Immunoassays

Question 1

Def of immunoassay

Answer 1

sensitive analytical test that uses highly specific antibody- antigen complexes to produce signal that can be measured and related to concentration (semi-qauntitative)

Question 2

Avidity

Answer 2

Combined strength of multiple sites on Ag, IgM has more avidity than IgG

Question 3

Cross reactivity

Answer 3

Aby can bind with low affinity to closely related epitopes

Question 4

Precipitin line

Answer 4

Line of Aby-Ag complexes that precipitate and form a visible line

Question 5

Aby concentration/ complex curve

Answer 5

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

Question 6

Immunodiffusion

Answer 6

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.

Question 7

Agglutination

Answer 7

Uses latex coated Aby to make a visible clumped complex when Ag binds (uses IgM)

Question 8

Competitive assay

Answer 8

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)

Question 9

Non-competitive assay

Answer 9

Antigen binds to immobilized Aby and is able to produce signal, from secondary labeled Aby or otherwiseH

Question 10

Homogeneous assay

Answer 10

Only uses one type of Aby and does not require rising

Question 11

Heterogeneous assay

Answer 11

Uses many types of Aby and requires rinsing between steps to separate immune-complex from free/unbound ones

Question 12

Hapten

Answer 12

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)

Question 13

Radiolabel RIA

Answer 13

very sensitive but dangerous and hard to work with
Can be competative via displacement
or non using and radio labeled secondary antibody

Question 14

Enzyme EIA

Answer 14

ELISA enzyme linked immunosorbent assay is the most common

Question 15

Direct ELISA

Answer 15

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

Question 16

Indirect ELISA

Answer 16

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”

Question 17

Test tube or multi well plates

Answer 17

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)

Question 18

Lateral flow type

Answer 18

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