Purification of Lymphocytes & Cell Based Immunoassays
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What are four ways of purification of lymphocytes?
- Centrifugation (buffy coats, density gradients)
- Magnetic bead separation
- Flow cytometry
What is the buffy coat?
Thin layer of WBC after the centrifugation of whole blood
Is the buffy coat pure?
- will have RBC contamination
What is the separation based on in density gradients?
sedimentation rates of cells
What does sedimentation rate of cells correlate to?
What are the (4) steps in magnetic bead separation?
- Small beads are coated with monoclonal antibodies
- Added to blood and incubated
- Magnet is placed near tube and cell rosettes with beads migrate to magnet
- Other cells and serum can be removed
Why is CD8 used to collect lymphocytes by CD3 and CD4 aren't?
CD8 is used to collect lymphocytes because CD3 and CD4 are present in monocytes
What is Panning? (4)
- Monoclonal antibody coated on bottom of petri dish
- Blood/cell suspension placed into petri dish to flood bottom
- Incubation then decant fluid & wash
- Specific cells trapped on bottom of petri dish by monoclonal antibody
What is flow cytometry also known as?
flurenscent activated cell sorting (FACS)
What is flow cytometry? (3)
- Specific antibodies labelled with fluorescent dyes react with blood/cell suspension
- Cells passed through flow cytometer which identifies labelled cells by the use of laser
- Cell becomes charged and can be separated by an electric field
What are the 2 cell based assays?
- Tissue typing
- Functional assays
What is involved in tissue typing?
human leucocyte antigen (HLA)
What is involved in functional assays?
- Lymphocyte proliferation
- Cytotoxic T cell function
Why do we use HLA typing? (5)
- Disease association
- Vaccine development
What is HLA?
- Human Leucocyte Antigen
- cell surface proteins encoded by genes within the MHC
- Inherited co-dominantly
- Belong to Ig super family of proteins
What are the various names for serology based typing? (3)
- Complement Dependent Cytotoxicity
T cells are used for what class?
What are the class I HLA?
HLA- A, B, C
B cells are used for what class?
What are the class II HLA?
HLA - DR, DQ
What is Serology Based Typing?
- Serum contain known specific anti-HLA antibodies in tray wall
- Lymphocytes added to serum
- Rabbit complement added
- Detect killing by addition of eosin dye, ethridium bromide
What is neutrophil function assays? (3)
- Intracellular killing
- Directional migration
What does neutrophil function assays measure?
Measure up-regulation of surface markers using monoclonal antibodies
How does lymphocyte proliferation occur?
Lymphocytes stimulated by antigen/other activators and undergo cell division (in vitro)
How is lymphocyte proliferation measured?
- Measuring release of cytokines by immunoassay (ELISA)
- Incorporation of radio labeled thymidine into DNA of dividing cells
- Incorporation of fluorescent dyes into plasma membrane of daughter cells from parent cell - measure by flow cytometry
Cytotoxic T cells kill target cells. How is the function of cytotoxic T cell measured?
- Labelled with radioisotopes (51Cr)
- Mixed with lymphocytes and incubated
- Release 51Cr into surrounding media - measured T cell ability to kill target
- Similar assay used for NK cells
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