Labeled immunoassay Flashcards

Optical density.

At the beginning of the reaction and one is taken at a set time later in the reaction.

The rate of the complex formation is continuously monitored and the peak rate is determined.

By the label used.

they are compounds that absorb radiant energy of one wavelength and emit radiant energy of a longer wave length in less than 10^-4 seconds.

it emits a photon of light as the result of an electrical, biochemical, or chemical reaction.

an analyte and a labeled antigen compete for a limited number of antibody binding sites

designed to have excess antibody binding sites and produce a signal directly proportional to the amount of analyte in the sample. AKA a sandwich immunoassay

a method using two antibodies, which bind to different sites on the antigen or ligand

( AKA prozone) is an immunologic phenomenon that occurs when excess analyte overwhelms the test system causing a a false result.

Any form of immunoassay that involves physical separation, at some stage of the procedure, of antibody‐bound antigen from remaining free antigen.

an assay format allowing to make an assay‐measurement by a simple mix and read procedure without the necessity to process samples by separation or washing steps.

quantitative immunological techniques employing a solid phase.

A solid phase is composed of a solid support on which an immunologically active substance has been fixed. The substance immobilized on the solid phase is often called an immunoadsorbent.

The production of linear calibration curves.

they are subject to false-positive and false negatives.

They are used for quantitative measurement of proteins, pathogens, and other molecules in body fluids, such as plasma, serum and urine

Enzyme-linked immunosorbent assay . They are a labeled immunoassay that is considered the gold standard of immunoassays. This immunological test is very sensitive and is used to detect and quantify substances, including antibodies, antigens, proteins, glycoproteins, and hormones

Enzyme Multiplied Immunoassay Technique.

• Competitive binding
• Concentration of the drug proportional to free enzyme activity
• Drug of abuse and therapeutic drug monitoring

Cloned Enzyme Donor Immunoassay.

• Homogenous EIA
• One enzyme fragment is attached to analyte. If it binds to antibody, then the other fragment cannot combine to form active enzyme
• Directly proportional
• Drug of abuse and therapeutic drug monitoring

Fluorescence polarization immunoassay

• Homogeneous, competitive, quantitative immunoassay method
• Fluorescein emits light in the same plane
• Concentration of the drug is inversely proportional to free enzyme activity
• Drug of abuse and therapeutic drug monitoring

(dissociation-enhanced lanthanide fluorescence immunoassay) AKA time-resolved fluorescence (TRF) intensity technology. ie they measure time delay.

They are a time-resolved fluorescence (TRF) intensity technology. Assays are designed to detect the presence of an analyte of interest using lanthanide chelate labeled reagents.

• Immune complexes formed
• Light detector is placed at 180 degrees
• Amount of light transmitted is proportional to concentration

• Immune complexes formed
• Light detector is placed at an angle
• Measures light scatter
• More sensitive than turbidimetry

Endogenous antibodies that can bind to animal antigens including immunoglobulins of other species.
Interferes with the assay when bound to assay antibodies.

High prevalence: polyspecific and low specificity, low to medium affinity, can
bind to multiple human and animal antigens. Example, Paul Bunell-type
antibodies

Low prevalence, high specificity, medium to high affinity, binds to antigen
from a single species specifically (constant region of FAB). Example, Human
anti-mouse antibodies (HAMA)

Antibodies to immunoglobulins, can bind to both human and animal
immunoglobulins (mostly to Fc portion) . Example, Rheumatoid factor

immunoglobulins: any of a class of proteins present in the serum and cells of the immune system, which function as antibodies

IgG, IgM, IgA, IgE, and IgD

Affinity- strength of binding
- Higher, binds more antigen
- interaction between epitope and antibody binding site, property of antigen

Avidity- total binding strength of the complex
- Based on affinity and number of binding sites
- Overall binding strength, property of the antibody

Microparticle Enzyme Immunoassay

• Solid-phase support consists of very small microparticles in liquid suspension.
• Specific reagent antibodies are covalently bound to the microparticles.
• Beads to isolate antigen-antibody complex

• Autoantibodies with lesser affinity than assay
antibody
• Autoantibodies that do not bind to capture or
detection epitopes
• However, causes prolonged antigen half-life by binding

• Autoantibodies that mask capture or detection antibodies
• Detection fails