Western blot of purified VHHs against BthTX-I.

<p>15% SDS-PAGE was carried out to resolve BthTX-I and BthTX-II under reducing conditions. After electrophoresis, BthTX-I and BthTX-II were electrotransferred to a nitrocellulose membrane and probed with selected VHHs (KF498607, KF498608, KC329715, and KC329718). Samples were incubated with mouse anti-His antibody followed by HRP-conjugated anti-mouse IgG produced in goat. Reactive signals were detected by DAB staining in the presence of hydrogen peroxide. <i>Lama glama</i> pre-immune serum was used as negative control (-), and <i>Lama glama</i> immune serum, as positive control (+).</p

<i>In vitro</i> inhibition of phospholipase activity.

<p>The quantification of phospholipase activity inhibition by selected VHHs was assayed using synthetic fluorescent phospholipid. To verify the ability of VHHs to inhibit the phospholipase activity of BthTX-II, the toxin was pre-incubated with selected VHHs for 30 minutes at 37°C in different proportions (1:5; 1:10 and 1:40 w/w). Inhibition of phospholipase activity by (A) KF498607; (B) KF498608; (C) KF329715; (D) KC329718. BthTX-II activity on the phospholipid, in the absence of VHH, was used as a positive control, and considered as having 100% activity. The negative controls were carried out using medium reaction without BthTX-II. All measurements were performed in duplicate. Error bars represent standard deviation.</p

Selection of anti-BthTX-I and anti-BthTX-II VHHs.

<p>VHHs from the recombinant phage displayed library were submitted separately against immobilized BthTX-I or BthTX-II. After one round of panning, 82 and 28 clones selected for BthTX-I and BthTX-II, respectively, presented VHHs. ELISA assays were performed to verify clone reactivity. Twenty-eight (28) and six (06) clones recognized BthTX-I and BthTX-II. (A) Clonal reactivity of VHHs (1–40) selected against BthTX-I, NC–negative control; (B) Clonal reactivity of VHHs (41–83) selected against BthTX-I, Cross-reactivity of VHH 82 with BthTX-II, NC–negative control; (C) Clonal reactivity of VHHs (1–37) selected against BthTX-II, Cross-reactivity of 02, 09, 20, 21, 28 and 30 VHHs with BthTX-I, NC–negative control. All clones that showed an absorbance value (OD 450nm) higher than the stipulated cut-off point (2 mean OD from negative samples plus 2 standard deviations) were considered positive. All measurements were performed in triplicate. The negative control was performed using the llama pre-immune serum. Error bars represent standard deviation.</p

VHHs affinity to BthTX-I and BthTX-II.

<p>Representative sensorgrams of the interaction were measured in a Biacore T200 system (GE Healtcare). Purified VHHs (KF498607, KF498608 and KC329718) at concentrations of 6.25 to 0.049 μg/mL were flowed over a BthTX-I-coated CM5 chip and 25 to 0.006 μg/mL were used for immobilized BthTX-II. (A) KF498607 x BthTX-I; (B) KF498607 x BthTX-II; (C) KF498608 x BthTX-I; (D) KF498608 x BthTX-II; (E) KC329718 x BthTX-I; (F) KC329718 x BthTX-II. Assays were injected in a flow rate of 30 μl/min at 37°C. RU indicates resonance units.</p

Amino acid sequence alignment of anti-BthTX-I and BthTX-II VHHs.

<p>The clones were clustered in four groups based on sequence homologies. The framework regions (FR) as well as the complementarity determining regions (CDR) are indicated with arrows; two conserved cysteines are shaded; VHH hallmark substitutions in FR2 and FR4 are bolded and underlined. Cluster I (KF498607, KF498608, KF498609, and KC329718), Cluster II (KC329709, KC329714), Cluster III (KC329713), and Cluster IV (KC329710, KC329711, KC329712, KC329715, KC329716, KC329717). The colon (:) represents highly conserved amino acids; the asterisk (*) represents identical amino acid residues; the period (.) means somewhat similar but different amino acids and blank represents dissimilar amino acids or gaps.</p

Docking results showing binding sites of VHHs on surfaces of PLA₂s.

