Glossary of 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/
- 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.
- What is immunoelectrophoresis?
- a 2-step precipitation reaction.
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?
- Done with PRECIPITATION, on to AGGLUTINATION!
- 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
- 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
- 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:
-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
- 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
- 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
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