Venom Antigens Research Articles

Prognostic significance of clinical grading of patients envenomed by Bothrops lanceolatus in Martinique

The correlation between clinical grading of patients bitten by Bothrops lanceolatus and the subsequent development of their envenoming was examined. Severity of envenoming was graded using a 1–4 scale (minor to major). Patients were classified into 2 groups according to the time elapsed between bite and treatment with a specific purified equine F(ab′)2 antivenom. The late/no treatment group ( n = 33) was characterized by a systemic thrombotic complication rate of 14 33 (42·4%) leading to 4 deaths, which increased with the maximum severity assessed on the first day following the bite ( P = 0·003). However, infarctions could develop in patients who presented initially with signs of moderate envenoming, normal blood clotting and low serum levels of venom antigens. No such complication or fatality occurred in the early (0·5–6 h) treatment group ( n = 70). Multiple regression analysis showed that duration of stay in hospital in this group increased with the length of the snake ( P = 0·017), venom antigenaemia ( P = 0·016), initial grading ( P < 0·001), and with the need for surgical debridement ( n = 10 70 , P < 0·001 ). Outcome was correlated with initial severity of envenoming. However, the only factor with a positive prognostic significance for the individual envenomed patient was the early infusion of specific antivenom, which led to 100% recovery in our series.

Development of an ELISA for the detection of scorpion venoms in sera of humans envenomed by Androctonus australis garzonii (Aag) and Buthus occitanus tunetanus (Bot): correlation with clinical severity of envenoming in Tunisia

A sandwich ELISA was set up for measuring scorpion venom levels in sera of accidentally envenomed humans with the aim to establish a quantitative relationship between these levels, envenoming severity and clinical symptoms. This assay used equine polyclonal F(ab′) 2, specific to two North African scorpion ( Androctonus australis garzonii: Aag and Buthus occitanus tunetanus: Bot) venoms. The test proved to be simple, reproducible, very sensitive (detection limit=0.9 ng/ml) and linear between 0.5 and 15 ng/ml of venom concentrations. A large survey on scorpion sting envenomings was conducted from 1993 to 1996 in Tunisia to gather accurate epidemiological, clinical and biological data from victims as well as informations on the treatment that they had received. Victims were classified into three grades (GI, GII and GIII) of increasing severity according to clinical signs of envenoming. Blood samples were collected from victims and tested by ELISA for their content of Aag and Bot venoms. A strong correlation was found between clinical symptoms of envenoming and the level of scorpion venom antigens in serum ( r=0.980). Mean serum venom concentrations were: 2.65±0.81 ng/ml in GI envenoming, 9.79±4.08 ng/ml in GII and 21.7±6.51 ng/ml in GIII. The difference between each group was statistically significant ( p<0.01). This ELISA may prove to be helpful to establish a rationale approach of specific antivenom therapy.

ELISA for the detection of venom antigens in experimental and clinical envenoming by Loxosceles intermedia spiders

Enzyme linked immunosorbent assays (ELISA) were developed to detect antigens from Loxosceles intermedia spider venom. Hyperimmune horse anti- Loxosceles intermedia IgGs were prepared by immunoaffinity chromatography and used to set up a sandwich-type ELISA. The specificity of the assay was demonstrated by its capacity to correctly discriminate the circulating antigens in mice that were experimentally inoculated with L. intermedia venom from those inoculated with L. gaucho, L. laeta, and Phoneutria nigriventer spider venoms, Tityus serrulatus scorpion venom and Bothrops jararaca, Crotalus durissus terrificus, Lachesis muta muta and Micrurus frontalis snake venoms. Measurable absorbance signals were obtained with 0.8 ng of venom per assay. The ELISA also detected antigens in the sera of patients envenomed by L. intermedia. Therefore, after standardization for clinical use this ELISA may be a valuable tool for clinicians and epidemiologists.

Evolution of lymphocyte transformation to wasp venom antigen during immunotherapy for wasp venom anaphylaxis.

