Antibodies as diagnostic tools

Question 1

Which part of antibody is constant?

Answer 1

Fc

Question 2

What can you do because the Fc part is constant?

Answer 2

Attach molecules (reporters) to this constant part without affecting the binding ability (specificity) of the antibody to the antigen

Question 3

Which part of the antibody is variable?

Answer 3

Fab- antigen binding part

Question 4

What sorts of things are reporters?

Answer 4

1. Enzymes- peroxidase, alkaline phosphatase etc
2. Fluorescent probes- dyes, beads of different sizes
   3, Magnetic beads: e.g. purification of cell types
3. Drugs e.g Kadcyla, anti-HER 2

Question 5

Why are antibodies used in diagnostic tests?

Answer 5

Their unique specificity for their target antigens

Question 6

Where do the antibodies that are used come from?

Answer 6

1. Patient
   - In autoimmune disease
   - Defence against infection
2. Manufactured
   - Antisera from immunised animals (polyclonal)
   - Monoclonal antibodies
   - Genetically engineered antibodies

Question 7

How do you generate monoclonal antibodies?

Answer 7

• take a normal B lymphocytes from spleen (limited cell devision, HGPRT+ve) which produces the antibody of interest
• fuse it with a myeloma cell line (immortal, no AB production,HGPRT-ve) which gives you a hybridoma
• hybridomas can produce antibodies indefinitely

Question 8

What are manufactured antibodies used for therapeutically?

Answer 8

1. Prophylactic protection against microbial infection
2. Anti-cancer therapy
3. Removal of T-cells from bone marrow grafts
4. Block cytokine activity

Question 9

How do manufactured antibodies act as anti-cancer therapy?

Answer 9

Monoclonal antibodies target molecules that are over-expressed on certain types of tumours

Question 10

Why is removal of T cells from bone marrow grafts important?

Answer 10

T cells cause graft versus host disease in transplants

Question 11

For the nomenclature of therapeutic monoclonal antibodies, what does the suffix -omab mean?

Answer 11

• removal of T-cells
• mouse monoclonal
  e. g. muronomab (anti CD3, transplant immunosuppression)

Question 12

What does the suffix -ximab mean?

Answer 12

• block cytokine activity

- Chimeric or partly humanised e.g. Anti TNFalpha

Question 13

What does the suffix -umab mean?

Answer 13

• human origin

e. g. palivizumab (anti-RSV)

Question 14

Which ones are ideal that are used therapeutically?

Answer 14

-umab

Question 15

What are manufactured antibodies used diagnostically?

Answer 15

1. Blood group serology
2. Immunoassays- hormones, antibodies and antigens
3. Immunodiagnosis- infectious diseases, autoimmunity, allergy and malignancy

Question 16

What does ELISA mean?

Answer 16

Enzyme
Linked
ImmunoSorbent
Assay

Question 17

How does ELISA work?

Answer 17

1. Two samples are used to coat the walls of some wells.
2. Anti-A antibody is covalently linked to a reporter (enzyme) that when it binds (whilst any non-binding antibody is washed away), a colourless substrate is added and turns colour if the antibody is still present in the well after washing.
3. Absorbance of the light can then be measured.

Question 18

What is an immune complex?

Answer 18

Antibody bound to antigen

Question 19

What governs the size of an immune complex?

Answer 19

Ratio of antigen to antibody

- excess of antigen to antibodies leads to smaller complexes

Question 20

What is the difference in response to larger or smaller immune complexes?

Answer 20

Larger immune complexes are recognised by immune system and cleared more easily but activate platelets and neutrophils freely

Smaller immune complexes get trapped in sub-endothelial layer. It will only activate complement when it is bound to a surface so attracts neutrophils what can dame kidney function

Question 21

What is a particular problem related to immune complexes?

Answer 21

Glomerulonephritis

Question 22

What is the difference between someone developing an acute response and healthy person in terms of serum electrophoresis?

Answer 22

At top of healthy person, there is a diffuse smear which is the gamma globulin region- diffuse because many different antibodies with different charges

If someone is developing an active immune response, there’s a lot more gamma globulin so smudge will be much darker

Question 23

What does a very sharp single band in serum electrophoresis indicate?

