A Rapid, Multiplexed, High-Throughput Flow-Through Membrane Immunoassay: A Convenient Alternative to ELISA

Abstract

This paper describes a rapid, high-throughput flow-through membrane immunoassay (FMIA) platform. A nitrocellulose membrane was spotted in an array format with multiple capture and control reagents for each sample detection area, and assay steps were carried out by sequential aspiration of sample and reagents through each detection area using a 96-well vacuum manifold. The FMIA provides an alternate assay format with several advantages over ELISA. The high surface area of the membrane permits high label concentration using gold labels, and the small pores and vacuum control provide rapid diffusion to reduce total assay time to ~30 min. All reagents used in the FMIA are compatible with dry storage without refrigeration. The results appear as colored spots on the membrane that can be quantified using a flatbed scanner. We demonstrate the platform for detection of IgM specific to lipopolysaccharides (LPS) derived from Salmonella Typhi. The FMIA format provides analytical results comparable to ELISA in less time, provides integrated assay controls, and allows compensation for specimen-to-specimen variability in background, which is a particular challenge for IgM assays.

Lateral and Vertical Flow Assays for Point‐of‐Care Diagnostics

“…To address multiplexing limitations of LFAs, VFAs have been developed . VFAs with porous membranes have been demonstrated to detect multiple nucleic acids, proteins and cells in parallel without contamination .…”

AuNP Coupled Rapid Flow-Through Dot-Blot Immuno-Assay for COVID-19 (SARS-CoV-2) Explicit IgG Antibody Revelation

“…[55] A comparative study conducted between conventional ELISA and developed assay revealed a high correlation, which others can find. [56] Dot intensity seemed to increase with corresponding ELISA value upon disease progression (Figure 5, Supplementary Tables 1 and 2). Statistical analysis revealed equivalent clinical agreement between the two techniques as well as with the gold-standard method of RT-PCR, with a Cohen's Kappa value of 0.84 (strong agreement) and 0.94 (robust agreement), respectively, in between < 14 and >14 days (Table 2).…”

Polymeric-Based In Vitro Diagnostic Devices

“…Lamination of polymer layers has been used for the fabrication of IVD devices based on its ease of operation and low cost [15,111]. Microchannels can be formed directly in a pressure-sensitive adhesive (PSA) or thermobond adhesive (TBA) film using a desktop cutting instrument such as a CO 2 laser, die cutter, or vinyl cutter followed by application of two plastic sheets pressed onto both PSA surfaces.…”

“…LFA strips adopt dried AuNP-conjugated antibody regents at the conjugation pad for rapid pregnancy, drug abuse, and other diagnostic tests [220,243]. Flowthrough immunoassays are another option that can be performed using on-chip reagent storage that employs a porous membrane or porous monoliths patterned with capture molecules to capture biomolecules for further tests [111,115].…”