Venom immunotherapy (VIT) has proven to be safe and effective in wasp venom anaphylaxis. However, there are no good parameters to indicate when to stop venom immunotherapy. To evaluate the relationship of the lymphocyte transformation test (LTT) to history and specific IgE determination, and to address the time course of lymphocyte transformation responses to wasp (Vespula) venom during VIT and the possible utility of LTT to determine the duration of therapy. Peripheral blood mononuclear cells (PBMCs) of 18 individuals with a history of wasp sting anaphylaxis and a positive serum-venom-specific IgE, were stimulated with wasp venom before immunotherapy, at the end of a 5-day semi-rush immunotherapy and at 24 months during venom immunotherapy. Results, expressed as stimulation index (SI), were compared with the SI in seven asymptomatic stung controls. In controls the median (minimum-maximum) of the SI were 2.39 (0.52-3.39) before therapy and 2.39 (1.12-6.02) when repeated after 24 months. For patients the median (minimum-maximum) of the SI were 10.13 (1.19-44.88) before immunotherapy (d0), 2.73 (0.67-12.03) at the end of the build-up immunotherapy (d5) and 4.21 (0.88-14.66) at the end of 24 months of maintenance therapy (m24). The proliferation responses in vespid-allergic patients were significantly higher than in stung controls (P = 0.006) but only 13/18 patients showed a positive LTT result before the start of immunotherapy (sensitivity of the LTT 72%). When the LTT was repeated after a 5 day build-up hyposensitization course the SI significantly dropped as compared to the pre-treatment levels (P = 0.002). The SI of the LTT was negative in eight out of 18 patients at 24 months and the median values were significantly lower than before therapy (P = 0.03). Although, in the absence of sting challenge data it is not possible to draw conclusions about the predictive value of the LTT, our data may suggest that abolition of the LTT during VIT might indicate clinical insensitivity. Further studies, comparing the results of sting challenges, with the results of lymphocyte transformation will be necessary in order to evaluate the role of LTT in stopping immunotherapy.

VIPER ENVENOMING: EVALUATION OF TREATMENT BY RESTORATION OF HAEMOSTASIS AND VENOM CLEARANCE

Treatment of systemic envenoming in snakebite victims has, in the past, depended almost entirely on the individual clinician's experience in assessing the severity of envenoming. The efficacy of treatment is obviously related to the neutralising potency of the antivenom used, the route by which it is administered and the dose. The use of techniques for evaluating the efficacy of antivenoms has proved to be very useful, as an adjunct to recognised clinical observations, for a more objective evaluation of antivenom efficacy and dosage. In patients bitten by many vipers, including the Brazilian pit vipers, the reversal of the venom-induced coagulopathy provides an ideal indicator as to the efficacy of an antivenom. Likewise, the development of enzyme immunoassay has permitted us to estimate levels of circulating specific venom and antivenom levels at any time after the bite in the patient's blood; the efficacy of an antivenom can thus be objectively assessed by measuring the neutralisation and clearance of venom antigen. In Brazil, it appears that clinicians treat patients with excessive amounts of highly efficient antivenoms, which results in an unacceptably high incidence of reactions. In Sri Lanka, the use of imported Indian antivenom is relatively ineffective in neutralising the procoagulant and other effects of the venoms of Sri Lankan snakes, demonstrating the real problem of venom variability within individual species. In West Africa, the improved restoration of blood coagulability, the resolution of haemorrhagic disturbances and the increased rate of clearance of venom following treatment of Echis victims with a monospecific as opposed to a polyspecific antivenom has been demonstrated, and new smaller fragment Fab antivenoms have been developed and are now under clinical assessment. Such clinically based coagulation and immunological studies should result in more efficient and controlled use of expensive antivenoms for treatment of systemic envenoming and the accurate assessment of newly designed products. Such studies also emphasise the importance of individual countries producing their own antivenoms for treatment of systemic envenoming. Likewise, the use of such objective systems now enable us to assess the use of first aid measures such as tourniquets.

The Use of Liposomes as Snake Venom Vehicles. Application in Protective Immunization

AbstractLiposomes (phospholipid-based vesicles) have been widely studied as drug delivery systems due to their structural versatility concerning size, composition, bilayer fluidity and their ability to incorporate almost any molecule regardless of its nature. Liposomes are successful for inducing potent immunity in vivo and they are now being employed in numerous irnmunization procedures and as vehicles for vaccines. This is a brief overview of the use of liposomes as carriers of venom antigens and immunomodulators, a safe and potent alternative for the production of antivenom and for the active immunization of dommestic animals in areas of high incidence of envenomation and mortality. Other potential applications of venom-containing liposomes are also presented.

