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Advancing the immunoaffinity platform AFFIRM to targeted measurements of proteins in serum in the pg/ml range.

ABSTRACT: There is a great need for targeted protein assays with the capacity of sensitive measurements in complex samples such as plasma or serum, not the least for clinical purposes. Proteomics keeps generating hundreds of biomarker candidates that need to be transferred towards true clinical application through targeted verification studies and towards clinically applicable analysis formats. The immunoaffinity assay AFFIRM (AFFInity sRM) combines the sensitivity of recombinant single chain antibodies (scFv) for targeted protein enrichment with a specific mass spectrometry readout through selected reaction monitoring (SRM) in an automated workflow. Here we demonstrate a 100 times improved detection capacity of the assay down to pg/ml range through the use of oriented antibody immobilization to magnetic beads. This was achieved using biotin-tagged scFv coupled to streptavidin coated magnetic beads, or utilizing the FLAG tag for coupling to anti-FLAG antibody coated magnetic beads. An improved multiplexing capacity with an 11-plex setup was also demonstrated compared to a previous 3-plex setup, which is of great importance for the analysis of panels of biomarker targets.

SUBMITTER: Sall A

PROVIDER: S-EPMC5810979 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Advancing the immunoaffinity platform AFFIRM to targeted measurements of proteins in serum in the pg/ml range.

Säll Anna A Corbee Daniel D Vikström Sara S Ottosson Filip F Persson Helena H Waldemarson Sofia S

PloS one 20180213 2

There is a great need for targeted protein assays with the capacity of sensitive measurements in complex samples such as plasma or serum, not the least for clinical purposes. Proteomics keeps generating hundreds of biomarker candidates that need to be transferred towards true clinical application through targeted verification studies and towards clinically applicable analysis formats. The immunoaffinity assay AFFIRM (AFFInity sRM) combines the sensitivity of recombinant single chain antibodies ( ...[more]

PMID: 29438379

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Project description:BackgroundSepsis is a common worldwide health condition with high mortality. It is caused by a dysregulated immune response to the pathogen. Severe infections resulting in sepsis can be also determined by monitoring several bloodstream biomarkers, one of them being pro-hormone procalcitonin (PCT). PCT concentration in the bloodstream correlates well with sepsis and in severe cases increases up to a thousand times from the healthy physiological values in a short time. In this study, we developed a rapid technique for PCT detection by MALDI-TOF mass spectrometry, that uses in-situ enrichment directly on the specialized immuno MALDI chips that are utilized as MALDI plates. The method's ability to detect PCT was confirmed by comparing the results with LC-MS bottom-up workflow. The new method detects intact PCT by its m/z and uncovers its alternations in septic serum.MethodsThe MALDI chips used for the detection of PCT were prepared by ambient ion soft landing of anti-PCT antibody on an ITO glass slide. The chips were used for the development of the rapid MALDI-TOF MS method. A parallel method based on affinity enrichment on magnetic beads followed by LC-MS/MS data-dependent peptide microsequencing was used to prove PCT presence in the sample. All samples were also tested by ELISA to determine PCT concentration prior to analyzing them by mass spectrometry methods.ResultsThe MALDI chip method was optimized using recombinant PCT spiked into the human serum. The PCT detection limit was 10 ng/mL. The optimized method was used to analyze 13 sera from patients suffering sepsis. The PCT results were confirmed by LC-MS/MS. The measurement of the intact PCT by the MALDI chip method revealed that sera of patients with severe sepsis have other forms of PCT present, which show post-processing of the primary sequence by cleavage of PCT, resulting in the formation of N and C termini fragments.ConclusionsProcalcitonin from human serum was successfully enriched and detected using immunoaffinity MALDI chips. The intact PCT was characterized in 13 septic patients. The method is more specific compared to non-MS-based immunoaffinity techniques and allows observation of different variants of PCT in septic patients.

Project description:Magneto-inertial measurement units (MIMU) are a suitable solution to assess human motor performance both indoors and outdoors. However, relevant quantities such as step width and base of support, which play an important role in gait stability, cannot be directly measured using MIMU alone. To overcome this limitation, we developed a wearable platform specifically designed for human movement analysis applications, which integrates a MIMU and an Infrared Time-of-Flight proximity sensor (IR-ToF), allowing for the estimate of inter-object distance. We proposed a thorough testing protocol for evaluating the IR-ToF sensor performances under experimental conditions resembling those encountered during gait. In particular, we tested the sensor performance for different (i) target colors; (ii) sensor-target distances (up to 200 mm) and (iii) sensor-target angles of incidence (AoI) (up to 60 ∘ ). Both static and dynamic conditions were analyzed. A pendulum, simulating the oscillation of a human leg, was used to generate highly repeatable oscillations with a maximum angular velocity of 6 rad/s. Results showed that the IR-ToF proximity sensor was not sensitive to variations of both distance and target color (except for black). Conversely, a relationship between error magnitude and AoI values was found. For AoI equal to 0 ∘ , the IR-ToF sensor performed equally well both in static and dynamic acquisitions with a distance mean absolute error <1.5 mm. Errors increased up to 3.6 mm (static) and 11.9 mm (dynamic) for AoI equal to ± 30 ∘ , and up to 7.8 mm (static) and 25.6 mm (dynamic) for AoI equal to ± 60 ∘ . In addition, the wearable platform was used during a preliminary experiment for the estimation of the inter-foot distance on a single healthy subject while walking. In conclusion, the combination of magneto-inertial unit and IR-ToF technology represents a valuable alternative solution in terms of accuracy, sampling frequency, dimension and power consumption, compared to existing technologies.

Dataset Information

Advancing the immunoaffinity platform AFFIRM to targeted measurements of proteins in serum in the pg/ml range.

Publications

Advancing the immunoaffinity platform AFFIRM to targeted measurements of proteins in serum in the pg/ml range.

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