Antibodies as Diagnostic Tools

Question 1

Why are antibodies used in diagnostics?

Answer 1

The unique specificity of antibodies for their target antigens is the basis of many diagnostic tests

Antibodies can be raised against almost any antigen (often but not always proteins)
Including immunoglobulins from other species
=anti-antibodies

Question 2

What can you attatch to an antibody?

Answer 2

• enzyme (e.g. peroxidase, alkaline phosphatase)
• Fluorescent probes: dyes, beads of different sizes
• Magnetic beads: e.g. purification of cell types using a magnet essentially.
• Drugs: e.g. Kadcyla (used in breast cancer) , anti-HER2 linked to emtansine

Question 3

What is a primary and secondary antibody in indirect labelling using anti-antibodies?

Answer 3

• the primary antibody is the antibody that binds to the antigen
• the secondary antibody bids to the primary antibody. It may have a reporter molecule bound to it.

Question 4

When are antibodies produced by a patient? When are antibodies manufactured?

Answer 4

Produced by the patient

• in AI disease
• for defence against infection

Manufactured Antibodies

• antisera from immunised animals (polyclonal)
• monoclonal antibodies (large amount of a single antibody)
• “genetically engineered” antibodies

Question 5

How can you generate monoclonal antibodies? Also for recombinant DNA technology antibodies?

Answer 5

-> see PPT

Question 6

What are some therapeutic uses of manufactured antibodies?

Answer 6

• prophylactic protection against microbial infection (e.g. IVIG, synagis = anti-RSV)
• Anti cancer therapy (anti-HER2)
• removal of T-cells from bone marrow grafts (Anti CD3)
• block cytokine activity (e.g. anti-TNF-alpha)
• Anti-calcitonin gene related peptide (CGRP) for migraine.

=> all these treatments are very expensive.

Question 7

What are the main 2 clinical uses of manufactured antibodies? (general)

Answer 7

• therapeutic

- diagnostic

Question 8

What are some diagnostic uses of manufactured antibodies?

Answer 8

• blood group serology
• quantitative immunoassays (hormones, antibodies, antigens)
• Immunodiagnosis (infectious diseases, autoimmunity, Allergy (IgE), malignancy (myeloma)

Question 9

ELISA

Answer 9

Enzyme linked ImmunoSorbent Assay

• (AB on membrane, target can bind, wash over to remove unbound targets, wash over with AB that binds to target and has a detection molecules bound.
• amount of signal you get is proportional to the amount of antigen

Question 10

Lateral Flow Assay

Answer 10

• > Rapid testing (point of care)
• > strip assay
• there are mobile ABs conjugated with a marker (e.g. gold nanoparticles) on the conjugated pad.
• there are 2 test lines with immobile antibodies: first a row that binds the antigen and second a row of anti antibodies that bind the ABs from the conjugated plate.
• test sample (analyte) is added and travels down, also moving the ABs on the conjugated pad.
• Positive binds to both lines; negative: only the anti-antibodies bind to the second line.

-e.g. pregnancy test

Question 11

Flow Cytometry

Answer 11

• mixture of cells labelled with a fluorescent AB

- stream of fluid containing marked antibodies passes and lasers detect fluorescent marking