Additional file 1: of Tityus serrulatus envenoming in non-obese diabetic mice: a risk factor for severity

Representative comparison of eyeball of Ts envenomed and non-envenomed mice. Mice were injected with Ts venom (1 mg/kg). (A) Envenomed NOD mice with glucose basal level ≥ 200 mg/dL, retinopathy indication. (B) Envenomed BALB/c mice, typical ptosis. (C) BALB/c mice control (non-envenomed), normal eyeball. (PPTX 81 kb

Molecular and electrophysiological characteristics of the neurotoxins used in the study.

Molecular and electrophysiological characteristics of the neurotoxins used in the study.</p

Nociception assays with Ts5 response neutralized.

(A) Nociceptive behavior was evaluated in BALB/c male mice (n = 6) using 0.5 μg/animal paw of TsV as positive control animals. In the tested group of animals, TsV (1 μg/animal paw) was pre-incubated 1 h at 37° C with 2 eqM of purified IgGs anti-Ts5 (considering the % of Ts5 from the whole venom). The same rate was used in the positive control group, using 0.5 μg of Ts5/animal paw pre-incubated 1 h at 37° C with 2 eqM of IgGs anti-Ts5. Animals were observed for 35 min to record the time the animals spent either licking or lifting/shaking the injected paw. (B) Area under curve (AUC) over any individual parameter in the evaluation of nociceptive response with Ts5 neutralized. Data was analyzed using one-way ANOVA and Tukey’s post hoc test. * p < 0.05, when compared to the control group. Graphs were generated using GraphPad Prism Version 9.3.1.</p

Nociception video.

(WMV)</p

Ts5-immunization assay.

(A) Male BALB/c mice (n = 3) received intramuscularly (i.m.) 7.5 μg of Ts5 antigen emulsified in a solution containing complete Freunds’ adjuvant (CF), followed by two sequential i.m. boosts with 3-week intervals of 7.5 μg and 5 μg/animal, respectively. The second boost with Ts5 was diluted in a solution with incomplete Freunds’ adjuvant (IF), and the third boost consisted only by the antigen (Ts5). Control animals (n = 2) received solely saline solution (vehicle). Figure was created with BioRender.com (Agreement number: BO23GVKOCK). (B) Immunorecognition of Ts5 toxin (2 μg/well) by animal’s sera used in experimental immunization. C (-): mice sera without immunization; M1(-): Mouse 1 serum before immunization; M1: Mouse 1 immunized with Ts5; M2 (-): Mouse 2 serum before immunization; M2: Mouse 2 immunized with Ts5; M3(-): Mouse 3 serum before immunization; M3: Mouse 3 immunized with Ts5. The ELISA assay was performed in duplicate, and results are plotted as mean ± SD. Data was analyzed using one-way ANOVA and Tukey’s post hoc test. Graph was generated using GraphPad Prism Version 9.3.1. *p < 0.05, when compared to negative controls.</p

Nociceptive assays.

(A) Nociceptive behavior was evaluated in C57BL/6 male mice (n = 6) using 0.5 μg paw injections of TsV and isolated toxins (Ts1, Ts5, Ts8, Ts19 frag II). Animals were observed for 35 min to record the time the animals spent either licking or lifting/shaking the injected paw. (B) Area under curve (AUC) over any individual parameter in the evaluation of nociceptive response. Data was analyzed using one-way ANOVA and Tukey’s post hoc test. * p p (C) Mice nociceptive behavior following paw injection with different quantities of Ts5 (0.1–4 μg). Graphs were generated using GraphPad Prism Version 9.3.1.</p

Ts5 recognition by scorpion antivenom and predicted epitopes.

(A) TsV + NI (-): TsV + non-immune horse serum; TsV + AS: TsV + scorpion antivenom (Butantan Institute); Ts5 + NI (-): Ts5 + non-immune horse serum; Ts5 + AS: Ts5 + scorpion antivenom (Butantan Institute); C (+): wells coated with non-immune horse serum. Rabbit anti-horse-HRP antibodies were used for the ELISA detection. The ELISA assay was performed in duplicate and results are plotted as mean ± SD. Data was analyzed using one-way ANOVA and Tukey’s post hoc test. Graph was generated using GraphPad Prism Version 9.3.1. *p (B) Ts5 primary sequence (P46115) [32] with the B-cell epitopes predicted by ABCpred Server tool highlighted and their scores. (C) Ts5 molecular model from AlphaFold Protein Structure Database [38, 39], modified by PyMol tool [40], with front and back view. Colors (blue, red, orange, and magenta) are not related to the alpha-helix and beta-pleated sheets, they represent the four B-cell epitopes predicted in different types of 3D representation: surface (up) and ribbon (down).</p

Purification of IgG anti-Ts5 antibodies.

(A) SDS-PAGE (8%) under reducing conditions following M2 mice-derived serum IgG isolation. Lane 1: mice serum (M2); Lane 2: void; Lane 3: washing; Lane 4: Molecular mass marker (97.0–14.4 kDa); Lane 5: washing 2; Lane 6: washing 3; Lane 7: Proteins (IgGs) eluted on tube 1; Lane 8: Proteins (IgGs) eluted on tube 2; 9: Proteins (IgGs) eluted on tube 3. SDS-PAGE gel was stained with Coomassie brilliant blue. (B) Immunorecognition of Ts5 toxin by the pooled anti-Ts5 IgGs’ eluted (tubes 1–3). C1(-): wells non sensitized; C2(-): wells sensitized with Ts5 (2 μg) and tested with non-immune mouse serum; C3(+): wells sensitized with mouse non-immune serum; 1:100 and 1:50: wells sensitized with Ts5 (2 μg) and tested with purified mouse anti-Ts5 IgGs. Rat anti-mouse-HRP antibodies were used in the ELISA detection. The ELISA assay was performed in duplicate, and results are plotted as mean ± SD. Data was analyzed using one-way ANOVA and Tukey’s post hoc test. Graph was generated using GraphPad Prism Version 9.3.1. *p < 0.05, when compared to negative controls.</p

<i>T</i>. <i>serrulatus</i> venom and toxins: Mass and molarity used for nociceptive assays.

T. serrulatus venom and toxins: Mass and molarity used for nociceptive assays.</p