Immunoassay Testing Methods

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Naming and Describing the different immunoassay Testing

Immunofixation Electrophoresis (IFE)

Type I Immunoassay, single site, Heterogenous, Noncompetitive
Methodology: Identification procedure that combines electrophoresis and an Ab-Ag reaction.

Enzyme-Linked Immunosorbant Assay (ELISA)

Type II, Two-site, Noncompetitive
Methodology: An enzyme/label is conjugated with an Ab that reacts with a colorless substrate to generate a colored reaction product. The "Sandwich" Assay.
Heterogenous, non-isotropic assay that utilizes an enzyme-labeled ligand and an Ab immobilized onto a solid support.

Typical Enzyme Labels:

Alkaline phosphatase
Horseradish Peroxidase (used to enhance the reaction)
Beta-galactosidase
G6PD

Frequently used to detect Multiple Myeloma

Immunofixation Electrophoresis (IFE)

Western Blot

Methodology: Technique in which proteins are separated by gel electrophoresis, transferred to a membrane, and identified through the use of labeled Abs specific for the protein of interest.
Current standard test for the confirmation of human immunodeficiency virus HIV

Enzyme-Multiplied Immunoassay Technique EMIT

Type III Immunoassay
Methodology: Limited Ab is mixed with patient Ag and enzyme labeled Ag. The Ags complete for Ab binding sites and Ab-Ag bindig inactivates enzyme label. Substrate is then added to the assay and only free, unbound Ag* -enzyme can react with substrate. Enzyme product is quantified.

Fluorescence Polarization Immunoassay FPIA

Generally Type III Immunoassay
Methodology: Based on the amount of polarized fluorescent light detected when the fluorescent label is excited with polarized light. Fluorescent label can be attached to Ag or Ab.
Interpretation: Amount of polarized fluorescent light resulting from a competitive binding reaction is inversely proportional to the concentration of unlabeled ligand.

Chemiluminescence

Methodology: Technique based on quantitation of an analyte based on emission of light resulting from a chemical reaction. Label is not inactivated by binding to Ab.
Firefly luciferase
Peroxidase
Often measured with a luminometer. The amount of light emitted is inversely proportional to the amount of analyte present in sample.

Indirect Immunofluorescence Analysis

Methodology: Based on the fact that Abs not only react with specific Ags, but can themselves be Ags. Typically second Abs recognizes an entire class of Igs. i.e. anti-immunoglobulins
Used extensively in the detection of auto-antibodies, antibodies on or in tissue and to identify cellular Ags.
Often used in the cytotechnology and histotechnology

Turbidometry

Methodology: A measurement of light transitted through a suspension of particles which can be measured by a spectrophotometer.
Absorbance measurements are made at 180 degrees to the incoming light source beam, unscattered light.

Nephelometry

Methodology: A direct measurement of light scattered by particles suspended in solution. Detects scattered light at a 90 degree angle from the incoming light source.
Common Analyte: Albumin
Can detect analytes at 1-10 mg/dL

What is an immunoassay?

Procedure that relies on the use of antibodies as "specific" binding Reagents.

What is another name for an epitope?

Antigenic Determinant Site

Antibody

an immunoglobulin protein produced by a lymphocyte in a response to an unique antigen

Affinity

Refers to the thermodynamic quantity defining the energy of interaction of a single antibody-binding site and it's corresponding epitope on the antigen "Random Attraction"

Avidity

Refers to the overall strength of the binding of antibody and antigen includes the sum of the binding affinities of all the individual combining sites on the antibody

The Heavy Chain

The chain of an antibody that determines the class of that antibody

How many chains does an antibody have?

4. Two identical heavy chains and two identical light chains.

What is the region of the antibody that binds antigens?

Antigen Binding Site, or S-S Bridge

What are the 5 classes of immunoglobulins?

IgA IgG IgE IgD IgM

This Ig can bind up to 10 antigens:

IgM

This Ig is the major serum Ig:

IgG

This Ig is a pentamer:

IgM

These Ig's are Monomers:

IgE, IgG, IgD

This Ig accounts for only 1% of serum Ig:

IgD

This Ig is responsible for the manifestation of allergies:

IgE

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