Immunological assays Flashcards
(27 cards)
Function of adjuvant
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
Adjuvant that trap antigens and form a depot in tissue from which antigen is slowly released
Adjuvant used in used in the Cervarix papilloma vaccine
Monophosphoryl lipid A
Factors that impact immunogenicity of an antigen
Factors that influence an immune response
How is selection achieved for monoclonal antibody production
Spleen cells from exposed rat and myeloma tumor cells lacking HGPRT
What is quantitative precipitation
Three zones of antigen titration curve
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
What is radial immunodiffusion
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
What is Double Immunodiffusion (Ouchterlony test)
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.
What is nephelometry
Direct, passive, and reverse passive agglutination
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.
Direct (noncompetitive) ELISA
- Antigen is bound to wells, patient serum added (if antibodies, will bind to antigen)
- Anti-human antibodies added with enzyme attached (often horseradish peroxidase)
- Substrate of enzyme is added added
- Light emmision or color is detected
Two-site or Sandwich ELISA
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.
Competitive ELISA or RIAs
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
Direct Immunofluorescence
Indirect Immunofluorescence
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.
Immunohistochemistry
Marker?
Delayed-type hypersensitivity (Skin testing)
T cell Proliferation test
Cytotoxicity test
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.
What stimulates B cell proliferation?
