Crotoxin stimulates an M1 activation profile in murine macrophages during Leishmania amazonensis infection.

American tegumentary leishmaniasis is caused by different species of Leishmania. This protozoan employs several mechanisms to subvert the microbicidal activity of macrophages and, given the limited efficacy of current therapies, the development of alternative treatments is essential. Animal venoms are known to exhibit a variety of pharmacological activities, including antiparasitic effects. Crotoxin (CTX) is the main component of Crotalus durissus terrificus venom, and it has several biological effects. Nevertheless, there is no report of CTX activity during macrophage - Leishmania interactions. Thus, the main objective of this study was to evaluate whether CTX has a role in macrophage M1 polarization during Leishmania infection murine macrophages, Leishmania amazonensis promastigotes and L. amazonensis-infected macrophages were challenged with CTX. MTT [3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl tetrasodium bromide] toxicity assays were performed on murine macrophages, and no damage was observed in these cells. Promastigotes, however, were affected by treatment with CTX (IC50 = 22·86 µg mL-1) as were intracellular amastigotes. Macrophages treated with CTX also demonstrated increased reactive oxygen species production. After they were infected with Leishmania, macrophages exhibited an increase in nitric oxide production that converged into an M1 activation profile, as suggested by their elevated production of the cytokines interleukin-6 and tumour necrosis factor-α and changes in their morphology. CTX was able to reverse the L. amazonensis-mediated inhibition of macrophage immune responses and is capable of polarizing macrophages to the M1 profile, which is associated with a better prognosis for cutaneous leishmaniasis treatment.

Expanding anti-venom strategies: Camelid polyclonal antibodies with high capacity to recognize snake venom.

Camelid immunoglobulins represent a unique facet of antibody biology, challenging conventional understandings of antibody diversification. IgG2 and IgG3 in particular are composed solely of heavy chains and exhibit a reduced molecular weight (90 kDa); their elongated complementarity determining region (CDR) loops play a pivotal role in their functioning, delving deep into enzyme active sites with precision. Serum therapy stands as the primary venom-specific treatment for snakebite envenomation, harnessing purified antibodies available in diverse forms such as whole IgG, monovalent fragment antibody (Fab), or divalent fragment antibody F (ab')2. This investigation looks into the intricacies of IgGs derived from camelid serum previously immunized with crotamine and crotoxin, toxins predominantly in Crotalus durissus venom, exploring their recognition capacity, specificity, and cross-reactivity to snake venoms and its toxins. Initially, IgG purification employed affinity chromatography via protein A and G columns to segregate conventional antibodies (IgG1) from heavy chain antibodies (IgG2 and IgG3) of camelid isotypes sourced from Lama glama serum. Subsequent electrophoretic analysis (SDS-PAGE) revealed distinct bands corresponding to molecular weight profiles of IgG's fractions representing isotypes in Lama glama serum. ELISA cross-reactivity assays demonstrated all three IgG isotypes' ability to recognize the tested venoms. Notably, IgG1 exhibited the lowest interactivity in analyses involving bothropic and crotalic venoms. However, IgG2 and IgG3 displayed notable cross-reactivity, particularly with crotalic venoms and toxins, albeit with exceptions such as PLA2-CB, showing reduced reactivity, and C. atrox, where IgGs exhibited insignificant reactivity. In Western blot assays, IgG2 and IgG3 exhibited recognition of proteins within molecular weight (≈15 kDa) of C. d. collilineatus to C. d. terrificus, with some interaction observed even with bothropic proteins despite lower reactivity. These findings underscore the potential of camelid heavy-chain antibodies, suggesting Lama glama IgGs as prospective candidates for a novel class of serum therapies. However, further investigations are imperative to ascertain their suitability for serum therapy applications.

Immunoprotection against lethal effects of Crotalus durissus snake venom elicited by synthetic epitopes trapped in liposomes.

