Production of Monoclonal Antibodies (mAbs) Purified Anti- metalloprotease from the Venom of the Serpent Bitis arietans

The African snake Bitis arietans is of great medical importance and is found in sub-Saharan Africa and in savannas and pastures of Morocco and western Arabia. Contributes complacently in the epidemiology of snakebites in humans and animals. The lack of specific antivenoms aggravates this situation. Identifying toxins, knowing their toxic properties and developing antitoxins are the goals of emerging projects. This project aims at monoclonal anti-metalloprotease (mAbs) from the toxin of Bitis arietans venom. mAbs will serve as sources of complementarity determining regions (CDR). The main applied methodologies will not develop the project fit mainly in the purification of metalloprotease and immunization of mice to obtain lymphocytes and replicate it. So far, we have been able to highlight a metalloprotease of interest, which will be properly identified for the production of antibodies, a finding that was confirmed by proteomic and transcriptomic analyzes. The next step will be to immunize mice and validate the antibodies produced.


Introduction
Ophidian accidents represent a major public health problem, especially in tropical and subtropical countries in Asia, Africa, Oceania and Latin America [1,2]. An epidemiological analysis of underreported snakebite accidents on data collected from 160 publications from 68 countries worldwide estimates that there are about 1,800,000 accidents with 94,000 deaths per year [1]. Besides deaths, survivors with sequelae, disabilities and lack of antivenoms, these factors are highly reported. In sub-Saharan Africa, there are an estimated 250,000 accidents, with 12,000 deaths and 9,000 complications, of which 95% occur in rural regions [1].
The World Health Organization (WHO) faced the association between the severity of snakebite accidents and the lack of medical care for victims, decided to put the snakebite accident back to the condition of neglected disease [3].
Antiophidic sera of recurrent use in the immunotherapy of humans bitten by snakes are, in general, produced in horses. The production process must follow the standards established by WHO [11]. In the final product, there is a predominance of fragments F (ab) 2, pH 7.0 ± 0.5, sterile, without endotoxin or pyrogenic factor, and containing the desired amount of LD50/mL. The final product is expensive, an impacting factor for developing countries. These general observations, plus about two decades of involvement in projects on animal venoms and antivenoms stimulated the development of experiments aimed at obtaining antivenoms of high specificity, avidity, affinity, specific activity and neutralizing potency of toxins at low cost, but with high quality. Our project proposal includes these objectives.
The main strategies used in the development of the project were: #1 use of established knowledge about the molecular and cellular mechanisms involved in the differentiation of B, T lymphocytes and memory plasma cells [12], in addition to carrying out preliminary experiments aimed at identifying points in the appropriate immunization protocols to stimulate memory plasma cells; #2 isolate and characterize relevant toxins, develop antitoxin mAbs, isolate hypervariable regions of these mAbs, identify and sequence CDRs and model scFv antitoxins.
PhD student Â ngela Alice Amadeu Megale in the development of her thesis [10], started to identify parameters identifying the local and systemic inflammation produced by Bitis arietans intoxication [13]. Analysis of the venom proteome and transcriptome, which revealed that the venom is made up of proteins belonging to a restricted number of families, of which metalloproteases and serinoproteases are the most abundant as shown in Figure 2. She purified these two proteases from Bitis arietans venom, which were identified as rhinocerase, being classified as serinoprotease (SVSP) [14] and metalloprotease (SVMP) [15]. Figure 3 confirms that biological activity tests of this venom carried out by our group demonstrated, based on the selective inhibition with EDTA, PMSF and PHE, the presence of metalloproteases and serinoproteases, corroborating the previous results of the proteome and transcriptome. In the process of purifying the proteases, the author must identify the fractions of SVSP and SVMP substrates "Fluorescent Resonance Energy Transfer" (FRET), with Abz-RPPGFSPFR-QEDDnp to identify SVSP and SVMP and Abz-FRSSR-QEDDnp that identifies SVSP [16].  SVSPs have a highly conserved catalytic triad. They have a high identity: 51-98% among themselves, 26-33% with human thrombin and 34-40% with human plasma kallikrein. They are generally presented as a single chain with 26-33 kDa, depending on the glycosylation content, which can vary from 0-30%. In general, they affect the coagulation cascade and blood pressure; they are fibrinogenolytic and cleave the kininogen culminating in the release of vasoactive kinins.
SVMPs, representing almost 40% of this venom, are zinc dependent endopeptidases and phylogenetically related to mammalian metalloproteases and ADAM proteins. They have binding domain to the conserved substrate and variable molecular weight, depending on their composition. They are classified into three classes, according to the composition of their domains. The PI class is the least complex and in its active form it is formed only by the catalytic domain. Class P-II have a disintegrin domain after the catalytic domain. It is important to note that few class P-II SVMPs are found in venoms, as they undergo auto-proteolysis, releasing a catalytic domain and a free disintegrin domain. Class P-III, in addition to the catalytic and disintegrin domains, have a domain rich in cysteine. SVMP are the main components of venoms involved in local and systemic hemorrhage. In general, hemorrhagic or not, they are fibrinogenolytic and cleave components of the extracellular matrix [17,18] (Figure 4). The purification of metalloproteases in this project aims to develop specific mAbs for epitopes related to the toxic domains of the snake venom Bitis arietans. Preliminary results indicate the relevance of this criterion in the appropriate selection of antitoxin hybridomas.

