Antigen-Antibody Interactions: Selected Tests (Contd.)

5. Tests for Cell Associated Antigens Immunofluorescence – Immunofluorescence is a technique whereby an antibody labeled with a fluorescent molecule (fluorescein or rhodamine) is used to detect the presence of an antigen in or on a cell or tissue by the fluorescence emitted by the bound antibody. The emitted light can be viewed with fluorescence microscope, which is equipped with a UV light source. Fluorescein, absorbs blue light (490 nm) and emits an intense yellow-green (517 nm). Rhodamine, absorbs in the yellow-green range (515 nm) and emits a deep red fluorescence (546 nm). Fluorescent-antibody staining of cell membrane molecules or tissue sections can be: a) Direct b) Indirect

a) Direct Immunofluorescence – In direct immunofluorescence the antibody specific to the antigen is directly tagged with the fluorochrome. Cut thin sections of tissue, Cells are affixed to a microscopic slide. Probe with labelled antibody. Wash away unbound labelled Ab. Add substrate look for colour development or examine under UV microscope

b) Indirect Immunofluorescence – In indirect immunofluorescence, the antibody (primary) specific for the antigen is unlabeled. cut thin sections of tissue, cells are affixed to a microscopic slide. cells are incubated with unlabeled antibody wash away unbound Ab and stained with a fluorochrome- labeled secondary antibody that binds to the primary antibody. Indirect fluorescence is more sensitive than direct immunofluorescence since there is amplification of the signal. Application: the localization of antigens in tissue sections or subcellular compartments. to identify a number of subpopulations of lymphocytes, notably the CD4+ and CD8+ T-cell subpopulations. for identifying bacterial species, detecting Ag-Ab complexes in autoimmune disease, detecting complement components in tissues, and localizing hormones and other cellular products stained in situ.

2. Western Blotting The technique for the Identification of a specific protein in a complex mixture of proteins. In this technique a protein mixture is treated with SDS, a strong denaturing detergent which also provide net negative charge to protein molecules. proteins/antigens are then separated by electrophoresis in an an SDS polyacrylamide gel (SDS-PAGE), which separates the components according to their molecular weight. The gel is removed from the apparatus and applied to a protein-binding sheet of nitrocellulose or nylon. The proteins in the gel are transferred to the sheet by the passage of an electric current. The individual protein bands are identified by flooding the membrane with radiolabeled or enzyme-linked polyclonal or monoclonal antibody specific for the protein of interest. Radiolabeled --- identified by exposing the membrane to a sheet of X ray film ----- autoradiography. Enzyme labeled ---- identified by colored product at the site of reaction

Western Blotting Applications: identify the components within a complex mixture. also determine their molecular weights. can detect immunologically similar components (breakdown products or related proteins) can also identify a specific antibody in a mixture. e.g- confirmatory testing for HIV

3. Flow Cytometry (Cell Sorting and Counting) Flow cytometry is commonly used in the clinical laboratory to identify and enumerate cells bearing a particular antigen. Cells in suspension are labeled with a fluorescent tag. The cells are then analyzed on the flow cytometer. Principle In a flow cytometer the cells exit a flow cell and are illuminated with a laser beam. The amount of laser light that is scattered off the cells as they passes through the laser can be measured, which gives information concerning the size of the cells. In addition, the laser can excite the fluorochrome on the cells and the fluorescent light emitted by the cells can be measured by one or more detectors. It can analyze cell populations that have been labeled with two or more different fluorescent antibodies. fluorescein-tagged antibody specific for T cells phycoerythrin-tagged antibody specific for B cells

The type of data that is obtained from the flow cytometer is: In a one parameter histogram, increasing amounts of fluorescence (e.g. green fluorescence) is plotted on the x axis and the number of cells exhibiting that amount of fluorescence is plotted on the y axis. The fraction of cells that are fluorescent can be determined by integrating the area under the curve. In a two parameter histogram the x axis is one parameter (e.g. red fluorescence) and the y axis is the second parameter (e.g. green fluorescence). The number is cells is indicated by the contour and the intensity of the color.

Fluorescence-activated cell sorter (FACS)

Applications of Flow Cytometry: A common clinical use is to determine the kind and number of white blood cells in blood samples. Flow cytometry occupies a key position in immunology and cell biology as an indispensable clinical tool. The flow cytometer is one of the essential tools for the detection and classification of leukemias. The choice of treatment for leukemia depends on the cell types involved, and precise identification of the neoplastic cells is an essential part of clinical practice. The rapid measurement of T-cell subpopulation is routinely done by flow-cytometric analysis as an important prognostic indicator in AIDs. Labeled monoclonal antibodies against T-cell subtypes bearing CD4 and CD 8 antigens ---- are used to determine their ratios (CD4/CD8 cells) in the patients blood. The number of CD4 T cells falls below a certain level, the patient is at high risk for opportunistic infections.

Cluster of Differentiation/Designation (CD) Differentiation antigens, surface molecules of leukocytes. Expressed by leukocytes at distinct stages of differentiation having different functions. CD number was assigned according to the invention during generation of many monoclonal antibodies against epitopes of these cell surface markers. Uses of CD: The CD system is commonly used as cell markers, allowing cells to be defined on what molecules are present on their surface (identification and investigation). Most of CD molecules are critical in antigen recognition. CD molecules can act as receptors or ligands. Some CD proteins have the property of cell adhesion.

CD molecules are utilized in cell sorting using Flow Cytometry. Cell population are usually defined as ‘+’ or ‘–’ symbol to indicate whether a certain cell fraction expresses or lacks a CD molecule. e.g. a “CD34+, CD31–” cell --------- cells expressingCD34 but not CD31 This CD combination corresponds to a Stem cell. Type of cell CD markers stem cells CD34+, CD31– all leukocyte groups CD45+ Granulocyte CD45+,CD15+ Monocyte CD45+,CD14+ T lymphocyte CD45+,CD3+ T helper cell CD45+,CD3+,CD4+ Cytotoxic T cell CD45+,CD3+,CD8+ B lymphocyte CD45+,CD19+ or CD45+,CD20+ Thrombocyte CD45+,CD61+ Natural killer cell CD16+,CD56+,CD3-

Stem cell Granulocyte Monocyte T Lymphocyte B Lymphocyte Helper T cell Thrombocyte Helper T cell Cytotoxic T cell