Abstract
Immunochemical methods are used not only in clinical practice for the diagnosis of a wide range of diseases but also in basic and advanced research. Based on the unique reaction between the antibody and its respective antigens, it serves to specifically recognize target molecules in biological complex samples. Current methods of labelling antibodies with elemental labels followed by detection by inductively coupled plasma mass spectrometry (ICP-MS) allow detection of multiple antigens in parallel in a single analysis. Using the laser ablation (LA) modality (LA-ICP-MS), it is also possible to monitor the spatial distribution of biogenic elements. Moreover, the employment of metal nanoparticle-labeled antibodies expands the applicability also to molecular imaging by LA-ICP-MS. In this work, conjugates of model monoclonal antibody (DO-1, recognizing p53 protein) with various metal nanoparticles-based labels were created and utilized in dot-blot analysis in order to compare their benefits and disadvantages. Based on experiments with the p53 protein standard, commercial kits of gold nanoparticles proved to be the most suitable for the preparation of conjugates. The LA-ICP-MS demonstrated very good repeatability, wide linear dynamic range (0.1–14 ng), and limit of detection was calculated as a 1.3 pg of p53 protein.
Highlights
Up to recently, inductively-coupled laser ablation plasma mass spectrometry (LA-ICPMS) has definitely not been promoted in bio-applications, because of its primary orientation on detection of sample elemental composition, whereasanalytical fields are interested more in molecular composition and interactions
The biggest advantage of imaging mass cytometry is that a single tissue sample can be imaged for 40+ markers simultaneously. This multiplexing capability given by Inductively coupled plasma mass spectrometry (ICP-MS) is significantly improved compared to optical fluorophores which are extremely useful in disease and biomarker research
Antibodies labelled by an elemental tag—chelate binding rare earth elements [3,4] enabled to detect specific proteins (e.g., p53 [5], ferroportin [6], MMP-11 [7], etc.) in levels of pg [8] by LA-ICP-MS
Summary
Inductively-coupled laser ablation plasma mass spectrometry (LA-ICPMS) has definitely not been promoted in bio-applications, because of its primary orientation on detection of sample elemental composition, whereas (bio)analytical fields are interested more in molecular composition and interactions. Antibody conjugates with several metal nanoparticles (Au (AuNPs), Ag (AgNPs), Eu (EuNPs), and CdTe quantum dots (CdTe QDs)) were tested to evaluate their benefits and disadvantages in the immunochemical analysis (dot-blot assay) coupled to LA-ICP-MS using p53 protein as a model analyte. This arrangement is beneficial as only 0.5 μL of antigen can be applied on the membrane. Based on2.o1u. rLapsreervBioeaums eFxlpueenricme Oenpttsimanizdateioxnperience [8], the laser beam size of 150 μm atewhaneecdrhleatshsspeeporostbattwmeeadapmalssalpnoesaofdneblfturtettloBehaeebdtdnaeeteshcasdoreemaeandmutwtPefloonpVanudotlshDeefeoonerf5Fuepc0Pdreet0mVidipfwmμDferreeamimaFrzvsteeeb/imonosdroptuae.wftnmlsFi5aemeie0sbrv.xe0eirezpAraμceesnbfhmdirteemeoi..m/arsFiAmseleidanvwfnrtofeetflet0suro-sr.beiare5dmlnncotoμhdchittlLe-eoabeusr(fsxlse0oap0pin.stn.5oe5tufg–terosμis5seriAtLonnJtpf/uhgcscoeppeNmAs5o[fPs28au3ti)ss]sb.N,p-tolTDetePrhfshoOstaepet-pne-D5l1diaoa3nOslsecpyesp-(oisrr14ebnoibsnclntje.ueoedgaOinage/nmnjμannu(atLclg4eslye)y,ainzstgeeis,/o.eμfOaL1cn5)hl0wysμptehmoreet of the measurwedasAaubliantteednsuitniedseorndtihffeeruesnetdllaasseerr bbeeaamm fflluueennccee i(s0s.5h–o5wJn/icnmF2i)g.uTreh1e.dItewpeansdence of the found that themmeaossutrseuditAabuleinistetnosuitsieesaolnastehrebuesaemd fllauseenr cbeeoamf 3flJ/ucemn2c,easisthsheoswignnainl iFsihgiugrhee1s.t It was found under these ctohnadt itthioenms.ost suitable is to use a laser beam fluence of 3 J/cm, as the signal is highest under these conditions
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