First chromatography step
Bitis arietans venom (BaV) is composed by a complex mixture of toxins. The analysis of the electrophoretic profile of BaV revealed the presence of proteins with molecular weight from 180 to 6 kDa. ( Figure 5). The first step of the BaV fractionation by molecular exclusion chromatography gave rise to 9 subfractions, denominated F1-F9 ( Figure 6A), which the electrophoretic profile is observed in SDS gel ( Figure 6B). Considering the molecular weight, proteolytic activity and stability of these fractions, fractions 2, 3, 4 and 5 were selected for the inhibition steps.  Figure 6A. 20 mg of lyophilized venom were subjected to a phase of molecular exclusion chromatography in a Superose 12 HR column, balanced and eluted with ammonium acetate (50 mM) in a climate-controlled environment (22 ± 2ºC). The samples were collected in a flow and 0.4 mL/min. and the protein content was monitored under 280 nm absorbance on the UPC-900 reader. Figure 6B. The electrophoretic profile was evaluated on gel with a gradient of 8-16% acrylamide and the bands revealed by impregnation with silver nitrate.

SVMP purification and characterization
The second chromatographic step of fraction 5, selected to obtain SVMP, originated 7 subfractions. Among them, F5-4 was the one with the highest proteolytic activity ( Figure  8A). This fraction was completely inhibited by selective SVMP inhibitors and the identification by mass spectrometry revealed that the peptides originated from the hydrolysis of this subfraction have similarity with two class I SVMP of botropic venoms ( Figure 8B). The SVMP of Bitis arietans presented itself as a single band of 23.5 kDa in the reduced gel ( Figure 8C). Like the SVSP, this SVMP was able to hydrolyze human fibrinogen, indicating its participation in hemostatic disorders, as well as fibronectin, indicating its participation in tissue damage and hemorrhage caused by the venom (manuscript in preparation.) Figure 8A. The fifth chromatographic peak (F5), obtained from the first purification step, was subjected to a second cycle of molecular exclusion chromatography on a Superdex 75 column, equilibrated and eluted with ammonium acetate (50 mM) in an air-conditioned environment (22 ± 2ºC). The samples were collected in a flow of 0.4 mL/min. and the protein content was monitored under 280 nm absorbance in a UPC-900 reader. A) Fraction 5.4 (F5-4) was shown to contain the highest proteolytic activity. B) The proteolytic activity of the fractions obtained from the F5 fractionation by molecular exclusion chromatography was evaluated by the cleavage of FRETs substrates. In 96well white plates, the fractions (0.5 -2 µ g/well) were incubated with 10 µ M of FRET subtract (Abz-RPPGFSPFR-EDDnp) in PBS (100 µ L final volume). Proteolytic activity was measured by spectrofluorimetry for 10 minutes. Representative result of two independent tests expressed as mean ± SD and analyzed by One-Way Anova followed by the Tukey post-test (* p <0.05). UF: Arbitrary fluorescence unit; N.D.: not detectable. Figure 8B. The fraction 5-4 (F5-4; 0.5 ug/well), was incubated with EDTA (100 mM/well), PMSF (2 mM/well) and 1.10-PHE (2mM/well) for thirty minutes at room temperature. Then, 10 µ M of FRET substrate (Abz-RPPGFSPFR-EDDnp) was added in a final volume of 100 µ L and the proteolytic activity was measured by spectrofluorimetry for 10 minutes. The calculation of the percentage of inhibition was performed considering the specific activity of F5-4 incubated only in PBS as 100% activity. Representative result of two independent tests expressed as the mean percentage of inhibition ± SEM and analyzed by One-Way ANOVA followed by Tukey's post-test. p <0.001 (***). Figure 8C. The fraction 5-4 (4 ug/well), corresponding to the metalloprotease isolated from fraction 5, was submitted to 12% polyacrylamide gel under reducing (R) and non-reducing (N/R) conditions. The band revealed by impregnation with silver nitrate.

