▪ It involves measuring the number of residual non-agglutinating particles in a specimen

These particles are counted by means of a laser beam in an optical particle counter similar to the one that is designed to count blood cells

▪ As agglutination occurs, clumps of antigens increase in size, these large clumps are not counted. The amount of unknown antigen in a patient sample is therefore INDIRECTLY PROPORTIONAL to the number of unagglutinated particle

Specimen is incubated with latex beads coated with the analyte of interest and reagent antibody

If patient’s antigen is low, reagent antibody will bind to the latex beads, resulting to aggregate formation and high level of turbidity

COMPETITIVE ASSAY, INVERSELY PROPORTIONAL, USED ON THERAPETIC DRUGS

▪ If patient’s antigen is high, the antigen will bind to reagent antibody and prevent it from binding to latex beads, resulting in less turbidity

Antigen consists of very fine particles that clump together in a positive reaction

Specific type of precipitation that occurs over a narrow range of antigen concentrations

Examples: VDRL, RPR

It measures the ability of the patient’s antibody to neutralize infectivity and protect cells from infection

Antigenic activity is stopped by its specific antibody

To detect toxins, viral agents, or antibodies to the toxin or viral agents

Types ✓ Toxin neutralization (Schick test, Dick test, ASO titration test) ✓ Virus neutralization

It is designed for antigens and antibodies that may be small in size or present in very low concentrations

It is determined indirectly by using a labeled reactant to detect whether or not specific binding has taken place

Most immunoassays use a solid phase vehicle for separation such polystyrene test tubes, microtiter plates, glass, or polystyrene beads, magnetic beads, plastic beads, and cellulose membrane

No separation step is necessary

Enzyme-multiplied immunoassay technique (EMIT)

Cloned-enzyme donor immunoassay (CEDIA)

Fluorescence polarization immunoassay (FPIA)

Enzyme immunoassay (EIA)

Rapid immunoassays

Fluorescence immunoassay (FIA

Radioimmunoassay (RIA)

Electrochemiluminescence immunoassay (ECLIA)

Chemiluminescent microparticle immunoassay (CMIA)

Enzyme-linked immunosorbent assay (ELISA)

Emission of light caused by a chemical reaction, not from the light source

▪ Chemiluminescence substances are luminol, acridinium esters, ruthenium derivatives, and nitrophenyl oxalates

Measured in luminometer Advantages: ✓ Excellent sensitivity ✓ Reagents are stable and relatively nontoxic ✓ Inexpensive to perform ✓ Faster TAT

Membrane-based, easy to perform, and give reproducible results

Designed primarily for point-of-care or home testing

Lateral flow assay

Uses fluorescent compounds known as fluorophores or fluorochromes as labels

It uses enzyme labels immunoassays such as Horseradish peroxidase (HRP/APO), Glucose-6- phosphate dehydrogenase, Alkaline phosphatase, β-d-galactosidase

Reacts with suitable substrates to produce breakdown products: Chromogenic, fluorogenic, or luminescent

Pioneered by Yalow and Berson in the late 1950s

It uses a radioactive substance label

Gamma counter in Radioimmunoassay

Beta counter in Radioimmunoassay

Amount of bound labeled antibody is DIRECTLY PROPORTIONAL to the amount of patient analyte

Analyte being detected competes with a radiolabeled analyte for a limited number of binding sites

Amount of label in the bound phase is INVERSELY PROPORTIONAL to the amount of patient antigen present

Most sensitive for drug assay and hormone

based on the principles of RIA

Enzyme activity is INVERSELY PROPORTIONAL to the concentration of the test substance

Referred as indirect ELISA tests because the enzyme-labeled reagent does not participate in the initial antigen-antibody binding reaction

Indirect ELISA

Amount of enzyme label is DIRECTLY PROPORTIONAL to the amount of the test substance

Solid phase support: microtiter plates, nitrocellulose membranes, magnetic, latex plastic beads

Used to measure antibody production to infectious agents that are difficult to isolate in the laboratory and for autoantibody testing

If antibody is bound to the solid phase, these assays are often called sandwich assays or immunometric assays. ▪ Enzymatic activity is directly proportional to the amount of antigen in the test sample

Capture assays

Absorbs maximally at 490 to 495 nm and emits a green color at 517 to 520 nm

Absorbs at 550 nm and emits red light at 580 to 585 nm

Antibody that is conjugated with a fluorescent tag is added directly to unknown antigen that is fixed to a microscope slide

Slide is read using Fluorescence microscope

Example of antigens detected by ____ include bacterial pathogens such as Legionella pneumophila and Chlamydia trachomatis

2-step procedure ✓ Incubation of patient serum with a known antigen attached to a solid phase ✓ The slide is washed, and then an antihuman immunoglobulin containing a fluorescent tag is added

▪ It is the principle employed in FANA testing and FTA-ABS test

Can be used for either antigen or antibody identification

It is a BLOCKING test in which antigen is first exposed to unlabeled antibody, then to labeled antibody, then to labeled antibody, and is finally washed and examined. If the unlabeled and labeled antibodies are both homologous to the antigen, there should be no fluorescence

It is based on the change in polarization of fluorescent light emitted from a labeled molecule when it is bound by antibody

Labeled antigens compete with unlabeled antigen in the patient sample for a limited number of antibody binding sites

Degree of fluorescence polarization is INVERSELY PROPORTIONAL to concentration of the analyte