Cellulose immobilization strategies for paper-based immunoassays

Paper-based analytical devices (PADs) enable the affordable, easy, rapid, and reliable detection of a range of analytes at the point-of-care. Cellulose is a versatile substrate for antibody attachment. However, how immunoreagents are immobilized onto cellulose plays an important role in assay performance. To provide an overview of the different antibody immobilization strategies used for developing PADs, we here critically review the existing literature from the last decade (2013-2023). First, we introduce cellulose as a substrate and summarize the different fabrication techniques for developing PADs. Thereafter, we delve into how antibodies are immobilized onto cellulose passively, covalently, or by affinity linkage. Affinity-based immobilization is further explored in a case study focused on using recombinant bifunctional proteins with cellulose-binding modules to anchor immunoreagents to cellulose. The final section discusses the validation and sustainable development of PADs addressing the existing and future challenges associated with developing such devices

A comparative study of the performance of <i>E. coli</i> and <i>K. phaffii</i> for expressing α-cobratoxin

Three-finger toxins (3FTxs) have traditionally been obtained via venom fractionation of whole venoms from snakes. This method often yields functional toxins, but it can be difficult to obtain pure isoforms, as it is challenging to separate the many different toxins with similar physicochemical properties that generally exist in many venoms. This issue can be circumvented via the use of recombinant expression. However, achieving the correct disulfide bond formation in recombinant toxins is challenging and requires extensive optimization of expression and purification methods to enhance stability and functionality. In this study, we investigated the expression of α-cobratoxin, a well-characterized 3FTx from the monocled cobra (Naja kaouthia), in three different expression systems, namely Escherichia coli BL21 (DE3) cells with the csCyDisCo plasmid, Escherichia coli SHuffle cells, and Komagataella phaffii (formerly known as Pichia pastoris). While none of the tested systems yielded α-cobratoxin identical to the variant isolated from whole venom, the His6-tagged α-cobratoxin expressed in K. phaffii exhibited a comparable secondary structure according to circular dichroism spectra and similar binding properties to the α7 subunit of the nicotinic acetylcholine receptor. The findings presented here illustrate the advantages and limitations of the different expression systems and can help guide researchers who wish to express 3FTxs

Multiplex lateral flow assay development for snake venom detection in biological matrices

Abstract Bothrops and Lachesis are two of Brazil’s medically most relevant snake genera, causing tens of thousands of bites annually. Fortunately, Brazil has good accessibility to high-quality antivenoms at the genus and inter-genus level, enabling the treatment of many of these envenomings. However, the optimal use of these treatments requires that the snake species responsible for the bite is determined. Currently, physicians use a syndromic approach to diagnose snakebite, which can be difficult for medical personnel with limited training in clinical snakebite management. In this work, we have developed a novel monoclonal antibody-based multiplex lateral flow assay for differentiating Bothrops and Lachesis venoms within 15 min. The test can be read by the naked eye or (semi)-quantitatively by a smartphone supported by a 3D-printed attachment for controlling lighting conditions. The LFA can detect Bothrops and Lachesis venoms in spiked plasma and urine matrices at concentrations spanning six orders of magnitude. The LFA has detection limits of 10–50 ng/mL in spiked plasma and urine, and 50–500 ng/mL in spiked sera, for B. atrox and L. muta venoms. This test could potentially support medical personnel in correctly diagnosing snakebite envenomings at the point-of-care in Brazil, which may help improve patient outcomes and save lives

Prototyping of a lateral flow assay based on monoclonal antibodies for detection of Bothrops venoms

Background: Brazil is home to a multitude of venomous snakes; perhaps the most medically relevant of which belong to the Bothrops genus. Bothrops spp. are responsible for roughly 70% of all snakebites in Brazil, and envenomings caused by their bites can be treated with three types of antivenom: bothropic antivenom, bothro-lachetic antivenom, and bothro-crotalic antivenom. The choice to administer antivenom depends on the severity of the envenoming, while the choice of antivenom depends on availability and on how certain the treating physician is that the patient was bitten by a bothropic snake. The diagnosis of a bothropic envenoming can be made based on expert identification of the dead snake or a photo thereof or based on a syndromic approach wherein the clinician examines the patient for characteristic manifestations of envenoming. This approach can be very effective but requires staff that has been trained in clinical snakebite management, which, unfortunately, far from all relevant staff has. Results: In this article, we describe a prototype of the first lateral flow assay (LFA) capable of detecting venoms from Brazilian Bothrops spp. The monoclonal antibodies for the assay were generated using hybridoma technology and screened in sandwich enzyme-linked immunosorbent assays (ELISAs) to identify Bothrops spp.-specific antibody sandwich pairs. The prototype LFA is able to detect venom from several Bothrops spp. The LFA has a limit of detection (LoD) of 9.5 ng/mL in urine, when read with a commercial reader, and a visual LoD of approximately 25 ng/mL. Significance: The work presented here serves as a proof of concept for a genus-specific venom detection kit that could support physicians in diagnosing Bothrops envenomings. Although further optimisation and testing is needed before the LFA can find clinical use, such a device could aid in decentralising antivenoms in the Brazilian Amazon and help ensure optimal snakebite management for even more victims of this highly neglected disease.ISSN:0003-2670ISSN:1873-432