Lab 1: Serological Testing A) Explain the principle of the Direct Fluorescent antibody Technique.

The principle of the direct fluorescent anti body, antibody that is conjugate with a fluorescent tag is added directly to unknown antigen that is fixed to microscope slide. Direct immune-fluorescent assay is best suited to antigen detection in tissue or body fluids while indirect assays can be used for both antigen and antibody identification B) Differentiate on the ouchterlony diffusion test the identity, partial identity and nonidentity C) Differentiate between direct and indirect ELISA testing. In a direct ELISA the antibody bound to the enzyme will be bound directly to the antigen of interest. In an indirect ELISA the antibody that is bound to the enzyme will bind to a different antibody and the different antibody will be bound to the antigen D) Explain the principle of the Western Blot test For HIV antibodies was introduced in 1984 and has been used for systematic conformation of positive ELISA results since 1985. The test is to detect protein in a given sample of tissue homogenate or extract. Western blot kits are prepared commercially as nitrocellulose or nylon strips containing individual HIV proteins that have been separated by polyacrylamide gel electrophoresis and blotted onto the test membrane. The protein antigens are derived from HIV virus grown in cell culture. Antigens with low molecular weight migrate most rapidly and are therefore positioned toward the bottom of the test strip, while antigens of high molecular weight remain toward the top of the membrane. 2. Lab #2 A) What does it mean if only lymphocytes are found on the 5-minute incubation tube with bacteria? There should be a noticeable difference between the 0- and the 5-minute slide.

B) In looking at in-vitro phagocytosis what cells engulfed the bacteria?

If lymphocytes are the only white blood cell seen, the bacterial suspension was too heavy, and the phagocytic cells destroyed themselves in attempting in attempting to engulf the bacteria present.

C) Why did you get different patterns of agglutination when you, mixed serum and cells of individuals together?

D) What is agglutination? The process by which particulate antigens such as cells aggregate to form large complexes when specific antibody is present E) What is an allo antibody? Antibody that reacts with an antigen occurring in genetically different member of the same species F) What is the importance of using antibodies to CD markers in the identification of lymphocytes ? CD molecules can be visualized by immunehisto-chemical methods using monoclonal antibodies and are useful in identifying specific subtypes of lymphatic or hematopoietic cells G) In using histopaque and separating the mononuclear layer, sheeps red cells were added. Describe the rosette in relation to cells and CD receptor. Lymphocytes are separated from whole blood and then mixed with a suspension of sheep red blood cell. If three or more red blood celled are attached to a lymphocyte, it is considered a rosette. Sheep cells attach to the CD2 antigen, found only on T cells. Using a counting chamber, 200 cells are counted and the percent forming rosettes is calculated. This represents the percentage of T cells, and the percent of B cells is calculated by subtracting this number from 100. There should be approximately twice as many T cells as B cells.

H) How would you differentiate between Tc cells, Th cells, and B cells today?

I) What is the interpretation and basic principle of sedimentation rate tests ?

Results from your sed test will be reported in the distance in millimeters red blood cells have descended in one hour. The normal range is 0-22mm /hr for men and 0-29mm/hr for women. The upperthreshold for a normal sed rate value may vary somewhat from one medical practical to another J) Identify the components on the plasma that cause an increased sedimentation rate. 3. Laboratory three: Serological testing for SLE testing for SLE, RPR, Mono, CRP,DD dimer, RF mono: Paul and Bunneli in 1932 discovered that serum samples from patients with IM have heterophile antibodies to sheeps red cells. Detect infectious mononucleosis heterophile antibodies in whole blood, serum and plasma for professional in vitro diagnostic use only. Heterophile antibodies are antibodies that react with different species red blood cells IM is caused by Epstein-Barr virus, which is a member of the herpes virus family. RA: Use of rheumatoid factor to determine/aid the diagnosis of Rheumatoid Arthritis in patient with inflammatory arthritis Mono: The main purpose of this experiment is to detect LE cells using the antinuclear antibodies(ANA) test. The LE cell was discovered by Malcolm Hargraves in 1948. The name lupus is derived from the Latin term meaning wolflike because a butterfly rash across the nose and cheeks may develop. In SLE there is a possible interplay between genetic susceptibility and environmental factors. c) How do B cells become infected with EBV (Epstein Bar Virus)? EBV causes polyclonal activation of B-lymphocytes, it infects and HIV suppresses the function of the CD 4 + T helper cells. d) What is the principle of the CRP test? What is the antigen and the antibody in the test? To test if CRP is active in the serum, which would indicate inflammation or infection in the body. The antigen used was the serum and the antibody was the latex reagent. e) Explain the principle of the D-dimer test. The d-Dimer test is a immunochromatography test used to rule out active blood clot (thrombus) formation. (detects fibrin degradation products)

- monoclonal antibody (specific for D-dimer) - D-dimer (antigen) 4) Laboratory Four: ABO, Rh and Antibody Screen c) Why is ABO typing done at room temperature? d) Why would you incubate the Rh o (D) typing tube if the reaction is negative? e) Explain why one washes the cells 3 times and not one time before addition of AHG serum. f) Why are group O cells used for antibody screening? g) Why do you read antibody screening reactions at 37 C only and then AHG? H) Interpret ABO Rh and antibody screening results.