Serologic reactions Flashcards by PATRICIA ONG

Contaminated equipment or
reagents may cause particles
to clump.

False positive

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Autoagglutination: causes what reaction

False-positive

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Delay in reading slide reactions results in drying out of mixture.

false positive

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Overcentrifugation causes
cells or particles to clump too
tightly.

False positive

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Type of reaction:
Inadequate washing of red blood cells in antihuman globulin (AHG) testing* may result in unbound immunoglobulins neutralizing the reagent.

False negative reaction

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Failure to add AHG reagent

False negative

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Type of reaction: Contaminated or expired reagents

False negative

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Type of reaction: Improper incubation

False negative

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Type of reaction:Delay in reading slide reactions

false negative

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Undercentrifugation

false negative

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Type of reaction: Prozone phenomenon

false negative

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Particulate antigens such as red cell aggregate to form large complexes when a specific antibody is present

Agglutination

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Combining soluble antigen with soluble antibody to produce insoluble complexes that are visible

Precipitation

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Overall strength of antigen-antibody binding and is the sum of the affinities of all the individual antigen-antibody combining gsite

Avidity

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Initial force of attraction that exists between single Fab site of antibody molecule and single epitope or determinant on
corresponding antigen

Affinity

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Antibodies capable of reacting with antigens resembling the original phenomenon
Resembles the original antigen, stronger the bond will be between the antigen and binding site
Describes detection of a substrate other than analyte of interest
It is typically observed as a false-positive results.

Cross reactivity

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Zone of antibody excess
Abs conc greater than the conc of antigen

Prozone

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High abs concentration in an unknown sample such as patient serum causes ____ reaction

false negative

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Number of multivalent sites of antigen and antibody are equal

Zone of equivalence

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Precipitation is the result of random, reversible reactions, forming a stable network or _______

Lattice

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Zone of antigen excess

post zone

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Type of agglutination

Widely used for bacterial agents:

Widal Test: S. typhii for detection of typhoid fever

Agglutination indicates the presence of patient antibody to a natural antigen

Direct Agglutination

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Reaction seen in ABO blood group typing of RBCs

Hemagglutination

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Microhemaagglutination assay for Treponema Pallidum

MHA-TP

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This test uses fluorescein isothiocyanate requires a fluorescent labeled-immunoglobulin to detect antigens or antibodies according to test systems Herpes Virus IgM Dengue Virus Rabies Virus Scrub and Murine Typhus

Immunofluorescence

cells in suspension are labeled with fluorescent tag

Flow Cytometry

radioactively labelled-antibody (or antigen) competes with the patient's unlabeled antibody (or antigen) for binding sites on a known amount of antigen (or antibody) HBsAg, Thyroid Function Test

Radio-Immunoassays

use of enzyme-labeled immunoglobulinto detect antigens or antibodies signals are developed by the action of hydrolyzing enzyme on chromogenic substrate optical density measured by micro-plate reader

Enzyme Linked Immuno-Sorbent Assay

Antigen or antibody are labeled with enzyme and allowed to compete with unlabeled ones (in patient serum) for binding to the same target

Competitive ELISA

Test that must remove excess/unbound Ag or Ab before every step of reactions

Non-competitive ELISA

Process for the quantitative and qualitative determination of antigens, antibodies and their complexes

Chemiluminescent Immunoenzymatic Assay

involves combining soluble antigen with soluble antibody to produce insoluble complexes that are visible

Precipitation

Soluble Antigen + Antibody (in proper proportions) → Visible Precipitate

Precipitation

(antigen binds with Fab sites of 2 antibodies)

Lattice Formation

antibody is incorporated into the agar gel as it is poured and different dilutions of the antigen are placed in holes punched into the agar ring of precipitation is formed

Radial Immunodiffusion (Mancini)

used more often Ag and Abs allowed to diffuse towards each other in an agar medium thin line of precipitate

Double Immunodiffusion by Ouchterlony

Diffusion Patterns: serologic identity / presence of common epitope

Fusion of Lines at their junction to form an Arc S

Diffusion Patterns: demonstrates 2 separate reactions compared antigens shared no common epitopes

Crossed lines

Diffusion Patterns: partial identity

Fusion of 2 lines with Spur

precipitate at junction layering of antigen solution over a column of antiserum in a narrow tube

Ring test

grouping of streptococci by lancefield test

Ascoli's Thermoprecipitin Test

Used only when Ag and Ab have opposite charges

Counter Immunoelectrophoresis

Pairs of wells are punched in agarose plates in which Ag is placed in one well of each pair and Ab in the other

Counter Immunoelectrophoresis

Reactions occur between migrating Ags and Abs

Counter Immunoelectrophoresis

precipitin lines will be visible between the wells of a pari of wells of matching specificity

Counter Immunoelectrophoresis

Antigen is electrophoresed into gel containing antibody Also known as rocket shape

Rocket Immunoelectrophoresis

The total distance of Ag migration and precipitation is directly proportional to Ag concentration

Rocket Immunoelectrophoresis

Polar antigen (miscible in water/hydrophilic)

SOLUBLE ANTIGEN

– Nonpolar antigen (immiscible in water/hydrophobic)

