Polymer-based elemental tags for sensitive bioassays - PubMed (original) (raw)

Polymer-based elemental tags for sensitive bioassays

Xudong Lou et al. Angew Chem Int Ed Engl. 2007.

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Figures

Figure 1

Figure 1

Experimental design for tagging antibodies with metal-chelating polymers. The antibody of interest is subjected to selective reduction of -S—S-groups to produce reactive -SH groups, which are reacted with the terminal maleimide groups of a polymer bearing metal-chelating ligands along its backbone. The polymer-bearing antibodies are purified, treated with a given lanthanide ion, and then purified again. Each type of antibody is labeled with a different element.

Scheme 1

Scheme 1

a) Et3N, DMF, benzylamine or 2, 14 h; b) TFA 95 %, 14 h; c) 1) DTT (20 m

m

), Na2HPO4 (50 m

m

, pH 8.5), 50 °C, 1 h; 2) 2,2′-(ethylenedioxy)bis(ethylmaleimide), DMF/H2O, 1 h, 22 °C.

Figure 2

Figure 2

1H NMR spectrum of maleimide conjugate 6 in D2O prior to (top) and after (bottom) reaction with 2-aminoethanethiol.

Figure 3

Figure 3

Titration of element-tagged antibody against cell surface antigen. KG-1a cells (1 × 106 cells per sample, run in triplicate) were incubated with increasing concentrations of CD45-Eu antibody. Separately, the same number of KG-1a cells were treated with mouse IgG-Eu.

Figure 4

Figure 4

Multiplex analysis of antigen expression by two acute leukemia cell lines. a) KG-1a cells were probed with five element-tagged antibodies to cell surface antigens: CD33-Pr, CD34-Tb, CD38-Ho, CD45-Eu, and CD54-Tm. Background controls included element-tagged mouse IgG-Pr, IgG-Tb, IgG-Ho, IgG-Eu, and IgG-Tm. Triplicate samples with 1 × 106 cells per tube were set up for reaction with each antibody separately and with a mix of all five antibodies together as well as controls. The cells were stained, fixed, and then treated with a RhIII-containing metallointercalator for cell enumeration and signal normalization. Washed cell pellets were dissolved in concentrated HCl, combined with an equal volume of Ir standard solution (1 ppb), and analyzed by ICP-MS. Results are presented as normalized response with respect to Ir, Rh, and background signals from nonspecific IgG binding. b) THP-1 cell line treated as described for KG-1a.

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