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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
Panning
Flow cytometry
What is the buffy coat?
Thin layer of WBC after the centrifugation of whole blood
Is the buffy coat pure?
No
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?
cell size
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?
Neutrophils
Lymphocyte proliferation
Cytotoxic T cell function
Why do we use HLA typing? (5)
Transplantation
Disease association
Forensic/Paternity
Anthropology
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)
Mircolymphocytotoxicity
Complement Dependent Cytotoxicity
Lymphcytotoxicity
T cells are used for what class?
class I
What are the class I HLA?
HLA- A, B, C
B cells are used for what class?
Class II
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)
Phagocytosis
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 (
51
Cr)
Mixed with lymphocytes and incubated
Release
51
Cr into surrounding media - measured T cell ability to kill target
Similar assay used for NK cells
Author
J.Seet
ID
247259
Card Set
Purification of Lymphocytes & Cell Based Immunoassays
Description
Purification of Lymphocytes & Cell Based Immunoassays (Immunobiology)
Updated
11/17/2013, 6:49:08 AM
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