RESUMEN
Stinging ants represent a wide range of over 200 different species across the world, of which Solenopsis, Myrmecia, Pogonomyrmex, and Brachyponera genera account for a substantial economic and healthcare burden. S. invicta (red imported fire ant [IFA]) and M. pilosula (jack jumper ant [JJA]) are 2 species of high clinical importance, known to cause anaphylaxis in humans, with numerous reported fatalities. Diagnostic testing should be performed in patients with a history of a systemic reaction with skin testing and/or in vitro specific immunoglobulin E (IgE) testing. In vitro testing is commercially available for IFA through whole-body extract specific IgE and JJA venom-specific IgE, but not widely available for other stinging ant species. Commercial venom component testing for IFA and JJA is currently not available. Patients with a clinical history and positive specific IgE testing should undergo treatment with specific immunotherapy, which is currently available for IFA and JJA. Buildup may be performed using conventional, semi-rush, rush, or ultra-rush schedules with similar risk profiles for IFA. Optimal duration for whole=body extract immunotherapy for IFA and specific JJA venom immunotherapy is not well studied, but generally recommended for at least 3 to 5 years. Sting challenges are used in research settings, primarily to assess treatment efficacy of immunotherapy.
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Jack Jumper ant venom allergy is a uniquely Australian medical issue. The stinging ant is a leading cause of insect venom allergy in south-eastern Australia. An effective venom immunotherapy-based treatment was successfully developed by the Tasmanian Jack Jumper Allergy Research group. This paper provides a synopsis of our 25 years' research journey in developing this evidence-based treatment modality.
Asunto(s)
Hormigas , Hipersensibilidad , Humanos , Animales , Australia , Desensibilización Inmunológica , Hipersensibilidad/terapia , DolorRESUMEN
Venom immunotherapy is the standard of care for people with severe reactions and has been proven to reduce risk of future anaphylactic events. There is a moral imperative to ensure production, supply and worldwide availability of locally relevant, registered, standardized commercial venom extracts for diagnosis and treatment. Insects causing severe immediate allergic reactions vary by region worldwide. The most common culprits include honeybees (Apis mellifera), social wasps including yellow jackets (Vespula and Dolichovespula), paper wasps (Polistes) and hornets (Vespa), stinging ants (Solenopsis, Myrmecia, Pachycondyla, and Pogonomyrmex), and bumblebees (Bombus). Insects with importance in specific areas of the world include the Australian tick (Ixodes holocyclus), the kissing bug (Triatoma spp), horseflies (Tabanus spp), and mosquitoes (Aedes, Culex, Anopheles). Reliable access to high quality venom immunotherapy to locally relevant allergens is not available throughout the world. Many current commercially available therapeutic vaccines have deficiencies, are not suitable for, or are unavailable in vast areas of the globe. New products are required to replace products that are unstandardized or inadequate, particularly whole-body extract products. New products are required for insects in which no current treatment options exist. Venom immunotherapy should be promoted throughout the world and the provision thereof be supported by health authorities, regulatory authorities and all sectors of the health care service.
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BACKGROUND: Allergic reactions to Hymenoptera insect stings remain a major global clinical problem. Although effective, parenteral desensitization regimens require use of costly venom extracts and require frequent visits over extended periods of time. OBJECTIVE: Adjuvants are commonly used to enhance the efficacy of infectious disease vaccines, and this study asked whether Advax (Vaxine Pty Ltd, Adelaide, Australia), a novel noninflammatory polysaccharide adjuvant, might provide similar benefits for allergy desensitization. METHODS: A randomized, controlled phase 1/2 trial was undertaken in 27 adults with a history of rapid-onset systemic allergic reactions to honeybee stings and positive specific IgE levels to evaluate the safety and efficacy of honeybee venom immunotherapy (HBVIT) combined with Advax adjuvant. Venom immunotherapy (VIT) was administered monthly for 30 months after achievement of maintenance doses. RESULTS: Advax-adjuvanted HBVIT was well tolerated. Around week 14 of VIT, specific IgG4 responses peaked in both groups but increased earlier, peaked higher, and were better maintained through the end of the study in the Advax-adjuvanted arm. Several different patterns of serologic response to VIT were seen; some subjects had a dominant IgG4 response, some had a combined IgG4 and IgG1 response, and some had an exclusively IgG1 response. In some subjects specific IgE levels increased during the induction phase and then decreased, whereas in others specific IgE levels progressively decreased from the start of VIT. CONCLUSION: Advax adjuvant favorably enhanced the immunogenicity of HBVIT, with an early and prolonged switch to specific IgG4 production. The ability of Advax adjuvant to enhance VIT efficacy warrants further study.