SVMP Activity Screening
The substrate Abz-RPPGFSPFR-QEDDnp, already defined as selective for the SVMPs present in the venom of B. arietans [16], was used for the selection of the chromatographic peaks of interest. EDTA, a site inhibitor targeting SVMP, was also used in the selection step. 5 µM of FRET substrate and 0.5 µg of the fractions in PBS (100 µL final volume) were used. The tests were performed in a fluorimeter (Hidex Sense, Hidex, Finland) in 96-well plates in a thermostabilized compartment (37 ° C) and under agitation and adjusted for excitation and emission reading 320 and 420 nm, respectively. All experiments were performed in duplicates (Table 1) [15]. Table 1. Specific activity (UF/min /µ g) on FRET substrates. Proteolytic activity on the substrate Abz-FRSSR-EDDnp (venom¹, fraction 3 and Kn-Ba) and Abz-RPPGFSPFR-EDDnp (venom², fraction 5 and F5-4).

Discussion
The global mortality rate caused by snake poisonings is extremely high, especially in underdeveloped countries located in Africa, Asia and Latin and South America [1]. The snake Bitis arietans is one of the main species of medical interest and also the most responsible for an accident. It is endemic to Sub-Saharan Africa, but is also found in Morocco and Saudi Arabia. Its venom can cause local and systemic effects, such as pain, fever, leukocytosis and cardiovascular and hypothermic disorders, which can culminate in the individual's death [4,19] So far, according to the literature, toxins from the Bitis arietans venom have been isolated and characterized, such as hemorrhage [20,21] and non-hemorrhagic [22] metalloproteases, fibrinogenolytic serinoproteases, kinin-releasing and acting on insulin [23], serinoproteases with the ability to release kallidin [24], phospholipases A2 as bitanarin [23,25], Ba25, as primary structure similar to venom type C lectins [26] and PAL (Puff adder lectin) that induces the release of Ca +++ in the sarcoplasmic reticulum [27] and bitistatin (P17497) also known as arietin [28].
In the present study, two specimens of the main classes of toxins present in the Bitis arietans venom were isolated and characterized: an SVSP of 33 kDa, and an SVMP of 23.5 kDa. The molecular mass of the two toxins is close to those found in the proteome and transcriptome experiments of the venom [29,30].
Tests of biological activity of this venom were performed and demonstrated that the proteolytic activity of SVSP was completely inhibited by PSMF, while the proteolytic activity of SVMP was completely inhibited by EDTA and PHE (1,10-phenanthroline).
The SVMP purified from the venom in the due study, had a molecular weight of 23.5 kDa, similar to other class P-I SVMPs, such as insularinase A [31] and BaP1 [32].

Conclusion
To date, two toxins from the venom of the snake Bitis arietans have been purified, a SVSP of 33 kDa that makes up about 19.5% of the venom and an SVMP of 23.5 kDa that represents 38.5%. Tests of the biological activity of the two toxins using the EDTA, PMSF and PHE inhibitors confirmed the results of the proteome and the transcriptome performed, demonstrating the presence of both toxins in the venom. The first stage of molecular exclusion chromatography resulted in 9 subfractions that were directed to analysis by electrophoresis, demonstrating the presence of proteins from 180 to 6 kDa. The purification and characterization of the SVSP F3 fraction found gave rise to the F3-1 fraction, selected from its inhibition by PSMF, which has fibrinogenolytic action and is capable of promoting the release of vasoactive kinins. The second stage of the SVMP F5 fraction, selected from the inhibition by EDTA and PHE, gave rise to the F5-4 fraction, similar to other class PI SVMPs of botropic venoms. This SVMP is capable of hydrolyzing human fibrinogen and participating in hemostatic disorders. The selection of the chromatographic peaks was made using specific substrates for SVSP (Abz-FRSSR-QEDDnp) and SVMP (Abz-RPPGFSPFR-QEDDnp).

