Antibodies as diagnostic tools

Question 1

what are the types of reporters/drugs used with antibodies?

Answer 1

reporters bind covalently by don’t affect antigen binding to the antibody

o Enzymes – e.g. peroxidase, alkaline phosphatase.
- Antigens washed over antibodies with enzyme, colourless substrate added which turns colour.
o Fluorescent probes – e.g. dyes and beads of various size.
o Magnetic beads – e.g. purification of cell types.
- Magnet-antibody attaches to receptor then run sample over a magnet
only linked-cells bind.
o Drugs – e.g. Kadcyla, anti-HER2 linked to emtansine.
o Radioisotopes

Question 2

how can antibodies be produced?

Answer 2

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

Question 3

how can monoclonal antibodies be produced?

Answer 3

normal B cells from spleen (limited cell division) fused with myeloma cells to create hybridomas that produce antibodies indefinitely

Question 4

how is recombinant DNA tech used to produce antibodies?

Answer 4

1) isolate required V segments of antibodies
2) display segments on protein or bacteriophage (they all display different specificities)
3) screen plates with phage collection, the complimentary phage will stick while others are washed off

Question 5

what are the therapeutic uses of manufactured antibodies?

Answer 5

oProphylactic against microbial infection – e.g. Synagis (anti-RSV).
-Suffix “-umab” – human – e.g. Synagis (anti-RSV).

o Anti-cancer therapy – e.g. anti-HER2.

o Removal of T-cells (bone marrow transplant) – e.g. anti-CD3.
- Suffix “-omab” – mouse monoclonal – e.g. anti-CD3.

o Block cytokine activity – e.g. anti-TNF-alpha.
- Suffix “imab” – chimeric/partly humanised – e.g. anti-TNF-alpha.

Question 6

how are antibodies used in making a diagnosis?

Answer 6

o Blood group serology.
o Immunoassays – hormones, antibodies, antigens.
o Immunodiagnosis – infectious disease, autoimmunity, allergy (IgE), malignancy (myeloma).

Question 7

what is the ELISA process?

Answer 7

• coat walls of wells
• anti-antigen antibody is linked to a enzyme (reporter) covalently
• wash away under bound antibodies
• add colourless substrate
• if antibody is present, due to binding with antigen, it will turn colour

Question 8

what is the lateral flow assay process and name an example of its usage?

Answer 8

• 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 9

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

Answer 9

• passed from mother via placenta (maternal antibodies)

- volunteers in clinical trials

Question 10

what is an immune complex?

Answer 10

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

Question 11

how are immune complexes (small and large) cleared?

Answer 11

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 12

how are serum Ig levels detected?

Answer 12

serum electrophoresis
ELISA
nephelometry

Question 13

how can specific antibodies be detected?

Answer 13

ELISA

Question 14

how can lymphocyte subsets be detected?

Answer 14

flow cytometry

	CD3+ All T-Cells.
	CD4+ T-helper Cells
	CD8+ Cytotoxic T-Cells.
	CD19+ B-Cells.
	CD56+ NK-Cells.
 use specific ABs against these receptors

Question 15

what can a single sharp band in serum electrophoresis indicate?

Answer 15

monoclonal expansion of B cells due to possible B cell malignancy e.g. myeloma

Question 16

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

Answer 16

the gamma globulin region becomes darker due to more gamma globulins

Question 17

how can you measure cell populations using fluorescence?

Answer 17

fluorescent dyes bound to monoclonal antibodies attach to the specific cells type

cells pass through a stream with a laser and the dye is detected.

cell is categorised depending on the fluorescence

Question 18

how does CD4 T cell and viral load change in HIV infection?

Answer 18

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

Question 19

what sort of infections can you get with a low CD 4?

Answer 19

mycobacterium avium complex

<100

Question 20

what are the therapeutic uses of manufactured antibodies?

Answer 20

• prophylactic protection against infection
• anti-cancer therapy
• removal of T-cell from bone marrow grafts
• block cytokine activity

Question 21

what are the diagnostic uses of manufactured antibodies?

Answer 21

• Blood group serology
• Immunoassays e.g. ELISA
• Hormones
• Antibodies
• Antigens
• Flow cytometry (cell subset identification)
• Immunodiagnosis (patient’s antibodies)
• Infectious diseases e.g. viral infection
• Immunodeficiency
• Malignancy e.g. myeloma
• Autoimmunity e.g. SLE

Question 22

what is flow cytometry used for?

Answer 22

measurement of cell subsets