Validation of antibody tagging protocol
The tagging protocol needs to be validated and represents a separate challenge. During the tagging procedure it is necessary to ensure that the antibody retains specificity toward its antigen (antigen binding sites remain intact), as well as be recognizable by anti-species specific antibodies for secondary immunoassays. First, tag conjugation was verified by performing the reaction in reducing and non-reducing conditions. Equal amounts of mouse IgG were labeled with thulium-containing tag (Tm-tag) in the presence (+) or absence (-) of TCEP. The resultant antibody conjugates were standardized to 0.58 mg ml-1 and serially diluted to 10, 5, 2.5, 1.25, 0.63 and 0.31 μg ml-1 in PBS. Similar reactions were set up for bovine serum albumin (BSA+ and BSA-). To assess tag binding and structural integrity of the immunoglobulins, we used 96-well plates coated with goat anti-mouse polyclonal antibodies (Pierce Biotechnology Inc.), which are specific for the Fc fragment of mouse IgGs. The goat α-mouse antibodies are immobilized on the bottom of plate wells. For every element separately, 30μL of each dilution are transferred into wells and incubated overnight at 4 °C. If the mouse IgG conjugated to the elemental tags were denatured during labeling, the goat α-mouse antibodies would not recognize them. In the case of the 96-well plate, the washing cycle consists of repeated aspiration and the addition of 200 μL PBS per well. Unbound IgG, tag and excess metal should be washed thoroughly to reduce non-specific background. The number of washes is usually determined experimentally. Finally, 85 μL of 37% HCl (Seastar Chemicals Inc.) was added per well and incubated for 30 min. Subsequently, 80 μL from each well were transferred into Eppendorf tubes containing 80 μL of 1 ppb Ir in 10% HCl as an internal standard (160 μL total). Alternatively, without re-formatting, the standard can be added directly to the wells, and the plate analyzed by ICP-MS via an autosampler.
As is evident from , the increase in Tm concentration for IgG(+) follows a distinct saturation curve, whereas IgG(-) displays an order of magnitude lower response. This can be explained by the fact that some amount of tag non-specifically (electrostatic interaction) attaches to the immunoglobulin. On the other hand, values for BSA(+) and (-) are similar and very low, indicating that the unreacted tag and/or Tm do not comprise a significant background in the assay.
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.
Furthermore, the influence of different metals on IgG structure and on tag loading was analyzed. In the following experiment, 13 aliquots of 50 μg of mouse IgG were labeled with the element tag containing polymer bearing DTPA metal-chelating ligands according to the previously described antibody labeling protocol. Each antibody-polymer-chelate preparation was loaded separately with 5 μL of a 0.1 M solution of Tb, Er, Lu, Tm, Ho, Eu, Pr, Dy, Yb, Nd, Gd, Sm, or Ce. Therefore, each antibody sample is labelled with a unique elemental tag independently according to the same procedure. The resultant antibody conjugate was standardized to 0.7 mg mL-1 and serial dilutions of 10, 5, 2.5, 1.25, 0.63, 0.31, and 0.16 μg mL-1 were prepared. The results of the ICP-MS analyses presented in and demonstrate that IgGs are not denatured after tagging. The capacity of the goat α-mouse 96 well plates is quite limited (12 pmol IgG per well, according to the manufacturer), which can be seen in as a tendency towards saturation of the signal in the range of high IgG concentrations. One would expect that the results represent differences in metal uptake by the tag. Unfortunately, the exact order of effectiveness of tagging from Ce (highest) to Dy (lowest) is not very reproducible. As we noted before, this effect may be the result of deficiencies in estimating IgG concentrations, as well as losses that occur during sample preparation (washes, filtration and so on). At this stage we are unable to delineate uncertainty in the sample preparation from binding efficiency of different metals.
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.
