Heterologous Venoms Research Articles

Cross-neutralization of Thai cobra ( Naja kaouthia) and spitting cobra ( Naja siamensis) venoms by Thai cobra antivenom

The neutralizing capacity of antivenom prepared against Thai cobra ( Naja kaouthia) venom was compared in mice using the homologous venom and that of spitting cobra ( Naja siamensis). The amounts of antivenoms neutralizing a dose of 4 ld 50 of the test venom were determined. Four antivenom preparations were used: three purified antivenoms and a crude antivenom, which were made using N. kaouthia venom only. Almost the same neutralizing capacity was obtained with the purified antivenoms, whereas a slightly lower capacity was seen with the crude antivenom. However, ratios of the amounts of four antivenoms neutralizing the homologous and heterologous test venoms were almost constant. These results indicated that Thai cobra antivenom possesses neutralizing capacity against spitting cobra venom.

Cross-reactivity of monovalent and polyvalent Trimeresurus antivenoms with venoms from various species of Trimeresurus (lance-headed pit viper) snake

N.-H. Tan, S.-K. Choy, K.-M. Chin and G. Ponnudurai. Cross-reactivity of monovalent and polyvalent Trimeresurus antivenoms with venoms from various species of Trimeresurus (lance-headed pit viper) snake. Toxicon 32, 849–853, 1994.— Trimeresurus bite is a serious medical problem in Asia. However, at present only a few monospecific Trimeresurus antivenoms are available. Investigation of the cross-neutralization capacity of three Trimeresurus antivenoms indicates that the antivenoms exhibit broad cross-reactivity. A polyvalent Trimeresurus antivenom was also found to be effective in neutralization of the haemorrhagic, necrotizing and thrombin-like activities of heterologous Trimeresurus venoms.

Selection of Hymenoptera venoms for immunotherapy on the basis of patient's IgE antibody cross-reactivity

Background: Positive skin test results to multiple venoms in patients with Hymenoptera venom allergy may result from IgE antibody cross-reactivity among venom proteins. To avoid treatment with unnecessary and costly venoms, we have developed a venom RAST inhibition test that identifies individuals in whom a positive venom IgE RAST result is due to cross-reactive venom-specific IgE antibody. Methods: Serum samples ( n = 412) were collected over 5 years from patients with clinically characterized Hymenoptera venom allergy who had positive skin test results to more than one venom. Venom allergosorbent was added to serum containing IgE antivenin and buffer or 100 μg of homologous or heterologous venom. Bound IgE was detected with radiolabeled anti-human IgE. Intraassay variation less than 10% coefficient of variation and homologous venom inhibition greater than 80% were required for acceptance of data. A “cross-reactivity index” (CRI) was computed as a ratio of percent inhibition produced by heterologous versus homologous venom. Results: Of the 412 sera-venom combinations analyzed, 41 (10%) were excluded because of incomplete homologous venom inhibition. Of the 371 remaining sera, 82% ( n = 305) were studied for IgE anti- Polistes wasp venom (PWV) cross-reactivity with yellow jacket venom (YJV) and the other 66 for other venom specificity cross-inhibitions. Of the serum samples tested, 36.4% (111 of 305) contained IgE anti-PWV venom of which the binding to solid-phase PWV was inhibited with soluble YJV to a level that produced CRIs greater than 95%. We believe that this constitutes complete inhibition and demonstrates exclusively YJV cross-reactive antibody in these samples. The remaining 63.6% had CRIs from 0% to 95%, indicating IgE specific for a spectrum of unique and cross-reactive PWV allergens. Only 4.3% (13 of 305) had CRIs less than 5%, which is consistent with IgE restricted to PWV unique allergens. The degree of the IgE anti-PWV inhibition to solid-phase PWV by YJV was independent of the IgE anti-PWV level. Conclusions: This study shows that one third of patients with Hymenoptera venom allergy evaluated with positive YJV- and PWV-reactive IgE in the skin and/or serum were identified as candidates for exclusion of PWV from their immunotherapy regimen because their IgE anti-PWV was more than 95% cross-inhibitable with YJV. Cost analysis of the venom RAST inhibition test and a conventional 5-year Hymenoptera venom immunotherapy program indicates that this serologic evaluation is cost-effective.

