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We have developed lanthanide labeling strategies for antibodies to adapt conventional biochemical workflows like Western blot immunoassays for detection by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) analysis with a special interest to apply the multi-element capabilities of ICP-MS for the design of multiplexed immunoassays. In this paper the lanthanide labeling of antibodies with MeCAT was investigated and the reaction conditions were optimized for application in a Western blot immunoassay analyzed by LA-ICP-MS. Furthermore, the MeCAT labeling strategy was compared with two other commercially available labeling reagents, MAXPAR and SCN-DOTA. As a proof-of-principle experiment chemically induced alterations of cytochrome P450 protein expression were investigated and the suitability of the differentially labeled antibodies for Western blot immunoassays of a complex liver microsomal protein fraction was tested. Limits of detection (LODs) in the lower fmol range were reached in the Western blot application using MeCAT and MAXPAR as element labeling reagents, whereas even sub-fmol LODs can be achieved in a dot blot experiment for the pure antibodies.
The development of iron oxide nanoparticles for biomedical applications requires accurate histological evaluation. Prussian blue iron staining is widely used but may be unspecific when tissues contain substantial endogenous iron. Here we tested whether microscopy by laser ablation coupled to inductively coupled plasma mass spectrometry (LA-ICP-MS) is sensitive enough to analyze accumulation of very small iron oxide particles (VSOP) doped with europium in tissue sections.
We developed new procedures for internal standardization and calibration to be used for laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) for elemental micro mapping imaging of biological samples like Western blot membranes and tissue sections. These procedures are based on printing of metal spiked inks onto the top of thin layer samples for simultaneous internal standardization and calibration of LA-ICP-MS. In the case of internal standardization the ink is spiked with indium as an internal standard and homogenously printed over the entire membrane (size 56 cm2) prior to LA-ICP-MS detection, a standard deviation (RSD) value of 2% was achieved. In the second approach the metal content of lanthanide tagged proteins and antibodies after biological work flows was quantified by LA-ICP-MS on nitro-cellulose membranes. In this case the inks spiked with varying metals were printed with different densities on the same nitrocellulose membranes in well-defined squares to produce matrix-matched calibration standards. For validation and calibration the ink squares were excised and the specific metal content was measured by liquid ICP-MS after solubilization of the membrane slice. For the printed calibration standard limits of detection (LOD) of <4 fmol for different metals and relative process standard deviations of 12% only were determined via LA-ICP-MS.
The analysis of biomolecules requires highly sensitive and selective detection methods capable of tolerating a complex, biological matrix. First applications of biomolecule detection by ICP-MS relied on the use of heteroelements as a label for quantification. However, the combination of immunoassays and ICP-MS facilitates multiparametric analyses through elemental tagging, and provides a powerful alternative to common bioanalytical methods. This approach extends the detection of biomarkers in clinical diagnosis, and has the potential to provide a deeper understanding of the investigated biological system. The results might lead to the detection of diseases at an early stage, or guide treatment plans. Immunoassays are well accepted and established for diagnostic purposes, albeit ICP-MS is scarcely applied for the detection of immune-based assays. However, the screening of biomarkers demands high throughput and multiplex/multiparametric techniques, considering the variety of analytes to be queried. Finally, quantitative information on the expression level of biomarkers is highly desirable to identify abnormalities in a given organism. Thus, it is the aim of this review to introduce the fundamentals, and to discuss the enormous strength of ICP-MS for the detection of different immunoassays on the basis of selected applications, with a special focus on LA-ICP-MS.