Demonstration of element tagged antibody reactivity on cells
Washing cell samples is a critical step in the reduction of non-specific background and variability between replicates. The cell washing cycle is time consuming but has to be done carefully, avoiding cell loss and damage. Normally, cells in suspension are distributed into Eppendorf tubes and centrifuged at low speed (300g) for 5 min. Liquid is carefully aspirated from the cell pellet. The pellet is sharply flicked to break up clumps of cells, and 1 ml of fresh wash buffer (PBS) is added. The tube is briefly vortexed (shaken), and washed cells are pelleted by centrifugation for further steps.
For the analysis of intra-cellular markers fixation/permeabilization of cell samples is required. The following is one of many described methods which we found particularly useful (see ). 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). Finally, cells were made permeable with ice-cold 90% methanol (10 min). After low speed centrifugation, cells were re-suspended in blocking buffer (10% serum-PBS) and incubated for 15 min.
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 (more ...)
The immuno-labeling procedure finalizes sample preparation for ICP-MS analysis (see ). The blocked cells were counted in a hemocytometer and equal numbers (usually 105
cells per replicate) were distributed into triplicate tubes to which a mixture of all the element tagged antibodies was added. (We recommend using the triplicate format to reduce variability in sample preparation at the exploratory stage of research. The number of samples should be reduced for routine analysis.) 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. Washed cells were stained with 1 μM Rh3+
-containing DNA-metallointercalator for normalization.29,30
Finally, the washed cells were spun down, and the cellular pellets were dissolved in ultra-pure concentrated HCl. An equal volume of internal standard (1 ppb Ir in 10% HCl) was added to each tube to compensate for possible long term sensitivity drift, and samples were analyzed by ICP-MS.
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 (more ...)
Example of multiplexed assay
The following is an example of a multiplexed cellular assay using the KG-1a, acute myelogenous leukemia, and THP-1, acute monocytic leukemia, cell lines. The cell lines were purchased from ATCC, Manassas,VA. Cells were grown in alpha-MEM, supplemented with 10% FBS (HyClone) and 2 mM l-glutamine (Invitrogen), in a humidified incubator at 37 °C and 5% CO2. Cells were split every 3-4 days. Monoclonal antibodies to surface antigens (CD33, CD34, CD38, CD45, CD64, HLA-DR) were purchased from BD Biosciences, San Jose, CA. Mouse immunoglobulins were from Biomeda Inc.
The two cell lines (KG1a and THP-1) represent different types of acute human myeloid leukemia and are characterized by the expression of specific surface markers. Seven cell surface antigens were detected simultaneously using specific antibodies labeled with element tag loaded with isotope enriched lanthanides (CD33-141Pr, CD34-169Tm, CD38-165Ho, CD45-159Tb, CD64-153Eu, CD44-151Eu, HLA-DR-147Sm). Live suspension growing cells were collected by low speed centrifugation and washed in phosphate buffered saline. Equal cell numbers were distributed into triplicate tubes (105 cells per tube). All seven lanthanide tagged antibodies were mixed into one tube at approximately 1 μg ml-1 each. To control for non-specific immunoglobulin binding to live cells, we used mouse IgGs prepared simultaneously with specific antibodies and labeled with the same lanthanide isotopes. The cells were incubated with either antibodies or IgGs for 30 min at room temperature, then washed several times with PBS by low speed centrifugation. Cells were post-fixed in 1% formaldehyde-PBS for staining with a Rh3+-containing DNA-metallointercalator for cell number normalization. Finally, the cellular pellets were dissolved in concentrated HCl, and the solution was analyzed by ICP-MS (see ).
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 (more ...)
The less differentiated hematopoietic progenitor cell line KG1a is known to express high levels of hematopoietic precursor marker (CD34), hyaluronic acid receptor (CD44) and the leukocyte common antigen (CD45), and very low levels of myeloid precursor marker (CD33), monocyte marker (CD64) and the human leukocyte antigen HLA-DR.31-35
On the other hand, THP-1 cells display characteristics of more differentiated monocytes being positive for CD33, CD64, CD38, CD45, as well as HLA-DR (low) and negative for CD34.36-38
The results, presented as a polar diagram in , clearly demonstrate the differences in surface marker expression between KG1a and THP-1, and are consistent with the above referenced fluorescent flow cytometry analyses.