<p>(A) KF498607 (blue ribbon) and BthTX-I (red surface); (B) KC329718 (blue ribbon) and BthTX-I (red surface); (C) KF498607 (blue ribbon) and BthTX-II (orange surface); (D) KC329718 (blue ribbon) and BthTX-II (orange surface). The interaction sites were enlarged to show hydrogen bonds formed between the amino acid residues. Both VHHs covered the enzymatic groove surface areas of the modeled BthTX-II and inserted the CDR3 into the catalytic cleft, as well as being able to interact with amino acid residues of the PLA₂ C-termini.</p

Interaction analysis by SPR.

<p>Interaction analysis by SPR.</p

<i>In vivo</i> neutralization of <i>B</i>. <i>jararacussu</i> venom and PLA₂-induced myotoxicity by VHHs.

<p>Plasma creatine kinase (CK) levels were measured at 340 nm to determine VHH ability to inhibit the myotoxicity caused by <i>B</i>. <i>jararacussu</i> venom or PLA₂s, or by a related toxin, MTX-I, a PLA₂ isolated from <i>B</i>. <i>brazili</i>. For this, groups of 5 animals were injected with PBS, VHH KF498607, VHH KC329718, BthTX-I, BthTX-I + VHH KF498607, BthTX-I + VHH KC329718, BthTX-II, BthTX-II + VHH KF498607, BthTX-II + VHH KC329718, <i>B</i>. <i>jararacussu</i> venom, <i>B</i>. <i>jararacussu</i> venom + VHH KF498607, <i>B</i>. <i>jararacussu</i> venom + VHH KC329718, MTX-I, MTX-I + VHH KC329718, MTX-I + VHH KC329718. Before administration to mice, venom or PLA₂s and antibody preparations were pre-incubated at 37°C for 1 h, in a proportion 1:5 or 1:10 (w/w). The negative control was performed with PBS or VHHs, and as a positive control, animals were injected with <i>B</i>. <i>jararacussu</i> venom, BthTX-I, BthTX-II or MTX-I without the addition of VHH. Bonferroni’s test was used for significance analysis. (*) P <0.05. Error bars represent standard deviation.</p

PDB code, resolution, references, percent similarity and RMSD of templates for modeled VHHs.

<p>PDB code, resolution, references, percent similarity and RMSD of templates for modeled VHHs.</p

Cross-reactivity of anti-BthTX-I and BthTX-II VHHs.

<p><i>In vitro</i> reactivity showing different levels of interaction of selected VHHs (A) KF498607; (B) KF498608; (C) KF329715; (D) KC329718 with a variety of snake venoms (<i>Bothrops alternatus</i>, <i>Bothrops atrox</i>, <i>Bothrops bilineata</i>, <i>Bothrops brazili</i>, <i>Bothrops diporus</i>, <i>Bothrops insularis</i>, <i>Bothrops jararaca</i>, <i>Bothrops leucurus</i>, <i>Bothrops marajoensis</i>, <i>Bothrops matogrossensis</i>, <i>Bothrops moojeni</i>, <i>Bothrops pauloensis</i>, <i>Bothrops pirajai</i>, <i>Bothrops urutu</i>, <i>Calloselasma rhodostoma</i>, <i>Crotalus atrox</i>, <i>Crotalus durissus cascavella</i>, <i>Crotalus durissus collilineatus</i>, <i>Crotalus durissus cumanensis</i>, <i>Crotalus durissus terrificus</i>, <i>Micrurus spixii</i>), and isolated toxins (PLA₂ from <i>Bothrops atrox</i>, and PLA₂-I, convulxin, crotamin crotapotin, crotoxin, and giroxin from <i>Crotalus durissus terrificus</i>). After being coated on the wells, venoms and toxins were probed with selected VHHs. Samples were incubated with mouse anti-His antibody and the reactive signals were detected after incubation with HRP-conjugated anti-mouse IgG produced in goat and TMB. All clones that showed an absorbance value (OD 450nm) higher than the stipulated cut-off point (2 mean OD from negative samples plus 2 standard deviations) were considered positive. The dashed lines represent the cut off. All measurements were performed in triplicate. For the negative control (NC), wells were not coated with venoms or toxins. Error bars represent standard deviation.</p