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Alergije so v razvitih državah vedno bolj razširjene. Zdravimo jih lahko na tri načine: z izogibanjem alergenom, s farmakoterapijo in z imunoterapijo. Alergensko specifična imunoterapija je edini kurativni pristop k zdravljenju alergij. V imunoterapiji lahko uporabimo različne pristope zdravljenja. Trenutno se uporabljajo predvsem ekstrakti alergenov ali rekombinantno pridobljeni alergeni. Ker lahko uporaba celotnega alergena vodi do pojava anafilaksije, je vedno več raziskav usmerjenih v razvoj krajših peptidov, ki predstavljajo ali posnemajo dele alergena. Takim peptidom pravimo mimotopi. V diplomski nalogi smo poskušali določiti vezavo specifičnih protiteles razreda E na mimotope glavnega mačjega alergena Fel d 1, izražene na nitastih bakteriofagih M13, ter primerjali, katera metoda imunodetekcije je najprimernejša in najbolj uporabna. Bakteriofage z izraženimi mimotopi smo pomnožili. Pripravili smo vzorce s primerno koncentracijo. Kot kontrolo smo uporabili bakteriofag brez izraženih peptidov na plaščnih proteinih. Z metodo poliakrilamidna gelska elektroforeza v prisotnosti natrijevega dodecilsulfata smo po velikosti ločili plaščne proteine bakteriofaga. Proteine smo nato s prenosom western prenesli na nitrocelulozno membrano. Primerjali smo dva načina imunodetekcije - klasično metodo inkubacije membrane s protitelesi ter metodo z uporabo aparature iBind. Ugotovili smo, da se protitelesa razreda E vežejo na vse izražene peptidne mimotope. V majhni meri so se protitelesa vezala tudi na kontrolni bakteriofag. Predpostavljamo, da zaradi nespecifične vezave protiteles na bakteriofagne plaščne proteine. Ugotovili smo, da predhodno čiščenje seruma in predhodna inkubacija seruma s kontrolnimi bakteriofagi ne izboljšata rezultatov imunodetekcije. Izkazalo se je, da imajo tudi zdravi prostovoljci majhne količine specifičnih imunoglobulinov E, ki se v majhni meri vežejo na peptidne mimotope oziroma direktno na plaščne proteine bakteriofagov. Ugotovili smo, da je imunodetekcijska metoda z aparaturo iBind bolj uporabna kot klasična metoda inkubacije membrane s protitelesi. Metoda iBind se je izkazala za enostavnejšo in hitrejšo. Allergies are very common in developed countries. They can be treated by avoidance of allergens, with pharmacotherapy or with immunotherapy. Allergen-specific immunotherapy is the only curative therapy. Different approaches have been used. Through the years whole allergens or recombinant allergens were used. As the use of whole allergens can lead to anaphylaxis research is focused on developing short peptides that mimic parts of allergens. Those peptides are called mimotopes. The goal of our work was to determine binding of immunoglobulin E from allergic patients sera to mimotopes of the major cat allergen Fel d 1 and to optimize the method of immunodetection. Peptides that mimic major cat allergen were expressed on immunological carrier – filamentous bacteriophage M13 as fusions with p3 coat proteins. We multiplied bacteriophages with expressed mimotopes and prepared samples with appropriate concentration. Bacteriophages without expressed mimotopes were used as control. We separated bacteriophage coat proteins by size using poliacrylamide gel electrophoresis with sodium dodecyl sulfate and blotted them to nitrocellulose membrane. For immunodetection we used and compared two different methods- classic and iBind. Immunoglobulin E from patients’ sera bound all peptide mimotopes including control bacteriophage, but with less affinity. We assume that it is because of the nonspecific binding of primary and secondary antibodies directly on phage coat proteins. Different attempts to increase selectivity of sera immunoglobulin binding did not improve the immunodetection. Moreover, even healthy volunteers’ sera showed some degree of binding to phage coat proteins. Our results also showed that iBind is more useful for immunodetection than classic method. It is faster and easier to use. |
