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Sequential Protein Capture in Multiplex Single Molecule Arrays: A Strategy for Eliminating Assay Cross-Reactivity.

ABSTRACT: Measurements of multiple biomolecules within the same biological sample are important for many clinical applications to enable accurate disease diagnosis or classification. These disease-related biomarkers often exist at very low levels in biological fluids, necessitating ultrasensitive measurement methods. Single-molecule arrays (Simoa), a bead-based digital enzyme-linked immunosorbent assay, is the current state of the art for ultrasensitive protein detection and can detect sub-femtomolar protein concentrations, but its ability to achieve high-order multiplexing without cross-reactivity remains a challenge. Here, a sequential protein capture approach for multiplex Simoa assays is implemented to eliminate cross-reactivity between binding reagents by sequentially capturing each protein analyte and then incubating each capture bead with only its corresponding detection antibody. This strategy not only reduces cross-reactivity to background levels and significantly improves measurement accuracies, but also enables higher-order multiplexing. As a proof of concept, the sequential multiplex Simoa assay is used to measure five different cytokines in plasma samples from Coronavirus Disease 2019 (COVID-19) patients. The ultrasensitive sequential multiplex Simoa assays will enable the simultaneous measurements of multiple low-abundance analytes in a time- and cost-effective manner and will prove especially critical in many cases where sample volumes are limited.

SUBMITTER: Gilboa T

PROVIDER: S-EPMC8238389 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The SlipChip platform was tested to perform high-throughput nanoliter multiplex PCR. The advantages of using the SlipChip platform for multiplex PCR include the ability to preload arrays of dry primers, instrument-free sample manipulation, small sample volume, and high-throughput capacity. The SlipChip was designed to preload one primer pair per reaction compartment and to screen up to 384 different primer pairs with less than 30 nanoliters of sample per reaction compartment. Both a 40-well and a 384-well design of the SlipChip were tested for multiplex PCR. In the geometries used here, the sample fluid was spontaneously compartmentalized into discrete volumes even before slipping of the two plates of the SlipChip, but slipping introduced additional capabilities that made devices more robust and versatile. The wells of this SlipChip were designed to overcome potential problems associated with thermal expansion. By using circular wells filled with oil and overlapping them with square wells filled with the aqueous PCR mixture, a droplet of aqueous PCR mixture was always surrounded by the lubricating fluid. In this design, during heating and thermal expansion, only oil was expelled from the compartment and leaking of the aqueous solution was prevented. Both 40-well and 384-well devices were found to be free from cross-contamination, and end point fluorescence detection provided reliable readout. Multiple samples could also be screened on the same SlipChip simultaneously. Multiplex PCR was validated on the 384-well SlipChip with 20 different primer pairs to identify 16 bacterial and fungal species commonly presented in blood infections. The SlipChip correctly identified five different bacterial or fungal species in separate experiments. In addition, the presence of the resistance gene mecA in methicillin resistant Staphylococcus aureus (MRSA) was identified. The SlipChip will be useful for applications involving PCR arrays and lays the foundation for new strategies for diagnostics, point-of-care devices, and immobilization-based arrays.

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Sequential Protein Capture in Multiplex Single Molecule Arrays: A Strategy for Eliminating Assay Cross-Reactivity.

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