LABELED IMMUNO ASSAY

BY; JOHN ALFREY D. PUEBLO

INTRODUCTION
Clinical

Laboratory must haven a Rapid, specific, and sensitive assay. LI designed for antigens and antibodies that may be small in size or present in very low concentrations. Analyte- a substance to be measured Helps in rapid monitoring and diagnosis of numerous disease prompt treatment for many such conditions

Immunoassays

are tests that use antibody and antigen complexes (also called immunocomplexes) to measure the presence of a specific analyte in a sample. Antibodies are proteins that are normally produced by the immune system in response to a foreign substance. Antigens are the molecules that antibodies bind to, which in the body could be an invading pathogen, or the foreign molecules injected into an animal to trigger the immune response.

CHARACTERISTICS OF LABELED IMMUNOASSAYS

COMPETITIVE VS. NONCOMPETITIVE ASSAYS

CURRENT

TECHNIQUES

1. 2. 3. 4.

Fluorescent Radioactive Chemiluminescent Enzyme labels

COMPETITIVE IMMUNOASSAY
unlabelled

analyte (usually antigen) in the test sample is measured by its ability to compete with labeled antigen in the immunoassay. The unlabeled antigen blocks the ability of the labeled antigen to bind because that binding site on the antibody is already occupied.

The

amount of bound label is inversely proportional to the concentration of labeled antigen More label detected, less patients antigen Thus, in a competitive immunoassay, less label measured in the assay means more of the unlabeled (test sample) antigen is present. The amount of antigen in the test sample is inversely related to the amount of label measured in the

competitive format (Figure 1-7).

One step competitive format

In

the one step competitive format (see Figure 1-8), 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

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 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 IMMUNOASSAY

Capture

antibody

The

amount of label is directly proportional to the amount of patient antigen

Unknown patient antigen is then allowed to react with and be captured by antibody. After washing, a second antibody with label is added to the reaction.

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

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. This format is referred to as a sandwich assay because analyte is bound (sandwiched) between two highly specific antibody reagents (Figure 1-10).

Homogeneous and Heterogeneous Immunoassay Methods

Immunoassay

methods that require separation of bound Ab-Ag* complex 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.

ANTIBODIES
Essential

to have high affinity for the antigen. Competitive binding sites Random interaction between individual Ag and Ab molecules THE Higher the affinity of antibody for antigen, the larger the amount of antigen bound to antibody and the more accurately specific binding can be measured. Ab used should also be very specific for Ag involved in the reaction. Monoclonal Ab---leads to highly specific antibodies to increase the detection of a mall amount of analyte with great accuracy

STANDARD OR CALIBRATORS

Known

concentrations of a substance to be measured. Used to established a relationship between the labeled analyte measured and unlabeled analyte that might be present in patient specimen.

SEPARATION METHOD
Separating

reacted from unreacted analyte. Solid-phase vehicle for separation. Polystyrene test tubes, microtiter plates, glass or polystyrene beads, magnetic beads and cellulose membrane. Great carte must be observe.

DETECTION OF LABEL
The

last step common for all immunoassays is detection of the labeled analyte. The method depends on the label; Enzymes are generally used to produce coloured products from colourless substrates that can be determined easily in a spectrophotometer or colorimeter. Automated plate readers are commercially available which make reading large numbers of samples relatively easy.

QUALITY CONTROL
It

is essential that quality control procedures be established. This is done to limit random errors, such as
temperature

fluctuations, minor changes in the concentration of reagents, and changes in detector efficiency.

negative control and a positive control should be run. This serves as a check on the quality of the reagents to make sure that the label is readily detectable under current testing conditions.