Development of analytical methods for multiplex bio-assay with inductively coupled plasma mass spectrometry

Abstract

Advances in the development of highly multiplexed bio-analytical assays with inductively coupled plasma mass spectrometry (ICP-MS) detection are discussed. Use of novel reagents specifically designed for immunological methods utilizing elemental analysis is presented. The major steps of method development, including selection of elements for tags, validation of tagged reagents, and examples of multiplexed assays, are considered in detail. The paper further describes experimental protocols for elemental tagging of antibodies, immunostaining of live and fixed human leukemia cells, and preparation of samples for ICP-MS analysis. Quantitative analysis of surface antigens on model cell lines using a cocktail of seven lanthanide labeled antibodies demonstrated high specificity and concordance with conventional immunophenotyping.

Fig. 1

Validation of antibody conjugation using Tm-containing tag. BSA is used as a non-specific background control. (+) and (-) marks indicate the presence and absence of reducing reagent, respectively.

Fig. 2

Results of multiple tagging of IgG. Every element tagged IgG was prepared and analyzed independently. Only Dy, Er, Ho, Tm, and Ce containing IgG are presented explicitly. The data from other tagged IgGs are all closely packed in the highlighted area.

Fig. 3

Preparation of cells for analysis of intra-cellular markers. The washed cell pellet was re-suspended in 1% formaldehyde fixation buffer, incubated for 15 min, washed in PBS, and blocked in 100 mM glycine (10 min). After additional washing, cells were made permeable with ice-cold 90% methanol (10 min) (not shown here). After low speed centrifugation, cells were re-suspended in blocking buffer (10% serum-PBS) and incubated for 15 min.

Fig. 4

Preparation of cellular samples for ICP-MS analysis. The blocked cells were distributed into triplicate tubes to which a mixture of all the element tagged antibodies was added. Another set of triplicate tubes was incubated with a mixture of element tagged mouse IgGs as a control against the non-specific binding of antibodies to cells. Cells were incubated for 30 min for 1 h at room temperature and washed in PBS. Finally, the washed cells were spun down, and the cellular pellets were dissolved in ultra-pure concentrated HCl.

Fig. 5

Cellular assay of KG-1a and THP-1 cell lines by ICP-MS analysis. Normalized response on selected antigens. The detector signal is normalized to the detector signal of the internal standard 1ppb of Ir (instrument sensitivity) and Rh metallointercalator (cell number). Tagged antibodies: CD33-¹⁴¹Pr, CD34-¹⁶⁹Tm, CD38-¹⁶⁵Ho, CD45-¹⁵⁹Tb, CD64-¹⁵³Eu, CD44-¹⁵¹Eu, HLA-DR-¹⁴⁷Sm. Connecting lines are plotted for clarity and do not have analytical significance, except for recognition of the expression patterns. Notice that HLA-DR-¹⁴⁷Sm was not detected for KG-1a cells and CD34-¹⁶⁹Tm was not detected for THP-1 cells.