Detection of Infertility-related Neutralizing Antibodies with a Cell-free Microfluidic Method

Abstract

The unwanted emergence of neutralizing antibodies (nAbs) against an endogenous or a therapeutic protein can result in deficiency diseases or therapy failure. Here, we developed a cell-free microfluidic method for the sensitive detection and quantification of nAbs in human serum that are associated with infertility. We used cell-derived vesicles containing the luteinizing hormone (LH)/choriogonadotropin receptor (LHHCGR) to detect nAbs against LH. The method exploits the entire cellular signal amplification mechanism, and facilitates the detection of as little as 0.44 nM of LH-nAb (Kd 1.5 nM) in human serum matrix within only 15 minutes. In addition, dose-response curves can be generated in less than 2 hours to evaluate the nAB concentration and dissociation constant. The developed system is devoid of problems associated with cell-based assays and we believe that this simple effect-directed analysis can be used in clinical environments, and is adaptable to other hormones or cytokines and their respective nAbs.

Cell‐Derived Vesicles as TRPC1 Channel Delivery Systems for the Recovery of Cellular Respiratory and Proliferative Capacities

“…Previously published protocols for producing cell‐derived vesicles have rendered important information about cellular signal transduction as well as revealed a provocative potential for such platforms to serve as biological‐based sensors or as vehicles for drug delivery. [ 6–9,54,55 ] Here, we reveal a novel potential of CDVs as biological sensors for temperature (menthol‐sensitivity) or magnetic fields and furthermore demonstrated that CDVs possess the capacity to recover sensory transduction of developmental importance for potential therapeutic use in regenerative medicine. Importantly, these signaling and developmental attributes of CDVs can be ascribed to their expression of TRP channels associated with the surface membrane.…”

“…The cytochalasin B method of producing CDVs is an established protocol. [6][7][8][9] Nonetheless, vesicle yields employing this method vary with cell type, culture conditions or subtle variations in the preparation protocol. [8,9] Dynamic light scattering (DLS) measurements indicated an ≈10-fold greater yield of CDVs with the addition of a shaking step (300 rpm, 37 °C, 15 min) implemented during the incubation with cytochalasin B.…”

“…[5] The use of cell-derived vesicles (CDVs) as delivery vehicles for cellular sensory apparatuses is thus appealing as they have been shown in some cases to retain the necessary molecular components to form functioning signaling apparatuses capable of conferring cellular responses to environmental stimuli. [6][7][8][9] CDV-based platforms may hence be applicable to reverse states of cell senescence or quiescence as a result of mitochondrial dysfunction. [2,3] Pulsed electromagnetic fields (PEMFs) are capable of specifically activating a TRPC1-mitochondrial axis underlying cell expansion and mitohormetic survival adaptations.…”

Microfluidics‐Implemented Biochemical Assays: From the Perspective of Readout

“…The luminescence signals will generate upon different conditions. Reproduced with permission . Copyright 2015, Springer Nature.…”

“…The target bacteria could be then detected based on the bioluminescence generation from the biochemical reactions, allowing an accurate detection of Escherichia coli with 10 −4 CFU mL −1 within 20 min. Dittrich's group has reported a cell‐free microfluidic platform to detect neutralizing antibodies (nAbs) with high sensitivity and ease of use based on cell‐derived vesicles allowing for the biochemical reactions that generated bioluminescence (Figure C) . The nAbs in human serum against luteinizing hormone has been detected with a straightforward way.…”