Development of immunoassays for determination of circulating venom antigens during envenomations by coral snakes ( Micrurus species)

A reverse agglutination assay and two capture enzyme-linked immunoassays (ELISAs) for the quantitative determination of Micrurus nigrocinctus nigrocinctus venom antigens in fluids were developed using affinity-purified polyclonal antibodies and a cocktail of three monoclonal antibodies. The lower detection limit was 0.3 mg/ml for the reverse agglutination assay and 4 ng/ml for the capture ELISAs. The optical densities of both ELISAs correlated very well with venom concentrations in the range 4-333 ng/ml ( r = 0.99). The ability of these assays to detect venoms of several medically important Micrurus species was studied. Besides detecting homologous venom, both ELISAs were also useful to quantitate venom from M. fulvius, M. dumerilii carinicauda and M. alleni. Using biotinylated polyclonal antibodies, M. n. nigrocinctus venom antigens were detected in sera or plasma from rabbits and mice during experimental envenomations with lethal and sublethal venom doses. The assays described in this work are promising tests to estimate the severity of poisoning in envenomations by the most important coral snakes of North and Central America.

An objective approach to antivenom therapy and assessment of first-aid measures in snake bite

Treatment of systemic envenoming in snake-bite victims has, in the past, depended almost entirely on the individual clinician's experience in assessing the severity of envenoming. The efficacy of treatment is obviously related to the neutralizing potency of the antivenom used, the route by which it is administered and the dose. The development of enzyme immunoassays has permitted a more scientific appraisal, allowing estimation of circulating specific venom and antivenom concentrations at any time after the bite in the patient's blood. It is therefore possible to measure accurately the efficacy of antivenom in the neutralization and clearance of venom antigen. In Brazil, it appears that clinicians treat patients with excessive amounts of highly efficient antivenoms and this results in an unacceptably high incidence of reactions. In Sri Lanka, the use of imported, Indian antivenom is relatively ineffective in neutralizing the venoms of Sri Lankan snakes, demonstrating the real problem of venom variability within individual species. In West Africa, the improved clearance of venom following treatment of Echis victims with a monospecific as opposed to a polyspecific antivenom has been demonstrated, and new, smaller fragment, Fab antivenoms have been developed and are now under clinical assessment. Such clinically-based immunological studies should result in more efficient and controlled use of expensive antivenoms for treatment of systemic envenoming and the accurate assessment of newly designed products. Such studies also emphasise the importance of individual countries producing their own antivenoms for treatment of systemic envenoming. Likewise, the use of such objective systems now enables the use of first-aid measures such as tourniquets to be properly assessed.

An objective approach to antivenom therapy and assessment of first-aid measures in snake bite.

Treatment of systemic envenoming in snake-bite victims has, in the past, depended almost entirely on the individual clinician's experience in assessing the severity of envenoming. The efficacy of treatment is obviously related to the neutralizing potency of the antivenom used, the route by which it is administered and the dose. The development of enzyme immunoassays has permitted a more scientific appraisal, allowing estimation of circulating specific venom and antivenom concentrations at any time after the bite in the patient's blood. It is therefore possible to measure accurately the efficacy of antivenom in the neutralization and clearance of venom antigen. In Brazil, it appears that clinicians treat patients with excessive amounts of highly efficient antivenoms and this results in an unacceptably high incidence of reactions. In Sri Lanka, the use of imported, Indian antivenom is relatively ineffective in neutralizing the venoms of Sri Lankan snakes, demonstrating the real problem of venom variability within individual species. In West Africa, the improved clearance of venom following treatment of Echis victims with a monospecific as opposed to a polyspecific antivenom has been demonstrated, and new, smaller fragment, Fab antivenoms have been developed and are now under clinical assessment. Such clinically-based immunological studies should result in more efficient and controlled use of expensive antivenoms for treatment of systemic envenoming and the accurate assessment of newly designed products. Such studies also emphasise the importance of individual countries producing their own antivenoms for treatment of systemic envenoming. Likewise, the use of such objective systems now enables the use of first-aid measures such as tourniquets to be properly assessed.

Wasp venom immunotherapy changes IgG antibody specificity.

