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Flashcards in Immunodiagnosis Deck (31):
1

What is serum protein electrophoresis?

Measures globulin proteins in the blood (tests how humoral portion of immune system is doing)

2

Procedure for serum protein electrophoresis

1) blood collected
2) serum (fluid after blood clots) placed on paper with agarose gel
3) current passed through to separate serum into 5 classes

3

5 classes of serum proteins

serum albumin
alpha-1 globulins
alpha-2 globulins
beta globulins
gamma globulins

4

Adv/Disadvantage of serum protein

Adv: Cheap, easily quantify

Disadv: not sensitive to small abnormalities (IgA deficiency b/c IgA runs with IgG gamma glob)

5

Serum protein electrophoretic pattern:
Selective IgA deficiency

would not pick up this b/c IgA runs together with IgG (gamma)

6

Serum protein electrophoretic pattern:
Multiple myeloma

cancer of B cells

urine electrophoresis shows Bence Jones proteins (free immunoglob light chains)

7

Serum protein electrophoretic pattern:
Severe pyogenic infection

Polyclonal hypergammaglobulinemia

Looks just like multiple myeloma (peak at albumin and gamma)

8

Serum protein electrophoretic pattern:
Hypogammaglobulinemia

underproduction of IgG

9

Serum protein electrophoretic pattern:
Agammaglobulinemia

no IgG production

10

Serum protein electrophoretic pattern:
monoclonal hypergammaglobulinemia

1 B cell going crazy, making exact same antibody

11

Serum protein electrophoretic pattern:
multiple sclerosis

look at CSF

see oligoclonal (few B cell clone) peaks in IgG b/c a few clones enter CNS and making antibodies

12

What is single radial immunodiffusion?

Measures levels of individual immunoglobulin class

Can measure any other multivalent antigen (can form precipitate w/ appropriate antibody)

13

Examples of multivalent antigen

individual complement/clotting components

14

How to run single radial immunodiffusion?

1) gel contains rabbit anti-serum to human IgA (specific for alpha heavy chains)

2) check if patient's serum (single radial immune diffusion) match which IgA standard

15

What is mixed cryoglublin test?

immune complexes in serum are insoluble in cold

If type 3 suspected, put serum sample in fridge and wait 1-7 days for precip

16

What is RF test?

Based on agglutination of latex particles if coated with IgG (passive agglutination)

17

What is anti-nuclear antibodies?

1) humans cells on slide
2) permeabilize plasma membrane so Abs can enter
3) add patient serum
4) add anti-human IgG

18

What is direct immunofluorescence?

1) pt tissue on slide
2) add fluorescent antibodies to known bacterial antigens

tests for bacterial antigen

19

What is indirect immunofluorescence?

1) known bacteria on slide
2) add pt serum
3) wash and add fluorescent anti-human Ig

test for antibody

20

What is purpose of passive agglutination and precipitation?

Measure amount of specific antibody in patient's serum

21

Procedure for precipitation

1) Mix patient's serum with antigen and look for precip

22

Disadvantage of precipitation

very insensitive

23

Why is passive agglutination better than precipitation?

Sensitivity increases as size of antigen increases

You couple antigen to RBCs or beads and add patient's serum

Look for highest dilution that still produces agglutination

24

What is ELISA?

1) add antigen to plate
2) add pt's serum
3) if ab in pt serum, it will bind and you can visualize with fluorescent antibody to pt antibody

DETECTS ANTIBODY

25

What is sandwich or capture ELISA?

DETECTS ANTIGEN (MUST BE AT LEAST DIVALENT)

1) add monoclonal ab to plate
2) pour in pt serum (antigen binds to ab on plate)
3) wash and add second ab that binds a diff epitope on antigen (antigen = sandwich)

second ab is couple to perodxiase to visualize using spectrophotometer

26

How to evaluate T cell immunocompetence?

Best test for Th1 = skin test with common antigens for delayed hypersensitivity

Read 24-48 hrs after skin test

Challenge DTH using dinitrofluorobenzen
98% of T cells sensitized to DNFB in 10 days

27

What happens with positive skin test to tuberculin?

1) antigen endocytosed by APC
2) antigen present on MHC II
3) Th1 anti-TB cells come by and recog antigen + MHC II
4) Th1 cells activated, secrete IFN-g
5) Macrophages attracted/activated

28

How to count T cell #s?

1) add mAbs to CD3 or CD4 or CD8 and count fluorescent cells using UV or flow

29

What is flow cytometry?

1) cells organized into single file stream
2) shoot lasers at cells
3) light scatter = size/cytoplasmic granularity

Quantify light and count # of cells with diff markers (mAb to CD8/CD4/CD3)

30

How to test T cell function?

1) Stimulate T cells in mononuclear leukocyte prep (lymphocytes + monocytes) with T cell mitogens PHA or Con A

2) Observe production of IL-2, IL-4, IFN-g

31

What organ

CD8 and CD4
CD8+/CD4+ cell = thymus

KNOW FOR EXAM!!