Lecture 15 ELISA and IMMUNOASSAYS

Question 1

What is an immunoassay?

Answer 1

This is a sensitive analytical test utilizing the highly specific binding between Aby and Ag to produce a signal that can be qualified/semi-quantified.
More useful to indicate the presence or absence of an analyte rather than the amount.

Question 2

What does affinity mean in terms of Ab-Ag interaction?

Answer 2

The strength of the interaction between the epitope and paratope which can be influenced by weak non-covalent bonding forced:
ionic, H, van der waals, hydrophobic bonds

Question 3

What does avidity mean in terms of Ab-Ag interaction?

Answer 3

This is the combined strength or binding intensity of multiple binding sites for Ag.
Pentameric IgM has 10 Ag binding sites which result in a high avidity whereas monomeric IgD only has two Ag-binding sites and thus has less avidity.

Question 4

What does specificity mean in terms of Ab-Ag interaction?

Answer 4

There is unique recognition of an Ag by an Aby. Aby will only bind with ag with very specific antigenic determinants. There can be cross reactivity between closely related epitopes in low affinity aby.

Question 5

What does sensitivity mean in terms of Ab-Ag interaction?

Answer 5

The detection limit of an Aby for an Ag.

Question 6

What is immune precipitation and how can it be observed?

Answer 6

Precipitates formed by cross-linking of Aby-Ag usually at equimolar ratios.
Ag and Aby are placed in wells. Diffusion creates a concentration gradient through the gel between Aby and Ag.
Precipitin formation observed as white bands in agarose gel at a certain distance between the wells.

Question 7

What are some detection methods for Aby-Ag interaction?

Answer 7

Agglutination (aby cross links Ag on target cell / particle)
IgM: highly effective at cross linking and agglutination of Ag.
Agglutination can be measured by changed in scattered light due to change in particle size.

Question 8

What are the different assay format types?

Answer 8

Competitive vs Non-competitive.
Homogenous vs heterogeneous.
Direct vs Indirect.

Question 9

What is a competitive assay?

Answer 9

Unlabelled analyte (ag of interest) in test sample is measured based on its ability to compete with labeled Ag in the immunoassay. 
The greater the amount of Ag in the test sample, the laser the signal (inverse correlation) as more of the labled ag is washed out.

Question 10

What is a non-competitive assay?

Answer 10

Sandwich assay.
Ag usually sandwiched between aby specific to two different epitopes.
Signal is directly proportional to the amount of target Ag present in the sample.

Question 11

What is a heterogenous assay?

Answer 11

This requires separation of the immune-complex from the free-unbound form.
Separation is accomplished by immobilization of Ab on a matrix so that unbound ag can be washed away.

Question 12

What is a hapten?

Answer 12

A small molecule that can elicit an immune response only when attached to a larger carrier such as a protein like albumin.

Question 13

What is a homogenous immunoassay?

Answer 13

This does not require separation of the immunocomplexes from the reaction mix like in point-of-care testing.

Question 14

What are the types of labels that can be used in immunoassays?

Answer 14

Radiolabel
Enzyme-label
Hemagglutination inhibition

Question 15

What is a radioimmunoassay (RIA?)

Answer 15

This is a competitive or non-competitive protein binding assay.
Heterogenous.
The competitive version involves competition of un-labled Ag for radio-labeled ag.
The non-competitive format is like a sandwich.

Question 16

What is an enzyme immunoassay?

Answer 16

Bound Aby conjugated to enzyme that converts a substrate of yield a measurable read out.
ELISA
Usually heterogenous involving washing out.

Question 17

What is Direct ELISA and its advantages / disadvantages?

Answer 17

Ag coated onto surface.
Enzyme-conjugated Ab recognizes Ag.
This is quicker as there are fewer steps.
Cross reactivity of secondary Ab is eliminated.
conjugation may Compromise immunoreactivity.
Expensive to label primary antibodies, labeled compounds cannot be used for other experiments as with 2ndary labled aby.
Signal amplification less.

Question 18

What is indirect ELISA and its advantages / disadvantages?

Answer 18

surface-ag-primary-secondary enzyme
surface-capture aby - Ag - primary - secondary enzyme
Enzyme is used to obtain a readout
immunoreactivity of primary aby not compromised due to conjugation
signal amplification can occur (increased sensitivity)

Non specific signal may result from cross-reactivity
Additional blocking step using albumin to prevent non-specific adsorption of labelled secondary aby needed.

Question 19

What is a test tube type immunoassay?

Answer 19

Capture abys coated onto test tube.
Sample containing ag added and allowed to bind.
non-bound stuff washed out.
Signal ab added, incubated with assay solution, and monitored (e.g. colour intensity)
Time-sensitive colorimetric quantitative measure.

Question 20

What is a coated dip-stick type of assay?

Answer 20

Capture abys coated on surface of plastic at one end.
Stick dipped into sample.
Stick dipped into solution of enzyme-Aby conjugate.
Rinsing done to remove unbound signalling aby
Stick dipped into solution for enzymatic assay
Appearance of colour indicates positive result.

Question 21

What is a lateral flow type assay?

Answer 21

Sample migrates from dipped end. It encounters soluble signalling enzyme as it migrates then immobilized second anti-antigen antibody which immobilized antigens bound to the first signal enzyme. Unbound first anti-antigen antibodies are immobilized last as a control by an anti-ig antibody.

Question 22

Where to OTC Pregnancy Kits fit into this section on assays?

Answer 22

Based on ELISA where hCG is the Ag. (beta subunit)

hCG produced by placenta during pregnancy in urine 4-5 days post conception/implantation.

Question 23

What are immunoswabs?

Answer 23

Useful for SARS. ID presence of antigen in body fluid by colorimetric assay.