Immunodiagnosis

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antiglobulin

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antibodies that react with immunoglobulins of different species.

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Immunodiagnosis is used in

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detection of antigens of infectious agents
detection of Ab formed in response to infect. agents
diagnosis of immunologic diseases
measurement of blood concentration of drugs or hormones

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Monoclonal antibodies are derived from

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a single B cell producing a single Ab recognizing a single epitope

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Titration and titers report

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the most dilute sample that gives a positive result

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Testing for Antibodies

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Test provides Ag, sample provides Ab
determine if patient exposed to pathogen and if patient vaccinated for pathogen.

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Test for Antigen

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Test provides Ab
Patient specimen is source of antigen
used to detect antigens of infectious agents and levels of hormones and drugs

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Ag:Ab binding produces these effects

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Agglutination
Hemagglutination Inhibition
Precipitation
complement activation
virus/toxin neutralization

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Ab or Ag is labeled

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Fluroescent antibody
enzyme-linked antibodies
Gold-labeled antibodies

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Enzyme-linked antibody

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western blot (immunoblot)
immunohistochemistry

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Agglutination

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IgG molecule binds multiple (2) Ag and clumps can be seen grossly. Normally detects Ab. Latex particles used for visualization

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Hemagglutination Inhibition
positive test

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sample antibodies bind virus (provided). When RBC's added, virus can't bind RBC's, so no agglutination

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Hemmagglutination inhibition negative test

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No Ab present so virus binds RBC;s and agglutination occurs

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A positive hemmaglutination test looks like

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RBC's in suspension.

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Agar Gel immunodiffusion has a positive test of

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precipitation line where Ab diffusing from sample well meet diffusion of Ag at ideal concentration.

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AGID is used to test for

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Coggin's in horses.

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Complement fixation

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Ag + patient sample, wait, + complement + sheep RBC's + Ab to sheep RBC's. If complement is available to lyse RBC's, patient does not have Ab to Ag.

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Complement fixation positive test

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Complement bound to sample Ab so RBC's not lysed and in bottom of well

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Complement fixation negative test

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No Ab/Ag complex for complement to bind in initial test, so binds Ab to sheep RBC's and eventually lyzes RBC's

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Serum Neutralization

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Virus + patient sample, wait, + cultured cells, and see if virus can infect cultured cells. virus infected cells change color. reported as titer

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Serum Neutralization positive test

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Ab bind virus and it cannot infect cell. (neutralization)

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Serum Neutralization negative test

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Ab not present to bind virus, so virus infects cultured cells and plaques form. (no neutralization)

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Direct Fluorescent Antibody Test (for Ag)

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Slide has sample with or without Ag, fluorescent tagged Ab added, look to see where Ab ends up . (Rabies)

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Direct Fluorescent Ab test positive

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Fluorescent tagged Ab color shows up on slide and appears as fluorescent spot

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Direct Fluorescent Ag test negative

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Fluorescent tagged Ab to Ag (on slide) washed away so no fluroescences shown.

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Indirect Fluorescent Ab test

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Ag fixed to slide, sample added, Fluorescent Ab to sample Ab added, wash.

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Indirect Fluorescent Ab test positive

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Patient Ab binds fixed Ag on slide, Fluorescent Ab bound to patient Ab, fluorescent area glows

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Indirect fluorescent Ab test negative

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Patient Ab not available to bind fixed Ag. Fluorescent Ab washes away from slide

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ELISA detecting Ags

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Ab for Ag fixed in well, patient sample added, Enzyme linked Ab added, Enzyme substrated added to see if Enzyme present. (Ab sandwhich)

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ELISA detecting Ags positive

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Patient sample has Ag bound to Ab in well, Enzyme linked Ab binds Ag, Enzyme catalyzes substrate reaction = color change

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ELISA detecting Ag negative

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patient sample lacks Ag, Enzyme linked Ab fails to bind and substrate stays the same color

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ELISA detecting Ab

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Ag fixed to slide, sample added, Enzyme linked Ab added, Substrate for enzyme added

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ELISA detecting Ab positive

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patient sample has Ab to bind Ag on slide, Enzyme binds Ab, Enzyme catalyzes substrate, sample changes color

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ELISA detecting Ab negative

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Ab from patient not available. Enzyme linked antibody washed away, no change in color.

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monoclonal antibodies

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Antibodies from single clone of B lymphocytes.

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clone is a

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collection of cells, all derived from a single cell

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Monoclonal Ab are are made by

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fusing B cells from an immunized mouse with cultured myeloma cells that can grow in the lab

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Monoclonal Ab are useful because

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they recognize a single epitope
They can have greater consistency than immunizing animal
spurious reactions due to other antibodies don't occur

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Hemagglutination inhibition test

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Patient sample + virus, wait, + RBC's
clinically used to detect Ab

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Immuno-precipitation

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Ag is in solution
Ab crosslinks soluble Ag forming complexes that precipitate

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Precipitation of Immuno-precipitation occurs when

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Ags and Abs are at equivalent molar concentrations

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AGID

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Agar Gel Immunodiffusion Test

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AGID has Ags, and Abs

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placed in wells cut in agar,
they diffuse into agar,
precipitate when meet and react.

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Serum Neutralization Test for viruses

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Virus is grown in tissue culture cells, growth detected by changes in, death of cells

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Serum Neutralization for toxins

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Toxin added to cells that are affected by the toxin. e.g. RBC's lysed by a hemolysin

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Direct fluorescent antibody test

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test for the presence of an Ag on slide using a labeled Ab to the Ag

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Indirect fluorescent antibody test

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test for the presence of Abs to an Ag on a slide using a labeled antiglobulin reagent.

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serology

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The study of serum. Applied to the detection of antibodies or antigens using immunodiagnostic test.

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Test for antigen are commonly used to

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detect antigens of infectious agents and to measure levels of hormones and drugs

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Test for antibodies are commonly used to determine

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whether an animal has been exposed to a pahtogen and sometimes whether the animal has been vaccinated for a pathogen.

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Antibody detection indicates

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exposure to the agent at some time prior to the test.

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Monoclonal antibodies are useful because they can be prepared

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with greater consistency than antibodies produces by immunizing animals

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Western blot

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Ags from infectious agent separated by electrophoresis, transfered to paper, add serum, add Enzyme linked Ab to serum Ab

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Western blot used to confirm

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FIV

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Immunohistochemistry

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unknown sample fixed to slide, enzyme linked Ab added, look for color change

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Immunohistochemisty positive test

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Enzyme linked Ab binds unknown tissue sample fixed on slide and Enzyme changes color with substrate

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Immunohistochemistry example used to detect

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CD 79 and CD 3 cells

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Immunochromatography

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Gold-labeled Ab, and unlabeled Ab added to filter paper. Sample added and Gold lab Ab washed to site of reg Ab where they bind Ab with GL Ab and are visualized.

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Bovine tuberculosis testing is an example of

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Intradermal test for activated T cells (delayed hypersensitivity)

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Tests that report results as titers

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Hemmoagglutanin
Complement fixation
Serum neutralization

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Sources of immunodiagnostic testing error

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specimen appropriateness
specimen quality
testing error