The evolution of the IgG response during venom immunotherapy (VIT) has been previously investigated in terms of antibody titres and subclasses. The present work studied the evolution of IgG antibody fine specificity in wasp allergic patients treated with rush VIT. Antibody specificity was evaluated in 51 wasp allergic patients in competitive ELISA using streptavidin biotin technology. Patients were tested before and during specific rush immunotherapy (at 15 days, 6 months, 12 months) and compared with 44 patients treated by venom injections for at least 2 years. The capacity of sera to prevent the antigen binding of pooled IgG from allergic patients changed rapidly with mean percentage inhibitions (+/-SD) falling from 70+/-11-51+/-18% after 15 days of treatment (P<0.001 by one way ANOVA). Similarly, the antigen binding capacity of pooled IgG from VIT patients was differently prevented by sera with mean percentage inhibitions increasing from 37+/-12-65+/-8 after 15 days of treatment (P< 0.0001 by one-way ANOVA). The immunodominance pattern of IgG epitopes recognized on wasp venom antigens by sera from wasp allergic patients changes soon after initiating rush VIT. Further studies will indicate whether, instead of measuring IgG titres, this marked change could be used as the basis of a new test for monitoring the outcome of VIT.

Relationship of Venom Effects to Venom Antigen and Antivenom Serum Concentrations in a Patient With Crotalus atrox Envenomation Treated With a Fab Antivenom

Study objective: To described the association among venom antigenemia, serum antivenom concentrations, and venom effects in a 53-year-old woman who was bitten by a Western diamondback rattlesnake ( Crotalus atrox). Methods: The patient was enrolled in a multicenter trial of an investigational Fab antivenom. Her clinical condition and coagulation parameters were monitored for 2 weeks after the bite. Results: After antivenom administration, the progression of the venom's effects was arrested. The antivenom reversed some local venom effects, caused venom antigens to disappear from the blood, and resolved the patient's profound thrombocytopenia (before antivenom, 12,000/mm 3; 1 hour after antivenom, 227,000/mm 3). Local venom effects recurred twice in the 24 hours after antivenom administration but were easily managed with additional Fab antivenom. Venom antigenemia was detected on days 5 and 8 after the initial treatment and was accompanied in one instance by the new onset of hypofibrinogenemia (119 mg/L) that resolved spontaneously and in both instances by renewed profound thrombocytopenia. Repeat Fab antivenom doses on days 6 and 9 were followed by increases in platelet count (from 16,000 to 40,000/mm 3 and from 11,000 to 20,000/mm 3, respectively) and by the reduction or disappearance of venom antigenemia. The patient sustained no significant bleeding complications, and all laboratory values had returned to normal 2 weeks after the bite. Conclusion: Initial control of local symptoms and coagulopathy was prompt after the administration of Fab antivenom. Repeat doses during the 24 to 36 hours after a bite may be necessary for local control. Recrudescence of coagulopathy was likely due in part to renewed venom antigenemia after clearance of Fab antivenom. The role of Fab antivenom in the treatment of recurrent coagulopathy requires further study. [Seifert SA, Boyer LV, Dart RC, Porter RS, Sjostrom L: Relationship of venom effects to venom antigen and antivenom serum concentrations in a patient with Crotalus atrox envenomation treated with a Fab antivenom. Ann Emerg Med July 1997;30:49-53.]

A novel cDNA from Drosophila encoding a protein with similarity to mammalian cysteine-rich secretory proteins, wasp venom antigen 5, and plant group 1 pathogenesis-related proteins

The CAP protein family is made up of a group of secreted proteins that share sequence similarity. Members of this family are found in animals, plants, and fungi, and their shared sequence similarity suggests that members share a common, but as yet unknown, molecular function. As a first step in defining the function of CAP family proteins, an 878 bp partial cDNA encoding a novel member of the CAP family was cloned by the polymerase chain reaction (PCR) from total RNA of adult Drosophila. The cDNA contained the complete coding sequence for a protein 256 amino acids in length, as well as the complete 3′ untranslated region (UTR) and a portion of the 5′ UTR. The protein, named Antigen 5-related (Agr), was most similar in sequence to antigen 5 (Ag5), a CAP family member found in social wasps and ants. The corresponding Agr RNA is about 1 kb in length and is present at all stages of development, with highest levels observed in adults. Agr RNA is transcribed from a single gene that is located within region 12F of the X chromosome. The identification of Agr in Drosophila expands the number of known CAP family members to well over four dozen. Further studies of Agr and the gene which encodes this protein using the Drosophila model system may help provide important insight into the molecular functioning of this little known, but increasingly significant protein family.