Asunto(s)
Adyuvantes Inmunológicos/administración & dosificación , Venenos de Abeja/administración & dosificación , Hipersensibilidad , Inmunoglobulina E/inmunología , Inmunoterapia , Mordeduras y Picaduras de Insectos , Inulina/análogos & derivados , Adulto , Anciano , Femenino , Humanos , Hipersensibilidad/inmunología , Hipersensibilidad/patología , Hipersensibilidad/terapia , Mordeduras y Picaduras de Insectos/inmunología , Mordeduras y Picaduras de Insectos/patología , Mordeduras y Picaduras de Insectos/terapia , Inulina/administración & dosificación , Masculino , Persona de Mediana EdadRESUMEN
A major challenge in broader clinical application of Jack Jumper ant venom immunotherapy (JJA VIT) is the scarcity of ant venom which needs to be manually harvested from wild ants. Adjuvants are commonly used for antigen sparing in other vaccines, and thereby could potentially have major benefits to extend JJA supplies if they were to similarly enhance JJA VIT immunogenicity. The purpose of this study was to evaluate the physicochemical and microbiological stability and murine immunogenicity of low-dose JJA VIT formulated with a novel polysaccharide adjuvant referred to as delta inulin or Advax™. Jack Jumper ant venom (JJAV) protein stability was assessed by UPLC-UV, SDS-PAGE, SDS-PAGE immunoblot, and ELISA inhibition. Diffraction light scattering was used to assess particle size distribution of Advax; pH and benzyl alcohol quantification by UPLC-UV were used to assess the physicochemical stability of JJAV diluent, and endotoxin content and preservative efficacy test was used to investigate the microbiological properties of the adjuvanted VIT formulation. To assess the effect of adjuvant on JJA venom immunogenicity, mice were immunised four times with JJAV alone or formulated with Advax adjuvant. JJA VIT formulated with Advax was found to be physicochemically and microbiologically stable for at least 2 days when stored at 4 and 25 °C with a trend for an increase in allergenic potency observed beyond 2 days of storage. Low-dose JJAV formulated with Advax adjuvant induced significantly higher JJAV-specific IgG than a 5-fold higher dose of JJAV alone, consistent with a powerful allergen-sparing effect. The pharmaceutical data provides important guidance on the formulation, storage and use of JJA VIT formulated with Advax adjuvant, with the murine immunogenicity studies providing a strong rationale for a planned clinical trial to test the ability of Advax adjuvant to achieve 4-fold JJAV dose sparing in JJA-allergic human patients.
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Adyuvantes Inmunológicos/administración & dosificación , Alérgenos/administración & dosificación , Venenos de Hormiga/administración & dosificación , Desensibilización Inmunológica/métodos , Hipersensibilidad/terapia , Inulina/análogos & derivados , Alérgenos/inmunología , Animales , Venenos de Hormiga/inmunología , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Estabilidad de Medicamentos , Femenino , Humanos , Hipersensibilidad/inmunología , Inulina/administración & dosificación , Ratones , Modelos AnimalesRESUMEN
BACKGROUND: Patients who suffer from acute IgE-mediated allergy to a cephalosporin antibiotic are frequently assumed to be at high risk of allergy to other cephalosporins and penicillins. AIM: To define cross-reactivity patterns in patients with confirmed allergy to a cephalosporin. METHODS: Subjects presenting with a history of immediate allergy to a cephalosporin-family antibiotic between March 2009 and July 2017 were investigated with specific IgE testing to penicillin, amoxycillin and cefaclor, followed by skin prick testing, intradermal testing and drug provocation testing with a panel of penicillins and cephalosporins. RESULTS: Out of 564 subjects with a reported beta-lactam allergy, 90 identified a cephalosporin as their index drug. Fifty-five (61.1%) of the 90 subjects tested had a history consistent with an IgE-mediated reaction, of whom 24 (43.6%) were proven to be allergic to their index cephalosporin. Twenty (83.3%) of the 24 were allergic only to their index cephalosporin. Of the four remaining subjects, two were co-sensitised to another beta-lactam with a similar side chain, while the other two had no specific cross-reactivity pattern. Major and minor penicillin determinants were negative for all cephalosporin-allergic individuals. CONCLUSION: In our cohort, cephalosporin allergy does not appear to be a class effect, with most cases found allergic only to their index cephalosporin. Co-sensitisation to other cephalosporins or penicillins was uncommon, and when it occurred, was usually consistent with side chain cross-reactivity.
