Immunodiagnosis

Question 1

What is serum protein electrophoresis?

Answer 1

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

Question 2

Procedure for serum protein electrophoresis

Answer 2

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

Question 3

5 classes of serum proteins

Answer 3

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

Question 4

Adv/Disadvantage of serum protein

Answer 4

Adv: Cheap, easily quantify

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

Question 5

Serum protein electrophoretic pattern:

Selective IgA deficiency

Answer 5

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

Question 6

Serum protein electrophoretic pattern:

Multiple myeloma

Answer 6

cancer of B cells

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

Question 7

Serum protein electrophoretic pattern:

Severe pyogenic infection

Answer 7

Polyclonal hypergammaglobulinemia

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

Question 8

Serum protein electrophoretic pattern:

Hypogammaglobulinemia

Answer 8

underproduction of IgG

Question 9

Serum protein electrophoretic pattern:

Agammaglobulinemia

Answer 9

no IgG production

Question 10

Serum protein electrophoretic pattern:

monoclonal hypergammaglobulinemia

Answer 10

1 B cell going crazy, making exact same antibody

Question 11

Serum protein electrophoretic pattern:

multiple sclerosis

Answer 11

look at CSF

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

Question 12

What is single radial immunodiffusion?

Answer 12

Measures levels of individual immunoglobulin class

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

Question 13

Examples of multivalent antigen

Answer 13

individual complement/clotting components

Question 14

How to run single radial immunodiffusion?

Answer 14

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

Question 15

What is mixed cryoglublin test?

Answer 15

immune complexes in serum are insoluble in cold

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

Question 16

What is RF test?

Answer 16

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

Question 17

What is anti-nuclear antibodies?

Answer 17

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

Question 18

What is direct immunofluorescence?

Answer 18

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

tests for bacterial antigen

Question 19

What is indirect immunofluorescence?

Answer 19

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

test for antibody

Question 20

What is purpose of passive agglutination and precipitation?

Answer 20

Measure amount of specific antibody in patient’s serum

Question 21

Procedure for precipitation

Answer 21

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

Question 22

Disadvantage of precipitation

Answer 22

very insensitive

Question 23

Why is passive agglutination better than precipitation?

Answer 23

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

Question 24

What is ELISA?

Answer 24

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

Question 25

What is sandwich or capture ELISA?

Answer 25

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

Question 26

How to evaluate T cell immunocompetence?

Answer 26

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

Question 27

What happens with positive skin test to tuberculin?

Answer 27

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

Question 28

How to count T cell #s?

Answer 28

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

Question 29

What is flow cytometry?

Answer 29

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)

Question 30

How to test T cell function?

Answer 30

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

Question 31

What organ

Answer 31

CD8 and CD4
CD8+/CD4+ cell = thymus

KNOW FOR EXAM!!