Sensitivity to bee and wasp venoms: association with specific IgE responses to the bee and wasp venom and HLA DRB1 and DPB1

Stings from bees and wasps can cause systemic reactions which can be fatal in some individuals. In these venom-sensitive patients, specific IgE to the venom is produced and is considered to participate in the adverse reactions. This immune response requires antigen presentation by human leucocyte antigens (HLA) class II molecules, which includes DR and DP, which are present on antigen presenting cells. To test for associations between HLA class II DRB1 and DPB1 alleles and life-threatening sensitivity to both bee and wasp venoms. To establish further whether any associations are independent of the atopy phenotype. A total of 33 bee- and 44 wasp-venom-sensitive patients was studied. DRB1 genotypes were determined by single stranded oligonucleotide (SSO) probing of PCR products, and DPB1 genotypes by amplified fragment length polymorphism (AFLP) analysis. Total and specific IgE were measured using the Pharmacia Immunocap, FEIA. Patients with specific IgE to the venom antigens only were termed monosensitive and those with additional specific IgE to HDM and/or GP were termed polysensitive. Allele frequencies were compared to an unrelated control population. The 33 bee-sensitive patients had a greater prevalence of DRB1*07 alleles than the control subjects, 26% vs 14%, with an odds ratio (OR) of 2.1 (95% CI, 1.2-3.7, P = 0.015, corrected for multiple comparisons, Pc = ns). This association was confined to the 15 monosensitive bee patients, who had a 43% DRB1*07 allele frequency when compared with 11% in the 18 polysensitive bee patients, OR 6.1 (95% CI, 1.73-22, P = 0.004, Pc = 0.05), and when compared with a control group of non-venom subjects, 43% vs 16%, OR 3.9 (95% CI, 1.72-9.0, P = 0.002, Pc = 0.02). The 44 wasp-sensitive patients had an increase in the DRB1*11 allele when compared with the control subjects, 13% vs 6%, with an OR 2.2 (95% CI, 1.0-4.6, P = 0.04, Pc = NS), and a decreased prevalence of DRB1*04 alleles, 10% vs 19%, with an OR 0.33 (95% CI, 0.24-0.99, P = 0.04, Pc = NS), but these were not significant when multiple comparisons were taken into account. The DPB1 alleles were not significantly different between the venom sensitive patients and the controls. Patients monosensitive to bee venom had a significantly greater prevalence of DRB1*07 alleles than the non-venom, control population suggesting that IgE responses in these patients may, in part be controlled by immune response HLA class II genes. These results are also suggestive of wasp-sensitive patients having a higher prevalence of DRB1*11 and a lower prevalence of DRB1*04 than the control population.

Clinical indicators of envenoming and serum levels of venom antigens in patients bitten by Bothrops lanceolatus in Martinique

An enzyme-linked immunosorbent assay was developed to measure venom antigen levels in the serum of 40 patients bitten by Bothrops lanceolatus. The grading system used for the severity of envenomation (grades 1 to 4, minor to major) was predominantly based on the presence of local signs. Serum venom levels increased with the grade of severity ( P < 0·001, by Spearman's rank correlation test); they were 6 ± 6 ng/mL (mean ± SD) in clinically non-envenomed patients (grade 1, n = 3), 7·6 ± 11·7 ( n = 17), 44·3 ± 41·8 ( n = 17), and 80·3 ± 34·1 ng/mL ( n = 3) in patients diagnosed as grade 2, 3 and 4 respectively. However, venom antigens could not be detected in the serum of 54% of patients who showed clinical signs of envenomation. Most patients diagnosed as grade 2, 3 or 4 were given 20, 40 and 60 mL of a monospecific F(ab′) 2 antivenom, respectively. Venom concentrations ≥+15 ng/mL were observed in all patients with progressive aggravation of swelling despite the use of early antivenom therapy. No venom was detectable in blood samples taken after completion of serotherapy. All patients recovered. These results confirm the efficacy of both the clinical severity scoring system used and the therapeutic regimen.

An enzyme-linked immunosorbent assay (ELISA) that discriminates between the venoms of Brazilian Bothrops species and Crotalus durissus

Enzyme-linked immunosorbent assays (ELISAs) were developed to detect specific antigens from Bothrops sp. and Crotalus durissus snake venoms in Brazil. Cross-reactive immunoglobulins from hyperimmune horse anti- Bothrops and anti- Crotalus sera were removed by immunoaffinity chromatography. Specific IgGs for Bothrops sp. and C. durissus venom antigens were prepared and used to set up a sandwich-type ELISA. The specificity of the assay was demonstrated by its capacity to identify correctly the circulating antigens in mice experimentally inoculated with both venoms. Measurable absorbance signals were obtained with 5 ng of venom per assay. The ELISA was also used to identify circulating antigens in the sera of humans bitten by Bothrops sp. and C. durissus. These ELISAs could be valuable for clinicians and epidemiologists if they prove to have both the high sensitivity and specificity required for such tests.

