Antigenic reactivity of selected monoclonal antibodies (mAbatrs).

<p>(A) Reactivity of purified mAbatrs (5 µg/mL) against several P-I SVMPs and <i>B. atrox</i> venom and BSA (negative control-not shown) was measured by ELISA. (B) Reactivity of mAbatr1, 2, 3 and 6 against <i>B. atrox</i> (from Brazil and Peru), <i>B. barnetti</i>, <i>B. brasili</i>, <i>B. casteunaldi</i>, <i>B. chloromelas</i>, <i>B. hyoprora</i>, <i>B. microphtalmus</i>, <i>B. peruvianus</i>, <i>B. pictus</i>, <i>B. taeniata</i> e <i>Lachesis muta muta</i> venoms (40 µg/mL) was accessed by ELISA. (C) Reactivity of polled mAbatr1, 2, 3 and 6 in sandwich ELISA against Peruvian <i>B. atrox</i> venom, <i>L. muta muta</i>, <i>C. durissus</i> and <i>M. frontalis</i> venoms (10 µg/mL) diluted in mice sera simulating experimental envenoming. Positive controls were performed using diluting <i>B. atrox</i> venom at the same concentration diluted in a PBS buffer (not shown). Threshold absorbance is represented as at least double that obtained from the blank wells. (*p<0.001). Results are expressed as mean of the absorbance value of triplicates.</p

Neutralization of Atr-I or <i>B. atrox</i> venom enzymatic activities on Abz-LVEALYQ-EDDnp substrate.

<p>Purified mAbatrs were pre-incubated with Atr-I or <i>B. atrox</i> venom at 37°C for 30 minutes previously to addition of the FRET substrate. Results are normalized to Atr-I or <i>B. atrox</i> venom alone (positive control) and represent means ±S.D. of triplicates.</p

mAbatrs affinity to Atr-I.

<p>Kinetic parameters were measured in a ProteOn system. mAbatrs were immobilized on a chip and different concentrations of Atr-I were injected in a flow of 30 µL/min. Binding was evaluated at room temperature.</p

Neutralization of Atr-I-induced hemorrhage in mice.

<p>Residual hemorrhage was evaluated after incubating mAbatr1, mAbatr2 or mAbatr3 with 1 MHD of Atr-I (<b>A</b>) or 1.8 MHD of <i>B. atrox</i> crude venom (<b>B</b>) and injecting subcutaneously in mice. After 3 hours mice were euthanized and skins removed. Negative control was saline and positive controls were Atr-I or <i>B. atrox</i> venom alone.</p

Cross-reactivity of mAbatrs with different toxins from <i>B. atrox</i> venom analyzed by western blotting.

<p><i>B. atrox</i> crude venom was transferred to a nitrocellulose membrane and incubated with rabbit polyclonal anti-Atr-I serum (<b>C</b>) as control, or mAbatr1 (<b>1</b>), mAbatr2 (<b>2</b>), mAbatr3 (<b>3</b>) or mAbatr6 (<b>6</b>). All mAbatrs recognized bands around 55, 30, 23 and 15 kDa.</p

Enzymatic hydrolysis of Abz-LVEALYQ-EDDnp by Atr-I.

<p>Cleavage of Abz-LVEALYQ-EDDnp by Atr-I results in a fluorescent emission dependent of the Atr-I enzymatic activity. Hydrolysis of the substrate was assessed after incubation of 11 ng of Atr-I alone (▾) or mixed with EDTA 2 mM (▪) with the FRET substrate for 30 minutes at 37°C. Abz-LVEALYQ-EDDnp alone was used as negative control (•).</p

Molecular pattern of mAbatrs recognition.

<p>Reactivity of 8-mer overlapping peptides derived from the amino acid sequence of Atr-I. Peptides were prepared by the Spot method on cellulose membranes and binding of mAbatrs (1 µg/mL) to cellulose-bound peptides was detected by an alkaline phosphatase-conjugated anti-mouse antibody (diluted 1∶1000). None of mAbatrs showed reactivity against linear sequence of Atr-I.</p