Antibodies as Diagnostic Tools

Question 1

How can Abs be used as diagnostic tools?

Answer 1

Abs can be raised against almost ANY antigen!

The Fc-region is CONSTANT
o this is where you attach molecules (REPORTERS/DRUGS)
o it does NOT affect the specificity of the Ab

Question 2

What types of Reporters/Drugs can you attach to the Ab?

Answer 2

o Enzymes

e. g. peroxidase, ALP
- Ags washed over Abs w. enzymes
- colourless substrate then added which turns colour

o Fluorescent probes
e.g. dyes, beads to different sizes

o Magentic beads

e. g. purification of cell types
- magnet-Ab attaches to receptor
- then runs sample over a magnet
- only linked cells bind

o Drugs
e.g. Kadcula - anti-HER2 linked to emtansine

Question 3

What is an anti-antibody?

Answer 3

Used to detect existing Abs in the body

Question 4

2 ways Abs can be produced?

Answer 4

1. Produced by PATIENT

o for autoimmune disease
o for defence against infection

2. Produced ARTIFICIALLY

o ANTISERA from immunised animals (polyclonal so multiple specifities BUT limited amount)
o MONOCLONAL Abs
o GENETICALLY ENGINEERED Abs

Question 5

How are monoclonal Abs generated?

Answer 5

1. Take normal B-lymphocytes from spleen
   - producing Ab of INTEREST
   - have LIMITED cell division
2. FUSE it with myeloma cells
   - these cells do NOT produce own Ab
3. This gives you HYBRIDOMA
   - these can produce Ab on interest indefinitely (as fused w. tumour cell)

Question 6

How can you produce Abs using recombinant DNA technology?

Answer 6

1. Isolate all V-segments of the needed Abs
2. Display all V-segments
   - on protein
   - OR on bacteriophage so each one display different specificity V segments
3. Use the phage-display library to screen plates for Ags
   - complimentary phage will STICK to plate
   - other phages will WASH off

Question 7

2 broad uses of Manufactured Abs?

Answer 7

Therapeutic

&

Diagnostic

Question 8

Therapeutic uses of manufactured Abs?

Answer 8

1. Prophylactic - against microbial infection
   e. g. Synagic (anti-RSV)
2. Anti-cancer therapy
   e. g. anti-HER2
3. Removal of T-cells from bone marrow graft (avoid attacking own cells)
   e. g. anti-CD3
4. Block cytokine activity
   e. g. anti-TNFalpha

Question 9

Diagnostic uses of manufactured Abs?

Answer 9

1. Blood group serology
2. Quantitative immunoassays
   e. g. hormones, Abs, Ags
3. Immunodiagnosis
   e. g. infectious disease, autoimmunity, Allergy (IgE), Malignancy (myeloma)

Question 10

ELISA?

Answer 10

Enzyme Linked Immuno-Sorbent Assay

Question 11

How does ELISA work?

Answer 11

1. Two samples are used to coat the walls
2. Anti-A Ab is covalently linked to a reporter (enzyme)
3. When it binds, colourless substrate is added
4. If Ab is still present, it turns it into a colour in the well after washing
5. Absorbance of the light can then be measured

Question 12

Lateral Flow Assay?

Answer 12

LITERALLY the hCG protein in pregnancy test kits!!

Question 13

How can you diagnose HIV?

Answer 13

HIV-Ab test

Question 14

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

Answer 14

o Maternal Abs

o Volunteers in clinical trial

Question 15

What can cause ‘vague aches and pains’?

Answer 15

Immune complexes!

• Inflammation / complement activation
• Serum sickness (I.Cs in circulation)
• I.C glomerulonephritis
• I.C deposits at other sites (skin, joints, lungs)

Question 16

Immune complex glomerulonephritis?

Answer 16

Glomerular diseases

in which kidney biopsy samples show granular deposits of immuno- globulin

Question 17

Large vs. small immune complexes?

Answer 17

Large I.C:

o recognised more EASILY by I.S
o activate platelets & neutrophils that release mediators
o cleared QUICKLY

Small I.C:

o get TRAPPED in sub-endothelial layer
o can ACTIVATE COMPLEMENT when stuck to surface
o attracts NEUTROPHILS that can damage kidney function

Question 18

What 3 things can you detect to determine immunodeficiency?

Answer 18

1. Serum Ig levels (e.g. IgG, IgM etc)
   o via. serum electrophoresis/ELISA/nephelometry)
2. Specific Abs (e.g. protein & polysaccharides)
   o ELISA
3. Lymphocyte subsets (e.g. CD3, CD4, CD8)
   o Flow cytometry - can use SPECIFIC Abs against these receptors)

Question 19

Immunodefieicny seen in electrophoresis?

Answer 19

ONENOTE!!

 In a healthy person, smear at top is gamma globulin region

 In someone with an active immune response, this region becomes darker due to more gamma globulins

 If you see a single sharp band, this indicates a monoclonal expansion of b-cells – e.g. myeloma

Question 20

How are cell populations measured?

Answer 20

FLOW CYTOMETRY!!

1. Attach different monoclonal Abs w. fluorescent dyes
2. Pass cells into a stream through a laser
3. Dyes are detected
4. Each cell categorised based on its fluorescence

Results shown on a flow cytrometry graph (ONENOTE!!)

Question 21

Order of HIV infection?

Answer 21

 Antibody HIV test –> Low CD4, high viral load –> commence ART (1st line) –> monitor CD4/viral load –> CD4 down or viral load up –> ART (2nd line) increase.
o This increases life expectancy.

Question 22

What happens when CD4 becomes too low in HIV?

Answer 22

Patient more prone to opportunistic disease
AND
Viral load shoots up!

At varying [CD4] prone to certain infections (ONENOTE!!)

Question 23

What infection can people w. HIV get that is not normal for normal people?

Answer 23

MAC - mycobacterium avium complex

This is an envrionmental mycobacterium that is everywhere and normal people can deal w. easily