diagnostic testing - lab evaluation of immunity

Question 1

what property of monoclonal antibodies makes them so useful in diagnostic testing?

Answer 1

each has one unique specificity

Question 2

b cell hybridoma

Answer 2

used to generate a large amount of an antibody with a known specificity:
- many b cells begin making antibodies to different epitopes on an antigen => one is selected for clonal expansion

Question 3

polyclonal antibodies and their use

Answer 3

mixture of many different antibodies against different epitopes on one molecule or organism, produced by many different B cells

anti-sera can be used for passive immunity

Question 4

nephelometry

Answer 4

• measurement of light scattered by precipitate in a liquid
• antigen and antibody are soluble in solution, but upon binding, immune complexes precipitate
• light scattering increases proportionally to precipitate

Question 5

ELISA

Answer 5

• enzyme linked immunosorbent assay
• measures antigen or antibody in a patient specimen

antibody detection in patient serum:

1. fix known antigen to well
2. add patient serum => antibody binding
3. add enzyme linked antibody that will bind to patient antibody (ex: anti-IgM)
4. add enzyme substrate => color change if enzyme linked anti-IgM has bound patient IgM

antigen detection in patient serum (sandwich elisa):

1. fix antibody with known specificity to well
2. add patient sample => antigen-antibody binding
3. add enzyme-linked antibody with specificity for antigen
4. add enzyme substrate => color change if enzyme linked antibody has bound patient antigen

Question 6

western blot

Answer 6

used to identify protein specific antibodies in a patient sample

1. separate known proteins (ex: HIV) on a gel
2. transfer to filter paper
3. expose paper to patient sample => patient antibodies will bind paper
4. add colorimetric or chemiluminescent anti-human isotype antibodies (ex IgG) for detection

Question 7

immunofluorescence assays

Answer 7

used to evaluate for auto-antibodies, complement binding to patients own cells, or serum antibodies (titers) => common in kidney disease and autoimmunity evaluation

1. fix known antigen
2. add patient sample => antibody-antigen binding
3. add fluorescently labelled anti-human antibodies
4. confocal microscopy

Question 8

flow cytometry

Answer 8

used for immunotyping WBCs, identifying/quantifying WBC subsets, and evaluating for abnormal proteins

1. cells are passed single file in a fluid stream through a laser beam
2. detectors measure light scattered by cell as well as emitted fluorescence from bound labelled antibodies

Question 9

flow cytometry - determination of intrinsic cell properties

Answer 9

forward scatter => cell size

side scatter => complexity of cell

Question 10

flow cytometry - cell identification

Answer 10

cells reacted with antibodies labelled with fluorescent probes and run through a flow cytometer => evaluation for presence of 6-10 cell surface markers

Question 11

quantitation of antibody isotypes

Answer 11

• nephelometry
• used to evaluate immunodeficiency
• add monoclonal antibodies specific for an antibody isotype (IgG, IgM, or IgA) to a patient sample => immune complex formation and precipitation => scatter

Question 12

flow cytometry - functional assays

Answer 12

normal flow can identify presence of a protein but not functionality….

binding assay:
- fluorescently label CD40 => if T cell CD40L is functional and binds it => fluorescent T cells

NADPH oxidase assay:
- add cell permeable (non-fluorescent) dye to neutrophils => if NAPDH oxidase is active => oxidation of dye makes fluorescent cells