Antibodies as diagnostic tools

Question 1

What can you do because the Fc part is constant?

Answer 1

Attach various things to this constant part without affecting the binding ability of the antibody to the antigen

Question 2

What sort of things are reporters?

Answer 2

Enzymes- peroxidase, alkaline phosphatase etc
Fluorescent probes- dyes, beads of different sizes
Magnetic beads:e.g purification of cell types
Drugs

Question 3

Why are antibodies used in diagnostic tests?

Antibodies can be raised against almost any antigen– often made of what

Simple expl of indirect labelling using an anit antibody

Answer 3

Their unique specificity for their target antigens

protein–not always
including immunoglobulins from other species-antiantibodies

Antigen bound antibody Fc part binds to Fab region of another antibody that has a reporter molecule bound to its own fc

Question 4

Where do the antibodies that are used come from?

Answer 4

Patient-
In autoimmune disease
Defence against infection
Manufactured-
Antisera from immunised animals
Monoclonal antibodies
Genetically engineered antibodies

Question 5

How do you generate monoclonal antibodies?

Production of antibodies using recomb DNA technology

Answer 5

1) Animal you immunise with antigen youre making antibody against, take their spleen cells as a source of antibody produicin b cells.
2) Myeloma- tumour of b cells that dont produce antibody– divide indef indef. They only make a few replications
3) Fuse above 2 cells using detergent producing hybridomas.
4) Culture cells in HAT medium that only allows hybridomas to grow
5) Harvest from cells that produce monoclonal antibodies.

These cells have the ability to produce the antibody of interest, furthermore, as it is fused with a tumour cell it can divide indefinitely

Take gene encoding variable parts of antibodies. Enter into bacteriophage and are displayed on surface – Phage display library.

Molecule immobilised (against which you’re trying to make antibody against) . Add diverse range of bacteriophage. Those with light specificity will bind to antigen. Multiple cycle to enrich phage.– can be expanded a lot.

Question 6

What are manufactured antibodies used for therapeutically?

Answer 6

Prophylactic protection against microbial infection-cant make own antibodies
Anti-cancer therapy
Removal of T-cells from bone marrow grafts
Block cytokine activity
Anti-calcitonin gene-related peptide (CGRP) for migraine

Question 7

What are manufactured antibodies used diagnostically?

Answer 7

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

Question 8

What does ELISA mean?

Answer 8

Enzyme
Linked
ImmunoSorbent
Assay

Question 9

How does ELISA work?

Answer 9

Plastic plates w wells containing antibodies against what we’re looking for
Bind to antigen –> wash
2nd detection antibody which is conjugated e.g w enzyme that can easily be measured.

Quantitative assay as amount of signal=amount of antigen
e.g measure of inflamm

Question 10

What governs the size of an immune complex?

Answer 10

Ratio of antigen to antibody- excess of antigen (increased viral load) to antibodies leads to smaller complexes

Deposite

Question 11

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

Answer 11

Larger immune complexes are recognised by immune system and cleared more easily but can activate platelets and neutrophils freely
Smaller immune complexes don’t efficiently activate complement, it will only activate complement when it is bound to a surface

Question 12

What is a particular problem related to immune complexes?

sites of deposition

Answer 12

Glomerulonephritis (deposited at kidney)

Skin, lungs, joints

Question 13

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

Answer 13

At top of healthy person, there is a diffuse smear which is the gamma globulin region-
If someone is developing an active immune response, there’s a lot more gamma globulin so smudge will be much darker

Question 14

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

Answer 14

Monoclonal expansion of B cells e.g. myeloma

Question 15

How can you measure different cell populations simultaneously?

Answer 15

FLOW CYTOMETRY

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 16

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 16

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 17

Immunodeficiency can be confirmed using

Answer 17

Serum Immunoglobulin levels
IgG, IgM, IgA and IgG1, IgG2, IgG3, IgG4
(Serum electrophoresis / ELISA / Nephelometry)
Specific Antibodies (ELISA)
Protein antigens – Tetanus & Haemophilus
Polysaccharides antigens – Pneumococcus

Lymphocyte subsets	(Flow Cytometry)
CD3 / CD4 / CD8 / CD19 / NK cells

Question 18

Rapid testing order of antibodies it meets on assay

Answer 18

First meets antibodies that are conjugated with coloured nanoparticle

Second set
where 2 lines of immobilised antibodies
First- For antigen we’re looking for
Second- Anti antibody– +ve if test has worked
–Capilllary action draw liq across strip