Snakebites caused by Crotalus genus are the second most frequent in Brazil. Crotoxin is a beta-neurotoxin responsible for the main envenomation effects of Crotalus biting, while crotamine immobilizes the animal hind limbs, contributing to prey immobilization and to envenoming symptoms. As crotoxin and crotamine represent about 90% of Crotalus venom dry weight, these toxins are of great importance for antivenom therapy. In this sense, knowledge regarding the antigenicity/immunogenicity at the molecular level of these toxins can provide valuable information for the improvement of specific antivenoms. Therefore, the aims of this study are the identification of the B-cell epitopes from crotoxin and crotamine; and the characterization of the neutralizing potency of antibodies directed against the corresponding synthetic epitopes defined in the current study. Linear B-cell epitopes were identified using the Spot Synthesis technique probed with specific anti-C. d. terrificus venom horse IgG. One epitope of crotamine (F12PKEKICLPPSSDFGKMDCRW32) and three of crotoxin (L10LVGVEGHLLQFNKMIKFETR30; Y43CGWGGRGRPKDATDRCCFVH63 and T118YKYGYMFYPDSRCRGPSETC138) were identified. After synthesis in their soluble form, the peptides mixture correspondent to the mapped epitopes was entrapped in liposomes and used as immunogens for antibody production in rabbits. Anti-synthetic peptide antibodies were able to protect mice from the lethal activity of C. d. terrificus venom.

Engineered protein containing crotoxin epitopes induces neutralizing antibodies in immunized rabbits.

Crotoxin (Ctx) is the main lethal component of Crotalus durissus terrificus venom. It is a neurotoxin, composed of two subunits associated by noncovalent interactions, the non-toxic acid subunit (CA), named Crotapotin, and the basic subunit (CB), with phospholipase A2 (PLA2) activity. Employing the SPOT synthesis technique, we determined two epitopes located in the C-terminal of each Ctx subunit. In addition, 3 other epitopes were mapped in different regions of Ctx using subcutaneous spot implants surgically inserted in mice. All epitopes mapped here were expressed together as recombinant multi-epitopic protein (rMEPCtx), which was used to immunize New Zealand rabbits. Anti-rMEPCtx rabbit serum cross-reacted with Ctx and crude venoms from C. d. terrificus, Crotalus durissus ruruima, Peruvian C. durissus and Bothrops jararaca (with lower intensity). Furthermore, anti-rMEPCtx serum was able to neutralize Ctx lethal activity. As the recombinant multiepitopic protein is not toxic, it can be administered in larger doses without causing adverse effects on the immunized animals health. Therefore, our work evidences the identification of neutralizing epitopes of Ctx and support the use of recombinant multiepitopic proteins as an innovation to immunotherapeutics production.

Ability of rabbit antiserum against crotapotin to neutralize the neurotoxic, myotoxic and phospholipase A2 activities of crotoxin from Crotalus durissus cascavella snake venom.

The toxicity of crotoxin, the major toxin of Crotalus durissus terrificus (South American rattlesnake) venom, is mediated by its basic phospholipase A(2) (PLA(2)) subunit. This PLA(2) is non-covalently associated with crotapotin, an acidic, enzymatically inactive subunit of the crotoxin complex. In this work, rabbit antiserum raised against crotapotin purified from Crotalus durissus cascavella venom was tested for its ability to neutralize the neurotoxicity of this venom and its crotoxin in vitro. The ability of this antiserum to inhibit the enzymatic activity of the crotoxin complex and PLA(2) alone was also assessed, and its potency in preventing myotoxicity was compared with that of antisera raised against crotoxin and PLA(2). Antiserum to crotapotin partially neutralized the neuromuscular blockade caused by venom and crotoxin in electrically stimulated mouse phrenic nerve-hemidiaphragm preparations and prevented the venom-induced myotoxicity, but did not inhibit the enzymatic activity of crotoxin and purified PLA(2). In contrast, previous findings showed that antisera against crotoxin and PLA(2) from C. d. cascavella effectively neutralized the neuromuscular blockade and PLA(2) activity of this venom and its crotoxin. The partial neutralization of crotoxin-mediated neurotoxicity by antiserum to crotapotin probably reduced the binding of crotoxin to its receptor following interaction of the antiserum with the crotapotin moiety of the complex.

