Scorpion sting envenoming is a global public health problem. However, there are geographical areas such as Latin America, North Africa, the Arabian Peninsula and the Middle-East where the incidence is higher (Chippaux and Goyffon, 2008). The treatment focuses on the control of the signs and symptoms of intoxication, as well as the neutralization of toxins (Abroug et al., 2020). The use of different antibody formats for the neutralization of venom from deadly animals has been widely described (Alirahimi et al., 2018, Alvarenga et al., 2014, Bermudez-Mendez et al., 2018, Ljungars and Laustsen, 2023). For example, use of antivenoms from equine-origin in F(ab')2 format against scorpions sting has been already reported (Ismael et al., 2018, Martin-Eauclaire et al., 2019, Pepin-Covatta et al., 1996). The utilization of this kind of antivenoms against the sting of scorpions from Centruroides (C.) genus, has helped to treat scorpionism in Mexico (Jean-Philippe et al., 2020), a public health problem with more than 264,000 cases reported in 2021 (Salud, 2021).

Another antibody format of current interest for scorpion venoms neutralization is single chain fragment variable (scFv) (Alonso Villela et al., 2023, Devaux et al., 2001, Pucca et al., 2014), which is formed by a variable heavy (VH) and light (VL) chain domains of antibodies, connected by a peptide linker (Pirkalkhoran et al., 2023). Its smaller size compared to full antibody (∼28 kDa vs ∼150 kDa) confers it greater bioavailability and rapid renal elimination (Laustsen et al., 2018). Besides, their relatively easy genetic manipulation, biochemical and functional characterization as well as previous results, enable considering them suitable for neutralization of toxins present in scorpion venoms (Fernandez-Taboada et al., 2021, Gomez-Ramirez et al., 2023, Riano-Umbarila et al., 2021).

In order to have an alternative to equine antivenoms production, two families of scFvs (3F and C1) have been developed in our group (Riano-Umbarila et al., 2005) whose members are able to recognize and neutralize different toxins and venoms from Centruroides scorpions (Riano-Umbarila et al., 2011, Riano-Umbarila et al., 2019). On the scFv 3F family side, the scFv RAS27 has been developed through random and site-directed mutagenesis as well as combination of mutations. Cross-reactivity of this scFv facilitate neutralization of CeII9 toxin from C. elegans and Ct1a from C. tecomanus, among others (Romero-Moreno et al., 2023). In the case of scFv C1 family, scFv HV was recently developed by directed evolution of scFv 10FG2 toward Chui5 toxin of C. huichol, which is recognized but not neutralized by this scFv. The scFv HV was capable to neutralize Chui2, Chui3, Chui4 and Chui5 toxins, as well as the whole venom of C. huichol (Valencia-Martinez et al., 2022).

Due to wide diversity of medically important scorpion species in Mexico, there are venoms, such as C. tecomanus venom, that have not been fully characterized. C. tecomanus scorpion inhabits in the state of Colima where 9323 stings were reported in 2021 (Salud, 2021). Some components of its venom have been previously characterized at biochemical (Alamilla et al., 2021, Bermudez-Guzman et al., 2022, Olamendi-Portugal et al., 2016, Valdez-Velazquez et al., 2016) and transcriptomic (Valdez-Velazquez et al., 2013) levels, with Ct1a toxin being the main so far medically important component. In the present work, scorpions were collected from the coastal area of the state of Colima near the border with the state of Michoacán. From the venom of these scorpions, Ct71 toxin was identified. This toxin and Ct1a are the responsible for lethality of this venom. Here, we report the characterization of the venom (where Ct71 toxin is an abundant component) and their neutralization by scFvs HV and RAS27. Additionally, kinetic, thermodynamic and in silico analyses of Ct71-HV interactions were performed.