An investigation into the antigenic cross-reactivity of Ophiophagus hannah (king cobra) venom neurotoxin, phospholipase A 2, hemorrhagin and l-amino acid oxidase using enzyme-linked immunosorbent assay

The antigenic cross-reactivity of four Ophiophagus hannah (king cobra) venom components, the neurotoxin (OH-NTX), phospholipase A 2 (OH-PLA 2), hemorrhagin (OH-HMG) and l-amino acid oxidase (OH-LAAO) were examined by indirect and double sandwich ELISAs. The indirect ELISAs for OH-NTX, OH-PLA 2 and OH-HMG were very specific when assayed against the various heterologous snake venoms and O. hannah venom components, at 25 ng/ml antigen level. At higher antigen concentrations (100–400 ng/ml), there were moderate to strong indirect ELISA cross-reactions between anti- O. hannah neurotoxin and venoms from various species of cobra as well as two short neurotoxins. However, anti- O. hannah hemorrhagin did not cross-react with any of the venoms tested, even at these high antigen concentrations, indicating that O. hannah hemorrhagin is antigenically very different from other venom hemorrhagins. Examination of the indirect ELISA cross-reactions between anti- O. hannah PLA 2 and several elapid PLA 2 enzymes suggests that the elapid PLA 2 antigenic class has more than two subgroups. The antibodies to O. hannah l-amino acid oxidase, however, yielded indirect ELISA cross-reactions with many venoms as well as with OH-NTX, OH-PLA 2 and OH-HMG, indicating that OH-LAAO shares common epitopes even with unrelated proteins. The double sandwich ELISAs for the four anti- O. hannah venom components, on the other hand, generally exhibited a higher degree of selectivity than the indirect ELISA procedure.

Species-specific detection of venom antigens from snakes of the Bothrops and Lachesis genera

L. G. D. Heneine and D. Catty. Species-specific detection of venom antigens from snakes of the Bothrops and Lachesis genera. Toxicon 31, 591–603, 1993.—Venom-insoluble adsorbents were employed to absorb out the cross-reacting antibodies from monovalent polyclonal antivenoms. The absorbed antivenoms were tested by enzyme-linked immunosorbent assay against homologous and heterologous venoms and showed species-specificity throughout a range of venom concentrations. The same absorbed antisera were used in immunoblots under non-reducing conditions as probes to reveal species-specific antigens. In all cases studied this was achieved. The range of mol. wts of specific antigens was between 20,000 and 120,000, approximately. Venoms added to human serum experimentally were specifically detected by their homologous absorbed antivenom antibodies. The work here described could be important in the development of diagnostic assays for envenomings involving snakes from the Bothrops and Lachesis genera.

Neutralization of different activities of venoms from nine species of Bothrops snakes by Bothrops jararaca antivenom

Antivenoms are the usual treatment in cases of systemic envenoming by Bothrops snakes. However, the neutralization of each venom component by the antivenom is not well established. Bothrops jararaca antivenom, produced in rabbits, recognizes the venoms of nine different Bothrops species with high ELISA antibody titres. Western blot analysis showed that almost all antigens present on both homologous and heterologous venoms are recognized. Neutralization tests were performed using whole antivenom or its IgG fraction. The antivenom was able to neutralize the haemorrhagic, coagulant and necrotizing activities of the heterologous venoms in the same antivenom/venom proportion as for the homologous venom. Myotoxic activity was only partially neutralized. Neutralization of the proteolytic activity of heterologous venoms required higher amounts of antivenom than for the homologous venom. Phospholipase and oedema-inducing activities were completely neutralized only in the homologous system.

Diagnosis of Snake Venoms by a Reverse Latex Agglutination Test

A reverse latex agglutination test using protein A column purified rabbit antivenom IgG-sensitized latex particles was developed for the detection of the six medically important snake venoms of Thailand. The detection limit of the reverse latex agglutination test was 0.16 to 1.2 micrograms/mL of crude venoms. Cross-reactions with heterologous venoms were observed at concentrations 460 to 16000 times that of homologous venoms. Detection of various snake venoms in clinical specimens was carried out by the reverse latex agglutination test. The sensitivity was 52.5% of the 59 serum samples. There was one (1.69%) false positive sample. The positive detection of venom in wound swabs (26 cases) was 38.5% and was not statistically different from that observed in paired serum samples. Interference from human plasma, serum and urine on the reverse latex agglutination test could be eliminated by adsorption with normal rabbit IgG-coated latex suspension or by heat inactivation at 56 degrees C for 30 min. Prozone effect observed in some sera was eliminated by heat inactivation at 56 degrees C for 30 min. The sensitized latex particles were stable at 4 degrees C and -20 degrees C for at least 3 months. Cycles of freezing-thawing and lyophilization did not change their reactivities. The total test time was about 40 min.

