Lanthanide polymer-labelled antibodies were investigated to improve the analytical figures of merit of homogeneous immunoassays with inductively coupled plasma mass spectrometry (ICP-MS) detection for multiplexed biomarker analysis in human serum samples. Specific monoclonal antibodies against four cancer biomarkers (CEA, sErbB2, CA 15.3 and CA 125) were labelled with different polymer-based lanthanide group to increase the number of metal labels per binding site. After the immunoreaction of the biomarkers with the specific antibodies, antigen-antibody complexes were separated by size-exclusion chromatography followed by ICP-MS detection. The polymer label could be loaded with 30-times more atoms of the lanthanide that the lanthanide-DOTA complex traditionally used for this purpose elsewhere [1] which resulted in a 10-fold improvement in both sensitivity and detection limits. Analytical figures of merit obtained with the lanthanide polymer labelling strategy make the detection of the biomarkers feasible below the threshold reference values used in clinical analysis. This labelling method was successfully validated by analyzing a control human serum spiked with the four biomarkers at three different concentration levels. For all the biomarkers studied, the recovery values ranged from 95% to 110% whereas inter-assay and intra-assay precision were lower than 8%. Results obtained with this approach were equivalent to those obtained by heterogenous-based immunoassays based on the detection by electro-chemiluminescence or ELISA. However, the method developed offers better analytical figures of merit using a smaller amount of sample.
12Haptens (i.e. biomolecules which molecular weight is lower than 10 kDa) 13 determination by inductively coupled plasma mass spectrometry (ICP-MS) is 14 usually performed by means of competitive immunoassays. In these 15 immunoassays, analyte quantification is indirectly carried out using two different 16 tracer species (i.e. antibodies or antigen-protein conjugates). However, the 17 benefits (and drawbacks) derived from using a given tracer species have not 18 been systematically investigated so far. The goal of this work is to evaluate the 19 influence of the tracer species employed in competitive immunoassays on the 20 analytical figures of merit for aflatoxin M 1 (AFM 1 ) determination in milk samples. 21To this end, three different strategies have been developed and evaluated, 22 namely: (i) antibody binding inhibition assay (ABIA); (ii) capture inhibition assay 23 (CIA); and (iii) capture bridge inhibition assay (CBIA). Experimental results 24show that the use of the antibody as tracer species (as in the ABIA approach) 25 42 Keywords: hapten, competitive immunoassay, nanoparticle, inductively 43 coupled plasma mass spectrometry, aflatoxin M 1 . 44 45 [1]. The use of ICP-MS in this field offers several attractive features regarding 49 other established approaches: (i) high sensitivity; (ii) specificity; (iii) compound-50 independent detection sensitivity; (iv) multi-element capabilities; (v) robustness; 51 and (vi) easy coupling to separation techniques. Initially, the analysis was 52 limited to those species containing a heteroatom detectable by ICP-MS. 53 However, analytical figures of merit were severely compromised since the 54 heteroatoms, naturally present in biomolecules (e.g. P, S, Se, As, etc.), suffer 55 from low sensitivity and (spectral and non-spectral) interferences due to matrix 56 components in biological samples (e.g. carbon, chloride, etc.) [1,2]. To improve 57 the analytical figures of merit, as well as to address with the determination of 58 non-containing heteroatom biomolecules, different strategies have been 59proposed in the literature. First, the biomolecule can be derivatized through a 60 chemical reaction with a heteroatom or an organometallic compound [3,4]. The 61 main drawback of this approach is the low selectivity of the labelling procedure. 62Alternatively, the analyte of interest can be labelled by means of an 63 immunoreaction using a heteroatom-labelled antibody [5,6]. Because of the 64 great specificity of the antigen-antibody reaction, biomolecules can be 65 successfully determined in complex mixtures. Heteroatoms labels used in 66 immunoassay includes: (i) transition metals [7,8]; (ii) lanthanides [9-11]; (iii) 67 metal nanoparticles [12][13][14]; and (iv) Quantum-Dots [15]. A priori, the use of 68 metal nanoparticles is the most advantageous approach to improve analytical 69 figures of merit (e.g. sensitivity, LoD, etc.) due to the high number of atoms 70 present in each nanoparticle. 71Non-competitive immunoassays have been traditionally employed for the 72 analysis ...
An inductively coupled plasma mass spectrometry (ICP-MS)-based immunoassay has been developed to quantify aflatoxin M1 (AFM1) at ultra-trace levels in milk samples. AFM1 detection is carried out by means of a competitive immunoassay using secondary biotinylated antibodies and streptavidin-conjugated Au nanoparticles. After acid addition, nanoparticles are decomposed and Au signal is registered by means of ICP-MS. Results demonstrate that, under optimum conditions, the limit of detection of the immunoassay (0.005μgkg) is low enough to quantify AFM1 according to current international policies (including the more restrictive European one). Method accuracy and precision was checked by analyzing an AFM1 certified reference material and different milk samples spiked with known amounts of AFM1. AFM1 recovery values range from 80% to 102% whereas inter-assay and intra-assay precision are lower than 15%. Finally, this immunoassay methodology affords a higher dynamic working range (0.012-2.5μgkg) than other immunoassay methodologies described in the literature.
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