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0. Immunoserologic test principles


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What is serum inactivation?
How is it accomplished?
How long does it last?
Heat to 56'C for 30 min.
Lasts for 4 hours.
What are 2 reasons for serum inactivation?
1. Destroy pt complement for tests where known complement is added in.
2. To prevent hemolysis when looking for agglutination.
What is the calculation for an original dilution?
Vol of sample
Vol sample + Vol diluent
What is the calculation for subsequent dilutions?
Orig. diln x Vol transferred/
Total Vol
What is a titer?
The highest dilution giving a positive agglutination result.
How do you report a titer?
Look for the well/tube with highest dilution that is positive.
If all tubes/wells are negative, how do you report the titer?
"Less than" whatever the lowest dilution was.
If all tubes/wells are positive, how do you report the titer?
"Greater than or equal to" the highest positive dilution.
Prozone = excess antibody
Postzone = excess antigen.
What is the solution for prozone?
Dilute the patient sample more.
What is sensitivity?
True pos / All pos
What is specificity?
True Neg / All neg
When is a test good for
Screening = high sensitivity
Confirmation = high specfcty
What is paired sera testing?
Testing two sera samples, one from acute phase and one from convalescent phase of infection, to find evidence of disease.
When evaluating paired sera testing, what is evidence of diseease?
A 4 fold increase of Ab titer from acute - convalesc. stage.
How many weeks elapses between acute and convalesc. illness?
Approx. 3
What is precipitation?
Clumping between antibody and soluble antigen.
What is radial immunodiffusion?
Antibody within agar gel; antigen is introduced from wells, and precipitation is evaluated.
what are advantages of RID?
-no special equipment
-sensitive, rapid, accurate
-quantitative version of DD
what is the limitation of RID?
-limited by the assay ranges of the plate.
What is the Ouchterlony technique?
the classic, immunodiffusion - also called double diffusion
how does ouchterlony technique work?
1 well contains known Ag/Ab; Other well contains Pt serum.
The wells diffuse toward ea other; at equal concentrations form a line of precipitation.
-Strictly qualitative
What is immunoelectrophoresis?
a 2-step precipitation reaction.
1. Electrophoresis
2. Double diffusion of antisera in well parallel to electroph. line
Product: precipitin arcs at Ag-Ab equivalence points
What is Countercurrent immuno-electrophoresis? (CIE)
basically just immunodiffusino (ouchterlony) with charge applied to speed it up and increase sensitivity. Ag/Ab are oppositely charged, so propelled toward ea. other.
What is rocket electrophoresis?
Basically gel contains antibody; pt serum w/ Ag is applied, the distance it "rockets" is proportional to concentration.
What is immunofixation electrophoresis?
a powerful enhancement of immunoelectrophoresis; series of post-electrophoretic gel slabs are layered with cellulose-acetate gels saturated with specific Abs. Resulting immune complexes fixed on the second gel may then be stained, allowing sensitive and specific qualitative visual identification of paraproteins by electrophoretic position.
What is the western blot, if anything?
the gold standard confirmatory test for HIV.
What is the screen for HIV?
what essential antigen must be pos to confirm HIV?
What is agglutination?
complexing between antibody and insoluble antigen.
What are the 2 steps involved in agglutination?
1. Sensitization
2. LAttice formation
Give 4 types of agglutination tests:
1. Direct Agglutination (DA)
2. Antiglobulin testing (Coombs)
3. Particle-enhanced Immunoassay
4. Agglutination Inhibition
What are 2 types of DA?
1. ABO typing
2. Hemagglutination
What are particles used in enhanced agglutination?
latex, RBCs, Bentonite, charcoal, microorganisms.
What is passive agglutination used for?
Detecting patient antibody with antigens complexed on enhancment particles.
Why is it called passive?
Because it's not direct complexing with the actual particle (eg RBC).
What is reverse passive agglut.?
Detecting patient antigen with Antibody adsorbed onto the enhancement particle.
What is aggltination inhibition?
Interference by Ag or Ab with an Ag-Ab reaction which would have resulted in agglutination had the interference not occurred.
Describe the principle of detecting antibody with HAI
1. Mix patient serum + virus
2. If Ab pos, will complex.
3. Add RBCs; if complex, no agglutination. If free virus, visible reaction.
Describe the principle of detecting antigen with HAI
1. Mix pt serum + Ab
2. If pos, complex.
3. Add RBCs sensitized w/ Ag; complex inhibits anymore reaction.
Done with precip/agglutin; on to ligand assays.
what is a ligand?
any substance that complexes to another molecule.
What are four types of ligand assays?
1. chemiluminescent
2. enzyme immunoassay EIA
3. immunofluorescence assay IFA
4. radioimmunoassay RIA
The 2 basic types of EIA are:
1. Homogenous
2. Heterogenous
What's the difference between homo and hetero EIA?
Homo: labeled/unlabeled reactants are not seperated
Hetero: they are
what is EMIT?
enzyme multiplied immunoassay technique
how does emit work?
1. Patient antigen competes with enzyme-labeled antigen for a known amt of antibody.
2. After reaction, only enzyme that is uncomplexed can be detected.
3. More pt Ag leaves more enzyme free, and a bigger signal
How is emit interpreted?
The higher Ag concentration in patient, the stronger enzyme signal.
What are 2 types of heterogenous EIA?
1. Competitive binding EIA
What is Competitive binding EIA?
Same as EMIT, but must seperate out reactants with:
-Chemical precipitation
-Ab attachd to solid phase
-Ab to first Ab
What is indirect EIA?
what are 2 synonyms for ELISA?
Indirect EIA
Solid Phase Immunoassay SPIA
State the 4 steps involved in ELISA for detection of Antibody:
1. Incubate Antigen to adsorb to well. Wash
2. Add Patient serum. wash
3. Add labeled anti-human Ab; wash.
4. Add substrate; detect.
What is ELISA used for detecting patient antigen called?
-Sandwich EIA
-Double antibody EIA
-Immunoenzymetric assay
How does sandwich EIA work?
1. Adsorb Antibody to well
2. Add pt sample w/ Ag
3. Add labeled Antibody specific for the antigen
4. Add substrate, view reaction
what is MAC Elisa?
Membrane based cassette elisa
how does macelisa work?
-Cassette has antigen in well;
-add patient serum; complexes diffuse through a nitrocellulose membrane.
-Anti-human Ab will stop complex at POS site
-Neg gets stopped by Ab at neg site.
What is the advantage of MACelisa?
-Increased speed
-Increased sensitivity
ONWARD HOE to immunoflourescent assays
what is the tag used in IFA?
Fluorescein isothiocyanate
What is DIF/DFA?
Immunoflour. directly for antigen; add tagged Ab specific for a particular organism.
What is IIF/IFA?
Indirect; detects antibody.
-Known antigen attached to slide
-Add pt. serum
-Add anti-human tagged Ab.
Finally, Radioimmunoassays.
what is the tag used in RIA?
radionucleotide tag, often 125I
How do 1) RIA and 2) SPRIA work?
Same as EIA and SPEIA only with radiographic signal instead of enzymatic reaction
What is the indicator system used in complement fixation?
Sheep RBCs - they are lysed if complement is not fixed, and not lysed if it is.
what will be visible result in:
-Positive C' fixation
-Negative C' fixation
Pos: no hemolysis
Neg: hemolysis

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