Lecture 8.5: Immunoassays and ELISA

Question 1

Which are more specific, monoclonal or polyclonal antibodies?

Answer 1

Monoclonal

Question 2

How is the Ag-Ab binding detected?

Answer 2

1. Using a marker (enzymes or radioactive label) that is incorporated into the antibodies
2. Using agglutination

Question 3

What are the basic steps involved in immunoassays ?

Answer 3

1. Use a specific antibody to capture an antigen or use a specific antigen to capture an antibody
   2 . Detect the presence of the captured analyte using a measurable label (e.g. radioactive probe).

Question 4

What is the full form of ELISA?

Answer 4

Enzyme-linked immunosorbent assay

Question 5

What are the two kinds of ELISA methods and what are they used for?

Answer 5

1. Sandwich ELISA – (example: ART for Covid-19) employs an antibody for determining the presence of a specific antigen in a sample.
2. Indirect ELISA (example: detection of HIV infection) – employs a specific HIV antigen to determine the presence of antibodies against the antigen in a sample.

Question 6

What is the general process of a Sandwich ELISA?

Answer 6

1. Add primary antibody to capture the antigen
2. Add the solution that may or may not contain the antigen
3. Add the 2nd primary antibody that binds to the antigen bound to the first primary antibody
4. Add enzyme labelled secondary antibody that will bind to the 2nd primary antibody
5. Add the enzyme substrate and measure the colour change to determine the amount of antigen

Question 7

What is the general process of competitive ELISA?

Answer 7

1. Add primary antibody
2. Add sample containing antigen
3. Add enzyme labelled antigen that will compete with the antigen for the primary antibody
4. Add enzyme substrate
5. More signal = less antigen

Question 8

What are some of the challenges that can arise from ELISA?

Answer 8

1. Possibility of false positive results due to the use of of polyclonal antibodies, inadequate blocking, inadequate washing etc
2. Possibility of false negative results due to degraded materials

Question 9

What is the protein that is detected in a pregnancy test?

Answer 9

Human chorionic gonadotropin (hCG)

Question 10

How does the one step midstream hCG test work?

Answer 10

1. The urine is added to the test strip and moves up
2. The test strip contains an antibody bound to alkaline phosphatase which is responsible for the coloured lines
3. The first line contains the anti hCG antibody, the hCG antigen binds here if pregnant, and then the alkaline phosphatase antibody binds to the hCG creating the first pink line
4. The second line is a control that has the anti-mouse goat antibody, which binds to the alkaline phosphatase antibody, producing the second pink line
5. If the woman isnt pregnant then the first pink line won’t appear

Question 11

What is the principle behind using Agglutination/Hemagglutination as a detection method?

Answer 11

1. Ag-Ab binding: If both antigen and antibody is soluble, Ag-Ab binding is not visible.
2. However, if Agor Ab is present in a semi-solid or presented on the surface of a solid particle, Ag-Ab binding is visible as precipitation (cloudiness)
3. If either Ag or Ab is particulate (that also means semi-solid/solid) and the other is soluble,
   agglutination occurs.
4. If both Ag and Ab are particulate, agglutination occurs and the turbidity will be more intense than the turbidity produced from agglutination resulting from Ag-Ab binding between 1 soluble Ag/Ab and 1 particulate Ab/Ag.

Question 12

How does passive hemagglutination and hemagglutination inhibition work?

Answer 12

1. Passive hemagglutination
   - Erythrocytes are coated with a soluble antigen
   - The binding of an antibody to that antigen makes it insoluble
2. Hemagglutination inhibition
   - Erythrocytes are coated with a soluble antibody and there is free antibody present too
   - The soluble antibody inhibits agglutination
   of antigen-coated RBCs by the antibody.

Question 13

Explain how direct agglutination is used in the context of pregnancy tests.

Answer 13

1. Red blood cells or latex particles are coated with an antibody for hCG
2. If urine contains sufficient hCG, then agglutination occurs, and the positive test appears as aggregates (more turbidity) –> can measure the intensity

Question 14

Explain how inhibition of agglutination is used in the context of pregnancy tests.

Answer 14

1. Latex particles are coated with the hCG antibody and a standard amount of hCG agglutinator is added
2. Normally the agglutinator binds to the antibody –> agglutination
3. If the urine contains hCG, then the hCG competes with the agglutinator for the antibody, so there is less agglutination
4. The extent of agglutination is measured using absorbance (of light?)

Question 15

What is an agglutinator made on?

Answer 15

Particles coated with the antigen

Question 16

What method of immunoassay does Hb1Ac monitoring use?

Answer 16

Inhibition of agglutination

Question 17

What is the process of ELISA in the context of HIV infection?

Answer 17

1. The well contains HIV antigens bound to it
2. Human serum is added to the well
3. The HIV IgG antibodies bind to the antigen in the well and the rest of the serum is washed off
4. A rabbit anti IgG antibody conjugated with horseradish peroxidase is added to the well and if the HIV IgG is there then it’ll bind to the HIV IgG
5. The substrate for horseradish peroxidase (5-amonsalicylic acid and hydrogen peroxide) is added which is converted to a brown coloured substrate

Question 18

Why is ELISA so sensitive?

(in the context of HIV screening)

Answer 18

1. ELISA is sensitive because the HIV antigen-HIV antibody, and Anti-IgG(Rabbit)-HIV antibody affinity is very strong/specific and these components are likely to bind if they came into proximity,
   T2. he HRP-substrate reaction has high catalytic turnover rates, so even if the patient has very little HIV antibodies (likely in the early stage of HIV) and little Anti-IgG(Rabbit)-HRP binds and remains in the well, the little amount of HRP can still catalyse the substrate reaction substantially and colour change from colourless to brown can be detected.

Question 19

During HIV screening, why is anti-HIV IgG screened instead of antigens for HIV?

Answer 19

1. In the early stages of HIV infection, the virus itself is difficult to detect as the levels are so low. Rather than looking for the virus, HIV testing usually involves looking at the body’s reaction to the presence of the virus.
2. Only within 50 days of HIV infection, antigens for HIV such as p24 Antigen can be detected. Subsequently, the levels of HIV-1 p24 Antigen become undetectable in the body. Antibodies against HIV on the other hand remain detectable in the long term, from 20+ days of infection onwards.

Question 20

What is the purpose of the blocking steps in ELISA experiments?

Answer 20

Without the blocking steps, the detection reagents can bind non-specifically to surface areas of the well that are not occupied by the primary antibody (used for capture of antigen of interest). The non-specific binding of antigen and other sample components can result in false positive results

Question 21

What kind of agglutination method is used during Hb1Ac monitoring?

Answer 21

Agglutination inhibition