We'd like to understand how you use our websites in order to improve them. Register your interest. Snake bites by Bothrops jararacussu result in moderate to severe envenoming, characterized by hemorrhage, coagulation disorders, tissue necrosis, and death. Antivenom has been regularly used for more than a century but poorly neutralizes myonecrosis. And, as a consequence, victims may have their affected limbs amputated. Thus, the production of antivenom must be improved as well as alternative treatments investigated.
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Metrics details. Snakes of the genus Bothrops , popularly known as pit vipers, are responsible for most cases of snakebite in Brazil. Within this genus, Bothrops jararacussu and B. Regarding the treatment of snakebites by Bothrops jararacussu , questions have been raised about the effectiveness of the specific bothropic antivenom in neutralizing myotoxic effects; however, there are no accurate data for humans. Thus, the development of a differential diagnostic kit for this species would be of great interest because it provides, for healthcare professionals, a tool that would allow us to determine whether the accident was caused by B.
It would also make it possible to evaluate the specificity of the treatment and to provide data for epidemiological studies. Polyclonal antibodies against bothropstoxin-I could be separated into several species-specific immunoglobulins. Then, aiming to develop a system of safe and standardized immunoassay, we produced monoclonal antibodies. Seven hybridomas were obtained. Five of them were specific to the venom of B.
The use of monoclonal antibodies also made it possible to differentiate B. Analyzing the reactivity of monoclonal antibodies against other bothropic venoms, we found mAb Bt-3 to be more specific than others for B. These results show the potential of BthTx-I for producing monoclonal antibodies that differentiate between B. Among the snake species of the genus Bothrops , Bothrops jararacussu is remarkable for the low immunogenicity of its venom, which is highly myotoxic, leading to necrosis of striated muscle fibers and slowing tissue regeneration [ 1 , 2 ].
In all Brazilian states where B. This makes it difficult to differentiate between these two snakes when an accident occurs, due to extensive homology among envenomation symptoms [ 4 , 5 ]. In Rio de Janeiro, both species have clinical importance. In the envenomation context, the question of the efficiency of specific bothropic antivenom against B. For this reason, some researchers have suggested the use of a combinated bothropic-crotalic antivenom as a more appropriate treatment [ 1 , 6 — 10 ].
The proteomic characterization of B. This protein is the most abundant myotoxin that can be isolated from B. This toxin is able to promote injury in lipid bilayer of cell membranes through a calcium-independent mechanism, inducing myonecrosis [ 13 , 15 ]. Methods for differentiating the envenomation caused by B. In this context, the objective of the present study was to produce monoclonal antibodies from BthTx-I to be used as tools for the development of a differential diagnostic kit for bites provoked by B.
The venoms of Bothrops alternatus, B. Bothropstoxin-I was isolated following the description of Correa-Netto et al. Briefly, B. Blood samples were drawn after the 5th week and the immune serum was collected. Two columns of Sepharose 4B activated by cyanogen bromide were prepared, one with Bothrops jararacussu jararacussu-Sepharose and the other with Bothrops jararaca from the southeast region of the country jararaca-Sepharose.
The column preparation followed instructions from Amersham Biosciences. Immunoglobulins with affinity for the B. The immunoglobulins that did not bind to the column were collected and those with affinity for the venom of B. For the elution of immunoglobulins from both columns, 0. Four days after the last booster, animals were bled from the ophthalmic plexus and one animal immunized with BthTx-I was killed and popliteal lymph nodes were removed.
Supernatant fluids were screened for species-specific antibodies by ELISA, as described in the next section. Antibody-secreting cells were expanded and cloned twice at limiting dilution.
The proteins were eluted in 0. An isotyping kit Sigma was used to determine the heavy-chain isotype. Dot blot was carried out as described by Towbin et al. Antigen—antibody reaction was detected by addition of anti-mouse IgG-peroxidase conjugate and the reaction with a chromogenic substrate, 4-chloronaphthol 0.
We first tested whether the anti-BthTx-I polyclonal antibodies could give specific antibodies to recognize venoms from B. For this purpose, we produced polyclonal antibodies in rabbits and tested against purified BthTx-I, B.
As expected, the antibodies recognized both venoms when analyzed by ELISA and immunobloting data not shown.
With the goal of eliminating cross-reactive IgGs from polyclonal serum, these antibodies were subjected to affinity chromatography as described in Methods section, which yielded specific IgGs called here species-specific anti-BthTx-I.
This set of antibodies clearly recognized B. These data clearly showed the utility of BthTx-I in generating such specific antibodies. Species-specific recognition of anti-BthTx-I against B. The purified antibodies were tested against a pools, individual venoms of b B.
One microgram of each venom was applied to a nitrocellulose membrane and subjected to recognition by the polyclonal species-specific antibodies at a dilution of Since polyclonal antibodies produced against BthTx-I were able to differentiate between the venom of two species, we decided to produce monoclonal antibodies.
