Use of a synthetic biosensor for neutralizing activity-biased selection of monoclonal antibodies against atroxlysin-I, an hemorrhagic metalloproteinase from Bothrops atrox snake venom

Abstract

Background: The snake Bothrops atrox is responsible for the majority of envenomings in the northern region of South America. Severe local effects, including hemorrhage, which are mainly caused by snake venom metalloproteinases (SVMPs), are not fully neutralized by conventional serum therapy. Little is known about the immunochemistry of the P-I SVMPs since few monoclonal antibodies (mAbs) against these molecules have been obtained. In addition, producing toxin-neutralizing mAbs remains very challenging.

Methodology/principal findings: Here, we report on the set-up of a functional screening based on a synthetic peptide used as a biosensor to select neutralizing mAbs against SVMPs and the successful production of neutralizing mAbs against Atroxlysin-I (Atr-I), a P-I SVMP from B. atrox. Hybridomas producing supernatants with inhibitory effect against the proteolytic activity of Atr-I towards the FRET peptide Abz-LVEALYQ-EDDnp were selected. Six IgG1 Mabs were obtained (named mAbatr1 to mAbatr6) and also two IgM. mAbatrs1, 2, 3 and 6 were purified. All showed a high specific reactivity, recognizing only Atr-I and B. atrox venom in ELISA and a high affinity, showing equilibrium constants in the nM range for Atr-I. These mAbatrs were not able to bind to Atr-I overlapping peptides, suggesting that they recognize conformational epitopes.

Conclusions/significance: For the first time a functional screening based on a synthetic biosensor was successfully used for the selection of neutralizing mAbs against SVMPs.

Figure 1. Enzymatic hydrolysis of Abz-LVEALYQ-EDDnp by Atr-I.

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 (•).

Figure 2. SDS-PAGE of purified mAbatrs.

Six anti-Atr-I monoclonal antibodies, isotyped as IgG1, were purified on a protein A-Sepharose column. They appear as homogeneous fractions on SDS-PAGE using a gradient gel (4–15%) in non-reducing conditions.

Figure 3. Antigenic reactivity of selected monoclonal antibodies (mAbatrs).

(A) Reactivity of purified mAbatrs (5 µg/mL) against several P-I SVMPs and B. atrox venom and BSA (negative control-not shown) was measured by ELISA. (B) Reactivity of mAbatr1, 2, 3 and 6 against B. atrox (from Brazil and Peru), B. barnetti, B. brasili, B. casteunaldi, B. chloromelas, B. hyoprora, B. microphtalmus, B. peruvianus, B. pictus, B. taeniata e Lachesis muta muta venoms (40 µg/mL) was accessed by ELISA. (C) Reactivity of polled mAbatr1, 2, 3 and 6 in sandwich ELISA against Peruvian B. atrox venom, L. muta muta, C. durissus and M. frontalis venoms (10 µg/mL) diluted in mice sera simulating experimental envenoming. Positive controls were performed using diluting B. atrox 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.

Figure 4. Cross-reactivity of mAbatrs with different toxins from B. atrox venom analyzed by western blotting.

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

Figure 5. Molecular pattern of mAbatrs recognition.

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.

Figure 6. mAbatrs affinity to Atr-I.

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.

Figure 7. Neutralization of Atr-I or B. atrox venom enzymatic activities on Abz-LVEALYQ-EDDnp substrate.

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

Figure 8. Neutralization of Atr-I-induced hemorrhage in mice.

Residual hemorrhage was evaluated after incubating mAbatr1, mAbatr2 or mAbatr3 with 1 MHD of Atr-I (A) or 1.8 MHD of B. atrox crude venom (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 B. atrox venom alone.