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Antibacterianos/efectos adversos , Cefalosporinas/efectos adversos , Hipersensibilidad a las Drogas/diagnóstico , Hipersensibilidad Inmediata/diagnóstico , Penicilinas/efectos adversos , Adulto , Anciano , Reacciones Cruzadas/inmunología , Eritema/inducido químicamente , Femenino , Humanos , Inmunoglobulina E/inmunología , Masculino , Persona de Mediana Edad , Estudios Retrospectivos , Pruebas CutáneasRESUMEN
The prevalence of allergies, including rhinitis, eczema, and anaphylaxis, is rising dramatically worldwide. This increase is especially problematic in children who bear the greatest burden of this rising trend. Increasing evidence identifies neutrophils as primary perpetrators of the more severe and difficult to manage forms of inflammation. A newly recognized mechanism by which neutrophils are recruited during the early phase of histamine-induced inflammation involves the sphingosine kinase (SK)/sphingosine-1-phosphate axis. This study examines whether topical application of fingolimod, an established SK/sphingosine-1-phosphate antagonist already in clinical use to treat multiple sclerosis, may be repurposed to treat cutaneous inflammation. Using two mouse models of ear skin inflammation (histamine- and IgE-mediated passive cutaneous anaphylaxis) we topically applied fingolimod prophylactically, as well as after establishment of the inflammatory response, and examined ear swelling, SK activity, vascular permeability, leukocyte recruitment, and production of proinflammatory mediators. The present study reveals that when applied topically, fingolimod attenuates both immediate and late-phase responses to histamine with reduced extravasation of fluid, SK-1 activity, proinflammatory cytokine and chemokine production, and neutrophil influx and prevents ear swelling. Intravital microscopy demonstrates that histamine-induced neutrophil rolling and adhesion to the postcapillary venules in the mouse ears is significantly attenuated even after 24 h. More importantly, these effects are achievable even once inflammation is established. Translation into humans was also accomplished with epicutaneous application of fingolimod resolving histamine-induced and allergen-induced inflammatory reactions in forearm skin. Overall, this study demonstrates, to our knowledge for the first time, that fingolimod may be repurposed to treat cutaneous inflammation.
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Dermatitis/tratamiento farmacológico , Clorhidrato de Fingolimod/uso terapéutico , Esclerosis Múltiple/tratamiento farmacológico , Neutrófilos/efectos de los fármacos , Fosfotransferasas (Aceptor de Grupo Alcohol)/antagonistas & inhibidores , Piel/efectos de los fármacos , Administración Tópica , Animales , Permeabilidad Capilar/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Citocinas/metabolismo , Modelos Animales de Enfermedad , Clorhidrato de Fingolimod/farmacología , Histamina/metabolismo , Humanos , Inmunoglobulina E/sangre , Mediadores de Inflamación/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neutrófilos/inmunología , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Piel/inmunologíaRESUMEN
Hypersensitivity reactions including anaphylaxis have been reported for nearly all classes of therapeutic reagents and these reactions can occur within minutes to hours of exposure. These reactions are unpredictable, not directly related to dose or the pharmacological action of the drug and have a relatively high mortality risk. This review will focus on the clinical presentation, immune mechanisms, diagnosis and prevention of the most serious form of immediate onset drug hypersensitivity reaction, anaphylaxis. The incidence of drug-induced anaphylaxis deaths appears to be increasing and our understanding of the multiple and complex reasons for the unpredictable nature of anaphylaxis to drugs is also expanding. This review highlights the importance of enhancing our understanding of the biology of the patient (i.e. immune response, genetics) as well as the pharmacology and chemistry of the drug when investigating, diagnosing and treating drug hypersensitivity. Misdiagnosis of drug hypersensitivity leads to substantial patient risk and cost. Although oral provocation is often considered the gold standard of diagnosis, it can pose a potential risk to the patient. There is an urgent need to improve and standardize diagnostic testing and desensitization protocols as other diagnostic tests currently available for assessment of immediate drug allergy are not highly predictive.