Camelid Single-Domain Antibodies (VHHs) against Crotoxin: A Basis for Developing Modular Building Blocks for the Enhancement of Treatment or Diagnosis of Crotalic Envenoming.

Toxic effects triggered by crotalic envenoming are mainly related to crotoxin (CTX), composed of a phospholipase A2 (CB) and a subunit with no toxic activity (CA). Camelids produce immunoglobulins G devoid of light chains, in which the antigen recognition domain is called VHH. Given their unique characteristics, VHHs were selected using Phage Display against CTX from Crotalus durissus terrificus. After three rounds of biopanning, four sequence profiles for CB (KF498602, KF498603, KF498604, and KF498605) and one for CA (KF498606) were revealed. All clones presented the VHH hallmark in FR2 and a long CDR3, with the exception of KF498606. After expressing pET22b-VHHs in E. coli, approximately 2 to 6 mg of protein per liter of culture were obtained. When tested for cross-reactivity, VHHs presented specificity for the Crotalus genus and were capable of recognizing CB through Western blot. KF498602 and KF498604 showed thermostability, and displayed affinity constants for CTX in the micro or nanomolar range. They inhibited in vitro CTX PLA2 activity, and CB cytotoxicity. Furthermore, KF498604 inhibited the CTX-induced myotoxicity in mice by 78.8%. Molecular docking revealed that KF498604 interacts with the CA–CB interface of CTX, seeming to block substrate access. Selected VHHs may be alternatives for the crotalic envenoming treatment.

The co-purification of a lectin (BJcuL) with phospholipases A2 from Bothrops jararacussu snake venom by immunoaffinity chromatography with antibodies to crotoxin.

Antigens of Bothrops jararacussu snake venom cross-reacting with specific antibodies against crotoxin, an Asp49 PLA(2)-containing heterodimeric complex from Crotalus durissus terrificus snake venom, were purified by two steps of immunoaffinity chromatography. The resulting fraction (Bj-F) was shown to be non-toxic (to mice and rabbits) and immunogenic to rabbits. Antibodies raised against Bj-F were able to protect mice against the lethal effect of both B. jararacussu and Crotalus durissus terrificus snake venoms. Then, the procedure developed showed to be useful for the rapid preparation of an antigen able to elicit neutralizing antibodies against the lethal activities of both venoms. Further fractionation of Bj-F revealed the concomitant presence of two major components: BJcuL, a lectin present in B. jararacussu venom, and BthTX-I, a Lys49 PLA(2) homolog, besides other molecules in minor amounts. Our data are discussed and raise the point that the presence of unrelated molecules may be taken into account when immuno-based methods are considered for purification purposes.

Proteomic, toxicological and immunogenic characterization of Mexican west-coast rattlesnake (Crotalus basiliscus) venom and its immunological relatedness with the venom of Central American rattlesnake (Crotalus simus).

