Immunoassays

Question 1

What are the two ways to detect Ag-Ab binding in immunoassays?

Answer 1

1. Marker tagged onto antibodies - Enzyme immunoassays or Radioimmunoassay
2. Agglutination/hemagglutination

Question 2

Describe competitive ELISA (enzyme-linked immunosorbent assay)

Answer 2

1. Antibodies immobilized on solid surface
2. Incubated with known amount of enzyme-linked antigens (inhibitor antigens) + patient sample containing antigens
3. Competitive binding b/w enzyme-linked and unlinked Ag with Ab occurs
4. Wash away unbound antigens using buffer solution (phosphate buffered saline PBS)
5. Add enzyme substrate and determine enzymatic activity by measuring absorbance of colour pdt using UV spectrometry (lighter colour indicates less enzymatic activity, which means greater amount of antigen present in pt sample)

Question 3

What are the similarities and differences between direct and indirect ELISA?

Answer 3

Similarities:
Both involve patient Ag immobilized on solid surface
Enzymatic activity is proportional to the amount of antigen present in sample

Differences:
In direct ELISA, enzyme-linked Ab is added
In indirect ELISA, primary Ab is added, followed by secondary enzyme-linked Ab
Indirect ELISA more accurate as it involves specificity of 2 Abs (primary Ab specific for Ag, and secondary Ab specific for Fc domain of primary Ab)

Question 4

Describe sandwich ELISA, and explain why is it most specific

Answer 4

1. Primary capture Ab immobilized on solid surface
2. Patient sample Ag added
3. Secondary Ab specific to another epitope of the Ag is added
4. Enzyme-linked Ab specific to Fc domain of secondary Ab is added

Sandwich ELISA is most specific because it involves 2 Ab (primary and secondary) that are specific to 2 diff epitopes of the antigen

Question 5

What is unique about competitive ELISA?

Answer 5

Enzyme-linked antigens (aka inhibitor antigens) are added along with pt sample antigens

Greater amount of Ag is indicated by less enzymatic activity (less absorbance)
*whereas for other ELISA, enzymatic activity is proportional to amt of Ag present

Question 6

What contributes to the specificity and sensitivity of antibodies in ELISA?

Answer 6

Specificity: monoclonal or polyclonal Ab

Sensitivity: qty of Ab required to detect a sizable amount of Ag

Question 7

What are 3 possible causes of false positive results with ELISA?

Answer 7

1. Polyclonal Abs may cross react with multiple epitopes (therefore, recognize other Ags), Secondary Abs may display cross reactivity as well
2. Inadequate blocking (blocking agent bovine serum albumin BSA used as a silent binder to bind to extra capture Ab binding sites, prevent impurities from binding to those sites, get rid of background signals)
3. Inadequate washing (e.g., did not wash away unbound enzyme-linked Ab, resulting in additional enzymatic activity being detected)

Question 8

What is a possible reason for false negative results with ELISA?

Answer 8

Protein (Ab or enzyme) denaturation

Question 9

Which blood type does not have antibodies in plasma?

Answer 9

AB blood type. AB blood cell has both A and B antigens, no plasma antibodies
AB is universal recipient

Meanwhile O blood type has does not express antigens, anti-A and anti-B ab present in plasma
O is universal donor

Question 10

What happens when A blood type is mixed with B antigens?

Answer 10

Hemagglutination is observed - clustering of red blood cells occur due to cross binding by anti-B antibodies with antigen B

Question 11

Viral particles can interact with RBCs through a viral surface glycoprotein called hemagglutinin. How is this observed?

Answer 11

Clumping of RBCs forming a lattice (RBCs lose high surface tension and spreads across well) instead of a little red dot that is normally observed with RBCs

Question 12

Agglutination can occur without RBC as long as antigen and/or antibody is ____

This can be achieved by ____

Answer 12

Particulate in nature (semi-solid/solid).

Can be achieved by conjugation to a solid particle (e.g., Au, latex particles)

Question 13

If both Ag and Ab are soluble, does agglutination occur?

Answer 13

No, no agglutination, no visible clumping and change in turbidity

Question 14

How can agglutination (visible change in turbidity) be detected?

Answer 14

UV spectrophotometry

Question 15

Inhibition of agglutination resembles competitive ELISA.

Inhibition of aggutination has agglutinator, competitive ELISA has inhibitor enzyme-linked antigens to compete with patient sample antigens

Explain the principle of inhibition of agglutination.

Answer 15

Pt soluble Ag competes with Ag-coated RBCs (agglutinator) for binding to Ab. Hence, inhibiting agglutination.

If greater amount of pt antigen present, then lower turbidity/low scattering (decreased absorbance) compared to positive control.

Positive control: Ag-coated RBCs + Ab

Question 16

What is passive hemagglutination?

Answer 16

Resembles direct ELISA - requires enzyme-linked Ab.

Passive hemagglutination requires particulate Ab to bind with pt soluble Ag. Can be quantified by cloudy appearance, use UV absorbance.

Question 17

Immunoassay for HbA1c monitoring using latex immunoagglutination inhibition method. Describe the agglutinator and antibody involved.

Answer 17

Agglutinator: synthetic polymer containing copies of immunoreactive portion of HbA1c

Antibody: latex coated with anti-HbA1c mouse monoclonal antibody

If no HbA1c in sample, then high scattering and increased absorbance
If HbA1c in pt blood sample, then inhibition of agglutination and low scattering, decreased absorbance

Question 18

Blood glucose monitoring uses enzyme technology instead of immunoassay. Why is this so?

Answer 18

Glucose is not a protein (unlike glycated Hb), hence it is not antigenic in nature and the immune system is unable to recognize it as an antigen and develop antibodies against it.