Element-tagged immunoassay with ICP-MS detection: evaluation and comparison to conventional immunoassays

Abstract

We have investigated the possibility of using element-tagged antibodies for protein detection and quantification in microplate format using Inductively Coupled Plasma Mass Spectrometry (ICP-MS), and compared the results to conventional immunoassays, such as Enzyme-Linked Immunosorbent Assay (ELISA) and Western blotting. The technique was further employed to detect low levels and measure DNA-binding activity of transcription factor p53 in leukemia cell lysates through its interaction with immobilized oligonucleotides and recognition by element-tagged antibodies. The advantages of ICP-MS detection for routine performance of immunoassays include increased sensitivity, wide dynamic range, minimal interference from complex matrices, and high throughput. Our approach advances the ICP-MS technology and demonstrates its applicability to proteomic studies through the use of antibodies directly labeled with polymer tags bearing multiple atoms of lanthanides. Development of this novel methodology will enable fast and quantitative identification of multiple analytes in a single well.

Figure 1

Comparison of colorimetric and ICP-MS immunoassays for growth factor hPDGF-AA. ReactiBind microplates were coated with recombinant hPDGF-AA at different concentrations. A) Indirect ELISA colorimetric method using anti-PDGF antibody, and mouse IgG for negative control (mIgG). Single measurements. B) ICP-MS using primary terbium (Tb) labeled antibodies against hPDGF-AA (anti-hPDGF-Tb), and mouse IgG tagged with Tb for negative control (mIgG-Tb). Response was normalized to 1 ppb Ir/10%HCl standard. Assay was performed in triplicate

Figure 2

A) ICP-MS measurement of low amounts of hPDGF-AA. Plates were blocked with 5% BSA and 20 µg/mL IgG in PBS, then treated with terbium-tagged anti-PDGF-AA antibodies or terbium-tagged IgG1 immunoglobulins diluted 1:800. Response was normalized to 1 ppb Ir/10%HCl internal standard. Assay was performed in triplicate. B) Correlation analysis for conventional ELISA and ICP-MS measurements of rhPDGF-AA concentrations. Responses for the colorimetric and ICP-MS assay formats were compared at the same concentrations of growth factor.

Figure 3

p53 protein detection in cell lines. A) ICP-MS immunoassay. Cell extracts were analyzed using Tb-labeled antibodies against p53. Response was normalized to 1 ppb Ir/10%HCl standard. B) Western blotting with anti-p53 and anti-GAPDH antibodies; C) Relative density of bands from exposed X-ray film.

Figure 4

Demonstration of element-tagged p53(DO1)Tm antibody binding to p53 protein in different cell lysates electroblotted onto PVDF membrane. A) Western blotting and B) Relative density of bands from exposed X-ray film; C) Juxtaposition of X-ray film and membrane for delineation of immunoreactive bands (dashed line rectangles), which were cut out and soaked in concentrated HCl used ICP-MS analysis of Tm levels.

Figure 5

Competition assays for the detection of activated p53 in MCF-7 cells. Nuclear extracts were treated with consensus (con-oligo) or scrambled (scr-oligo) oligonucleotides. A) Colorimetric ELISA. B) ICP-MS immunoassay; response was normalized to 1 ppb Ir/10%HCl internal standard. Assays were performed in duplicate.

Figure 6

DNA-binding activity of p53 in cell lines. A) Indirect ELISA; B) ICP-MS assay using secondary Tm-tagged antibodies. Response was normalized to 1 ppb Ir/10%HCl standard. Assay was performed in duplicate.

Figure 7

DNA-binding activity of p53 in Tex cells. A) Detection of activated p53 in Tex cells by ICP-MS using secondary Tm-tagged antibodies. Response was normalized to a 1 ppb Ir/10%HCL internal standard. Assay was performed in duplicate B) Western blot analysis of p53 protein expression in Tex cells. Control cells (Tex-ShRFP) and those blocked for p53 gene expression (Tex-Shp53) were irradiated and harvested 5 h and 24 h post-irradiation (post-IR) along with non-treated (NT) cells. K562 cell extract was included as negative control‥