The venom of the Mexican west-coast rattlesnake (Crotalus basiliscus) was characterized for its protein composition, toxicological profile and immunogenic properties. This venom is composed of 68% Zn2+-dependent metalloproteinases (SVMPs), 14% phospholipases A2 (PLA2s), 11% serine proteinases, 4% SVMPs-inhibitor tripeptides (SVMP-ITs), 2% bradykinin-potentiating peptides (BPPs), 0.6% cysteine-rich secretory proteins (CRISPs), and 0.2% l-amino acid oxidases (LAAOs). SVMPs present in the venom are responsible for azocasein hydrolysis and hemorrhagic activity, but their contribution to the lethal activity of the venom in mice is masked by the neurotoxic activity of PLA2s, which in addition are also responsible for myotoxic activity. Despite its relatively high content of serine proteinases, the venom of C. basiliscus did not exert in vitro coagulant or in vivo defibrinogenating activities. The ability of antivenoms raised against the venoms of C. basiliscus and C. simus (from Costa Rica) to neutralize homologous and heterologous venoms revealed antigenic similarities between toxins of both venoms. Preclinical evaluation of an antivenom produced by using the venom of C. basiliscus as immunogen demonstrated that it is able to neutralize not only the most relevant toxic activities of C. basiliscus venom, but also those exerted by Costa Rican C. simus venom, including coagulant and defibrinogenating activities. BIOLOGICAL SIGNIFICANCE: The Central American rattlesnake (Crotalus simus) is widely distributed from Mexico to west central Costa Rica, and induces an important number of envenomations in this region. On the other hand, the immunogenic mixture used by Laboratorios de Biológicos y Reactivos de Mexico S.A. (Birmex) to produce the snake antivenom more frequently used in Mexico does not include the venom of C. simus. This immunogenic mixture is composed by the venoms of the Fer-de-lance (Bothrops asper) and the Mexican west-coast rattlesnake (Crotalus basiliscus). We studied the protein composition, toxicological profile and immunogenic properties of the venom of C. basiliscus, and evaluated the ability of the Birmex antivenom to neutralize the venom of C. basiliscus and whether it cross-neutralizes the venom of C. simus from Costa Rica. Using proteomics analysis, in combination with in vitro and mouse tests, we determined that the venom of C. basiliscus is mainly composed by SVMPs, which confer proteolytic and hemorrhagic activities to the venom. Other major components of the venom of C. basiliscus are PLA2s, which are responsible for the myotoxic activity and are the main contributors to the lethal activity. Non-clotting SVSPs correspond to 11% of the venom. Minor components include SVMP-ITs, BPPs, CRISPs and LAAOs, which have not been associated with toxicity. The antibodies induced in horses by the venom of C. basiliscus are able to neutralize not only the most relevant toxic activities of the homologous venom, but also those exerted by Costa Rican C. simus venom, including coagulant and defibrinogenating activities. Our preclinical evaluation suggests that Birmex antivenom can be used to treat envenomations by Costa Rican adult C. simus snakebites, despite this venom not being included in the immunizing mixture.

Cross-neutralization of the neurotoxicity of Crotalus durissus terrificus and Bothrops jararacussu venoms by antisera against crotoxin and phospholipase A2 from Crotalus durissus cascavella venom.

We have previously demonstrated that rabbit antisera raised against crotoxin from Crotalus durissus cascavella venom (cdc-crotoxin) and its PLA2 (cdc-PLA2) neutralized the neurotoxicity of this venom and its crotoxin. In this study, we examined the ability of these antisera to neutralize the neurotoxicity of Crotalus durissus terrificus and Bothrops jararacussu venoms and their major toxins, cdt-crotoxin and bothropstoxin-I (BthTX-I), respectively, in mouse isolated phrenic nerve-diaphragm preparations. Immunoblotting showed that antiserum to cdc-crotoxin recognized cdt-crotoxin and BthTX-I, while antiserum to cdc-PLA2 recognized cdt-PLA2 and BthTX-I. ELISA corroborated this cross-reactivity. Antiserum to cdc-crotoxin prevented the neuromuscular blockade caused by C. d. terrificus venom and its crotoxin at a venom/crotoxin:antiserum ratio of 1:3. Antiserum to cdc-PLA2 also neutralized the neuromuscular blockade caused by C. d. terrificus venom or its crotoxin at venom or toxin:antiserum ratios of 1:3 and 1:1, respectively. The neuromuscular blockade caused by B. jararacussu venom and BthTX-I was also neutralized by the antisera to cdc-crotoxin and cdc-PLA2 at a venom/toxin:antiserum ratio of 1:10 for both. Commercial equine antivenom raised against C. d. terrificus venom was effective in preventing the neuromuscular blockade typical of B. jararacussu venom (venom:antivenom ratio of 1:2), whereas for BthTX-I the ratio was 1:10. These results show that antiserum produced against PLA2, the major toxin in C. durissus cascavella venom, efficiently neutralized the neurotoxicity of C. d. terrificus and B. jararacussu venoms and their PLA2 toxins.

Immunochemical analysis of a snake venom phospholipase A2 neurotoxin, crotoxin, with monoclonal antibodies.