Venom
The venom of Bitis arietans (Venom Supplies, SA, Australia) will be provided by the Soros section hyperimmune the Butantan Institute, developed as of adult animals, males and developments, each measuring approximately 60 cm and from the south of the African continent. Lyophilized poisons are stored in a -20 ° C freezer. For the tests, the venom filter will be diluted in pyrogenic saline (0.15 M NaCl) and filtered through sterilizing filters, with 0.22 µ m pores, and stored in a freezer. -80ºC until use.

SVMP purification
Initially, 20mg of the lyophilized venom will be fractionated by molecular exclusion chromatography in a Superose 12 HR column, balanced and eluted with ammonium acetate (50 mM) in a climatized environment (22 ± 2 ºC) and the proteolytic activity of the peaks was determined on FRET substrates. After selecting the peak containing the SVMP of interest, it will be submitted to a new chromatographic step, using Superdex 75. Then, from the inhibition of proteolytic activity with EDTA, PMSF and phenatroline, selective SVMP and SVSP inhibitors, the enzymatic class of the fractions will be determined. The identification of the selected toxins will be performed by mass spectrometry and the activity of the proteases on the physiological substrates related to the poisoning will be evaluated.

Immunization of mice
Female BALB / c mice, aged 4 to 6 weeks and weighing 18 to 20 g, will be supplied by the Central Animal Farm, Instituto Butantan. Subcutaneous injections of SVMP in the presence or absence of SBA-15 and the collection of blood samples for testing for anti-SVMP Abs will be done under anesthesia. Samples of the maxillary sinus will be performed on days 0, 30 and 60, and the injection of purified SVMP will be performed every two weeks. Adverse reactions will be under constant observation. To collect lymphocytes, the animals will be anesthetized and euthanized by lethal combination of the anesthetics Xylazine (30 mg / kg) and Ketamine (300 mg / kg) via intraperitoneal.

Preparation of peritoneal cells
Peritoneal cells will be obtained from the peritoneal cavity of BALB / c mice. 5 mL of the medium "Dulbecco's Modified Eagles Medium F-12 (DMEM-F12, GIBCO, BRL)" will be injected intraperitoneally, and the peritoneal fluid collected after a delicate massage for 1.5 min. The animal will be euthanized as described in the topic "Immunization of mice". The collected peritoneal liquid will be centrifuged at 1200 rpm for 5 min, the supernatants will be discarded and the cells suspended in 10mL of "DMEM-F12 medium containing 10% fetal bovine serum. Samples of these cells will be distributed in the 96-well plate wells and incubated for 24 h, at 37ºC under a 5% CO2 atmosphere.

Identification of anti-SVMP mAbs isotypes
Wells of 96-well plates (Costar) will be coated with 5 µ g of the purified fraction of SVMP for 1 hour at room temperature, and the wells will be washed 3 times with PBS buffer, pH 7.4. Then, the plates will be blocked with PBS/BSA 5%, 200 µ L/well, for 2h at 37ºC. After 1 wash with PBS, pH 7.4, 100 µ L of purified SVMP anti-fraction mAbs diluted in a 1: 2 ratio from the plateau dilution obtained in the ELISA assays will be added and the plates incubated 1h at 37 ° C. After incubation, the wells will be washed 3 times with 0.05% PBS / Tween, 200 µ L / well. Next, 100 µ L of anti-horse or anti-mouse IgG produced in rabbit and labeled with peroxidase (secondary [Ab] antibody) will be added to each well and the plates incubated for 1h at 37ºC. Then the wells will be washed 3 times again with PBS / Tween 0.05%, 200 µ L / well, and 50 µ L / well of the substrate buffer for peroxidase, OPD (orthohyldiamine) (Sigma Chemical Co) and color development will be applied. observed after 15 min of reaction at room temperature under light. The reaction will be terminated with the application of 4 M sulfuric acid, 50 µ L/well, and the absorbances (ABS) read at 490 nm. The values obtained will be projected on a graph with the X axis representing the corresponding dilutions versus the Y axis representing the absorbance. Dilutions giving maximum values of ABS 490 nm in the obtained curve will be considered titers of the tested Abs.