PARTICULATE ANTIGEN

Insoluble in nature  Ex. Bacterial antigen, RBC membrane antigen (ABO)

Neutral particulate

Soluble in nature, but a carrier is added to make it insoluble (Promotes insolubilization of soluble antigen)

Artificial particulate

Reaction of Ouidin test; SINGLE DIFFUSION, SINGLE DIMENSION

(+) Precipitin line/band forms

SINGLE DIFFUSION, DOUBLE DIMENSION; Radial Immunodiffusion reaction

(+) Precipitin zone - Diameter of the pptn line is directly proportional to the concentration of the target antigen

Kinetic diffusion

Fahey method

End point diffusion

Mancini method

Longer incubation & cold/room temp

IgM

Shorter incubation & warm temp

IgG

agar with antibody is added into the test tube to solidify

Double diffusion, single dimension: Oakley Fulthorpe

bottom layer of oakley

Agar with antibody

top layer of oakley

serum of patient with soluble antigen

Ouchterlony technique: Ag in the sample is similar with known Ag

Smooth curve

Ouchterlony technique: Ag in sample is partially similar with known Ag

Spur formation

Ouchterlony technique: Ag in the sample different with known Ag

Intersects Double spur formation

Anti-human IgH (AHG) is added to cross bridge IgG incomplete antibodies to demonstrate Lattice formation

Indirect Agglutination

soluble Ag (reagent) is artificially attached to a particulate carrier to detect Ab

PASSIVE AGGLUTINATION

Homologous Ag inhibits agglutination of Ag coated particle

Agglutination inhibition

(+) for agglutination inhibition

No agglutination/inhibition of agglutination

Agglutination of rbc due to ab, viruses, bacteria, or other biological substances

Hemagglutination

not the antigen of RBC but the artificially attached Ag (after undergoing tx) that are made to react with the Ab

Hemagglutination

Bacteria (Bacteria cell) serves as the carrier for Ab to detect Ag

Coagglutination

TPCF (Treponema Pallidum Complement-Fixation) positive reaction

no hemolysis on indicatior

A type of label that emits radioactivity  Radioactivity is measured using a Gamma counter / Scintillating counter  Ex.: Carbon 14, Radioactive iodine

Istotopic labels

Do not emit radioactivity

Non-isotopic labels

Any substance that will complex to another substance; substance to be measured

ligand

A substance that is added to a reagent to facilitate the visualization/demonstration of a reaction and measurement of a reaction

label

The greater the fluorescent light emission, the higher the concentration of the target antigen or antibody being detected in the serum.

Fluorescence

FLUORESCENT PROBES USED: Emits green fluorescent light

FITC (Fluorescein Isothiocyanate)

FLUORESCENT PROBES USED: emits red fluorescent

Phycocyanin Texas Red

Fluorescent probe: red-orange fluorescent light

Tetramethyl rhodamine

antigen is the target in the serum, while the antibody is labeled/reagent targets antigen

Direct/Single Layer Immunofluorescent Assay

antigen is one of the reagents, while the antibody is the target in the serum, and AHG is the reagent with label

Indirect/Double Layer Immunofluorescent Assay

Radioactive isotopes

131 – Iodine 131 I125 – Iodine 125 H3 – Hydrogen 3 C14 – Carbon 14

The labeled reactant in the reagent competes with the unlabeled reactant in the sample/serum to the limited binding site of the antibody in the reagent

Competitive

Used to measure antigen-specific IgE (type 1 hypersensitivity)

RAST Radioallergosorbent test

Used to measure the total serum IgE (type 1 hypersensitivity)

RIST (Radioimmunosorbent test)

ENZYME IMMUNOASSAY end reaction

Colorimetric reaction – Chromogenic substrate is used

most commonly used enzyme reagent prepared from calf intestines

ALP

Commonly used enzyme from horseradisnh

Horseradish peroxidase

Enzymes used for immunoassays

Glucose oxidase B-galactosidase ALP Horseradish Peroxidase

The antigen is the target in the serum, while the antibody is labeled with enzyme

Direct

Antigen is part of the reagent, antibody is the target in the serum, AHG is labeled with enzyme

Indirect

Two antibodies are used. One antibody is attached to a solid phase, while the other antibody is used as the label (AHG) to capture a specific antigen

Sandwich/Double Ab/ Antigen capture technique

The labeled reactant in the reagent competes with the unlabeled reactant in the sample/serum to the limited binding site of the antibody in the reagent

Competitive binding

Enzyme Inhibition: reverse colorimetric reaction

No color reaction

True or False: Lighter colors indicate higher levels of antibody/antigen in ELISA plate

False: Darker color

Uses colloidal particles in a liquid consisting of metal or insoluble metal compound as label

Sol Particle immunoassay

Best because this is the only metallic/colloidal particle that produces magenta colored product when reacted in aqueous solution

Gold

Substitute for Gold for Sol Particle Immunoassay

Silver

Colloidal Particles used

Gold Silver Silver Iodide Barium Sulfate

Examples of Chemiluminiscent Immunoassay

Luminol, Acridinium phosphate ester