Crotoxin is the major neurotoxic component of the venom of the South American rattlesnake, Crotalus durissus terrificus. The crotoxin molecule is composed of two subunits: a basic and weakly toxic phospholipase A2 (PLA2) called component-B (CB), and an acidic, nonenzymatic and nontoxic subunit called component-A (CA). Crotoxin exists as a mixture of several isoforms (or variants) resulting from the association of several subunit isoforms. We prepared monoclonal antibodies (MAbs) against each isolated subunit. Six anti-CA MAbs and eight anti-CB MAbs were tested for their cross-reactivities with each subunit and with other toxic and nontoxic PLA2s. Four of the six anti-CA MAbs cross-reacted with CB, whereas only one of the eight anti-CB MAbs cross-reacted with CA. Two anti-CB MAbs were found to cross-react with agkistrodotoxin, a single chain neurotoxic PLA2 purified from the venom of Agkistrodon blomhoffii brevicaudus. We determined the dissociation constants of each MAb for CA and CB isoforms and their capacities to neutralize the lethality and to inhibit the catalytic activity of crotoxin. We defined three epitopic regions on CA and four on CB, and used a schematic representation of the two subunits to characterize these epitopic regions with respect to: (1) the "toxic" and the "catalytic" sites of CB, and (2) the zone of interaction between the two subunits. We propose three-dimensional structures of the crotoxin subunits in which we localize amino acid residues that might be involved in the epitopic regions described here.

P9a(Cdt-PLA2) from Crotalus durissus terrificus as good immunogen to be employed in the production of crotalic anti-PLA2 IgG.

Four proteins with phospholipase A2 (PLA2) activity, designated P9a(Cdt-PLA2), P9b(Cdt-PLA2), P10a(Cdt-PLA2) and P10b(Cdt-PLA2) were purified from the venom of Crotalus durissus terrificus by two chromatographic steps: a gel filtration and reversed phase HPLC. The profile obtained clearly shows that three of them have a similar abundance. The molecular mass, 14193.8340Da for P9a(Cdt-PLA2), 14134.9102Da for P9b(Cdt-PLA2), 14242.6289Da for P10a(Cdt-PLA2) and 14183.8730Da for P10b(Cdt-PLA2), were initially evaluated by SDS-PAGE and confirmed by ESI-Q-TOF spectrometry, and all of them displayed a monomeric conformation. Also, partial amino acid sequence of each protein was obtained and their alignments with other crotalic PLA2 revealed a high degree of identity among them. Additionally, we studied some pharmacological activities like neurotoxicity, myotoxicity and lethality, which prompted us to pick two of them, P9a(Cdt-PLA2) and P10a(Cdt-PLA2) that resulted to be less toxic that the others, and further characterize them to be used as immunogen. We next injected these last proteins in mice to produce antitoxins against them and ELISA and dot blots reveled that both toxins do not show immunogenic differences, unlike those other pharmacologic activities tested. Furthermore, the antibodies produced cross-reacted with all the isoforms purified demonstrating the feasibility of using only one of them and ensuring the cross-reaction of all. The results obtained show that P9a(Cdt-PLA2) isoform has the lowest toxicity and also a good purification performance; thus this protein may be a promising candidate to be employed in the production of crotalic antitoxins.

Neutralization of lethal potency and inhibition of enzymatic activity of a phospholipase A2 neurotoxin, crotoxin, by non-precipitating antibodies (Fab).

Rabbit antibodies were prepared against both purified catalytic (component-B) and purified non-catalytic (component-A) subunits of crotoxin, the major phospholipase A2 neurotoxin from the South American rattlesnake. They cross-react with crotoxin-like toxins from the venom of several Crotalus species as well as with single-chain phospholipase A2 neurotoxins from Crotalid and Viperid venoms (agkistrodontoxin and ammodytoxin A) but not from Elapid venoms (notexin). Immunological cross-reactions of anti-component-A and anti-component-B sera with crotoxin and with its isolated components A and B showed that component-A exposes determinants of low immunogenicity which are present on component-B, whereas the major antigenic determinants of component-B are not present on component-A. Anti-component-B antibodies, but not anti-component-A antibodies, neutralize the lethal potency of crotoxin and inhibit its enzymatic activity. Furthermore, non-precipitating anti-component-B Fab fragments were as potent as antibodies, indicating that crotoxin neutralization results from the binding of the antibodies to the catalytic subunit, rather than the formation of an immunoprecipitate.

