Immunoassays Flashcards
Def of immunoassay
sensitive analytical test that uses highly specific antibody- antigen complexes to produce signal that can be measured and related to concentration (semi-qauntitative)
Avidity
Combined strength of multiple sites on Ag, IgM has more avidity than IgG
Cross reactivity
Aby can bind with low affinity to closely related epitopes
Precipitin line
Line of Aby-Ag complexes that precipitate and form a visible line
Aby concentration/ complex curve
Forms a normal distribution with a middle amount of Aby being the most effective as all of the Ag doesn’t get bound right away
Immunodiffusion
Seen in plates where a Ag and Aby are diffusing toward each other, at the right concentration a precipitin line will form. Heavy Aby IgM form the line closer to the Aby well.
Agglutination
Uses latex coated Aby to make a visible clumped complex when Ag binds (uses IgM)
Competitive assay
Sample analyte and taged analyte are added to Aby solution, if sample analyze is present it will displace some labeled analyte and lower the signal. (inversely correlated)
Non-competitive assay
Antigen binds to immobilized Aby and is able to produce signal, from secondary labeled Aby or otherwiseH
Homogeneous assay
Only uses one type of Aby and does not require rising
Heterogeneous assay
Uses many types of Aby and requires rinsing between steps to separate immune-complex from free/unbound ones
Hapten
small molecule can’t trigger immune response alone but when attached to large carrier such as albumin it can. (used to make Aby in animals)
Radiolabel RIA
very sensitive but dangerous and hard to work with
Can be competative via displacement
or non using and radio labeled secondary antibody
Enzyme EIA
ELISA enzyme linked immunosorbent assay is the most common
Direct ELISA
Ag is bound to surface and tagged Aby is place over top
Pros: very few steps and cross reactivity of 2 Aby is eliminated
Cons: Labeling by chemical processes denatures Aby, labeling is time consuming, less signal amplification
Indirect ELISA
Uses primary and secondary labeled Aby to show signal, can be with Ag added to surface or caught on capture Aby in “sandwich”
Pros: primary Aby reactivity not lessened by labeling, signal amplification
Cons: Cross reactivity of secondary Aby, it can also stick to surface of test and provide false signal if not “blocked”
Test tube or multi well plates
Uses heterogenous enzyme linked Aby to bind to Ag, if present when colouring solution is added enzyme will make it coloured
Pros: large number and allows for controls, semi-quantitiative from spectrophotometer
Cons: many rinsing steps, low shelf life (needs 4 degrees)
Lateral flow type
Have labeled Aby that are picked up, flow towards test strip which had Aby that bind Ag and Control Strip which has Aby that binds the labeled Aby.
Cons:false positives form dead organisms, low variability for mutations in Ag, human error
