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What is ELISA used to detect?
presence of Ab in pt's sera
What are the steps for ELISA?
- 1. Ag coated onto surface of plastic well
- 2. pt's serum (w/ Ab) added to well; binding of Ab/Ag and excess washed away
- 3. enz-labeled anti-Ab added to well to bind to pt's Ab; excess washed away
- 4. colorless subst added to well; reacts w/ enz still there to produce a color
What does the Western Blot begin w/?
electrophoresis: separates mix of prot based on diff rates of migration in electric field (SDS-PAGE
What happens after electrophoresis w/ Western Blot?
pt antiserum incubated w/ blot, any Ab present that recog electrophoresed prot on blot bind; bound Ab can be detected w/ labeled anti-Ab to allow color rxns
What is the basic principle of Western Blot?
sep a standardized prepartion of Ag derived from a particular pathogen and demonstrate "pattern" of Ab reactivity that provides a fingerprint unique to that pathogen
What are the adv of Western Blot?
simultaneously demonstrate reactivities of mult Ab in polyclonal pt antiserum against mult Ag assoc w/ particular path
Direct Fluorescenct Ab test
primary Ab (of known Ag specificity and labeled w/ fluorescein) reacts w/ specific Ag in test specimen
Indirect Fluorescent Ab Test
presence of an unlabeled "primary" Ab bound to Ag in a clinical specimen is detected by fluorescein-labeled "secondary" Ab; secondary finds and binds to primary
What is the adv of IFA?
increased sensitivity of detection due to amplification effect as mult secondary Ab can bind to each single molecule of primary Ab
dep on bivalent characteristics of Ab that allows it to crosslink mol of Ag to each other; sol Ab bind to sol Ag molecules and lead to precip of lg insoluble Ag:Ab complexes...agglutination
RPR card test
agglutination test to detect reagin-type reactivity; uses carbon particles that are coated w/ cardiolipin Ag that reacts w/ reagin
Ab that react w/ lipid Ag assoc w/ Treponema
determines serum Ab titers in diagnosis of infectious diseases; demonstrates agglutination in tubes and prozone effect
conc of Ab is too high in relation to Ag (Ab interferes w/ its ability to crosslink); serum that is agglutination neg bc of prozone becomes pos when diluted
complement fixation test
Ag:Ab rxn can lead to fixation of complement which triggers complement cascade and leads to cell lysis
What is a positive CF test?
complement binds to Ag:Ab complex; no lysis of RBC bc no free complement in serum bc all bound to Ag:Ab complex
What is a negative CF test?
no Ab present so no Ag:Ab complexes; free complement binds to RBC antibody and MAC forms
Neutralization (Virus Plaque Inhibition) Assay
monolayers on culture plates exposed to pt specimens that may have virus; if virus in pt serum then it will infect cells in monolayer and make them lyse (CPE)...plaques form
viruses cannot infect monolayer bc of presence of virus-specfic Ab; no plaques; neutralizing Ab indicative of an Ab response against particular virus
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