Use LEFT and RIGHT arrow keys to navigate between flashcards;
Use UP and DOWN arrow keys to flip the card;
H to show hint;
A reads text to speech;
68 Cards in this Set
- Front
- Back
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.
|
|
Define
-Prozone -Postzone |
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 -confirmation |
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?
(IEP) |
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?
|
Elisa
|
|
what essential antigen must be pos to confirm HIV?
|
p24
|
|
Done with PRECIPITATION, on to AGGLUTINATION!
|
OK
|
|
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.
|
ok
|
|
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
2. ELISA |
|
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?
|
Elisa
|
|
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
|
ok
|
|
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.
|
ok
|
|
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 |