2. Basic Immunologic

Procedures

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

m

http://www.cellsalive.com/mac.htm

http://jeeves.mmg.uci.edu/immunology/Assays/Assays.htm

http://www.medschool.lsuhsc.edu/microbiology/DMIP/dmex03.htm

http://www.tulipgroup.com/Common/html/TurbidTech.pdf

http://departments.oxy.edu/biology/Franck/Bio222/Lectures/Feb1lecture.htm

http://www.mercodia.se/global/mainpage.asp?page_id=41 ELISA

http://www.clinprointl.com/technical.htm ELISA

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