Immunology

Question 1

How many immunological test types can most hospitals request? What are the immunological basis of these?

Answer 1

In immunology, there are 86 tests that you can request.
53 of these tests detect specific antibodies

Many non-immunolgical tests also USE antibodies in some way, this is due to their unique specificity and now antibodies against most molecules (including carbohydrates and fats) can be produced in labs

Question 2

What is the general structure of antibodies and what advantages does this grant pathologists?

Answer 2

Antibodies have a variable part (binds the antigen) and a constant part (has the biological activity). This means you can bind molecules to the constant region without disrupting the binding capability of the antibody.

Question 3

What sort of molecules are bound to the constant regions of antibodies therapeutically?

Answer 3

Attach enzymes e.g. peroxidase, alkaline phosphatase (can colour specimens)

Attach fluorescent probes e.g. dyes, beads of different sizes

Attach magnetic beads e.g. purification of cell types

Attach drugs e.g. kadcyla, anti-HER2 linked to emtansine

Question 4

What are anti-antibodies?

Answer 4

Antibodies that act against antibodies of a different class, these can be manufactured for therapeutic purposes or be naturally occurring in disease - E.G Rheumatoid Factor is an IgM antibody against IgG

In diagnostics this is very useful because one anti-antibody can bind to any antibody of the class it targets and therefore can be used as a reporter for many antigens that are bound by antibodies of that class.

Question 5

What are the two different categories of antibodies for therapeutic use?

Answer 5

Antibodies produced by the patient - can be used in diagnosis

Antibodies manufactured - Antisera for immunised animals (polyclonal), Monoclonal antibodies, and genetically manufactured antibodies - These can all be used in treatments or diagnoses)

Question 6

How are monoclonal antibodies produced?

Answer 6

Two different cell types are fused together using polyethylene glycol, making a hybridoma.

One of the cell types produce the antibodies of interest but have limited capacity for cell division.

The other will be a myeloma, capable of almost indefinite reproduction but not producing the antibody

When the cells are fused together, you end up with
immortal cells that produce the antibodies of interest, and positive for the enzyme

Screen clones for the AB you want and then filter it out

Question 7

What enzyme must the AB producing cells be +Ve in and the Myeloma cells be -Ve in for monoclonal antibody production to succeed? What culture medium is used that ensures this?

Answer 7

HGPRT -Hypoxanthine-guanine phosphoribosyltransferase

HAT medium used - in HAT medium only HGPRT +Ve cells can grow - preventing proliferation of the myeloma cells not hybridised and producing the AB

Question 8

How is recombinant DNA technique used to produce antibodies?

Answer 8

Isolation population of genes encoding antibody variable region. Produce fusion protein of V region with a bacteriophage coat protein.

Cloning a random population of variable regions gives rise to a mixture - a phage display library.

Select correct antibody by binding it to desired antigen

Question 9

What are the therapeutic uses of manufactured antibodies?

Answer 9

Prophylactic protection against microbial infection e.g. IVIG (pool of many antibodies), synagis (anti-RSV)

Anti-cancer therapy e.g. anti-HER2

Removal of T-cells from bone marrow grafts – Anti-CD3

Block cytokine activity e.g. anti-TNFα

Question 10

What do different suffixes mean for monoclonal antibodies?

Answer 10

“-omab” mouse monoclonal
E.g. Muronomab, anti-CD3, transplant immunosuppression (approved 1986)

“-imab” – chimeric or partly humanised antibody
E.g. Infliximab (Remicade) anti-TNFa, Rituximab anti-CD20

“-umab” – fully human antibody
E.g. Palivizumab, anti-RSV (Synagis)

Note: All can cause immune response but the more similar to host the less likely.

Question 11

How are monoclonal antibodies used diagnostically?

Answer 11

Blood group serology
Immunoassays – hormones, antibodies, antigens
Immunodiagnosis – Infectious diseases, Autoimmunity, Allergy (IgE), Malignancy (myeloma)

Question 12

What is ELISA? How does it work?

Answer 12

ELISA – Enzyme-linked Immunosorbent Assay

An antibody or antigen sample is immobilised (immunosorbent) in wells of a multi-well plastic plate.An antibody against the antigen is added. This antigen has a reported molecule on it (e.g. enzyme). Unbound antibodies are washed away (Any without antigen to bind to)

A substrate is added, and causes development of the colour (which can easily be measured by a spectrophotometer) in presence of enzyme. The level of the colour gives a quantitative measure of the amount of antigen.

Question 13

What is rapid testing?

Answer 13

Rapid testing (dipstick tests, strip tests) use antibodies
to develop coloured lines.

There is a sample pad that absorbs the liquid sample.
There are antibodies against the sample, conjugated to
small nanoparticles. The capillary flow along the
system moves the sample. If the antibody binds to the
analogue in the sample, there’s a complex.

There is a strip with a second antibody against the
thing you are trying to measure. If the sample contains
a complex, it will bind to that strip, to produce a strip
of colour that is detectable by eye.

The control strip is after this first strip – it contains the anti-antibody. The control strip is needed to show that
the sample has reached this region. It shows that the unbound antibody is there, and produces positive control.

Question 14

How are immune complexes used in diagnosis?

Answer 14

Depending on the ratio of antibody to antigen, you can get large immune complexes, or small ones.

The large immune complexes are good at directly activating complement, activating neutrophils and activating platelets to form thrombi.

The smaller complexes don’t activate cells efficiently on their own, but once they become immobilised on cell membranes, they become efficient at activating
immune components. Complement activation leads to peptide production.

(You can search for any of the products of this in blood or tissue)

Question 15

What can be done to diagnose immunodeficiency?

Answer 15

Serum Immunoglobulin levels (IgG, IgM, IgA and IgG1, IgG2, IgG3, IgG4) Done via Serum electrophoresis /ELISA/Nephelometry

Specific Antibodies (Protein antigens – Tetanus & Haemophilus, Polysaccharides antigens – Pneumococcus) Done via ELISA

Lymphocyte subsets (CD3/CD4/CD8/CD19/NK cells) Done via Flow cytometry

Question 16

How is serum electrophoresis to detect IGs interpreted?

Answer 16

All samples will have think band of albumin at the bottom due to its abundance in blood

Should see diffuse smearing - indicate polyclonal responses but no active infection

If stark bands (not smears) seen elsewhere this suggests monoclonal expansion - possible malignancy (Do biopsy for myeloma)

If denser smearing - Indicates active infection

If less dense or areas absent - possible immunodeficiency

Question 17

How is flow cytometry performed and interpreted?

Answer 17

Different cells have different proteins on surface, markers for antibodies to target

CD3+ T cells – pan T cell marker
CD4+ T cells – T helper/cells
CD8+ T cells – cytotoxic T cells
CD19+ B cells
CD56+ Natural Killer (NK) Cells

Mix antibodies with cells, and pass them through a
flow cytometer (one cell at a time). The cytometer detects the fluorescence emitted from the cells. It also detects the forward and side scatter (allowing information about size and granulation to be gathered)

Question 18

When is flow cytometry commonly used?

Answer 18

To quantify the number of CD4 T cells in patients with HIV.