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Anafilaxia , Hipersensibilidad a las Drogas , Anafilaxia/inducido químicamente , Anafilaxia/diagnóstico , Anafilaxia/genética , Anafilaxia/inmunología , Anafilaxia/prevención & control , Técnicas y Procedimientos Diagnósticos , Hipersensibilidad a las Drogas/diagnóstico , Hipersensibilidad a las Drogas/genética , Hipersensibilidad a las Drogas/inmunología , Hipersensibilidad a las Drogas/prevención & control , HumanosRESUMEN
A case of a 58-year-old with fatal anaphylaxis due to multiple bee stings is reported. Supportive evidence for anaphylaxis included post-mortem serum tests, which demonstrated a markedly elevated tryptase level and increased sensitivity to bees on radioallergosorbent test (RAST). At autopsy a previously undiagnosed esophageal adenocarcinoma involving the gastroesophageal (GE) junction was also identified. Histology of the tumor demonstrated significant numbers of mast cells, many of which were degranulating. Increased numbers of mast cells, as in mastocytosis, are known to predispose to an allergic sensitivity to Hymenoptera. The finding of a significant peritumoral mast cell population with degranulating forms in this case, therefore, raises the possibility that death due to anaphylaxis was contributed to by mast cell proliferation in an occult esophageal carcinoma.
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Anafilaxia/etiología , Abejas , Mordeduras y Picaduras/complicaciones , Mastocitos/patología , Adenocarcinoma/patología , Animales , Degranulación de la Célula , Neoplasias Esofágicas/patología , Patologia Forense , Humanos , Inmunoglobulina E/sangre , Masculino , Mastocitos/enzimología , Mastocitosis/etiología , Microscopía , Persona de Mediana Edad , Triptasas/metabolismoRESUMEN
BACKGROUND: Venom immunotherapy can be initiated by different schedules, but randomized comparisons have not been performed. OBJECTIVE: We aimed to compare the safety of 2 initiation schedules. METHODS: Patients of any age with prior immediate generalized reactions to jack jumper ant (Myrmecia pilosula) stings were randomized to venom immunotherapy initiation by a semirush schedule over 10 visits (9 weeks) or an ultrarush schedule over 3 visits (2 weeks). In a concurrent treatment efficacy study, the target maintenance dose was randomized to either 50 µg or 100 µg. The primary outcome was the occurrence of 1 or more objective systemic reactions during venom immunotherapy initiation. Analyses were by intention to treat. We also assessed outcomes in patients who declined randomization. RESULTS: Of 213 eligible patients, 93 were randomized to semirush (44 patients) or ultrarush (49 patients) initiation. Objective systemic reactions were more likely during ultrarush initiation (65% vs 29%; P < .001), as were severe reactions (12% vs 0%; P= .029). Times to maximal increases in venom-specific IgG(4) were no different between treatments, whereas the maximal increase in venom-specific IgE occurred earlier with ultrarush treatment. Similar differences between methods were observed in patients who declined randomization. One hundred seventy-eight patients were randomized to maintenance doses of either 50 µg (90 patients) or 100 µg (88 patients). The target maintenance dose had no effect on the primary outcome, but multiple-failure-per-subject analysis found that the 50 µg dose reduced the likelihood of reactions. CONCLUSION: Ultrarush initiation increases the risk of systemic reactions. A lower maintenance dose reduces the risk of repeated reactions, but the effect on treatment efficacy is unknown.
