Dual- and Triple-Co... （一）

实验概要

Cytokine ELISPOT  has become a powerful routine tool for the analysis of disease- as well  as vaccine-induced T-cell responses. The method is limited, however, in  that only one cytokine at a time is assessed. Fluorospot is based on the  principle of ELISPOT, but facilitates the analysis of single cells  secreting several cytokines, e.g., polyfunctional T cells, suggested to  be of protective importance in various infectious diseases. By detecting  each cytokine with a specific fluorophore and analyzing differentially  colored spots by fluorophore-­specific filter systems, cells producing  single or multiple cytokines are identified. Fluorospot maintains the  simplicity and sensitivity of the ELISPOT while taking the analysis a  step forward toward multiplex analysis.

实验原理

ELISPOT has, due  to its high sensitivity and simplicity, proven ­valuable for assessing  antigen-specific T-cell responses, both with regard to specificity and  magnitude. One limitation of the regular ELISPOT method is, however,  that only one cytokine at a time is measured. Still, in many settings,  it is desirable to measure the ­production of multiple cytokines in a  single well. For example, in studies of HIV, TB, and malaria infection  or in the development of vaccines against these and other diseases,  enumeration of antigen-specific T cells secreting, e.g., IFN-γ, may not  yield a complete picture of the quality of the immune response. Recent  studies in the field have highlighted the importance of polyfunctional T  cells that secrete multiple cytokines. The ability of CD4  and CD8  T  cells to respond to antigen with a combination of, e.g., IFN-γ, IL-2,  and TNF-α, rather than only one of the cytokines, has been associated  with enhanced protective immunity in viral, ­bacterial, as well as  parasitic diseases (1–4).

An ELISPOT-based assay analyzing multiple cytokines would not only be  useful for defining polyfunctional T cells, it may as well be used for  simultaneous measurement of functionally distinct cell populations of  various types, e.g., T-cell populations, predominantly secreting single  key cytokines representing, e.g., Th1, Th2, Th17, or Tregs. The  possibility to measure multiple cytokines simultaneously in the same  well also has other advantages, such as a need for less sample cells,  valuable, e.g., in studies of mucosa-derived cells or studies on  newborns and children (5).

Over the years, efforts to broaden the ELISPOT technique to include  staining with substrates of two colors have been made, first for B cells  secreting different Ig isotypes (6) and later for analysis of cytokines  (7). Although successfully used in several studies (8, 9), the dual  ELISPOT technique can be technically challenging to perform, suffers  from several inherent analytical difficulties, and is limited to the  analysis of two cytokines.

Building on the principle of ELISPOT, but using detection based on  fluorescence instead of substrates, the fluorospot assay was developed  (10). At present, two-color fluorospot has been described in several  publications and reagents/kits for various cytokine combinations and  species are commercially available. The most common protocol for  fluorospot includes the use of biotinylated detection antibodies for one  cytokine and an FITC-labeled detection antibody for the other cytokine  (Fig. 1). As a second step in the detection, streptavidin conjugated to a  red fluorophore (Cy3) and anti-FITC antibodies labeled with a green  fluorophore, respectively, are used (10, 11). The resulting spots are  subsequently analyzed using an automated reader equipped with filters  for FITC and Cy3.

Fluorescent detection  can be as sensitive as ELISPOT, or even more sensitive, and offers  several advantages compared to dual ELISPOT. Most prominently, by using  readers equipped with several narrow-band fluorophore filters, spots  derived from cells secreting multiple cytokines are identified by the  colocalization of single-colored spots in an overlay analysis of images  from different filters (Fig. 2). Importantly, this enables the analysis  of not only two, but also three and potentially even more cytokines  simultaneously.

Experimental systems for triple-color fluorospot have been described (12),  but the method may need further development before commercial reagents  become available. The major ­limitation is the availability of  additional amplification systems compatible with, e.g., FITC/anti-FITC  and biotin/avidin and, in particular, automated readers designed for the  analysis of three-colored spots. One amplification strategy that has  been evaluated is to use a third detection antibody from a unique  species that can be detected by fluorophore-labeled species-specific  anti-Ig antibodies, reactive only with the third detection antibody (12).  However, in a wider perspective, this may be a limiting factor since  most highly ­functional antibody pairs are derived from a few species.  Another ­strategy being employed for triple staining is the use of  additional tag/anti-tag systems other than FITC/anti-FITC (Fig. 1).