Immunology

Question 1

What is ELISA?

Answer 1

Enzyme Linked ImmunoSorbent Assay

Question 2

What is an ELISA?

Answer 2

An enzyme linked ImmunoSorbent assay is an immunological assay used to measure antibodies, antigens, proteins and glycoproteins in biological samples.

Question 3

Why do they use ELISA’s?

Answer 3

The samples used are measured to diagnose HIV, pregnancy, measuring of cytokines, supernatant, soluble receptors and supernatants

Question 4

How is an ELISA done?

Answer 4

They are done on a 96 well plate, allowing multiple samples to be measures. The plates need to be special absorbent plates to ensure antibodies and antigens stick to the surface. Each Elisa measure a specific antigen and multiple kits are available for different antigens.

Question 5

What is the method to do a sandwich ELISA?

Answer 5

1- two sets of antibodies are used to detect secreted products. You first coat the plate with capture antibodies and any excess are washed away.

2- next you add the sample, any antigens present bind with the capture antibodies. The sample is added in triplicate to ensure detection and analysis. Excess is washed away again.

3- next a detection antibody is added. This antibody is labelled with an enzyme. This antibody binds to the target antigen bound in the plate. Finally a substrate is added and a chromogenic reaction occurs, converting the substrate into a coloured product which can be measured

Question 6

How do you determine an antigen concentration?

Answer 6

Determining the antigen concentration in a sample requires a production of a standard curve using antigens of a known concentration.

Question 7

What are Antibodies and antigens

Answer 7

Antibodies are Y shaped proteins that recognise and bind antigens- they’re made by B cells.

An antigen is anything that can cause an immune response. It can be a pathogen component, or components of proteins, carbohydrates, lipids, drugs, toxins etc

Question 8

What is analytical sensitivity?

Answer 8

The ability of a test to detect small amounts of Ab and Ag

Question 9

What is clinical sensitivity?

Answer 9

Ability of a test to give positive results if patients have the disease (no false negatives)

Question 10

What is a false negative?

Answer 10

This is where the result is negative as the concentration of the Ab or Ag is too low to detect. In reality, the patient has the disease.

Question 11

What is analytical specificity?

Answer 11

The ability of a test to detect a substance without interference from cross-reacting substances

Question 12

What is clinical specificity?

Answer 12

The absolute of test to give a negative result if the patient doesn’t have the disease (no false positive)

Question 13

What is a false positive?

Answer 13

The test result is positive however the test is detecting a cross reacting substance. In reality the result should be negative as the patient doesn’t have the disease

Question 14

What is highly sensitive?

Answer 14

False negative reactions are absent or minimal

Question 15

What is highly specific?

Answer 15

False positive reactions are absent or minimal

Question 16

Why are antibodies used as research tools?

Answer 16

• They’re highly specific for corresponding antigens
• easy to isolate and study
• invaluable as probes of biological processes
• can be generated in large amounts
• have high affinity for their antigen

Question 17

Monoclonal vs polyclonal antibodies

Answer 17

Polyclonal (pAb)- Heterogeneous, secreted different by B cells, same antigen but different epitope.

Monoclonal (mAb)- Homogeneous, secreted by one B cell clone, has the same antigen and same epitope

Question 18

Monoclonal antibody production

Answer 18

1- specific antigen injected in mouse
2- spleen B cells producing desired antibodies are isolated
3- fuse the spleen cells with myeloma cancer cells which are immortal
4- produces Hybridoma- subclonal hybdrodoma cells produce antibody

Question 19

Polyclonal antibody production

Answer 19

1- inject antigen into rabbit
2- antigen activates B cells
3- Plasma B cells produce polyclonal antibodies
4- Obtain antiserum from rabbit containing antibodies

Question 20

ELISA controls

Answer 20

Positive control- Confirm the assay is working, can be done as part of the standards

Negative control- confirm a positive result is not due to a non-specific binding of Abs or faulty reagents

Question 21

How do you determine ELISA sensitivity?

Answer 21

As you dilute the standard further, it will eventually reach an Absorbance thag is undetectable above the negative control.
You identify the lowest possible concentration of the primary antibody that still gives a positive result.
This is the sensitivity limit of your assay.

High limit= low sensitivity = potential for false negatives as the concentration of the measured analysts is too low for detection