The Immunoassay

Definition
Immuno refers to an immune response that causes the body to generate antibodies, assay refers to a test. An immunoassay is an analytical method that uses antibodies or antigens as reagents to measure specific analytes. Most tests that are performed in the clinical chemistry laboratory measure antigen, using antibodies as the reagent.

History and Background

In the year 1959, Drs. Rosalyn Yalow & Soloman Berson invented the radioimmunoassay, which applied the use of radioisotopes in the measurement of insulin.
Dr. Rosalyn Yalow became the first female to win a Nobel Prize with her work on the radioimmunoassay

1972 First Hepatitis RIA 1972 EMIT Assay 1975 Bead-based Assays 1978 Coated-tube assays

1985 HIV Immunoassay 1993 Troponin Assays 1997 Automated Cytokine Assays

Principle
Is related to formation of immune complex between the antibody and a given antigen, followed by the discrimination between the amounts of bound versus free reactants .To aid this, either the antigen or antibody is labeled, most of them with an enzyme of radioisotope. For optimal sensitivity the concentration of antibody should be as low as possible. It require minimum sample preparation since antibodies bind only to specific antigens. Immunoassays are reliable and sensitive to very low levels of analyte.

THE COMPONENTS OF IMMUNOASSAY

1-Antibodies. 2- Sample : Contain the analyte , which may be hormone, tumor marker, drug, virus, and others. 3- Calibration curve: Standards are known concentrations of the analyte that are used to construct a standard curve over an appropriate range of values.

4-Controls: Controls are samples of known concentration that are assayed to check the accuracy of the standard curve. 5-Detection system: Detecting the quantity of antibody or antigen can be achieved by a variety of methods. One of the most common is to label either the antigen or antibody. The label may : 1-enzyme: (enzyme immunoassay )(EIA) 2-radioisotopes :( Radioimmunoassay )(RIA) 3-fluorescence. 4-Luminescence 5- Electrochemical 6-Visual - Colloidal gold- Colored latex

Detection
Directly visible agglutination Invisible requires specific probes (enzyme-labelled antiimmunoglobulin, isotope-labelled antiimmunoglobulin, etc.) binds Ag-Ab complex and amplifys signals signals can be measured by naked eyes or specific equipment e.g. in ELISA, RIA, IFA

Antibody Types
Two Types: Polyclonal Antibodies A pool of many antibody types that may detect different epitopes on the target protein Monoclonal Antibodies All antibodies produced are identical and stem from the same hybridoma cell.

Polyclonal Antibodies
Animals are injected with analytical target Many different antibody-producing cells make Polyclonal antibodies These antibodies are purified from a blood sample, validated for sensitivity and specificity, and then are used in immunoassays Antibodies are derived from several different cell lines and therefore have specificity for different epitopes .

Monoclonal Antibodies
A mouse is immunized with an antigen After repeated immunizations, antibody producing B-cells are removed from spleen Antibody-producing cells are fused with cells that grow continuously in culture to form Hybridomas Each hybridoma cell produces one monoclonal antibody. Living hybridomas are frozen indefinitely in liquid nitrogen. Hybridoma cells can replicate infinitely and produce the same antibody

Monoclonal vs. Polyclonal Antibodies

Monoclonal Higher initial costs Lot-to-lot consistency Indefinite supply Highly specific Longer lead time Polyclonal Lower initial costs Lot-to-lot variability Large quantities can be produced from serum More broadly reactive Shorter lead times

Categories of Immunoassay Methodologies

1- Noncompetitive and competitive immunoassays 2- Homogeneous and heterogeneous immunoassays

Competitive Format
In which patient antigen (unlabeled sample antigen) and labeled reagent antigen compete for the same site on the antibody. The amount of labeled antigen bound to the antibody site is then measured. In this method , the response will be inversely proportional to the concentration of antigen in the unknown.

One step competitive format

In the one step competitive format (see Figure below), both the labeled antigen reagent (Ag*) and the unlabeled specimen (or test sample analyte) compete for a limited amount of antibody.

Two step competitive format

In the two step competitive format, the antibody concentration of the reaction solution is present in excess in comparison to the concentration of antigen. Antibody reagent is first incubated with specimen containing antigens of interest; then in the second step, labeled antigen is added. Remember that in the competitive format, less bound labeled antigen indicates more antigen present in the test sample. Two step competitive assay formats provide several fold improved assay sensitivity compared to one step assay formats.

