Cellular Diagnostics - Practical II

Study Notes

Cellular diagnostics is a crucial area in immunology, focusing on detecting single cells.
CD (Cluster of Differentiation) molecules are used to identify specific cell types. These molecules are found on the membrane of cells. CD4 is specific to T Helper cells.
Polyclonal antibodies are a pool of antibodies targeting various epitopes (parts of a protein) on an antigen.
Different cells can be identified by evaluating antigens on the cell surface via the use of antibodies.
Polyclonal antibodies are produced by injecting an antigen into an animal (like a goat or sheep).
- Selecting the right animal is critical to determine the needed amount of antibodies.

Monoclonal antibodies target a single epitope. This process is more involved than polyclonal antibody production.
Identifying a single protein part of an antigen is more complex.
Identifying one part of a molecule (antigen), requires identification of 20 different epitopes from molecules from different species.
Cells from spleen (immunized mouse) are fused with cancer cells (myeloma cells).
The fused cells, called hybridomas, are able to produce and multiply continuously. Cancer cells (myeloma) are immortal.
Positive positive hybridomas are identified to detect the production of antibodies.
Selected clones of hybridomas are propagated to develop a large population of identical cells.
These antibody-producing cells are frozen for later use.

Antibodies conjugated with fluorescent dyes bind to specific CD molecules on cell surfaces.
This process allows for identification of targeted cells within a heterogeneous sample.
- Antibodies against a specific CD molecule can be conjugated with a florescent dye.
- This is added to a cell with that CD molecule.
- The florescent antibody is then attached to the CD Molecule, allowing it to be identified.
The cell is then labeled, showing which cell has the specific CD molecule, allowing the user to differentiate cells with differing CD Molecules.

Enzymes are linked to antibodies to amplify the detection signal.
Primary antibodies recognize the target antigen.
Secondary antibodies recognize primary antibodies and are coupled to the enzyme.
A chromogen, with an enzyme, develops a color reaction.

Dye-based, fluorescence-based, and luminescence-based assays are used for diverse applications.
Radioactive methods are also possible, for more detailed detection of cells.

Flow cytometry is a technique that analyzes and sorts cells based on their physical and chemical characteristics via single-file passage through a beam of light emitted from a laser or an impedance-based technology.
Cells are suspended in a fluid and passed through a laser beam.
Forward scatter indicates size, side scatter indicates complexity.
Fluorescence from labeled antibodies is measured.
Data can be used for cell counting and sorting.

Cells separated by characteristics such as size, charge, fluorescence, and color can be analyzed individually, based on the above information.

The cell identification process can continue with observation of tissues. A tissue sectioned into a slide is stained in order to identify antigens.
- Staining can be direct or indirect.
- Direct testing uses labeled antibodies to bind directly to the antigen of interest.
- Indirect testing uses unlabeled antibodies to bind to the antigen. This is followed by labeled antibodies specifically targeting those unlabeled antibodies.
Tissue sections are labeled according to which antigen the antibody is designed for.

Lymphocytes are extracted from blood samples using Ficoll density gradient centrifugation to remove other blood components (granulocytes, RBCs).
Different layers in the purification tube contain the target cell.