Rapid and Efficient Enrichment of Snake Venoms from Human Plasma Using a Strong Cation Exchange Tip Column to Improve Snakebite Diagnosis

Abstract

Snake envenomation is a serious public health issue in many tropical and subtropical countries. Accurate diagnosis and immediate antivenom treatment are critical for effective management. However, the venom concentration in the victims’ plasma is usually low, representing one of the bottlenecks in developing clinically applicable assays for venom detection and snakebite diagnosis. In this study, we attempted to develop a simple method for rapid enrichment of venom proteins from human plasma to facilitate detection. Our experiments showed that several major protein components of both Naja atra (N. atra) and Bungarus multicinctus (B. multicinctus) venoms have higher isoelectric point (pI) values relative to high-abundance human plasma proteins and could be separated via strong cation exchange–high-performance liquid chromatography (SCX-HPLC). Based on this principle, we developed an SCX tip column-based protocol for rapid enrichment of N. atra and B. multicinctus venom proteins from human plasma. Application of liquid chromatography-tandem mass spectrometry (LC-MS/MS) led to the identification of cytotoxin and beta-bungarotoxin as the major proteins enriched by the SCX tip column in each venom sample. The entire process of venom enrichment could be completed within 10–15 min. Combination of this method with our previously developed lateral flow strip assays (rapid test) significantly enhanced the sensitivity of the rapid test, mainly via depletion of the plasma protein background, as well as increase in venom protein concentration. Notably, the SCX tip column-based enrichment method has the potential to efficiently enrich other Elapidae snake venoms containing proteins with higher pI values, thereby facilitating venom detection with other assays. This simple and rapid sample preparation method should aid in improving the clinical utility of diagnostic assays for snakebite.

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

“…S2 and SI Table S1 , all sample matrices tested in the NaCl-free running buffers had non-specific binding, resulting in false positives; the most intense test line was observed using the running buffer with Tergitol. Adding NaCl to the running buffer reduced the false positives in serum samples, indicating it could inhibit non-specific binding caused by matrix effects, as others have reported 31 , 42 . It is essential to consider the intended sample matrix early in the LFA development process, as success in buffer systems rarely reflects how the assay will perform in biological matrices 26 .…”

“…S2 and SI Table S1, all sample matrices tested in the NaCl-free running buffers had non-specific binding, resulting in false positives; the most intense test line was observed using the running buffer with Tergitol. Adding NaCl to the running buffer reduced the false positives in serum samples, indicating it could inhibit non-specific binding caused by matrix effects, as others have reported 31,42 .…”

“… Asia, Africa Horse & rabbit pAbs AuNPs 20 min 5–500 ng/mL in serum depending on which of eight species was tested Spiked fetal bovine serum 34 Multiplex D. russelii Asia Horse & goose pAbs AuNPs 25 min 10 ng/mL in fetal bovine serum Spiked fetal bovine serum & serum from patients 35 Singleplex N. naja B. caeruleus Asia Mouse mAbs AuNPs Not reported 5000–250,000 ng/mL in buffer for recombinant toxin, 25,000–250,000 ng/mL in buffer for N. naja , 25,000–250,000 ng/mL in buffer for B. caeruleus Spiked fetal bovine serum 36 Multiplex B. multicinctus N. atra Asia Horse pAbs AuNPs 10–15 min (enrichment) + 15 min (LFA) 5 ng/mL (without enrichment) and 1 ng/mL (with enrichment) in plasma for B. multicinctus . 5 ng/mL (with enrichment) in plasma for N. atra Spiked human plasma 31 Singleplex B. multicinctus Asia Rabbit pAbs AuNPs 10–15 min 1 ng/mL in buffer for B. multicinctus Tissue homogenates, blood, and urine from envenomed rats 37 Singleplex Bothrops spp. Latin America Mouse mAbs AuNPs 5 min (pre-incubation) + 15 min (LFA) …”

“…Yet, detection limits are impacted by biological matrices where biomolecules can interact with the analyte or the binders, influencing the assay sensitivity by introducing non-specific matrix interactions 26 . Recently, a method was developed for eliminating plasma matrix effects by pre-enriching venoms using a cation exchange tip, but this method only works for elapid venoms 31 .…”

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

“…Generally, venom concentrations in patient samples are expected to vary depending on the sample matrix (e.g., urine or serum), time since the bite occurred, the body size of the patient, and other parameters. If the LoD of the prototype proves to be too high to detect venom in a certain sample type or at a certain time (e.g., in a urine sample immediately after the bite), it might be possible to improve the LoD by enriching the venom from the sample, or alternatively to measure another sample type (e.g., a wound swab or serum sample) [28,29]. Conversely, if the antigen concentration in the sample is high enough to bring the assay into the hook effect range, it might be possible to dilute the sample.…”