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Diagnostics > Antibodies as diagnostic tools > Flashcards

Antibodies as diagnostic tools Flashcards

1
Q

Which part of antibody is constant?

A

Fc

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2
Q

What can you do because the Fc part is constant?

A

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

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3
Q

Which part of the antibody is variable?

A

Fab- antigen binding part

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4
Q

What sorts of things are reporters?

A
  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
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5
Q

Why are antibodies used in diagnostic tests?

A

Their unique specificity for their target antigens

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6
Q

Where do the antibodies that are used come from?

A
  1. Patient
    - In autoimmune disease
    - Defence against infection
  2. Manufactured
    - Antisera from immunised animals (polyclonal)
    - Monoclonal antibodies
    - Genetically engineered antibodies
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7
Q

How do you generate monoclonal antibodies?

A
  • 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
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8
Q

What are manufactured antibodies used for therapeutically?

A
  1. Prophylactic protection against microbial infection
  2. Anti-cancer therapy
  3. Removal of T-cells from bone marrow grafts
  4. Block cytokine activity
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9
Q

How do manufactured antibodies act as anti-cancer therapy?

A

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

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10
Q

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

A

T cells cause graft versus host disease in transplants

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11
Q

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

A
  • removal of T-cells
  • mouse monoclonal
    e. g. muronomab (anti CD3, transplant immunosuppression)
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12
Q

What does the suffix -ximab mean?

A
  • block cytokine activity

- Chimeric or partly humanised e.g. Anti TNFalpha

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13
Q

What does the suffix -umab mean?

A
  • human origin

e. g. palivizumab (anti-RSV)

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14
Q

Which ones are ideal that are used therapeutically?

A

-umab

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15
Q

What are manufactured antibodies used diagnostically?

A
  1. Blood group serology
  2. Immunoassays- hormones, antibodies and antigens
  3. Immunodiagnosis- infectious diseases, autoimmunity, allergy and malignancy
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16
Q

What does ELISA mean?

A

Enzyme
Linked
ImmunoSorbent
Assay

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17
Q

How does ELISA work?

A
  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.
18
Q

What is an immune complex?

A

Antibody bound to antigen

19
Q

What governs the size of an immune complex?

A

Ratio of antigen to antibody

- excess of antigen to antibodies leads to smaller complexes

20
Q

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

A

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

21
Q

What is a particular problem related to immune complexes?

A

Glomerulonephritis

22
Q

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

A

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

23
Q

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

A

Monoclonal expansion of B cells e.g. myeloma

24
Q

How can you measure different cell populations simultaneously?

A

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

25
Q

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

A

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

26
Q

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

A

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

27
Q

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

A
  1. Maternal antibodies

2. Volunteers in clinical trials

28
Q

How does lateral flow assay work?

A
  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
29
Q

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

A

CD3+- T cells (pan T cell marker)

CD4+- helper T cells

CD8+ cytotoxic T cells

CD19+ B cells

CD56+ Natural Killer cells

30
Q

Give an example how detection of specific antibodies can diagnose immunodeficiency

A

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

31
Q

When might flow cytometry be used?

A

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

32
Q

What would be tested if immunodeficiency suspected?

A
  1. Serum Immunoglobulin levels
    IgG, IgM, IgA and IgG1, IgG2, IgG3, IgG4
  2. Specific Antibodies (ELISA)
  3. Lymphocyte subsets (Flow Cytometry)
33
Q

What is serum sickness?

A

immune complex mediated disease….. hypersensitivity type reaction

34
Q

What is ELISA sandwich?

A

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

35
Q

Give an example of immunoassay

A

Enzyme linked ImmunoSorbent Assay

36
Q

What is synagis?

A

Therapeutic monoclonal antibody against RSV for prophylaxis

37
Q

What is IVIG?

A

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

38
Q

Why are T cells removed from haemoatopoitic stem cell transplants?

A

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

39
Q

How can ABs be produced using recombinant DNA technology?

A

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)

40
Q

Outline structure of antibody

A

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

Diagnostics (5 decks)

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