Clearing of circulating whole venom, crotoxin and crotamine antigens after specific serotherapy in patients envenomed by Crotalus durissus snakebite

Clearing of circulating whole venom, crotoxin and crotamine antigens after specific serotherapy in patients envenomed by Crotalus durissus snakebite

Development of ELISA texts for the detection of Brazilian snakes, scorpions and spiders venom antigens

Development of ELISA texts for the detection of Brazilian snakes, scorpions and spiders venom antigens

USE OF AN ELISA ASSAY TO EVALUATE VENOM, ANTIVENOM, IgG AND IgM HUMAN ANTIBODY LEVELS IN SERUM AND CEREBROSPINAL FLUID FROM PATIENTS BITTEN BY Crotalus durissus terrificus IN BRAZIL

A sandwich-type ELISA technique for specific and sensitive detection of Crotalus durissus terrificus venom antigens, horse-antivenom, human IgG and IgM antibodies was set up. Sixteen patients, 13 males and 3 females aged between 13 to 63 years (mean 33 ± 15) bitten by Crotalus durissus terrificus snakes were studied. Of the 15 patients, 6 had previously received anti-Crotalus venom and no seric venom was detected. For the other 9 patients studied, the venom levels ranged from 2 to 108 ng/ml according to the severity of each case. Seric antivenom was detected up to 44 days after the bite. IgM human antibody levels against Crotalus venom were higher between 3 and 18 days after specific treatment. IgG human antibody levels against Crotalus venom were detected between 30 and 90 days after envenoming. Venom and antivenom levels in cerebrospinal fluid were not observed 24 h after the bite. This suggests that neither the venom nor the antivenom is capable of crossing the blood-brain barrier. In addition, when either the venom or the antivenom is presented to the immune system cells an immune response is prepared.

Local compression pads as a first-aid measure for victims of bites by Russell's viper ( Daboia russelii siamensis) in Myanmar

A prospective study of the efficacy of applying local pressure by compression pads in retarding spread of venom was carried out on 15 cases of bite by Russell's viper ( Daboia russelii siamensis) in Myanmar. A firm rubber pad was applied with cotton bandaging over the site of bite and the limb was immobilized with a splint. Serial monitoring of venom levels by enzyme immunoassay (EIA) was carried out at 15 min intervals for 1h (2h in one case) while the pad was in place and at 15 and 30 min after its removal. A rise of 10–40ng/mL in serum venom antigen level was observed in most cases after removal of the pad. The central movement of venom antigen was retarded in 13 of the 15 cases. Mild haemostatic changes (factor V and X assays and screening tests) were observed in 10 pad-treated cases measured at the time of onset of incoagulability of blood. The side effects observed while the pads were in place were minimal, consisting of swelling, pain and tenderness, and were well tolerated by most patients (for up to 2h by one patient), except for 2 who had incisions or bruising at the site of the bite.

King cobra ( Ophiophagus hannah) bites in Myanmar: Venom antigen levels and development of venom antibodies

Venom, venom IgG and IgM antibody and total serum IgG levels following king cobra bites in two reptile handlers were measured by enzyme immunoassay. The patient in case 1 received antivenom while the patient in case 2 did not. Case 1 made a complete recovery following the bite and produced a high titre short-lived antibody. Venom antigen was not detected in the sample taken 11 hr after antivenom. Case 2 had experienced two recent minor king cobra bites and had received traditional immunization 4 weeks before the accident reported here. He had developed only local swelling and suffered no neurological symptoms. Venom antigen measured at 1.45 hr after the bite was 132 ng/ml; this rapidly fell to 45 ng/ml over the next 30 min, and was no longer detectable 14 hr after the bite. The pattern of venom IgG and IgM antibody responses in both cases was comparable, except that in case 2 the venom IgG peak was maintained for 13 days, compared with 1 day in case 1; in case 2 it subsequently fell to low levels 8 weeks after the bite. Venom IgM appeared 1 day after the bite, peaked at day 7–9, rapidly tailed off on day 12–16 and was then undetectable from day 20 onwards in both. Total IgG level remained within normal limits in both. It is possible that previous bites and recent immunization contributed to the boosting of the venom IgG response in case 2.