Re: Nanoparticles in CyTOF
Posted: Mon Dec 07, 2015 10:05 pm
Hi all,
I am not sure whether comparing signals elicited by a particle (or cell) suspended in water to signals coming from metal salts or nanoparticles dissolved in acid may work very well. There is certainly a correlation, but I doubt that one can serve well as a standard for quantifying the other.
We ran two assays to analyze metal loading of Ab (which could be extrapolated to metal content of Nanoparticle-conjugates provided that they completely dissolve). i) We used Ab capture beads incubated with known concentrations of metal labeled Ab and ii) dissolved the metal labeled Ab in tuning solution or other acid and running them in “solution mode”. I estimate the bead-based assay to be more practically meaningful for the application of the Ab conjugates, since signal intensities elicited by the Ab-conjugate-loaded beads comprise both, the metal loading of the Ab but also incorporate differences in ion transmission efficiency across the mass spectrum of the individual machine. The approach appears to work well, but does not permit the estimation of the number of metal atoms per Ab. In order to get these numbers, we ran additional ‘solution mode’ experiments.
I first diluted Ab conjugates and metal solutions (which I intended to use as reference with known concentration of metal ions) in water, aiming at being as close as possible to cell sample conditions (Ab conjugate in water). This gave me, as expected, detectable signals at varying levels for the conjugates but signals for metal salts were unexpectedly low for some, but not all metals, using data of tuning solution as a 2nd reference. I repeated this with new dilutions but similar results.
Based on that, I compared signals obtained from metal salts (and cisplatin) diluted in water versus spiked into tuning solution at identical concentrations. This revealed dramatic differences between water vs tuning (nitric acid), but also between metals (different salts or cisplatin). Attached figures (raw data, all panels represent the identical injection series, examples highlight the different "types" of metals/ metal behavior I noted in the whole experiment) show some of the measurements (CyTOF v2), successive injections are differently colored. No HF/’wash’ but water washes between injections.
In order to circumvent the differences which occur based on which metal is measured in which matrix, we ended up using tuning as reference for lanthanide labeled Abs, but separate SPEX Pt and Pd ICP-MS standard solutions for Pt and Pd labeled Abs, respectively. These standards were in 2%HCl, so we used 2% HCl as a matrix for Pt and Pd conjugates.
Possibly, at least some of the difference arose from different distribution of metal ions into liquid vs tubing depending on whether the liquid was water or acid.
Notably, lanthanide signals were comparably very low when salt solutions were diluted in water as opposed to tuning, while lanthanide-conjugate-labeled cells in water routinely give principally useful signals, so that chelated metal appear to show behavior differing from metal salt solutions. In line with the differential kinetics of tuning isotopes that can be observed when injection tuning solution, I also consider the potential retention of a given metal in the tubing to depend on the overall composition of metal salts in the injected solution.
I also want to note that Fluidigm provided on request a protocol that describes quantification of metal ions per Ab molecule, including an Excel spreadsheet which does the calculations. It is however limited to lanthanide metals which can be obtained through Fluidigm in MAXPAR kits, so it didn’t help us much for the platinum-labeled Abs.
Henrik
I am not sure whether comparing signals elicited by a particle (or cell) suspended in water to signals coming from metal salts or nanoparticles dissolved in acid may work very well. There is certainly a correlation, but I doubt that one can serve well as a standard for quantifying the other.
We ran two assays to analyze metal loading of Ab (which could be extrapolated to metal content of Nanoparticle-conjugates provided that they completely dissolve). i) We used Ab capture beads incubated with known concentrations of metal labeled Ab and ii) dissolved the metal labeled Ab in tuning solution or other acid and running them in “solution mode”. I estimate the bead-based assay to be more practically meaningful for the application of the Ab conjugates, since signal intensities elicited by the Ab-conjugate-loaded beads comprise both, the metal loading of the Ab but also incorporate differences in ion transmission efficiency across the mass spectrum of the individual machine. The approach appears to work well, but does not permit the estimation of the number of metal atoms per Ab. In order to get these numbers, we ran additional ‘solution mode’ experiments.
I first diluted Ab conjugates and metal solutions (which I intended to use as reference with known concentration of metal ions) in water, aiming at being as close as possible to cell sample conditions (Ab conjugate in water). This gave me, as expected, detectable signals at varying levels for the conjugates but signals for metal salts were unexpectedly low for some, but not all metals, using data of tuning solution as a 2nd reference. I repeated this with new dilutions but similar results.
Based on that, I compared signals obtained from metal salts (and cisplatin) diluted in water versus spiked into tuning solution at identical concentrations. This revealed dramatic differences between water vs tuning (nitric acid), but also between metals (different salts or cisplatin). Attached figures (raw data, all panels represent the identical injection series, examples highlight the different "types" of metals/ metal behavior I noted in the whole experiment) show some of the measurements (CyTOF v2), successive injections are differently colored. No HF/’wash’ but water washes between injections.
In order to circumvent the differences which occur based on which metal is measured in which matrix, we ended up using tuning as reference for lanthanide labeled Abs, but separate SPEX Pt and Pd ICP-MS standard solutions for Pt and Pd labeled Abs, respectively. These standards were in 2%HCl, so we used 2% HCl as a matrix for Pt and Pd conjugates.
Possibly, at least some of the difference arose from different distribution of metal ions into liquid vs tubing depending on whether the liquid was water or acid.
Notably, lanthanide signals were comparably very low when salt solutions were diluted in water as opposed to tuning, while lanthanide-conjugate-labeled cells in water routinely give principally useful signals, so that chelated metal appear to show behavior differing from metal salt solutions. In line with the differential kinetics of tuning isotopes that can be observed when injection tuning solution, I also consider the potential retention of a given metal in the tubing to depend on the overall composition of metal salts in the injected solution.
I also want to note that Fluidigm provided on request a protocol that describes quantification of metal ions per Ab molecule, including an Excel spreadsheet which does the calculations. It is however limited to lanthanide metals which can be obtained through Fluidigm in MAXPAR kits, so it didn’t help us much for the platinum-labeled Abs.
Henrik