Antibodies & diagnostics [Complete]

Question 1

Give examples of what can be attached to antibodies for diagnostic purposes

Answer 1

Enzymes: e.g. peroxidase, alkaline phosphatase

Fluorescent probes: dyes, beads of different sizes

Magnetic beads: e.g. purification of cell types

Drugs: e.g. Kadcyla, anti-HER2 linked to emtansine

Question 2

Antibodies which bind to other antibodies are known as?

How can they be used in diagnostics?

Answer 2

Anti-antibodies

(Tend to target immunoglobulins from other species)

Can be used in indirect labelling of an antigen. They can bind to the primary antibody which binds to that specific antigen.

Question 3

How can antibodies be produced?

Answer 3

1) Humans due to autoimmune disease or defence against infection
2) Artificially by animals (antisera), monoclonal antibodies and genetically engineered antibodies

Question 4

List 3 ways antibodies can be artificiallyt manufactured

Answer 4

Antisera from immunised animals (polyclonal)

Monoclonal antibodies

“Genetically engineered” antibodies

Question 5

How are monoclonal antibodies produced?

Answer 5

Immunise animal such as a mouse

Take out spleen cells which produce HGPRT+ve antibodies.

As they have limited cell division, these cells are fused with immortal HGPRT-ve myeloma cells to form hybridomas

Culture in a medium selective for HGPRT+ve cells

Clone cells by limiting dilution (place individually and see if they display hybridoma characteristics)

Harvest antibodies produced by cells.

Question 6

How are genetically modified antibodies produced?

Answer 6

Take the V section gene of an antibody and fuse it with a bacteriophage coat protein - displays V section on its surface

Create a library of these

Add the library to the antigen and wash unbound phages away

Expand the right phage population (the ones that stick to the antigen)

Question 7

With examples list the therapeutic uses of antibodies

Answer 7

Drugs:

Targeting drugs to certain cell types e.g. breast cancer

e.g Kadcyla linked to emtansine (med used to treat HER2 positive breast cancer) (anti-HER2 antibodies)

Prophylactic protection against microbal infection: (Especially in immunocompresed)

E.g Synagis to protect from respiratory syncytial virus

Removal of t-cells from bone marrow grafts prevent graft vs host disease (Anti-CD3)

Block cytokine activity (anti-TNFα)

Treat migraines: Anti-CGRP (Calcitonin gene related peptide)

Question 8

State some uses of antibodies in diagnostics

Answer 8

Blood group serology

Immunoassay: hormones, antibodies, antigens

Immunodiagnosis: infectious disease, autoimmunity, allergy (IgE), malignancy (myeloma).

Question 9

What is ELISA and how does it work?

Answer 9

ELISA: Enzyme Linked ImmunoSorbent Assay

Captured antibody present on assay

Test sample added and if corresponding antigen present, binds to the antibody.

Another antibody which binds to different site of antigen added.

Sample is washed to remove unbound antibodies

It has an enzyme which can catalyse a substrate which leads to a color change.

Color change indicates positive result.

Question 10

With an example how does lateral flow assay work?

Answer 10

Provides rapid testing

Pre-made antibodies bound to gold nanoparticles have an analyte passed over them.

If the antibodies bind successfully to analyte (+ve result), the antibodies bind to positive strip and will show up.

E.G. hCG protein in pregnancy.

Question 11

In what cases do you have anti-HIV antibodies without an HIV infection

Answer 11

Passed from mother via placenta (maternal antibodies)

Volunteers in clinical trials

Question 12

What is an immune complex?

Answer 12

An antibody-antigen unit in the blood that make itself becomes an antigen

Question 13

What kind of conditions is immunodiagnosis useful for?

Answer 13

• Infectious diseases
• Autoimmunity Allergy (IgE)
• Malignancy (myeloma)

Question 14

Explain the clearance of immune complexes (small and large)

Answer 14

Large ones are recognised easily and cleared quickly as platelets are activated and netrophils release mediators

Small ones get trapped in sub-endothelial layers so can activate complement and attract neutrophils to cause damage.

E.g. in kidney –> glomerulonephritis

Question 15

How are serum Ig levels detected?

Answer 15

Serum electrophoresis

ELISA

Nephelometry

Question 16

How can specific antibodies be detected?

Answer 16

ELISA

Question 17

How can lymphocyte subsets be detected?

Answer 17

Flow cytometry

CD3+ All T-Cells.
CD4+ T-helper Cells
CD8+ Cytotoxic T-Cells.
CD19+ B-Cells.
CD56+ NK-Cells.

Use specific antibodies against these receptors

Question 18

What is serum electrophoresis?

What should a normal test result look like?

Answer 18

Electrophoresis of blood antibodies

Shouldnt see thin dense bands

Bands should be spread out more

Question 19

What can a single sharp band in serum electrophoresis indicate?

Answer 19

If a single band is very dense - indicates monoclonal expansion

Could be B-cell malignancy

Investigate for myeoma

Question 20

What happens to the top region of an electrophoresis in a person with an active immune response?

Answer 20

The gamma globulin region becomes darker due to more gamma globulins

Question 21

What happens in flow cytometry?

Answer 21

Antibodies to specific antigens labelled with florescent markers

Mixed with cells

Bind to cells with their antigen

Passed through machine

Laser meaures size granulation and floresence.

Question 22

Explain how CD4 T cell and viral load changes in HIV infection

Answer 22

T cell count drops rapidly at first with acute infection than drops slowly during latency period

Viral load increases at acute infection and drops right before the beginning of the latency period.

Once T cell is low enough, viral load increases rapidly at the AIDS stage