Biomedical Engineering Reference
In-Depth Information
isotopes can be detected simultaneously without any overlap between their resulting
mass signatures. In addition, their signal response in the CyTOF platform is within a
twofold difference from the best channel to the worst channel suggesting that they
could all be used relatively interchangeably.
15.7.2.1 Reagents
The affinity reagents used (e.g., antibodies) are labeled with
chelating polymers that can then be loaded with the desired elemental salt for mass
measurement [32]. The prototype tags that are currently available employ an acrylate
polymer backbone functionalized with chelators (e.g., diethyl-triamine pentacetic
acid (DTPA))
that
specifically bind trivalent heavy metal cations
tightly
10
16
M). The current generation of tag is designed for attachment to
antibodies, linking to the reduced disulfide bridges of the antibody Fc chains. For
this purpose, current commercial reagents are maleimide derivatized and,
consequently, the labeling procedures are similar to those used in labeling
antibodies with fluorophores. Therefore, it is anticipated that all affinity reagents
compatible with conventional cytometry platforms should translate into the mass
cytometry system.
It has already been demonstrated that the approach of using polymer-coupled
metal ligands can be used to effectively label antibodies [28, 31, 33]. Furthermore, the
signal generated by each metal isotope tag is highly and linearly correlated with the
amount of antigen detected, making these reagents at least as sensitive and quanti-
tative as radio-immuno or other colorimetric assays with the added benefit of high
multiplexing capacity [34].
With respect to sensitivity, the polymers produced to date bind approximately
30 atoms of metal per polymer label and 4-8 polymer tags can be linked to each
antibody without loss of function. This results in approximately 120-240 metal
isotope atoms per antibody. With the current mass cytometry efficiency of
approximately 1 ion detected per 4
(K
d
10
4
atoms introduced to the plasma, the
current limit of detection is as low as 100-400 copies of antigen per cell.
Certainly, the number of metal atoms attached to each detection reagent will
be a driving force behind the mass cytometry platform sensitivity. Increasing the
number of metal ions per tag will likely involve synthesizing either longer
polymers than those used now or polymers that are more densely functionalized
with metal atoms.
The other major reagent, which has to be considered in the case of cell analysis, is a
general cellular stain. Because of the lack of parameters such as forward and side light
scatter used in conventional fluorescence-based cytometry and the fact that the
majority of elemental ions inherent in biological substances are filtered out prior
to analysis, a cell negative for the antigens of interest in an experiment would appear
“invisible” in mass cytometry analysis. To address this, Rh (103 Da) and Ir (191 and
193 Da) DNA intercalators have been developed for the baseline detection of a cell in
the instrument, as well as to provide some information about DNA content [33]. The
algorithm currently used for the detection and extraction of cell-induced transients
from the mass analysis includes summation of the Ir responses allowing determina-
tion of single cell events.
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