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Hormigas/inmunología , Venenos de Artrópodos/administración & dosificación , Desensibilización Inmunológica/efectos adversos , Hipersensibilidad Inmediata/terapia , Mordeduras y Picaduras de Insectos/inmunología , Adulto , Animales , Venenos de Artrópodos/efectos adversos , Desensibilización Inmunológica/métodos , Esquema de Medicación , Femenino , Humanos , Hipersensibilidad Inmediata/etiología , Hipersensibilidad Inmediata/inmunología , Masculino , Persona de Mediana Edad , Resultado del TratamientoRESUMEN
OBJECTIVE: To determine the Australian native ant species associated with ant sting anaphylaxis, geographical distribution of allergic reactions, and feasibility of diagnostic venom-specific IgE (sIgE) testing. DESIGN, SETTING AND PARTICIPANTS: Descriptive clinical, entomological and immunological study of Australians with a history of ant sting anaphylaxis, recruited in 2006-2007 through media exposure and referrals from allergy practices and emergency physicians nationwide. We interviewed participants, collected entomological specimens, prepared reference venom extracts, and conducted serum sIgE testing against ant venom panels relevant to the species found in each geographical region. MAIN OUTCOME MEASURES: Reaction causation attributed using a combination of ant identification and sIgE testing. RESULTS: 376 participants reported 735 systemic reactions. Of 299 participants for whom a cause was determined, 265 (89%; 95% CI, 84%-92%) had reacted clinically to Myrmecia species and 34 (11%; 95% CI, 8%-16%) to green-head ant (Rhytidoponera metallica). Of those with reactions to Myrmecia species, 176 reacted to jack jumper ant (Myrmecia pilosula species complex), 18 to other jumper ants (15 to Myrmecia nigrocincta, three to Myrmecia ludlowi) and 56 to a variety of bulldog ants, with some participants reacting to more than one type of bulldog ant. Variable serological cross-reactivity between bulldog ant species was observed, and sera from patients with bulldog ant allergy were all positive to one or more venoms extracted from Myrmecia forficata, Myrmecia pyriformis and Myrmecia nigriceps. CONCLUSION: Four main groups of Australian ants cause anaphylaxis. Serum sIgE testing enhances the accuracy of diagnosis and is a prerequisite for administering species-specific venom immunotherapy.
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Anafilaxia/etiología , Hormigas , Mordeduras y Picaduras de Insectos/etiología , Adulto , Animales , Venenos de Hormiga/antagonistas & inhibidores , Antivenenos/uso terapéutico , Australia , Femenino , Humanos , Mordeduras y Picaduras de Insectos/diagnóstico , Mordeduras y Picaduras de Insectos/tratamiento farmacológico , Mordeduras y Picaduras de Insectos/inmunología , Masculino , Persona de Mediana EdadAsunto(s)
Anafilaxia/tratamiento farmacológico , Epinefrina/administración & dosificación , Gangrena Gaseosa/etiología , Clostridium perfringens , Resultado Fatal , Femenino , Gangrena Gaseosa/terapia , Humanos , Inyecciones , Persona de Mediana Edad , Prednisona/administración & dosificación , Prometazina/administración & dosificación , AutoadministraciónRESUMEN
Allergy to Myrmecia pilosula (Jack Jumper Ant) venom is common in Australia, affecting â¼2.7% of some communities. Venom immunotherapy is a highly effective treatment, but for the venom to be widely distributed for clinical use, the stability and shelf-life of formulated Jack Jumper Ant venom must be demonstrated. HPLC-UV, ELISA Inhibition, SDS-PAGE and SDS-PAGE Immunoblot were used to assess venom stability under conditions of varying temperature, pH and in the presence of various stabilising agents. Optimal stability occurred between pH 8 and 10, however the presence of benzyl alcohol within this pH range resulted in a cloudy appearance within 3 days, so a pH of 6 was used. Increasing polysorbate 80 concentrations accelerated the degradation of allergenic peptides in 100 µg/mL venom, but improved stability at concentrations of 1 µg/mL or less. Sucrose reduced degradation of allergens Myr p 1 and Myr p 3, whilst glycerol was destabilizing. In the presence of 22% sucrose, 1.1mg/mL Jack Jumper Ant venom was stable at -18 °C and 4 °C for 12 months; following dilution to 100 µg/mL with 0.9% sodium chloride, 10mM phosphate (pH 6), 0.05% polysorbate 80 and 0.9% benzyl alcohol (giving 2% sucrose), venom was stable for 7 days when stored at 4 °C. Concentrated Jack Jumper Ant venom can be stored in 22% sucrose for 12 months, and after dilution to 100 µg/mL for clinical use, it should be discarded after 7 days.
