Immunology: Precipitation, Agglutination, and Testing Methods

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51 Terms

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Precipitation

Combine soluble antigen and soluble antibody to produce insoluble complexes that are visible.

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Agglutination

Particulate antigens form visible aggregates when an antibody is present.

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Antigen-Antibody Binding Affinity

Initial attraction force between a Fab site on an antibody molecule and an epitope of determinant site on an antigen.

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Antigen-Antibody Binding: Avidity

Sum of the attractive forces between an antigen and an antibody that keeps the molecule together.

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Prozone phenomenon

Antibody excess where antigens combine with only one or two antibody molecules, resulting in a false negative.

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Postzone phenomenon

Antigen excess where small aggregates are surrounded by excess antigen, resulting in a negative test result.

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Zone of Equivalence

Point where the number of multivalent sites of antigen and antibody are approximately equal.

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Radial Immunodiffusion

Single diffusion technique where antigen diffuses out until the point of equivalence is reached.

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Ouchterlony Diffusion

Double diffusion method where antigen and antibody diffuse out radially to form a line of precipitation.

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Identity (Ouchterlony Diffusion)

Antigens placed in the wells are identical.

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Partial identity (Ouchterlony Diffusion)

Antigens in the wells are similar but not identical.

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Non-identity (Ouchterlony Diffusion)

Antigens in the wells do not share any similarity.

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Immunofixation Electrophoresis

Double-diffusion technique where unknown antigen is electrophoresed, and then antibody is applied directly to the gel.

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Nephelometry

Technique measuring light scattered at an angle to indicate the amount of antigen or antibody present.

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Radial Immunodiffusion (RID)

Technique where the square of the diameter of the ring of precipitation is proportional to the concentration.

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Ouchterlony Diffusion Patterns

Concept to match the precipitation pattern with the correct interpretation.

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Immuno-electrophoresis

Differentiation of serum proteins by electrophoresis followed by diffusion of antibody from the wells.

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Immunofixation electrophoresis

Technique used for identifying over- or underproduction of antibody by electrophoresis of serum followed by direct application of antibody to the gel.

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Direct Agglutination

Agglutination reaction using known bacterial antigens to test for unknown antibodies, such as in the Widal Test for Typhoid Fever.

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Hemagglutination

Agglutination involving red blood cells as particles with antigens, commonly used in ABO blood group typing.

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Passive agglutination

Agglutination method using particles coated with antigens not usually found on their surface, like erythrocytes or latex particles, to detect patient factors or antibodies.

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Reverse Passive Agglutination

Agglutination technique employing antibodies attached to a carrier particle to detect microbial antigens, used for rapid identification of infectious agents.

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Agglutination Inhibition

Agglutination reaction based on competition between particulate and soluble antigens for limited antibody-combining sites, used to detect antibodies to viruses like Rubella and Influenza.

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Competitive Immunoassays

Immunoassays where the labeled antigen competes with unlabeled patient antigen for a limited number of antibody binding sites, with the bound label inversely proportional to the labeled antigen concentration.

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Noncompetitive Immunoassays

Immunoassays where the antibody is first passively absorbed to a solid phase, allowing the unknown patient antigen to react and be captured by the antibody, with the measured label amount directly proportional to the patient antigen.

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Homogenous Immunoassays

Does not require a separation or washing step to separate the bound from the free reactants

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Heterogeneous Immunoassays

Often performed utilizing a solid phase for the reaction and washing away of unbound reactant

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Radioimmunoassay's (RIA)

Uses radioactive labels (125 I most popular) and emits gamma radiation detected by a gamma counter; measures trace amounts of analytes like hormones, serum proteins, and vitamins

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Enzyme Immunoassays (EIA)

Uses enzymes as labels that react with suitable substrates to produce breakdown products that may be chromogenic, fluorogenic, or luminescent; can be homogeneous or heterogeneous

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Capture Assays (Sandwich Immunoassays)

Best suited for antigens with multiple determinants like antibodies, cytokines, proteins, tumor markers, and microorganisms

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Fluorescent Immunoassay (FIA)

Involves qualitative observations using a fluorescent microscope, used for rapid identification of microorganisms, tumor-specific antigens, and transplantation antigens

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Direct IFA

Antibody conjugated with a fluorescent tag added directly to unknown antigen fixed to a microscope slide for pathogen presence demonstration

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Indirect IFA

Patient serum incubated with known antigen attached to a solid phase, then anti-human immunoglobulin with a fluorescent tag added to form a sandwich for antibody identification

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Chemiluminescent Immunoassays

Follows antigen-antibody combination with light emission from a chemical reaction, can be used in heterogeneous and homogenous assays, sensitive with a large linear range of measurements

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Rapid Immunoassays

Membrane-based, single-use, and disposable assays used for rapid strep, pregnancy, mono, etc., in home, clinic, or hospital settings

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Immunoassay

Antigen or antibody coupled to membrane, producing visible color change

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Colloidal immunoassay

Incorporates colloid for visible color change in reaction

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Hybridization Methods

Techniques like Southern blot, Northern blot, array, and solution hybridization

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Southern blot

Uses probe with known sequence to identify DNA sequence

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Northern blot

Identifies RNA sequences

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Array methods

Examines multiple genes simultaneously using specific probes on glass slides

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Solution hybridization

Probe and single-stranded target in solution form RNA:DNA hybrids

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FISH

molecular technique that uses fluorescent probes to detect specific DNA sequences or gene changes on chromosomes

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Polymerase chain reaction

A laboratory technique to rapidly amplify millions of copies of specific DNA segments

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PCR

In vitro replication process with oligonucleotide primers and synthetic nucleic acids

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Denaturation

First step of PCR cycle: DNA separation at 94-96°C

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Annealing

Second step of PCR cycle: Primer binding at 50-70°C

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Extension

Third step of PCR cycle: DNA synthesis at 68-72°C

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Transcription Based Amplification

Process focusing on making copies of RNA at a single temperature

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Probe Amplification

Extending primers into multiple copies of probes with known target sequence

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Signal Amplification

Binds large signal amounts to target sequences, detecting multiple genotypes