Noncompetitive (Sandwich) Method

Antigen in the unknown is bound to the antibody site , and then labeled antibody is bound to the antigen. The amount of labeled antibody on the site is then measured. In this method , the response will be dirrectly proportional to the concentration of antigen . Noncompetitive assay formats generally provide the highest level of assay sensitivity and specificity and are applied to the measurement of critical analytes such as cardiac and hepatitis markers

Noncompetitive Method
Noncompetitive assay formats can also utilize either one step or two step methods, as with the competitive assay. The two step assay format employs wash steps in which the sandwich binding complex is isolated and washed to remove excess unbound labeled reagent and any other interfering substances. The two step noncompetitive format usually offers the highest specificity and sensitivity of all the assay formats.

Homogeneous and Heterogeneous Immunoassay Methods

Immunoassay methods that require separation of bound Ab-Ag* complex before labeled is measured are referred to as heterogeneous immunoassays. Those that do not require separation are referred to as homogeneous immunoassays. Homogeneous methods have been generally applied to the measurement of small analytes such as abused and therapeutic drugs. Since homogeneous methods do not require the separation of the bound Ab-Ag* from the free Ag*, they are generally much easier and faster to perform.

Classification of immunochemical methods

Particle methods Precipitation Immunodiffusion Immunoelectrophore sis Light scattering Nephelometry Turbidimetry

Label methods Non-competitive One-site Two-site Competitive Heterogeneous Homogeneous

Types of Immunoassays
1- Immunoprecipitation: The simplist method measures the quantity of precipitate , which forms after the reagent antibody (precipitin) has incubated with the sample and reacted with it is respective antigen to form an insoluble aggregate. The reaction may be qualitative or quantitative. Adv: can be used for any assay Disadv: longer assay time; additional processing steps

2- Particle immunoassay: By linking several antibodies to the particle , the particle is able to bind any antigen molecules simultaneously. This greatly accelerates the speed of the visible reaction. Disadv: time of contact between & incubation mixture is critical. This allows rapid and sensitive detection of antibodies that are markers of such diseases, as infectious mononucleosis and rheumatoid arthritis.

3- Immunonephelometry. The immediate union of antibody and antigen forms immune complexes that are too small to precipitate. However, these complexes will scatter incident light and can be measured using nephelometer. The antigen concentration can be determined within minutes of the reaction.

4- Radioimmunoassay (RIA) Competitive immunoassay is a method employing radioactive isotopes to label either the antigen or antibody. This isotope emits gamma rays, which are usually measured following removal of unbound (free) radiolabel.

Advantages and disadvantages

Advantages higher sensitivity, easy signal detection, and well established , rapid assays. Disadvantages the health and safety risks posed by the use of radiation and the time and expense associated with maintaining a licensed radiation safety and disposal program. For this reason, RIA has been largely replaced in routine clinical laboratory practice by enzyme immunoassay.

5- The enzyme-linked immunosorbent assay (ELISA)

This method use an enzyme to label either the antibody or antigen. Uses an immune reaction like RIA Differs from RIA in detection method Detection based on Enzyme catalysed reaction OR Fluorescent probe NOT radioactivity [great advantage!]

Types of ELISA
Noncompetitive binding assay or Sandwich method Antigen measuring system [Titre wells coated with antibodies ; Enzyme labelled antibodies] Antibody measuring system [Titre wells coated with antigens ; Enzyme labelled antiantibodies] Competitive binding assay [Titre wells coated with antibodies ; Enzyme labelled antigens]

Noncompetitive or Sandwich Assay

Antigen measuring system - Titer wells coated with suitable antibody - Add patient sample containing the antigen - Incubate: till antigen antibody reaction is complete - Wash remove unbound antigen - Add Antibody labelled with Enzyme - Incubate till antigen binds labelled antibody - Wash remove unbound labelled antibody - Add substrate ; incubate - Enzyme + Substrate Product measure colour - Colour proportional to antigen in patient sample

Noncompetitive or Sandwich Assay

Antibody measuring system - Titre wells coated with suitable antigen - Add patient sample containing the antibody - Incubate: till antigen antibody reaction is complete - Wash remove unbound antibody - Add Antiantibody labelled with Enzyme - Incubate till labelled antiantibodies binds antigen-antibody complex - Wash remove unbound labelled antiantibody - Add substrate ; incubate - Enzyme + Substrate Product measure colour Colour proportional to antibody in patient sample

Competitive binding assay

Titre wells coated with antibodies Known quantities of patient sample containing antigen + antigen labelled with enzyme Incubate: till antigen antibody reaction is complete Wash remove unbound antigens Add substrate ; incubate Enzyme + Substrate Product measure colour Colour inversely related to antigen in patient sample

Enzyme labels
Enzyme labels should have high specific reactivity Should be easily coupled to ligands & the labelled complex must be stable The reactivity should be retained after linking of the enzyme to the antigen/antibody The chosen enzymes should not be normally present in the patient samples Examples of enzyme labels: Alkaline phosphatase, Glucose oxidase

6 - Fluorescent immunoassay (FIA)

Fluorescent immunoassay (FIA) refers to immunoassays which utilize a fluorescent label or an enzyme label which acts on the substrate to form a fluorescent product. Fluorescent measurements are inherently more sensitive than colorimetric (spectrophotometric) measurements. Therefore, FIA methods have greater analytical sensitivity than EIA methods, which employ absorbance (optical density) measurement.

