W11L15 Part 2 - Functional Assays

Question 1

EdU Assay

Answer 1

Alternative to CFSE
Thymidine analog: 5-ethynyl-2-deoxyuridine (Edu)
- add with fluorescent azide
Incorporated into DNA of proliferating cells
Fast and sensitive method
More photo stable than CFSE
Added to cells after stimulation
Gain of signal at end point

Question 2

Ki-67 Expression

Answer 2

Ki-67 is a nuclear protein involved in regulation of cell division
It is expressed in cells that are actively proliferating
Often used to detect cancer cells as they are continuously
proliferating
- usually by immunohistochemistry
Recently used to measure T cell proliferation after vaccination
- T cells should proliferate
Can be detected by flow cytometry
May replace thymidine method for assessing lymphocyte proliferation responses

Question 3

Cytokine Production

Answer 3

Alternative to proliferation assays
T cells produce cytokines in response to stimulation
Typically IL-2, IFN-ɣ, TNF-α
- can be measured internal to the cell by flow-cytometry
- stimulate cells - permeate cells - used labelled Abs to cytokines
Can be measured by release into media if cells cultured
- stimulate cells
- remove media
- measure cytokines by ELISA or bead array

Question 4

Cytotoxic Assays

Answer 4

The ability of Cytotoxic T cells or NK cells to destroy target cells
Labelled target cells mixed with CTL or NK cells
Release of label as target cells are destroyed by CTL or NK cells

Question 5

NK Cell Assay or Chromium Release Assay

Answer 5

K562 cells are labelled with 51Cr, washed to remove excess 51Cr
- K562 cells are a human chronic myelogenous leukaemia cell line that does not express MHC 1
Separate NK cells from blood (gradient, Dyna beads, flow-cytometry)
Mix NK cells with labelled K562 cells, incubate 37°C (various times, ratio of cells)
Centrifuge - remove supernatant
Measure/count 51Cr
More 51Cr = more lysis of target cells

Question 6

Alternative NK Cell Assay

Answer 6

Isolate NK cells (gradient, dynabead, flow cytometry)
K562 cells labelled with CMFDA
Green dye retained in living cells
Mix NK cells and K562
Incubated 37°C (time, ratio of cells)
Can stain NK cells specifically to view (anti CD56 monoclonal Ab)
Measure by flow cytometry
- reduction of cells with dye = killing of target cells

Question 7

Treg Function

Answer 7

Treg express CD4+, CD25high, CD127low, FoxP3+
Suppressing T cell activation and proliferation
Treg inhibit expression of CD69 and CD40L (CD154) on activated T cells
- stimulate T cells with anti-CD3 and anti-CD28 antibodies
- co-culture with Treg (7 hours)
- measure CD69 and CD40L expression by flow cytometry
- compare with cells not co-cultured

Question 8

B Cells

Answer 8

Measurement of B cells by flow cytometry for numbers (CD19, CD20) also measurement of immunoglobulin levels is what is usually done in diagnostic labs
Lymphocyte proliferation assay
- lymphocytes (PBMC) from patient
- culture with agents known to activate B cells (3-7 days)
- measure proliferation of B cells (flow cytometry)
- measure immunoglobulin production (ELISA since levels are low)

Question 9

Antibody Functional Assays - Bactericidal Assays

Answer 9

Patients serum mixed with known amount of bacteria (usually GNB)
Add complement (human or rabbit)
Plate out and count colonies to determine amount of bacteria killed
- use of dyes to quantify bacteria

Question 10

Antibody Functional Assays - Opsonophagocytosis Assay (OPA)

Answer 10

Killing assays involve coating bacteria (usually GPB) with Abs
Add phagocytic cells
Count surviving cells (plating out)
Uptake assays involve labelling bacteria with fluorescent dye and Abs
Add phagocytic cells
Measure number of granulocytes with fluorescent dye by flow cytometry

Question 11

Enzyme-Linked Immunospot Assay (ELISPOT)

Answer 11

Used to detect CD8+ and CD4+ T-cell responses to antigen
- function of cells - indicate number of cells responding
- currently the method of choice when measuring CD8+ and CD4+
function
Antigens from a wide range of infectious agents, vaccines and tumours
Modifications to enhance sensitivity by:
- adding exogenous cytokines
- adding co-stimulatory antibodies
- adding antigen presenting cells
- automatic readers
- multi-colour (fluorospot) assays for simultaneous detection of 2 or more secreted factors

Question 12

ELISPOT - Basic Principle

Answer 12

Antibodies against particular cytokine (INF-ɣ, TNP, IL-2, IL-4), on nitrocellulose membrane in plate well
Cells (PBMC) from patient stimulated with antigen/agent (infectious agent, vaccine, mitogens)
Cells added to well with antibodies
- only some of the cells directly stimulated and so only some produce cytokine
- only certain areas (spots) have cytokine release
Remove cells and proceed as for capture ELISA
- only spots where secreting cells were give positive reaction
Count spots to get estimate of number of cells responding