Full Neutralization of Centruroides sculpturatus Scorpion Venom by Combining Two Human Antibody Fragments

Abstract

A fundamental issue of the characterization of single-chain variable fragments (scFvs), capable of neutralizing scorpion toxins, is their cross-neutralizing ability. This aspect is very important in Mexico because all scorpions dangerous to humans belong to the Centruroides genus, where toxin sequences show high identity. Among toxin-neutralizing antibodies that were generated in a previous study, scFv 10FG2 showed a broad cross-reactivity against several Centruroides toxins, while the one of scFv LR is more limited. Both neutralizing scFvs recognize independent epitopes of the toxins. In the present work, the neutralization capacity of these two scFvs against two medically important toxins of the venom of Centruroides sculpturatus Ewing was evaluated. The results showed that these toxins are recognized by both scFvs with affinities between 1.8 × 10−9 and 6.1 × 10−11 M. For this reason, their ability to neutralize the venom was evaluated in mice, where scFv 10FG2 showed a better protective capacity. A combination of both scFvs at a molar ratio of 1:5:5 (toxins: scFv 10FG2: scFv LR) neutralized the venom without the appearance of any signs of intoxication. These results indicate a complementary activity of these two scFvs during venom neutralization.

Anti-Strychnine Immunoconjugate Reduces the Effects of Strychnine-Induced Toxicity in Mice

“…Synthesis of Strychnine Hapten and Conjugates. 1 H, 13 C, and 19 F NMR spectra were measured in DMSO-d 6 on a Bruker Avance 500 MHz spectrometer. Chemical shifts are reported in ppm employing the residual solvent resonance as the internal standard ( 1 H DMSO , δ 2.50 ppm; 13 C DMSO , 39.52 ppm).…”

“…1 H, 13 C, and 19 F NMR spectra were measured in DMSO-d 6 on a Bruker Avance 500 MHz spectrometer. Chemical shifts are reported in ppm employing the residual solvent resonance as the internal standard ( 1 H DMSO , δ 2.50 ppm; 13 C DMSO , 39.52 ppm). LC−MS analysis was performed on a Dionex Ultimate 3000 uHPLC system coupled to a Thermo Scientific TSQ Quantum Access MAX triple quadrupole mass spectrometer.…”

“…1 H NMR (500 MHz, DMSO-d 6 ): δ 11.17 (br s, 1H), 8.11 (t, J = 5.6 Hz, 1H), 7.96 (d, J = 8.4 Hz, 1H), 7.87 (t, J = 5.6 Hz, 1H), 7.78−7.62 (m, 4H), 7.54 (dd, J = 8.5, 1.4 Hz, 1H), 7.41 (d, J = 15.7 Hz, 1H), 6.56 (d, J = 15.7 Hz, 1H), 6.39−6.30 (m, 1H), 4.57−4.48 (m, 1H), 4.37 (dt, J = 7.5, 3.5 Hz, 1H), 4.23−4.08 (m, 4H), 3.82− 3.74 (m, 1H), 3.56 (d, J = 13.7 Hz, 1H), 3.37−3.30 (m, 1H), 3.24− 3.14 (m, 3H), 3.14−3.03 (m, 3H), 2.99 (dd, J = 17.2, 8.3 Hz, 1H), 2.85−2.73 (m, 2H), 2.66 (dd, J = 17.2, 2.7 Hz, 1H), 2.22 (dd, J = 13.0, 6.0 Hz, 1H), 2.14−2.06 (m, 2H), 1.94 (td, J = 13.4, 8.0 Hz, 1H), 1.67−1.46 (m, 8H). 13 sKLH-NH 2 -Strychnine (Strych-sKLH). To sKLH protein (19.7 mg) in H 2 O (1 mL) was added 1 mL of pH 6 1× PBS buffer.…”

“…This work investigated the feasibility of immunotherapy against strychnine to prevent symptoms of toxicity. Antibody-based treatments (antitoxins) including polyclonal sera, Fab, or scFv have been used historically, are currently in use, or are being studied to neutralize components of venom from scorpions, spiders and snakes, − and protein toxins such as botulinum, diphtheria, anthrax, tetanus, and ricin. , Antitoxins consisting of immune sera are generally produced from sheep or horses immunized with sub-lethal doses of these toxins and are employed to reduce or reverse symptoms by binding to toxins and preventing access to target receptors. These antibodies or derived antibody fragments (e.g., Fab and scFv) neutralize target proteins in circulation, reducing symptoms of toxicity and improving the probability of survival.…”

Characterization of Four Medically Important Toxins from Centruroides huichol Scorpion Venom and Its Neutralization by a Single Recombinant Antibody Fragment

“…Based on these observations, mix-type assays were carried out [16], where 1 LD 50 of fresh C. huichol venom was mixed with scFvs LR and 10FG2 at a molar ratio of 1:10:10 (venom: scFv LR: scFv 10FG2). To estimate the amount of scFv to be used, in this experiment it was assumed that the proportion of toxins corresponds to 10% of the venom (average value with respect to the venoms studied so far [16,18,20]).…”