Specificity and binding affinity of an anti-crotoxin combinatorial antibody selected from a phage-displayed library.

A crotoxin-specific, monoclonal, high-affinity, single-chain antibody variable region (scFv) was generated by combinatorial methods using Pharmacia's Recombinant Phage Antibody System. A high-affinity clone, designated A10G, was selected, and its DNA sequence was determined. Protein A10G showed high reaction specificity, with only the closely related rattlesnake neurotoxins, concolor toxin and Mojave toxin, showing cross-reactivity out of eleven group II phospholipase A2s (PLA2s) screened. No group I PLA2s cross-reacted in enzyme-linked immunosorbent assays. The gene coding for A10G was subcloned into an expression vector, and the resulting expressed nonfusion protein, designated A10GPE, was renatured and purified to apparent homogeneity. Dissociation constants of A10G with intact crotoxin and crotoxin basic subunit were determined to be 7 x 10(-10) and 6.8 x 10(-9) M, respectively. When A10GPE was preincubated with either the basic subunit or intact crotoxin at molar ratios of up to 5:1, no inhibition of phospholipase activity was observed. Expressed protein, however, could partially neutralize the lethality of Mojave toxin, a crotoxin homolog, in mice.

Effects of subunit cross-linking on the properties of crotoxin.

Crotoxin was cross-linked using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride. Cross-linked crotoxin had the expected amino-terminal amino acids, amino acid composition, behavior on SDS-PAGE and an 80% reduction of reactable lysine residues. It was also non-toxic, had reduced immunological cross-reactivity toward both poly- and monoclonal antibodies raised to the basic subunit of crotoxin and had lost greater than 95% of its phospholipase activity. Loss of toxicity is due to either subunit cross-linking or the modification of essential residues.

Preparation of a crotoxin neutralizing monoclonal antibody.

Crotoxin is a heterodimeric protein composed of an acidic and basic subunit from the venom of Crotalus durissus terrificus and is representative of a number of presynaptically acting neurotoxins found in the venom of rattlesnakes. Four different monoclonal antibodies, typed as IgG1 subclass, were raised against the basic subunit of this toxin. One was a potent neutralizing antibody of intact crotoxin, which could neutralize approximately 1.6 moles of purified crotoxin per mole of antibody. The monoclonal antibody enhanced the neutralizing ability of commercial polyvalent crotalid antivenom against the lethality of crude C. d. terrificus venom four-fold. Paradoxically, this monoclonal antibody by itself was ineffective against the lethality of crude C. d. terrificus venom. Using an enzyme-linked immunosorbent assay, we tested various proteins for competitive inhibition of binding of biotinylated-crotoxin to plates coated with the four individual monoclonal antibodies. Concolor toxin, vegrandis toxin, intact crotoxin, Mojave toxin, and the basic subunit of crotoxin showed increasing effectiveness as displacers of crotoxin from the neutralizing monoclonal antibody. None of the monoclonal antibodies reacted with purified phospholipase A2 enzymes from Crotalus atrox or Crotalus adamanteus, nor any of the components present in the crude venoms from four different elapids known to contain presynaptically acting neurotoxins, which show some sequence identity to crotoxin.

Protection against the lethal effects of Crotalus durissus terrificus (South American rattlesnake) venom in animals immunized with crotoxin.

Mice and rabbits were immunized against crotoxin (the neurotoxic component isolated from Crotalus durissus terrificus venom) using small amounts of antigen in a water-in-oil emulsion. Following boosting (three times at 21-day intervals) a high titre of antibodies against crotoxin was obtained. Crotoxin immunoglobulin G antibody recognizes whole venom antigen at a level comparable with that of crotoxin antigen, using the ELISA method for antibody detection. The antibodies generated by crotoxin were capable of providing 100% protection against challenge with 11 and 50 i.p. LD50 doses of whole venom in mice. When 100 i.p. LD50 doses of whole venom were injected survival was 77.8%.

Cross-reactivity and neutralization by rabbit antisera raised against crotoxin, its subunits and two related toxins.

