Scalable Conjugation and Characterization of Immunoglobulins with Stable Mass Isotope Reporters for Single-Cell Mass Cytometry Analysis

Abstract

The advent of mass cytometry (CyTOF^®) has permitted simultaneous detection of more than 40 antibody parameters at the single-cell level, although a limited number of metal-labeled antibodies are commercially available. Here we present optimized and scalable protocols for conjugation of lanthanide as well as bismuth ions to immunoglobulin (Ig) using a maleimide-functionalized chelating polymer and for characterization of the conjugate. The maleimide functional group is reactive with cysteine sulfhydryl groups generated through partial reduction of the Ig Fc region. Incubation of Ig with polymer pre-loaded with lanthanide ions produces metal-labeled Ig without disrupting antigen specificity. Antibody recovery rates can be determined by UV spectrophotometry and frequently exceeds 60%. Each custom-conjugated antibody is validated using positive and negative cellular control populations and is titrated for optimal staining at concentrations ranging from 0.1 to 10 μg/mL. The preparation of metal-labeled antibodies can be completed in 4.5 h, and titration requires an additional 3-5 h.

Keywords: Antibody; Bismuth; Conjugation; CyTOF; IgG; Immunoglobulin; Isotope; Lanthanide; Mass cytometry; Titration.

Figure 1 –

Sources of signal interference in mass cytometry assays as demonstrated by analyzing a mixture of metal standard (139La, 141Pr, 159Tb, 169Tm, and 175Lu) on a CyTOF mass cytometer. (A) Spectral overlap from the 169 Tm standard into the 170 Er measurement channel. 169 Tm is naturally mono-isotopic with no expectation of a constituent with mass 170. (B) Natural Lu, used to make the standard in this experiment, contains ~99% 175 Lu and 1% 176 Lu. A distinct peak corresponding to the 176 Lu isotopic ‘contaminant’ can be seen in the measurement window that would be used for 176 Yb labeled reagents (red arrow). The dashed black boxes in (A) and (B) represent the measurement window where all signals within that range would be attributed to the indicated elemental isotope. (C) La and Pr oxide formation indicated with the blue and red arrows, respectively. Here the resulting ‘+16Da’ oxide interference peak is indicated with a ‘*’ of a matching color.

Figure 2 –

A workflow summarizing the timing and coordination of steps for the conjugation of a purified immunoglobulin with a sulfhydryl reactive polymer pre-loaded with metal isotope reporters.

Figure 3 –

Anticipated results for the titration of metal reporter conjugated antibodies by CyTOF mass cytometry. Using the protocol described herein, antibodies against human CD95, CD21, and CD14 were labeled with ¹⁶⁴Dy, ¹⁵²Sm, and ¹⁴⁸Nd, respectively. Using the surface staining protocol and a methanol permeabilization, these antibodies were simultaneously titrated from 0.25–8 μg/ml on human peripheral blood mononuclear cells that were counterstained with CD45-¹⁵⁴Sm, CD3-¹⁷⁰Er, CD20-¹⁴⁷Sm, CD33-¹⁵⁸Gd, and CD16-¹⁶⁵Ho in order to identify positive and negative cell populations. (A) From data analysis at cytobank.org, the following populations were identified: 1) CD33-positive myeloid cells, 2) CD20-positive B cells, 3) CD3-positive T cells, and 4) CD16-positive NK cells. (B) Histogram overlays of CD95, CD21, and CD14 expression levels in the gated populations from (A) were created at cytobank.org. Histogram color scale indicates minimum (black) and maximum (yellow) median counts for each given antibody. (C) Dot plots summarizing the median counts of representative positive (blue) and negative (red) cell controls for each of the titrated antibodies across the range of 0.25–8 μg/ml. In (B) and (C) the red asterisk indicates the ideal antibody concentration with maximum signal-to-noise (the ratio of positive control to negative control signal) and lowest antibody concentration where the positive signal begins to saturate.