RESUMEN
Insect stings can cause life-threatening IgE-mediated anaphylactic reactions in venom-allergic patients. Although several compounds have already been described as venom allergens, prominent allergen candidates especially in the higher m.w. range have still remained elusive. Tandem mass spectrometry-based sequencing assigned a candidate gene to the most prominent putative high m.w. allergen Api m 5 (allergen C) in honeybee (Apis mellifera) venom and also allowed identification of its homologue Ves v 3 in yellow jacket (Vespula vulgaris) venom. Both proteins exhibit a pronounced sequence identity to human dipeptidyl peptidase IV or CD26. Reactivity of a human IgE mAb verified the presence of these proteins in the venoms. Both proteins were produced in insect cells and characterized for their enzymatic activity as well as their allergenic potential using sera and basophils from insect venom-allergic patients. Both Api m 5 and Ves v 3 were recognized by specific IgE of the majority of patients even in the absence of cross-reactive carbohydrate determinants. Serologic IgE reactivity closely matched activation of human basophils by Api m 5 or Ves v 3, thus underlining their relevance in functional assays. With Api m 5 and Ves v 3, a new pair of homologous allergens becomes available for future clinical applications in diagnosis and therapy that may also contribute to the understanding of molecular mechanisms of insect venoms. Moreover, the patient IgE reactivity together with the cellular activation demonstrates for the first time the relevance of high m.w. allergens in the context of hymenoptera venom allergy.
Asunto(s)
Alérgenos/química , Alérgenos/inmunología , Venenos de Abeja/química , Venenos de Abeja/inmunología , Dipeptidil Peptidasa 4/química , Dipeptidil Peptidasa 4/inmunología , Venenos de Avispas/química , Venenos de Avispas/inmunología , Alérgenos/genética , Secuencia de Aminoácidos , Animales , Venenos de Abeja/genética , Abejas/enzimología , Abejas/genética , Abejas/inmunología , Dipeptidil Peptidasa 4/genética , Humanos , Inmunoglobulina E/biosíntesis , Mordeduras y Picaduras de Insectos/inmunología , Mordeduras y Picaduras de Insectos/terapia , Proteínas de Insectos/química , Proteínas de Insectos/genética , Proteínas de Insectos/inmunología , Datos de Secuencia Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Spodoptera/genética , Spodoptera/inmunología , Venenos de Avispas/genética , Avispas/enzimología , Avispas/genética , Avispas/inmunologíaRESUMEN
Nowadays, recombinant antibody and phage display technology enable the efficient generation of immunotools and a subsequent manipulation for optimized affinity, specificity or overall performance. Such advantages are of particular interest for haptenic target structures, such as TNT (2,4,6-trinitrotoluene). The toxicity of TNT and its breakdown products makes a reliable and fast detection of low levels in aqueous samples highly important. In the present study, we aimed for the generation of scFvs (single-chain antibody fragments) specific for the TNT-surrogate TNP (2,4,6-trinitrophenyl) and their subsequent production as monoclonal avian IgY immunoglobulins providing improved assay performance. Therefore we subjected a human synthetic scFv library to selection following different strategies. TNP-specific human antibody fragments could be identified, characterized for their primary structure and evaluated for production as soluble scFv in Escherichia coli. Additionally, a murine TNP-specific antibody fragment was obtained from the hybridoma 11B3; however, the prokaryotic expression level was found to be limited. To generate and evaluate immunoglobulin formats with superior characteristics, all recombinant antibody fragments then were converted into two different chimaeric bivalent IgY antibody formats. After expression in mammalian cells, the IgY antibodies were assessed for their reactivity towards TNT. The IgY antibodies generated on the basis of the combinatorial library proved to be useful for detection of TNT, thereby emphasizing the high potential of this approach for the development of detection devices for immunoassay-based techniques.
