2. Basic Immunologic Procedures

Part 6 Labeled Immunoassays

Terry Kotrla, MS, MT(ASCP)BB

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.

“Sandwich” technique is an example.

“Sandwich” Technique ELISA

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.

Competitive Binding Add known labeled antigen Add unknown antigen Will compete with each other for sites on bound

antibody molecule. Must wash off unreacted substances. Type of label on known antigen will determine

method of detection.

Competitive Binding

Noncompetitive Binding Patient sample added. Will react with its homologous antigen or

antibody, depending upon what is being tested for.

The reaction is measured and the concentration is 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 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 Uses Iodine 125 (I 125) as label Radioactive label competes with patient for sites High radioactivity, small amount of patient

substance Low radioactivity high amount of patient

substance. 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 on

the 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 was bound to the reagent antibody.

Low radioactivity indicates a high concentration of patient antigen was bound to the reagent antibody.

Thus, the results are inversely related to the label detected. Standards are run and results read off of standard curve.

Radioimmunoassay

Radioimmunassay

Radioimmunoassay Competitive

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.

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.

Enzyme Immunoassay Techniques pose no health hazards. Little reagent enzyme necessary. Can be used for qualitative or quantitative

assays. Enzymes selected according to

Substrate molecules converted per molecule of enzyme.

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 alkaline phosphatase are the most popular. Highest turnover High sensitivity Easy to detect

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.

Competitive ELISA Unknown antigen competes with labeled known antigen Enzyme produces color reaction

Heterogenous EIA 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. 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

Positive Reaction = Color Change

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.

Rapid Immunoassays Membrane based cassettes are rapid, easy to perform and give

reproducible 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 immobilized

antigen or antibody. Color change occurs.

Homogeneous Enzyme Assay Reaction which requires NO separation of

reactants. 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.

Homogeneous Enzyme Assay Based on principle of change in enzyme activity

as specific antigen-antibody combinations occur. Reagent antigen labeled with enzyme tag. Antibody binds to specific determinant sites on

antigen, active site on enzyme blocked, causes measurable loss of activity.

Free antigen competes with enzyme-labeled antigen for limited number of antibody sites.

Enzyme activity directly related to patient antigen.

Fluorescent Immunoassay

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.

Fluorescence

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 bovine

immunoglobulin which can be seen as green light in the fluorescence microscope.

Fluorescent Immunoassay

Direct immunofluorescence Tagged antibody added to unknown antigen fixed to

slide 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 Immunoassay

Positive Immunofluorescence Cryptosporidium parvum oocysts Photo Credit: H.D.A Lindquist, U.S. EPA

Fluorescent Polarization Fluorescence polarization is a measure of the time-averaged rotational motion of fluorescent

molecules. A fluorescent molecule, when excited by a polarized light, will emit fluorescence with its

polarization primarily determined by the rotational motion of the molecule. Since the molecular rotation is inversely proportional to the molecular volume, the polarization is

in turn related to the molecular size. A small molecule rotates fast in solution and exhibits low value of polarization whereas a large

molecule exhibits a higher polarization because of its slower motion under the same conditions. Thus, changes in fluorescence polarization can reflect the association or dissociation between

molecules of interest.

Fluorescent Polarization Another picture to illustrate the principle. Measure polarized light.

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.

Chemiluminescent Immunoassay

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 Immunoassays

Best known application of chemiluminescense is luminol Luminol reacts with the iron in blood hemoglobin.

References http://web.indstate.edu/thcme/PSP/labtests/precip.htm http://www.gla.ac.uk/departments/immunology/education/nursing/lectures/antibody.ht

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http://www.nsbri.org/HumanPhysSpace/focus4/sf-hormonal.html http://ccm.ucdavis.edu/cpl/Tech%20updates/TechUpdates.htm molecular

diagnostics

References (Continued) http://www.liv.ac.uk/~agmclen/Medpracs/practical_5/theory_5.html http://www.fao.org/docrep/W0049E/w0049e06.htm

http://www.genwaybio.com/gw_file.php?fid=6056