Immunological assays

Question 1

Function of adjuvant

Answer 1

Adjuvants increase immunogenicity by slowly (and continuously) releasing an antigen (e.g., from a water/oil emulsion) and/or by nonspecifically stimulating antigen-presenting cells and production of costimulatory molecules and cytokines

Question 2

Adjuvant that trap antigens and form a depot in tissue from which antigen is slowly released

Question 3

Adjuvant used in used in the Cervarix papilloma vaccine

Answer 3

Monophosphoryl lipid A

Question 4

Factors that impact immunogenicity of an antigen

Answer 4

Question 5

Factors that influence an immune response

Answer 5

Question 6

How is selection achieved for monoclonal antibody production

Answer 6

Spleen cells from exposed rat and myeloma tumor cells lacking HGPRT

Question 7

Answer 7

Question 8

What is quantitative precipitation

Question 9

Three zones of antigen titration curve

Answer 9

Ab excess: not enough Ag has been added to precipitate all of the Ab

Equivalence: equal proportions of Ag and Ab, yielding optimal precipitation

Ag excess: too much Ag has been added; free Ag exists in supernatant

Question 10

What is radial immunodiffusion

Answer 10

Antigen (e.g., patient’s serum) is added to the center well in an agar plate containing specific antibody (e.g., anti-IgM). Antigen diffuses out of the well into the agar, establishing a concentration gradient. Precipitation occurs at the site where the antigen concentration in the gel reaches equivalence with the antibody concentration.

Diameter squared is directly proportional to Ag concentration

Question 11

What is Double Immunodiffusion (Ouchterlony test)

Answer 11

This technique is employed to measure the presence of antigen or antibodies and has been used to detect immunoglobulins and antibodies to extractable nuclear antigens (ENAs). In this technique, wells in an agar plate are filled with either an antibody or an antigen. In the outside wells serum from a goat, pig, cow, rabbit, and horse are added. One of the wells also contains cow albumin as a control. Both antigen and antibody diffuse toward each other and precipitation occurs where both attain the equivalence point.

Question 12

What is nephelometry

Question 13

Direct, passive, and reverse passive agglutination

Answer 13

Direct Agglutination : Tests for the reaction of soluble antibodies against antigens present on the surface of bacteria or cells. Blood group testing uses this technique.

Passive Agglutination: Test the patient’s serum for the presence of antibodies against antigens not normally present on the surface of cells, but that have been artificially bound to particles. Some screening test for syphilis, such as the VDRL or the RPR (detecting cardiolipin) use this technique.

Reverse Passive Agglutination: Tests the patient’s serum for the presence of a soluble antigen using insoluble particles coated with antibodies.

Question 14

Direct (noncompetitive) ELISA

Answer 14

1. Antigen is bound to wells, patient serum added (if antibodies, will bind to antigen)
2. Anti-human antibodies added with enzyme attached (often horseradish peroxidase)
3. Substrate of enzyme is added added
4. Light emmision or color is detected

Question 15

Two-site or Sandwich ELISA

Answer 15

The above technique can be adapted for detection of antigens in serum by coating a plate with antibody, adding the serum specimen, washing, and adding a second enzyme-conjugated antibody reactive with the same antigen. The antibody on the plate and the labeled antibody must react with different epitopes on the antigen in order for this to work.

Question 16

Competitive ELISA or RIAs

Answer 16

This extremely sensitive technique can detect picogram quantities of antigen. It is used for the assay of hormone levels, drug (e.g., morphine) levels, vitamins, viral proteins (Hepatitis B virus) etc. The basic principle is a competitive binding between a standard amount of a labeled antigen and unlabeled antigen (in the patient’s sample) to a high affinity antibody bound to the plate. Alternatively, a different format utilizes an antigen bound to the plate. Here, the ability of a labeled antibody to bind the immobilized antigen can then be competitively inhibited by soluble unlabeled antigen (in the patient’s specimen). The greater the concentration of the antigen in the patient’s sample, the lower the signa

Question 17

Direct Immunofluorescence

Question 18

Indirect Immunofluorescence

Answer 18

In this case, the cells are incubated with an unconjugated primary antibody (e.g., mouse anti-human CD4), washed, incubated with a fluorochrome-labeled secondary antibody (e.g., anti-mouse IgG-fluorescein), then washed again and analyzed.

Question 19

Immunohistochemistry

Question 20

Marker?

Answer 20

Question 21

Delayed-type hypersensitivity (Skin testing)

Question 22

T cell Proliferation test

Question 23

Cytotoxicity test

Answer 23

The cytotoxic activity of CD8 T cells or NK cells can be tested in vitro. Target cells (a virus infected cell line with the appropriate MHC class-I alleles for CD8 T cells or an NK-sensitive tumor cell line for NK cells are pre-incubated with radioactive chromate (Na51CrO4). This “loads” the cytoplasm with 51CrO4 . The labeled target cells are then incubated with peripheral blood mononuclear cells for 4-6 hours, after which the amount of 51CrO4 released into the culture supernatant is determined.

Question 24

What stimulates B cell proliferation?

Question 25

Nitro-Blue Tetrazolium (NBT) test

Question 26

Adhesion molecule expression disorders and test

Question 27

erythrocyte sedimentation rate (ESR)