Hybridomas
Activated B cells will be fused with myeloma cells in the presence of HAT medium containing hypoxanthine, aminopterin, and thymidine and the positive hybridomas, identified based on viability and the production and secretion of mAbs, will be propagated. Representative hybridomas will be submitted to three sequential rounds of cloning. Clonal propagation will be performed by cultivation in 25 cm 3 plastic bottle bioreactors containing DMEM-F12 medium supplemented with 10% FCS, 10% gentamicin and 5 mM of 2-β-mercaptoethanol at 35°C under a 5% CO2 atmosphere. The secreted mAbs will be collected, and the titers and affinities of these mAbs were measured using ELISA. Clonal propagation will be performed by cultivation in 25 cm 3 plastic bottle bioreactors containing DMEM-F12 medium supplemented with 10% FCS, 10% gentamicin and 5 mM of 2-βmercaptoethanol at 35°C under a 5% CO2 atmosphere. The secreted mAbs will be collected, and the titers and affinities of these mAbs were measured using ELISA. After the fusion of activated B cells with myeloma cells in the presence of HAT medium containing hypoxanthine, aminopterin and thymidine, positive hybridomas will be cloned by three successive limiting dilution steps. Clones resulting from the 3rd step will be expanded. The secreted mAbs will be harvested and the antibody titers, affinity, specificity, immunoglobulin isotypes and antigen recognition will be assayed [33,34].

Cell fusion
The spleen of mice producing purified SVMP anti-fraction Abs with desirable titers and affinities will be collected after euthanasia. Splenic extracts will be prepared, lymphocytes will be organized and prepared for fusion [34].

Identification of anti-SVMP myelomas
Myelomas will be multiplied in a culture medium containing hypoxanthine, aminopterin and thymine, conditions that select anti-SVMP hybridomas [33].

Expansion of hybridomas
Selected hybridomas will be grown in 26 ml plastic bottles containing DMEM-F12 medium supplemented with 10% fetal bovine serum, 10% gentamicin and 5 mM 2-β mercaptoethanol, at 35°C under 5% CO2 for 3 days. Viable hybridomas that are secreting mAbs will be collected, kept in liquid nitrogen and will be sources of anti-SVMP mAbs [33].

Characterization of anti-SVMP mAbs in the hybridoma culture medium
The culture medium will be concentrated, the mAbs purified by immunochemical methodology, their titers quantified by ELISA and isotypes of Igs identified [34].

Evaluation of the antitoxic activity of anti-SVMP mAbs
Samples of anti-SVMP will be incubated with standardized samples of B. arietans venom or purified SVMP under test conditions. 100 µ L of PBS containing 10 µ L of the Abz-RPPGFSPFR-QEDDnp substrates will be pre-incubated at 37°C with buffer (control) or with 0.5 µg samples of the anti-SVMP mAbs and the reactions continuously monitored by fluorescence emission at 420 nm after excitation at 320 nm in a fluorimeter (Victor3™ Perkin-Elmer, Boston, MA, USA or FLUO star ® Omega, BMG Labtech, HE, Germany). The Specific Activity Unit (UAE) will be expressed in fluorescence units of cleaved substrate per minute [14]. The antitoxic activities of the anti-SVMP mAbs will be reported in% inhibition using control results without anti-SVMP mAbs as 100%.

Protein quantification
The protein content of the purified venom and mAbs will be quantified by the bicinconinic acid method [35].

Titration of anti-SVMP mAbs by the ELISA method
96-well high-binding plates (Costar) will be incubated at room temperature for ± 12h with 100 µ L of PBS buffer, pH 7.4, containing 1 µ g of crude venom or purified fractions. Then, the plates will be blocked with PBS/BSA 5%, 200 µ L/well, for 2h at 37ºC. After 1 wash with PBS, pH 7.4, equal volumes of dilutions of equine anti-B arietans serum or mAbs will be added in PBS/BSA 0.1% (primary Ab) and the plates incubated for 1h at 37ºC. After that, the wells will be washed 3 times with PBS/Tween buffer 0.05%, 200 µ L/well. Next, 100 µ L of anti-horse or anti-mouse IgG produced in rabbit and labeled with peroxidase (secondary Ab) will be added to each well and the plates incubated for 1h at 37ºC. Then the wells will be washed 3 times again with 0.05% PBS/Tween 200 µ L/well and 50 µ L/well of the substrate buffer for peroxidase, OPD (Sigma Chemical Co) and the color development will be applied after 15 min of reaction at room temperature under light. The reaction will be terminated with the application of 4 M sulfuric acid, 50 µ L/well and the absorbances read at 490 nm. The values obtained will be projected on a graph with the X axis representing the corresponding dilutions versus the Y axis representing the absorbance. Dilutions giving maximum values of ABS 490 nm in the obtained curve will be considered titers of the tested Abs.