Purification and properties of an antivenom factor from the plasma of the South American rattlesnake (Crotalus durissus terrificus)

Purification and properties of an antivenom factor from the plasma of the South American rattlesnake ( Crotalus durissus terrificus). Toxicon 29, 997–1008, 1991.—The lethal toxicity of Crotalus durissus terrificus (Crotalinae, Viperidae) can be attributed mainly to the presence of a neurotoxic protein, crotoxin, which also shows phospholipase A 2 activity. It has been previously demonstrated that both lethal and phospholipase A 2 activities of crotoxin can be neutralized by an alpha 1-globulin factor that is present in the homologous blood. Crotalus durissus terrificus plasma also renders some degree of protection to mice against the lethal toxicity of heterologous venoms from snakes of the genus Bothrops (Crotalinae, Viperidae), but not of the genus Micrurus (Elapinae, Elapidae). An anti-toxic factor was purified to homogeneity from C.d. terrificus plasma after three chromatographic steps (DEAE-Sephacel anion exchange, CM-Sepharose cation exchange and Pro-RPC reverse phase chromatography); it is named CNF for Crotalus neutralizing factor. The purification process was accompanied by polyacrylamide gel electrophoresis in the presence of SDS and by measurements of phospholipase A 2 inhibition. After the first two purification steps, an 86-fold increase of the inhibitory activity of CNF was observed; however, the third step caused an apparent inactivation of the factor. The inactive CNF was shown to correspond to the previously active plasma material and to be homogeneous on electrophoresis, immunoelectrophoresis and partial amino-terminal sequence. The mol. wt of CNF was estimated as 23,600 by SDS-PAGE.

Antigenic relationship between the venom of the night adder Causus maculatus and venoms of other viperids

Monovalent antivenoms were raised in mice against the venoms of Causus maculatus, Vipera ammodytes, Echis carinatus, Cerastes cerastes, Bitis arietans, Agkistrodon rhodostoma and Bothrops atrox. These antivenoms as well as four commercially available antivenoms were tested against the venoms of 15 viperid species by means of immunoelectrophoresis and/or ELISA. Cross-reactive protein bands were determined by immunoblot. ELISA cross-reactions of C. maculatus antivenom were low with all heterologous venoms. When investigating the other viperine antivenoms in ELISA stronger cross-reactions were observed with several heterologous venoms. In immunoblot, two heterologous antivenoms cross-reacted with one or two protein bands of C. maculatus venom whereas there were at least four heterologous antivenoms cross-reacting with each of the other venoms. The findings indicate that there is little antigenic affinity between C. maculatus venom and the other venoms investigated. Broad in vitro cross-reactions between viperine antivenoms and Causus venom which were reported in literature seem to be attributable to the use of antivenoms of commercial grade. Specificity of commercially produced, mono- or polyvalent antivenoms may not be strictly limited to those venoms, against which potency is claimed on the label of the product.

Biotoxicology of sea snake venoms

Sea snakes are the most abundant venomous reptiles, found throughout the Indian and Pacific Oceans. Divided into two subfamilies, Laticaudinae and Hydrophiinae, all sea snakes are poisonous. Venoms are highly toxic, as indicated by low LD50 values in test animals. Toxic compounds include presynaptic and postsynaptic neurotoxins. Similarities in phylogeny are reflected in immunodiffusion patterns, immunoelectrophoresis, cross-neutralization by antivenin against heterologous venoms, and amino acid composition. The clinical syndrome following a bite is largely neurotoxic and myotoxic, with rare hepatotoxicity and nephrotoxicity. Proper emergency field therapy and timely administration of antivenin can be lifesaving. Hemodialysis may be useful when antivenin is not available.

Venom Toxins: Plausible Evolution from Digestive Enzymes

Some hydrolytic enzymes are common to the pancreas, the mammalian salivary glands and the snake venom glands. Phospholipase A, which is found in elapid and viperid venoms and in the mammalian pancreas, shows 29 common amino acid residues out of 118–125 positions. Presynaptic neurotoxins and other venom toxins are usually composed of 2–3 units or subumts,one of which is a phospholipase. The Vipera palaestinae two-component toxin retains its lethality when the enzyme is replaced by heterologous venom phospholipases, but not by the pig pancreatic enzyme. This toxin is neutralized by a factor found in the blood serum of snakes, which binds to the phospholipase and inhibits its activity. The blood serum of snakes also neutralizes hemorrhagins and inhibits the protease activity of the venom. It is hypothesized that the developing venom glands first produced enzymes that were already secreted by the pancreas and against which inhibitors were present in the blood. These inhibitors facilitated the evolution of enzyme-based toxins by neutralizing any damaging substances that might have escaped from the venom glands.