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Venenos de Hormiga/química , Desensibilización Inmunológica , Inmunoterapia , Animales , Venenos de Hormiga/inmunología , Venenos de Hormiga/aislamiento & purificación , Cromatografía Líquida de Alta Presión , Estabilidad de Medicamentos , Almacenaje de Medicamentos , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Congelación , Concentración de Iones de Hidrógeno , Hipersensibilidad/inmunología , Immunoblotting , Péptidos/inmunología , Péptidos/aislamiento & purificación , Polisorbatos/farmacología , Temperatura , Factores de TiempoAsunto(s)
Anticonvulsivantes/efectos adversos , Encefalopatía Hepática/inducido químicamente , Convulsiones/tratamiento farmacológico , Ácido Valproico/efectos adversos , Anciano , Anticonvulsivantes/uso terapéutico , Biopsia , Diagnóstico Diferencial , Electroencefalografía , Femenino , Estudios de Seguimiento , Encefalopatía Hepática/diagnóstico , Humanos , Hígado/diagnóstico por imagen , Hígado/patología , Tomografía Computarizada por Rayos X , Ultrasonografía , Ácido Valproico/uso terapéuticoRESUMEN
Ant sting allergy is relatively common within south-eastern Australia and is predominantly due to Myrmecia pilosula (Jack Jumper Ant, JJA). Venom immunotherapy has been shown to be effective in preventing anaphylaxis to the sting of the JJA, but analytical techniques to standardise the venom have not been validated. The purpose of this study was to develop assays to analyse JJA venom and apply these to the standardisation of venom prior to new batches being used for the diagnosis and treatment of JJA sting allergy. Venom was analysed by protein content, HPLC-UV, enzyme-linked immunosorbent assay (ELISA) inhibition, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and SDS-PAGE immunoblot. The protein content in JJA venom was adjusted so that all batches were equivalent. A HPLC-UV assay was used to quantify the relative amount of the major allergen Myr p 2 and two minor allergens Myr p 1 and Myr p 3 and allergenic potency was determined by ELISA inhibition. SDS-PAGE and SDS-PAGE immunoblot were used as qualitative tools to determine the protein profile and presence or absence of additional high molecular weight allergens not quantifiable by HPLC-UV. A standardisation procedure has been developed that complies with the requirements described in the European Pharmacopoeia. Techniques used to determine the content of some of the other minor allergens could be developed, which would further improve the standardisation methodology.
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Venenos de Hormiga/inmunología , Hormigas/química , Hipersensibilidad/inmunología , Vacunas/inmunología , Alérgenos/química , Alérgenos/inmunología , Alérgenos/aislamiento & purificación , Animales , Venenos de Hormiga/química , Venenos de Hormiga/aislamiento & purificación , Cromatografía Líquida de Alta Presión/métodos , Desensibilización Inmunológica , Electroforesis en Gel de Poliacrilamida/métodos , Ensayo de Inmunoadsorción Enzimática/métodos , Hipersensibilidad/prevención & control , Immunoblotting , Proteínas de Insectos/química , Proteínas de Insectos/inmunología , Proteínas de Insectos/aislamiento & purificación , Reproducibilidad de los Resultados , Espectrofotometría Ultravioleta/métodos , Vacunación/métodos , Vacunas/química , Vacunas/normasRESUMEN
Ant sting allergy in Australia is predominantly due to the Myrmecia pilosula species complex. Gel separation of M. pilosula venom is necessary so that the allergenic importance of each component can be defined by western blotting. However, previous PAGE methods produced suboptimal resolution and the components of each band were not precisely defined. Venom was resolved in both non-reduced and reduced form by one-dimensional acid urea PAGE, SDS-PAGE and two-dimensional acid urea-SDS PAGE. Resolved peptides were extracted and analysed by HPLC-MS. Acid urea PAGE and acid urea-SDS PAGE proved more effective than SDS-PAGE for resolution of peptides smaller than 10 kDa. All of the major peptides previously observed in M. pilosula venom were observed in gel resolved venom. Venom was found to primarily consist of peptides with molecular weight <10 kDa, most of which contain disulfide bridges. SDS-PAGE of non-reduced venom clearly defined six higher molecular weight proteins between 26 and 90 kDa. An 8546 Da dimer named pilosulin 5 was observed, but pilosulin 4, a peptide recently proposed to be present in venom was not. A variant of pilosulin 4 here named pilosulin 4.1a, existing as an 8198 Da dimer, was observed and has been characterised.