sandwich or Capture Assays Antibody bound to solid phase. If looking for antigen must have multiple epitopes, boundantibody specific for one epitope, second labeledantibody 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 differentepitope on the antigen. Add substrate, measure intensity of color.
Rapid Immunoassays Membrane based cassettes are rapid, easy to perform and givereproducible results. Popular in POCT and home use. Designed to be single use and disposable. Membrane coated with antigen or antibody produces color reaction.
Rapid Immunoassays Immunochromatography Apply sample to one end, migrates forward. Sample dissolves labeled antigen or antibody to which it binds. Migrates towards detection zone where it will bind to immobilizedantigen or antibody. Color change occurs.
Homogeneous Enzyme Assay Reaction which requires NO separation ofreactants. Less sensitive BUT rapid, easy to perform andautomate. Chief use is to detect low molecular weightanalytes such as: Hormones Therapeutic drugs Drugs of abuse Can use serum or urine.
Homogeneous Enzyme Assay Based on principle of change in enzyme activityas specific antigen-antibody combinations occur. Reagent antigen labeled with enzyme tag. Antibody binds to specific determinant sites onantigen, active site on enzyme blocked, causesmeasurable loss of activity. Free antigen competes with enzyme-labeledantigen for limited number of antibody sites. Enzyme activity directly related to patientantigen.
Fluorescent Immunoassay Markers Fluorophores or fluorochromes Ability to absorb energy and emit light Two most commonly used: Fluorescein – green Tetramethylrhodamine – red Tests may be qualitative or quantitative
Fluorescent Immunoassay Complex must form for fluorescence to occur.
Fluorescent Immunoassay Antibodies and bacteria are fixed on a glass-plate. The surplus i.e. non-bounded antibodies are washed out, antibody-bacteria-complexes ("sandwiches") remain. The "sandwich" becomes visible by adding fluorescent anti bovineimmunoglobulin which can be seen as green light in thefluorescence microscope.
Fluorescent Immunoassay Direct immunofluorescence Tagged antibody added to unknown antigen fixed toslide If patient antigen present = fluorescence Indirect immunofluorescence – sandwich assay Patient plus known fixed antigen Allow to react and wash off unbound reactants Add tagged anti-antibody Fluorescence
Fluorescent Polarization Fluorescence polarization is a measure of the time-averaged rotational motion of fluorescentmolecules. A fluorescent molecule, when excited by a polarized light, will emit fluorescence with itspolarization primarily determined by the rotational motion of the molecule. Since the molecular rotation is inversely proportional to the molecular volume, the polarization isin turn related to the molecular size. A small molecule rotates fast in solution and exhibits low value of polarization whereas a largemolecule exhibits a higher polarization because of its slower motion under the same conditions. Thus, changes in fluorescence polarization can reflect the association or dissociation betweenmolecules of interest.
Fluorescent Polarization Another picture to illustrate the principle. Measure polarized light.
Chemiluminescent Immunoassays The process of chemiluminescence occurs when energyin the form of light is released from matter during achemical reaction.
Chemiluminescent Immunoassays Large number of molecules capable ofchemiluminescence Luminol Acridium esters Ruthenium derivatives Nitrophenyl oxalates Use sodium hydroxide as a catalyst Light emission ranges from quick burst or flash to lightwhich remains for a longer time. Different types of instruments are required based onemission.
Chemiluminescent Immunoassays Can be used for heterogeneous orhomogeneous assays. Can attach label to antigen or antibody. Heterogeneous assays use competitive andsandwich assay. Competitive assays used to measure smalleranalytes. Sandwich assays are used to measure largeranalytes.
Chemiluminescent Immunoassay Many applications. Can measure antigen or antibody. Add chemiluminescently tagged analyte. Measure light which is emitted which is directly related toconcentration although competitive binding assays are available.
Chemiluminescent Immunoassays Best known application of chemiluminescense is luminol Luminol reacts with the iron in blood hemoglobin.