Specificity of antibodies to the reconstituted crotoxin complex, from the venom of South American rattlesnake ( Crotalus durissus terrificus), using enzyme-linked immunosorbent assay (ELISA) and double immunodiffusion

Some differences between the reaction of antiserum to reconstituted crotoxin complex, to ‘native’ crotoxin and to whole Crotalus durissus terrificus (South American rattlesnake) venom were detected by enzyme-linked immunosorbent assay (ELISA) and by immunodiffusion. ELISA showed the presence of antibodies to the components of the crotoxin complex, phospholipase A 2 and crotapotin. When the antiserum was tested against North American rattlesnake venoms some species gave a positive reaction ( C. horridus atricaudatus and C. basiliscus), whilst the venoms of other species were virtually negative ( C. durissus totonacus, C. horridus horridus, C. viridis viridis and C. atrox). Confirmation of antigenic similarities between crotoxin and Mojave toxin (from the venom of C. scutulatus scutulatus) was obtained with ELISA and some implications of these species differences in antigen-antibody reaction are discussed. No paraspecificity was observed with heterologous snake venoms from other Crotalidae, Elapidae, Hydrophiidae or Viperidae. ELISA also showed a lack of cross-reactivity of the antiserum to heterologous purified phospholipases A 2 from Enhydrina schistosa, Naja nigricollis or Apis mellifera venoms or from porcine pancreas. The antiserum reacted to the homologous phospholipase A 2 inactivated with p-bromophenacyl bromide as well as to the detoxified crotoxin complex reconstituted with this modified phospholipase A 2. This may be useful in the raising of high titres of antibody to crotoxin.

Action of antisera against homologous and heterologous snake venom phospholipases A 2

Studies on the inhibitory effects of a series of anti-snake-venom sera on the phospholipase A 2 activity of a number of snake venoms have been made using the suppression of phospholipase A 2 activity by the antiserum as an indicator of interaction between antibodies against phospholipase A 2 and snake venom phospholipase A 2. Specific antiserum against the venom of a particular species inhibited the phospholipase A 2 activity of the venoms of closely related species, but in most cases, failed to suppress phospholipase A 2 activity of venoms from other families. The amount of antiserum required for 50% inhibition of phospholipase A 2 activity varied with the species. The differences in the inhibitory properties of a particular antiserum on a heterologous phospholipase A 2 activity indicate variations in the antigenic sites of the enzyme molecule.

Coral Snake Venoms

Abstract Cross neutralization and precipitin tests were performed with coral snake venoms and rabbit antisera against five venoms to determine the most suitable venoms for producing a polyvalent antivenin useful in treating persons bitten by coral snakes. Antisera were produced against Micrurus species M. fulvius, M. frontalis, M. spixii, M. carinicauda, and M. nigrocinctus venom but not against M. mipartitus venom. The latter was the only venom not neutralized by heterologous antisera. M. spixii venom was neutralized by all antisera. Anti-M. frontalis serum neutralized more heterologous (M. spixii) venom than homologous (M. frontalis) venom. The cross reactions suggest that some venom antigens that elicit precipitating and neutralizing antibodies may be distinct. M. frontalis venom produced the most cross-reactive neutralizing antibody, having neutralized five of six venoms. Of the venoms tested, M. frontalis venom would be most suitable for use in the production of a polyvalent coral snake antivenin.

Use of Polistes Venom in Petrolatum: Arlacel Repositories to Immunize against Yellow Jacket Wasp-Sting Allergy

