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Coupling immuno-magnetic capture with LC-MS/MS(MRM) as a sensitive, reliable, and specific assay for SARS-CoV-2 identification from clinical samples.

ABSTRACT: Recently, numerous diagnostic approaches from different disciplines have been developed for SARS-CoV-2 diagnosis to monitor and control the COVID-19 pandemic. These include MS-based assays, which provide analytical information on viral proteins. However, their sensitivity is limited, estimated to be 5 × 10⁴ PFU/ml in clinical samples. Here, we present a reliable, specific, and rapid method for the identification of SARS-CoV-2 from nasopharyngeal (NP) specimens, which combines virus capture followed by LC-MS/MS(MRM) analysis of unique peptide markers. The capture of SARS-CoV-2 from the challenging matrix, prior to its tryptic digestion, was accomplished by magnetic beads coated with polyclonal IgG-α-SARS-CoV-2 antibodies, enabling sample concentration while significantly reducing background noise interrupting with LC-MS analysis. A sensitive and specific LC-MS/MS(MRM) analysis method was developed for the identification of selected tryptic peptide markers. The combined assay, which resulted in S/N ratio enhancement, achieved an improved sensitivity of more than 10-fold compared with previously described MS methods. The assay was validated in 29 naive NP specimens, 19 samples were spiked with SARS-CoV-2 and 10 were used as negative controls. Finally, the assay was successfully applied to clinical NP samples (n = 26) pre-determined as either positive or negative by RT-qPCR. This work describes for the first time a combined approach for immuno-magnetic viral isolation coupled with MS analysis. This method is highly reliable, specific, and sensitive; thus, it may potentially serve as a complementary assay to RT-qPCR, the gold standard test. This methodology can be applied to other viruses as well.

SUBMITTER: Schuster O

PROVIDER: S-EPMC8723902 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundHighly accurate and sensitive method to measure testosterone in hypogonadal male, female and children is vital for proper diagnosis of hormone-related conditions and their treatment.ObjectiveTo develop an accurate and robust total testosterone ESI-LC-MS/MS quantification method with a simple sample preparation workflow and sufficient sensitivity for serum or plasma samples of all gender and age groups, via ketone functional group derivatization (using Amplifex™ Keto Reagent).MethodA simple sample preparation method to accommodate both low and high numbers of samples was developed using simultaneous protein precipitation and derivatization with Amplifex™ Keto reagent, followed by centrifugation and direct injection of supernatant into an LC-MS/MS system (SCIEX Topaz™ IVD LC-MS/MS, in which MS is equivalent to a SCIEX 4500MD Mass Spectrometer). Total testosterone in human serum or plasma samples was quantified using an external calibration curve generated by calibrators spanning a broad concentration range of ∼1-2000 ng/dL (10-20,000 pg/mL), traceable to NIST 971 SRM. 13C3-enriched testosterone was used as an internal standard to correct for both analyte loss during sample preparation and matrix effect during analysis (Supplementary Information: SI Fig. 4C). Two methods, one using a 96-well filter plate and another using Eppendorf tubes, were developed. Both methods were certified by the Centers for Disease Control (CDC) hormone standardization (HoSt) program for total serum testosterone. The feasibility of implementing the method for plasma and serum samples was tested via a small-scale method comparison study between matched pediatric serum and plasma samples derived from the same donor. In addition, plasma samples originating from the same donor collected in two different anticoagulant tube types (Li-heparin and K2EDTA) were compared.ResultsUsing in-house formulated NIST 971-traceable calibrators, the method was linear (r2 > 0.999) between 1 and 2000 ng/dL (10 and 20,000 pg/mL) with a limit of detection of approximately 1 ng/dL (10 pg/mL). The testosterone concentration bias against 40 reference samples from the HoSt certification program was absolute <3% with an average %CV of ∼3-4%. More than 78% of samples passed the CDC bias criterion of ±6.4%. Comparison between pediatric matched serum and plasma samples resulted in high correlation (r2 = 0.997) and bias of <5%. The calculated % difference between matched adult serum and plasma samples was ∼1%.ConclusionsFeasibility for an accurate and streamlined method suitable for measuring total testosterone in all human samples was demonstrated with a choice of sample preparation workflow to suit low or high number of samples. The method can potentially be used for plasma matrix from different blood collection tubes (Li-Heparin and K2EDTA).

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Coupling immuno-magnetic capture with LC-MS/MS(MRM) as a sensitive, reliable, and specific assay for SARS-CoV-2 identification from clinical samples.

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