Antisera were raised against intact crotoxin (Crotalus durissus terrificus), Mojave toxin (Crotalus scutulatus scutulatus) and concolor toxin (Crotalus viridis concolor), as well as the subunits of crotoxin. Double immunodiffusion and enzyme-linked immunosorbent assays (ELISA) demonstrated antigenic similarity between these three purified toxins and their subunits. Additionally, when crotoxin antisera were pre-incubated with each of the three toxins before injection, the lethal activity of all were neutralized equally well. Antiserum was considerably more effective in neutralizing crotoxin in vivo when the toxin was injected i.m. than when injected i.v. Antisera against both intact crotoxin and its basic subunit were an order of magnitude more effective than crotoxin acidic subunit antiserum in crotoxin neutralization. Purified phospholipase A2 from Crotalus adamanteus and Crotalus atrox showed weak cross-reactivity with antisera raised against intact crotoxin and its subunits in the ELISA. Our results suggest that crotalid neurotoxins can be detected and neutralized by polyclonal antibodies raised against any intact toxin or basic subunit in this group of homologous toxins.

Immunochemical cross-reactivity of two phospholipase A2 neurotoxins, agkistrodotoxin and crotoxin.

Polyclonal rabbit antisera were raised against the phospholipase A2 neurotoxin agkistrodotoxin (AGTX) from Agkistrodon blomhoffii brevicaudus venom and against the phospholipase A2 subunit (component-B, CB) of crotoxin from Crotalus durissus terrificus venom. Anti-AGTX antibodies cross-reacted strongly with crotoxin and crotoxin-like molecules and more weakly with other phospholipases A2 from the venoms of Viperidae and Crotalidae. On the other hand, anti-CB antibodies cross-reacted with AGTX, and also recognized ammodytoxin A and the phospholipase A2 from Vipera berus venom, but not other phospholipases A2 from Crotalidae and Viperidae. Anti-AGTX and anti-CB antibodies were able to inhibit the phospholipase A2 activity and to neutralize the lethal potency of the homologous and heterologous toxins (AGTX or crotoxin). Immunoaffinity chromatography columns were used to isolate anti-AGTX antibodies which recognized CB (91% of the total anti-AGTX antibodies), and anti-CB antibodies which recognized AGTX (52% of the total anti-CB antibodies). Immunochemical investigations performed with each type of antibody indicated that the majority of AGTX antigenic determinants are present on crotoxin component-B and on phospholipases A2 from Viperidae venoms, and that some of these determinants are involved in the neutralization of lethal potency and in the inhibition of enzymatic activity of AGTX and crotoxin.

Primary sequence determination of the most basic myonecrotic phospholipase A2 from the venom of Vipera russelli.

The most basic phospholipase A2 (PLA2), VRV-PL-VIIIa, was purified from (Sri Lankan) Vipera russelli venom. It is a major component of the venom, contributing over 40% to the whole venom PLA2 activity. The purity of VRV-PL-VIIIa was ascertained by electrophoresis and by reverse phase high-pressure liquid-chromatography (RP-HPLC). VRV-PL-VIIIa had an apparent mol. wt of 13,000 and was a single polypeptide. The protein was reduced, pyridylethylated and subjected to sequence analysis. The N-terminal amino acid sequence was established up to the 39th residue. Pyridylethylated VRV-PL-VIIIa was digested with endoprotease Glu-C, and several peptides were purified by RP-HPLC; six purified peptides were sequenced. The sequence of the C-terminal was established by sequencing a CNBr-produced peptide purified by RP-HPLC. Several peptides were also generated by digestion with endoprotease Asp-N. Two peptides were sequenced to obtain overlapping regions. The complete structure was deduced from sequences of overlapping peptides and through homology with other group II PLA2 sequences. Sequence homology was greatest with ammodytoxin A: 99 amino acid residues out of 121 occurred in identical positions. Myotoxin III of Bothrops asper showed 73% homology, 89 out of 121 residues. In agreement with the sequence data, polyclonal antiserum against VRV-PL-VIIIa cross-reacted in ELISA with ammodytoxin A and, to a lesser extent, with caudoxin.