Answer 23

Monoclonal expansion of B cells e.g. myeloma

Question 24

How can you measure different cell populations simultaneously?

Answer 24

Have several different monoclonal antibodies and label each with a different coloured fluorescent dye
Add the mixture of antibodies to the cell mixture
Then pass the cells in a stream through the laser beam and detect fluorescent so each cell can be categorised based on fluorescence

Question 25

What is the natural progression of HIV in someone that hasn't had treatment in terms of CD4 T cell count and viral load?

Answer 25

Primary infection- CD4 will initially go down and then it will go up again after a few weeks The viral will remain controlled by immune system for some time (clinical latency) but CD4 will keep going down When CD4 gets very low, patient will show signs of opportunistic infection and viral load will go up

Question 26

What are MAC infections and what are there relation to HIV?

Answer 26

Mycobacterium avium complex - environmental mycobacterium that is everywhere and normal people can deal with it easily but it's an opportunistic infection that develops when you have a low CD4 count

Question 27

When may you have anti-HIV antibodies but NOT have HIV?

Answer 27

1. Maternal antibodies | 2. Volunteers in clinical trials

Question 28

How does lateral flow assay work?

Answer 28

1. Pre-made antibodies bonded to gold nanoparticles have an analyte passed over them. 2. If the antibodies do successfully bind to the analyte (+ve result), they antibodies will bind to the positive strip and it will show up. E.G. hCG protein in pregnancy

Question 29

Which antigens are used in flow cytometry to detect the different types of lymphocyte cell?

Answer 29

CD3+- T cells (pan T cell marker) CD4+- helper T cells CD8+ cytotoxic T cells CD19+ B cells CD56+ Natural Killer cells

Question 30

Give an example how detection of specific antibodies can diagnose immunodeficiency

Answer 30

You can give vaccination of an antigen (or the ABs to this antigen) Then do blood sample Do ELISA where you coat the well in antigens and see if any ABs attach by treating it with an enzyme If they are immune deficieny tey won't be able to make any of their own antibodies against the antigen

Question 31

When might flow cytometry be used?

Answer 31

You can count number of CD4+ T cells in HIV patient

Question 32

What would be tested if immunodeficiency suspected?

Answer 32

1. Serum Immunoglobulin levels IgG, IgM, IgA and IgG1, IgG2, IgG3, IgG4 2. Specific Antibodies (ELISA) 3. Lymphocyte subsets (Flow Cytometry)

Question 33

What is serum sickness?

Answer 33

immune complex mediated disease..... hypersensitivity type reaction

Question 34

What is ELISA sandwich?

Answer 34

When you add AB, which is absorbed to surface, then add antigen, then add AB with reporter

Question 35

Give an example of immunoassay

Answer 35

Enzyme linked ImmunoSorbent Assay

Question 36

What is synagis?

Answer 36

Therapeutic monoclonal antibody against RSV for prophylaxis

Question 37

What is IVIG?

Answer 37

Some patients have to have IVIG (intravenous immunoglobulin if they can't make their own)

Question 38

Why are T cells removed from haemoatopoitic stem cell transplants?

Answer 38

To avoid graft versus host disease (i,e, so the donor transplant, which would contain T cells, don't attack the host. Note that you don't need the T cells in the bone marrow transplant, you are doing the transplant for the haematopoietic stem cells

Question 39

How can ABs be produced using recombinant DNA technology?

Answer 39

You can take the parts of the human genome encoding variable regions of ABs and put them into bacteriophages. (Vl and Vh) Then, the bacteriophages will start expressing ABs with all the different variable regions in the genome on their cell surface Then you get an agar plate with the antigen you want an antibody against on it Then you apply the ABs. The AB with the correct specificity for the antigen will bind, and you wash the others away. Then you can clone this one, and repeat the process with the bound antigen to get even greater specificity BENEFIT: uses human AB from the beginning, so no immune response against (as there would be with foreign AB such as from animal)

Question 40

Outline structure of antibody

Answer 40

Heavy and light chains Classes differ by the constant part of their heavy chain The top part has variable and constant domains. The variable part binds antigen The bottom of the molecule is just the heavy chain, and this is the biological effector