Immunodiagnosis

Question 1

Describe the procedure used in serum protein electrophoresis, and the underlying principles

Answer 1

At pH 8.2, most –COOH groups are –COO- (ionized,) and most NH3+ are NH2 (not ionized)
Many proteins have a net negative charge and will migrate towards an anode.
Serum is separated in an electrical field, the proteins segregate into an albumin and several globulin bands.
Ab is found mostly in the gamma (γ) globulin zone (some in beta, too)

Question 2

Discuss the serum protein electrophoretic pattern which would be expected if a patient:
A. was normal
B. had selective IgA deficiency
C. had multiple myeloma
D. had severe pyogenic (pus-producing) infections
E. was hypogammaglobulinemic

Answer 2

A. Big narrow tall peak for Albumin, then 3 smaller bumps for a1, a2, and B, followed by a relatively short broad peak gamma.
B. Would still look normal because IgA runs pretty much together with the much larger IgG
C. Gamma would have a sharp tall peak (monoclonal hyper)
D. High a regions? esp a2?
E. Gamma would be very short (or even absent), still broad tho

Question 3

Discuss single radial immunodiffusion, with regard to the types of antigens that can be quantified with it, and the way that quantization is done.

Answer 3

Can be used to measure any other MULTIVALENT antigen (one that can form a precipitate with an appropriate antibody).

Compare to standards with known conc (mg/dL)

Question 4

Describe tests that are used for determining if a patient has antibody to a soluble or particulate antigen.

Answer 4

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Question 5

Distinguish between direct and indirect immunofluorescence techniques.

Answer 5

Direct is good to test for Ag (Ag coat, fluorescent primary Ab). Take swab of pt throat, add special Ab

Indirect has extra step. Ag –> Serum –> Sec. Fluor Ab

Question 6

Discuss the advantages of passive agglutination (e.g., with antigen-coated latex particles) over precipitation, and outline the technique.

Answer 6

HA assay, use big particle so it’s more sensitive

Question 7

Describe in principle the ELISA test. Diagram the reactions involved when the ELISA is used to measure antibody, and to measure antigen.

Answer 7

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Question 8

Describe a test which can be used to evaluate T cell immunocompetence in a clinic or on the ward.

Answer 8

1. Th1: Skin test with common antigens to which most people will have DTH. A good set is: Candida, streptokinase/streptodornase (SK/SD,) trichophytin, mumps, tetanus, tuberculin. Read at 24-48 hours.
2. Challenge DTH test: over 98% of normals will become “sensitized” (immunized) to dinitrofluorobenzene in about 10 days if it’s painted on their skin. This is like intentionally inducing poison ivy.
3. Stimulate T cells in mononuclear leukocyte preparations (lymphocytes + monocytes) with the T cell mitogens PHA or Con A, and observe either proliferation or IL-2, IL-4, or IFN production.
4. In infants, CXR for thymus (massive!)
5. Lymphoid biopsy may be necessary in suspected primary immunodeficiency. A biopsy of rectal mucosa, though it sounds nasty, is often less traumatic to the patient.
6. Killer cell assays are done in research lab

Question 9

Describe tests to evaluate T cell numbers and function in the lab. Describe flow cytometry.

Answer 9

Tag cell w/ fluoro Ab
Machine runs cells thru & shines laser on ‘em
Can do multiple Abs at once
Plots results w/ intensity