Assessment of avidity of anti-SVMP mAbs obtained by the ELISA method
96-well high-binding plates (Costar) will be incubated at room temperature for ± 12h with 100 µ L of PBS buffer, pH 7.4, containing 1 µ g of crude venom or purified fractions. Then, the plates will be blocked with PBS/BSA 5%, 200 µ L/well, for 2h at 37ºC. After 1 wash with PBS, pH 7.4, samples of anti-SVMP mAbs will be added in PBS/BSA 0.1% (primary Ab) and the plates incubated for 0 min, 20 min, 40 min or 60 min of incubation at 37ºC. After incubation, the wells will be washed 3 times with 0.05% PBS/ ween buffer, 200 µ L/well. Next, 100 µ L of anti-horse or anti-mouse IgG produced in rabbit and labeled with peroxidase (secondary Ab) will be added to each well and the plates incubated for 1h at 37ºC. Then the wells will be washed 3 times again with 0.05% PBS/Tween, 200 µ L/well, and 50 µ L/well of the substrate buffer for peroxidase, OPD (Sigma Chemical Co) and the color development observed after 15 minutes will be applied. min reaction at room temperature under light. The reaction will be terminated with the application of 4 M sulfuric acid 50 µ L/well, and the ABS read at 490 nm. The values obtained will be projected on a graph with X representing time and the Y axis representing absorbance. The avidity index indicates the shortest time for the toxin-antitoxin complexes to be formed.

Evaluation of the affinity of anti-SVMP mAbs obtained by the ELISA method
96-well high-binding plates (Costar) will be incubated at room temperature for ± 12h with 100 µ L of PBS buffer, pH 7.4, containing 1 µ g of venom or purified fractions. Then, the plates will be blocked with PBS/BSA 5%, 200 µ L/well, for 2h at 37ºC. After 1 wash with PBS, pH 7.4, the mAbs that gave maximum values of ABS 490 nm in the curve obtained in the "Identification of the anti-SVMP immunoglobulin isotypes" assay will be added to the wells as primary Ab and the plates will be incubated for 1h at 37ºC. After that, the wells will be washed 3 times with PBS/Tween buffer 0.05%, 200 µ L/well. Next, 100 µ L PBS/BSA 5% containing concentrations of 0.5 to 4.5 M of the potassium thiocyanate chaotropic agent (KSCN, Sigma Aldrich, St. Louis, MO, USA) will be added to each well, and the plates will be incubated for 10 minutes at room temperature, and washed 3 times with PBS pH 7.4. The ELISA assay continues as previously described. The affinity index will be defined by the highest mM concentration of the KSCN that breaks up the immune complexes.

Identification of SVMP protein bands recognized by mAbs
This "Western blotting" protocol will be conducted as originally described [20]. The SDS-PAGE analysis will be done according to the described methodology [35]. Samples of purified SVMP or B. arietans venom (5µ g) will be applied to SDS-PAGE gels (5% Upper, 10 to 15% Lower) in reducing and non-reducing conditions. The gels will be electrophoresed for 100V in a Power Pac Basic (Bio-Rad). The separated protein bands will be revealed by staining with Coomassie blue, and their molecular masses evaluated using the parallel band pattern profile in parallel. The identified bands will be electrotransferred "overnight" at 4ºC, in a transfer tank TE 22 (Hoefer Pharmacia Biotech, San Francisco, CA), under a current of 150 mA for nitrocellulose membranes. Then, the membranes will be washed quickly with PBS, and treated with anti-SVMP mAbs. The membranes will be incubated for 1h at room temperature, and then washed 3 times with PBS/0.05% Tween. After that, the membranes will be incubated with anti-mouse or anti-horse IgG produced in rabbit and marked with alkaline phosphatase (secondary Ab) diluted in PBS/BSA 0.1% for 1h at room temperature, and then washed 3 times with 0.05% PBS/Tween and developed by adding NBT/BCIP solution (Sigma Aldrich, St. Louis, MO). The development of the bands will be monitored visually, and the reaction will end with the addition of distilled water.

Statistical analysis
The data obtained will be analyzed by "one-way ANOVA", complemented by "Dunnetts Multiple Comparison Test (standard antivenom as comparison)", or by "two-way ANOVA", complemented by "Bonferroni Post Test (standard antivenom as comparison)". Differences will be considered significant if p <0.05. The data will be analyzed using the "Graphic PAD Prism 5" model (Graph Pad Software).