Seven stable, immortalized clones secreting anti-BthTx-I antibodies were obtained. Analysis of mAb recognition of Bothrops venoms. The test was performed in triplicate and serum polyclonal anti-BthTx-I produced in mice was used as positive control. Normal serum and supernatant of SP2-O cells provided negative controls. Since ontogenetic and individual variations are described in Bothrops venoms we analyzed the specificity with which mAbs could recognize individual venoms of ten neonates and adults of B.
On the other hand, mAbs Bt and Bt recognized both venoms B. Analysis of mAbs against neonates and adult individual venoms. In a individual venom of B.
One microgram of each venom was applied to a nitrocellulose membrane and incubated with mAbs, followed by anti-mouse IgG-peroxidase. The antigen-antibody reaction was developed.
Given the reproducibility of the mAbs against ten individual venoms neonates and adults of B. As soon as we discovered the presence of BthTx-I in the venom of B. First, a serial dilution of mAbs in PBS was added. With a high mAbs concentration, venom of southern B. However, as the mAbs concentration decreased, the intensity of the signal was gradually reduced until complete abrogation at 0. Hence, Bt and Bt do not lose reactivity to venom of southern B.
Titration of monoclonal antibodies. A well plate was sensitized overnight with one microgram of BthTx-I and venoms from B. The plate was then incubated with various concentrations of mAb and revealed with anti-mouse IgG labeled with peroxidase.
The arrow in a shows the concentration of mAb Bt-3 0. The venoms from B. In b , titration of mAb Bt To confirm these data, a dot-blot analysis was carried out in order to compare effects of different mAbs and dilutions against venom from B.
It was again demonstrated that by using 62 nanograms of mAbs it is possible to detect venom from B. Analysis of mAbs against B. One microgram of B. After blocking, or 62 nanograms of mAbs was applied. In a and b mAbs against B. Given the reproducibility of the mAbs against the B. We investigated whether these mAbs would recognize venoms of other Bothrops species from Brazil. However, Bt-3 was highly specific to the venom of B. The mAbs Bt and Bt showed cross-reactivity with the venoms of B.
Monoclonal 3 Bt-3 showed the highest specificity for B. The use of immunodiagnostic tests that allow for the elucidation of the pattern of envenomation caused by venomous animals is recommended by the World Health Organization. This emphasizes the need to improve the quality of epidemiological and clinical data on accidents caused by venomous animals, in order to improve the therapeutic approach [ 24 ].
Nevertheless, common antigens present in venoms from different snake species have shown to be a major problem in developing immunodiagnostic tests [ 25 — 27 ]. In this study, we produced polyclonal antibodies in rabbits against BthTx-I with the aim of differentiating between venoms from B. The produced antibodies showed extensive cross-reactivity with B. Then, the cross-reacting molecules were removed by affinity chromatography Fig.
Other authors have successfully used a similar approach to differentiate among the snake venoms from Bothrops, Lachesis, Crotalus and Micrurus due to their overlapping distribution in Brazil [ 28 — 31 ]. However, polyclonal antibodies resulting from immunization of animals vary in different matches, given the variability of animal and immunization protocols [ 32 ].
In contrast, the monoclonal antibody constitutes a valuable tool to develop methods for the identification of unknown antigens contained within a mixture of antigens, since each hybridoma is specific for a single antigenic determinant. Because of their high specificity, monoclonal antibodies are standardized reagents that can accurately point out differences in the same or in different molecules, making them important tools in the basic research, immunodiagnosis and clinical studies [ 33 ].
Nakamura et al. Later, Malli and et al. Such antibodies contributed to an increase in technical specificity of antibodies as immunodiagnostic tools and made it possible to discover the identity of the offending animal [ 35 ]. In our study, we showed that monoclonal antibodies raised against BthTx-I are capable of distinguishing between B. In Brazil, there is no commercial kit available for snake venom detection.
Only Australia produces a detection kit, which is based on polyclonal antibodies [ 36 — 40 ].
Metrics details. Snakes of the genus Bothrops , popularly known as pit vipers, are responsible for most cases of snakebite in Brazil. Within this genus, Bothrops jararacussu and B. Regarding the treatment of snakebites by Bothrops jararacussu , questions have been raised about the effectiveness of the specific bothropic antivenom in neutralizing myotoxic effects; however, there are no accurate data for humans.
Bothrops jararacussu. Family: Viperidae. Countries Argentina, Bolivia, Brazil, Paraguay. General Shape Large in length, heavy bodied pitviper. Can grow to a maximum of over 2.
Go to: main text of page main navigation local menu. Illustrations of reptiles. Fauna 1 1 : Amaral, A. A general consideration of snake poisoning and observations on neotropical pitvipers. Harvard Inst. Ambientais Blumenau 3 1 : Boulenger, G. Catalogue of the snakes in the British Museum, Vol.