Summary and Conclusions Twenty-four persons with extreme allergy toward yellow jacket wasp stings were immunized safely with single, yearly, subcutaneous repositories of emulsified heterologous venom from the polistes wasp, there being no significant adverse developments on treatment day and no longterm sequelae such as abscess, cyst or neoplasm in the next 3 to 4 years. By contrast, control groups with identical allergy exhibited two adverse responses among 111 single-visit courses of fluid venom prepared from the homologous wasp or from polistes wasps. Effectiveness of therapy was judged by the clinical response to later stingings, of which 60 took place by plan in the hospital and four in the field by accident. Aside from trivial focal developments in 12 instances, only two significant adverse episodes were engendered, one in a repository-treated man and one after an immunization with homologous fluid venom. Subsequent repetition of the challenge found both men fully tolerant, one having taken a booster of fluid polistes venom in the meantime while the second merely rested. It was deduced that factors other than specificity differences in the two immunizing venoms probably had been responsible for the clinical failure after repository. The 64 challenges involved one to four insects and post-treatment periods that ranged from 9 days to 21 weeks for courses of fluid venom, and from 15 to 27 weeks for depot therapy. All but a few of the sting tested subjects have been transferred to intermittent sting-therapy to maintain their demonstrated immunity and are now receiving 2-min stingings from 3 to 4 wasps—polistes in cold months, yellow jacket wasps in summer—at intervals approaching a year. Threshold tests of skin and eye may afford a clue, before immunization, to the patient's tolerance for injected venom. After therapy, there is a small but statistically significant elevation in requirement, due to induced blocking antibody. As to the indication for specific prophylaxis in sting allergy, probably only those victims whose next encounter threatens disaster merit immunization, in view of the implied long-term risk of using foreign proteins parenterally. Obviously, only the simplest preparations and the smallest effective amounts are justified in any case, viz., venom as opposed to whole body extracts, and a single annual treatment as opposed to weekly and monthly injections over protracted periods.

Heterologous Antivenin in Neutralization of North American Coral Snake Venom

NEUTRALIZATION of heterologous venoms by snake antivenins is well known. The importance of this paraspecific aiction in antivenin production has been discussed by Criley (1) and Grasset (2). It has also been shown, most recently by Keegan and Yoshino (3), that the relationship of snake species involved is not always a practical basis for accurate prediction of neutralization. Actual performance of a neutralization test or use of the antivenin in therapy are the only sure methods of determining whether paraspecificity exists. Presented here are results of neutralization tests conducted to determine whether an antivenin produced with venom of a South American snake of genus Ilicrurus would neutralize venom of the North American coral snake, Mierurzts fulviws fulvius (Linnaeus, 1766). Although Shannon (4), and undoubtedly many others, speculated as to whether such neutralization would take place, test results to support such speculation have never been published.

Immunological Studies on Scorpions

Abstract These investigations on scorpions constitute a further contribution to the experimental studies carried out by one of us (Grasset and Zoutendyk, 1931; Grasset, 1931) on the immunity of the lower vertebrata, such as reptiles, compared with mammals. The following immunological aspects were investigated: Susceptibility of scorpions to bacterial toxins such as tetanus, diphtheria, dysentery, and the persistence of the toxin in the system of the scorpion.The immunity response of scorpions to bacterial toxins and toxoids and to bacterial somatic antigens.Susceptibility of scorpions to homologous and heterologous scorpion venoms, and to a variety of snake venoms. The bulk of these investigations have been done on the 3 most common genera of South African scorpions: Hadogenes, Opisthophthalmus and Parabuthus, the venoms of which have made the subject of previous investigations by the authors (Grasset, Schaafsma and Hodgson, 1945). For zoological taxonomy see Hewitt (1918) and Lawrence (1942).

A comparative investigation into the antigenic properties of detoxicated Indian and African venoms and the cross action exerted by the respective antivenenes

The potency of samples of Indian Daboia (Russell's viper) and Naia tripudians (Indian cobra) venoms has been compared with that of African viperine and colubrine venoms. Strong solutions of both Indian venoms may be detoxicated and converted into anavenoms by the same methods evolved by us for treating African venoms. The anavenoms retain their antigenic properties as shown by the immunization of laboratory animals, but from the results obtained on a necessarily small number of animals it would appear that the antigenic value of the Indian cobra anavenom is not relatively as high as that of Cape cobra anavenom. The venoms of Daboia and Bitis arietans are so antigenically dissimilar that neither African not Indian concentrated antivenenes exert any appreciable neutralizing action on the heterologous venom. A comparable lack of action was observed when a sample of South American anti-rattlesnake serum was tested against puff adder venom. The colubrine venoms, Indian cobra and Cape cobra, appear to be very closely allied ; Indian concentrated antivenene exerts a moderate group action on Cape cobra and other African colubrine venoms, whilst concentrated African antivenene exerts an even more powerful neutralizing action against Indian cobra venom than it does against the Cape cobra venom from which it was produced. A final point of interest is the fact that antivenenes of a comparable cross potency, as far as colubrine venoms are concerned, may be produced on the one hand by the use of unmodified venoms and on the other by employing only formalized anavenoms ; this furnishes further evidence of the practical utility of the rapid method of producing African polyvalent antivenene of high potency, as successfully employed in our Serum Department during the past 3 years.