A universal nanoparticle cell-secretion capture assay for the study of HIV-1 infected

tissues.

A new tool to identify cytokine secreting cells

Wendy Fitzgerald and Jean-Charles Grivel

The Eunice Kennedy-Shriver National Institute of Child Health and Human Development, Bethesda, MD.

Identification of secreting cells

ELISPOT

Intracellular cytokine staining

Exocytosis assay

These assays have flaws:

They kill the cells being studied.

Cell fixation and permeabilization interfere with downstream molecular work.

They do not distinguish between pre-stored cytokines and secreted cytokine.

Elispot, doesn’t provide information on cell phenotype. 

Exocytosis is limited to certain cell types and doesn’t reveal the nature of the cytokine secreted.

The ideal assay:

Captures the secreted cytokine before it diffuses away from the producing cells.

Is compatible with cell isolation and culture.

Some assays have been developed:

Agarose cell encapsulation (One cell systems) Requires dedicated instrumentation to encapsulate the cells

Bi-specific antibody affinity matrix (Miltenyi Biotech)

Nanoparticle-based affinity matrix

Cell surface anchor

Cytokine of interest

Cytokine detection antibody

Cytokine capture antibody

Cell surface specific antibody

Nanoparticule

Functionalized Magnetic Nano Particles: MNPs

20 minutes

Cell and cytokine bi-specific affinity matrix

Cell surface targeting Ab

Secreted protein

capture Ab

+

Principle for the construction of an affinity matrix

Magnetic separation

10-20 minutes !

An

ti-C

D3

lab

eled

NC

CD3 Surface staining

Isot

ype

con

trol

Nanoparticles can be used to form an affinity matrix on the cells surface

CD3-MNPs

CD3 surface label

CD3 + Isotype MNPs

CD3 surface label

CD3-MNPs CD3 + Isotype -MNPs

IL-2

CD8

14.7%18.7%19.4%

IFN

-g

Capture assay

9.5%12%20%

Intracellular assay Commercial assay

Comparison of capture assay, intracellular cytokine staining and commercial secretion assay

19.4%

MIP-1b

13.7%

RANTES

16.4%

MIP-1 a

Capture of cytokine secretion:Non-commercially available assays

CD8

Cyt

okin

e

Separation on centrifugal concentration units

Pall Nanosep 300K : Pore opening ± 35nm

Underivatized nanoparticles are not retained

IgG Antibodies are not retained

Complexed nanoparticles are retained

Allows the use of non-magnetic nanoparticles, such as Qdots

15 nm MNPs CD45 Targeted

Qdots GAMCD45 Targeted

Comparison of several nanoparticles

CD45 + IL-2 MNPs

Labeled anti-IL-2 Ab

Labeled anti-IL-2 Ab

CD45 + IL-2 Qdot

Non-Activated cells Activated cells

ON Qdots Invitrogen Qdots15 nm MNPs

CD8

IL-2

11%

CD8

IL-2

QDot 620 (IL-2/CD45 QDot 620)

CD

45 (

surf

ace)

Qdots allow simultaneouscell labeling and cytokine capture

Cell labeling Cytokine capture

The cytokine capture assay can be multiplexed

IL-2

IFN-g

ConclusionsWe have designed a simple, fast and inexpensive method to capture and analyze cell secretion by flow cytometry.

This method allows the detection on virtually any secreted protein on any cell provided that antibodies are available.

Qdots can be used to form the affinity matrix allowing the simultaneous detection of the targeted population and the secreted protein.

We have measured Il-1a, Il-1 ,b IL-2, IL-17, MIP-1a, MIP-1b, RANTES, TNF-a, TGF-b, IFN-g

Flexibility in the choice of cell targeting reagents.

The capture assay can be multiplexed for the detection of several cytokines at once.

Thanks to

Wendy Fitzgerald

Leonid Margolis