7- Chemiluminescent immunoassays
Chemiluminescent immunoassays utilize a chemiluminescent label. Chemiluminescent molecules produce light when they are excited by chemical energy. These emissions are measured by a light detector.

Immunoassay Results
Qualitative Single point calibration at a specific cutoff Results are either positive or negative ; (i.e. above or below the cutoff) Possible false positives; monoclonal antibodies restrict this slightly.

Quantitative Provides numeric results that are an estimate of drug/compound concentration based on the measurement of labeled analyte in the solution, and taking into consideration the competitive/noncompetitive nature of the device. In terms of use on drugs, this is sometimes complicated by possible cross-reactivities.

Although immunoassays are both highly sensitive and specific, false positive and negative results may occur: False negative: may be caused by improper sample storage or treatment, reagent deterioration , or improper washing technique. False positive: some times seen in persons who have certain antibodies, especially to mouse immunoglobulins (immune cells) that may be used in the test. False-positive results have been reported for samples containing small fibrin strands that adhere to the solid phase matrix. False-positives may also be caused by substances in the blood or urine that cross-react or bind to the antibody used in the test.

Factors Affecting Immunoassay Results Temperature Antibodies have evolved to bind best at body temperature. Lower temperatures will effect antibody binding affinities Time Assay Time Extraction Time Longer extractions lead to more available Movement ELISA shaking during incubation Sample extraction movement

Assay interferences 1- venous stasis from prolonged application of tourniquet will increase plasma protien which lead to increase serum or plasma ligands concentration. 2- samples anticoagulated with EDTA or sodium fluoride un suitable for enzymatic immunoassays. 3- use of gel serum separators have been shown to interfere with serum peptide and protein assays. 4- sample contamination: - radioisotopes, given for diagnostic or therapeutic purposes can interfere with radiolabeled immunoassays.

- impurities in tracers interfere with direct analysis methods for free hormones. - the use of intravenous fluorophores for ophthalmic examination interfere with the therapeutic drug assays 5- haemolysis and hyper bilirubinaemia. 6- lipaemia interfere with some assays for fat-soluble compounds such as steroids. 7- stability and storage. 8- Excessive concentration of free fatty acids.

Assay problems and troubleshooting

Loss of binding: This can happen for a number of reasons, such as decomposition of tracer ,decomposition of the antisera, or separating agents going off . one or other of the reagents has become contaminated with analyte Shifted calibration curves Problems with standards Contamination problems Difficulties in transferring an assay to another laboratory

Keys to Good Immunoassay Results

Follow the manufacturer s directions Careful sample preparation Pipette accuracy Care in handling conjugates Care in handling positive samples and avoiding cross contamination to negative wells/samples Thorough washing

Applications Of Immunoassay
1) endocrine hormones: e.g. ACTH, FSH, T3,T4, Glucagon, Insulin, Testosterone, and hGH . 2) Diagnostic procedures for detecting infections: e.g. hepatitis A, B, C, D and E , Detect antibodies to HIV in the blood, urine or saliva , Rubella, CMV, chlamydia, and Helicobacter pylori antigens in the dental plaque. 3)Cancer tumor markers: Tumor cells can produce protein that are detected in serum by immunoassay, and these proteins are called tumor markers. e.g. AFP, CEA, PSA, and hGh.

4) hCG hormone test: This test measures human chorionic gonadotrophin (hCG), a hormone that is secreted in urine during pregnancy. can be done for urine or blood hCG level doubles approximately every 2 days and it can be detected in urine as early as 7 days following conception. Early immunoassay tests were done with blood because this type of test is the most sensitive and can detect minute quantities of hCG

5) Cardiac Markers, e.g. CK-MB, CRP, Myoglobin 6) Therapeutic drug monitoring, e.g. Barbiturates,
morphine, digoxin.

7) Drugs of abuse screening, e.g. cocaine, amphetamines. 8) Allergy , e.g. Histamines , egg , milk. 9) immunological screening . 10) Enzyme activity.

Advantages of Immunoassays
Diverse applications (screening or quantitative) High Sensitivity Low detection limit High Specificity - Detect specific compound User friendly and simple Safe to perform - extraction uses buffer or water Rapid testing time (5 minutes to 1 hour typically) Cost effective

Questions?