Venoms

The assay was run by measuring coagulation dynamically (in-time) in plate reader format (most experiments performed in 384-well plate), as an increase in absorbance (more precisely a decrease in transmittance) at a wavelength of 595 or 600 nm during fibrinous clot formation. The addition of an excess of CaCl₂ solution to titrate away the citrate in citrated plasma initiated clot formation. Different calcium concentrations, assay volumes and plasma-to-buffer ratios were evaluated for assay performance. Optimized conditions were then chosen and used to demonstrate the assay with anticoagulants (drugs) and procoagulants (Russell’s viper (Daboia russelii) venom (RVV)).
Assay preparation was performed at room temperature (unless stated otherwise). As coagulation is initiated by the mixing of plasma with the calcium solution, the assay preparation must be performed rapidly, and the plate reader measurement must be initiated, ideally within 5 min. For measurements, a fresh working solution of 20 mM CaCl₂ was prepared (2 weeks expiration time when kept at 4 °C) and frozen aliquots of plasma, either citrated bovine plasma or pooled human plasma, were rapidly defrosted in a water bath (37 °C). Within an hour of defrosting, the plasma was used. Before robotic pipetting (completed with a well plate ThermoScientific™ Multidrop™ 384 Labsystems (see supporting information S1 for the cleaning procedure)), the plasma was centrifuged for 20–30 s at 1400 rpm with an Eppendorf 5810 R centrifuge. The general assay procedure was performed by robotically pipetting CaCl₂ (20 µL/well) followed by plasma (20 µL/well) over a selected area of a 384- well plate.
The prepared plate, containing 40 µL CaCl₂ solution and plasma (1:1) per well, was always placed in the plate reader within 5 min after assay mixture pipetting (unless stated otherwise). The plate reader temperature was set at room temperature (unless stated otherwise). The absorbance was measured at a wavelength of 595 or 600 nm with a Thermo Fisher Scientific Laboratory Varioskan™ LUX Multimode Microplate Reader using SkanIt 4.1. Measurements were performed in one kinetic loop consisting of up to 80 readings, each 80 s long (65 s + 15 s interval time), resulting in a total measurement time of 6400 s per kinetic loop. Three data-processing options in the SkanIt 4.1—(1) single reading, (2) slope of a reading range, and (3) average rate in time per well—were used to determine both procoagulation and anticoagulation activity by processing the coagulation curves (further details in Section 2 and Section 3). The bioassay data is presented as a bioassay chromatogram when used for snake venom screening (Section 2.3). When there is a significant difference in signal intensity between anti- and procoagulation, it should be noted that both procoagulation and anticoagulation activities cannot be monitored in the same manner (details in Section 2.3).

Binding kinetics were analyzed by BLI utilizing the Octet Red 96 system (ForteBio). All assays were conducted in standard Greiner black 96 microtiter well plates. Analyte (venom) samples were diluted 1:20 from the working stock to make a final experimental concentration of 50 µg/mL in the well (10 µg per well). Mimotope aliquots were diluted 1:50 to have a final concentration of 1 µg/mL in the well (0.2 µg per well). Assay running buffer was 1X DPBS with 0.1% BSA and 0.05% Tween-20. This buffer inhibits non-specific binding to the surface of the sensor and other proteins. Prior to experimentation, streptavidin sensors were hydrated in the running buffer for 30–60 min, whilst being agitated at 2.0 RPM on a shaker. To regenerate the sensor tips during experimentation, the dissociation of analytes occurs using a standard acidic solution (glycine buffer), made up of 10mM glycine (pH 1.5–1.7) in deionized water.
Octet RED 96 assay methodology in the ForteBio Data Acquisition 9.0 program was set as follows: 60 s baseline, 50 s loading, 120 s baseline, 120 s association, 120 s dissociation, and 80 s regeneration/neutralization step. The regeneration/neutralization step consists of four cycles, lasting 10 s each, alternating between dipping in glycine buffer (regeneration) and then in running buffer (neutralization) per cycle. For each baseline step throughout the experiment the same running buffer was used a maximum of three times per well. Experiments were run at 30 °C with the orbital agitation of the microplate set to 1000 rpm. Experiments were limited to less than 1 h to limit the change in analyte concentration due to evaporation on the warmed plate [32 (link)].
Analytes were set up in rows A-H, with triplicates set up in columns 1–3 and 4–6. To account for any potential evaporation effect in the wells during experimentation, the column running order was set to 1, 4, 2, 5, 3, 6 (rather than 1, 2, 3, 4, 5, 6). The mimotopes were set in column 7, running buffer was set in columns 8–10, and regeneration step (glycine buffer) and neutralization step (running buffer) were columns 11 and 12 respectively.
Negative controls consisted of deionized water:glycerol 1:1 mix in replacement of the sample in the wells. Bungarus multicinctus venom was used as a positive control, as it has been shown to bind nAChR mimotopes [14 (link),15 (link),16 (link)].

Serum sIgE‐levels to honeybee venom (HBV), wasp venom (WV), hornet venom (HV), and recombinant allergens: rApi m 1, rApi m 2, icarapin (rApi m 10) from HBV, rVes v 5, phospholipase A1 (rVes v 1) from WV, and CCD were measured by Immunoblot (Euroimmun, Germany, Euroline DPA‐Dx insect venoms 3). Serum samples of 29/71 multiple‐positive patients were tested in the EUROLINE assay using the anti‐CCD Absorbent (Euroimmun). The absorbent (CCD‐inhibitor) for eliminating CCD‐sIgE was composed of glycoprotein bromelain extracted from pineapple. Serum samples were incubated with anti‐CCD Absorbent for 60 min at room temperature according to the manufacturer's instructions. The sera were retested using the same insect venoms profile, as previously mentioned.
The cut‐off value for positive sIgE‐test results was 0.35 kUA/L. The sIgE‐reactivity was categorized quantitatively into six classes: Class 1 (≥0.35 to <0.70 kUA/L), Class 2 (≥0.70 to <3.50 kUA/L), Class 3 (≥3.50 to <17.50 kUA/L), Class 4 (≥17.50 to <50.00 kUA/L), Class 5 (≥50.00 to <100.00 kUA/L), and Class 6 (≥100.00 kUA/L).

For this study, specimens were collected alive from Iran (Zamani et al., 2021 ). The Agelena labyrinthica was photographed with a camera. The specimens were kept under suitable conditions, humidity (60%), and temperature (25°C) and fed by crickets and mealworms. The specimens were identified using the taxonomic keys (Nentwig et al., 2017 ). Epigyne of the female specimen was visualized with a stereoscope.
Female spiders were separated to extract the venom. Specimens were anesthetized with CO₂ in a small chamber, and the opisthosoma and carapace were removed under the stereoscope. The fangs and venom glands of the female specimen were visualized by a loop. Venom glands were collected into 4°C Phosphate buffered saline (PBS) were prepared in the laboratory with this recipe (137 mM NaCl, 3 mM KCl, 10 mM Na₂PO₄, 2 mM KH₂PO₄, and pH 7.4) and gently crushed with a glass stirrer for 30 min. Then, pieces of the venom gland were removed from the solution by centrifugation at 13,000 rpm for 30 min at 4°C, and the supernatant was lyophilized and stored at −70°C. Protein concentration was measured by Bradford assay with bovine serum albumin as standard protein.