Browsing by Subject "antigeenit"

Anoplocephala perfoliata, an equine tapeworm, is an intestinal parasite of horses. Tapeworms colonize and live attached to the mucosal surfaces of ileum, caecum, and colon. The lifecycle of an equine tapeworm is indirect which means it requires an intermediate host in addition to its definitive host (a horse). The intermediate host is a mite (Oribatidae) which lives in pastures. The infection occurs when the horse digests the mites containing the infective larval forms while grazing. Infection is usually asymptomatic but can cause lesions in the intestines, and colic. The current coprological techniques often fail to recognise the infection, as they are based on observing the helminth eggs in faeces. Unlike many other intestinal parasites equine tapeworms do not release their eggs into the faecal matter. Instead, they are carried into the environment within the segments of the worm. This causes issues with diagnostics. In general, anthelmintics are recommended to be used only on horses that are diagnosed as having the need for a medication in order to prevent anthelmintic resistance from spreading. Problems in diagnosing the infections lead to decisions about medication having to be made without a proper diagnosis of a tapeworm infection. A reliable coprological test would identify the horses in need of medical care. Because of difficulties in detecting tapeworm eggs, antigens secreted by the worm could be used as a basis for test development. The aim of this study was to characterise secreted and somatic antigens and to localise them in the structures of the tapeworm, in order to obtain more information concerning the mechanisms of infection and to recognise potential antigen candidates for diagnostic purposes. Antigenic proteins were characterised using immunoblotting. Proteins were first separated based on their molecular weights and then treated with horse serum antibodies to indicate antigenic proteins. Immunohistochemical staining was used to localise the antigenic proteins in the structures of the tapeworm. Serum and worms collected from previously euthanised or slaughtered horses were used as material for this study. None of the horses were euthanised or slaughtered because of this study. Several of the secreted or somatic proteins were characterised as antigenic. The molecular weights of the observed characterised antigens ranged from 10 to 150 kDa (kilodaltons). Although these antigens were not specifically localised in the structures of the worm, the eggs and surface structures of the worm seemed to be antigenic. Exact locations could not be confirmed due to unspecific binding. Antigenic proteins characterised in this study by using immunoblotting will be identified using mass spectrometry in further studies. The end goal is to find a suitable antigen that can be used as a base for developing a diagnostic ELISA-test.

Kokeessa yritettiin selvittää sitä, toimivatko kaupallisesti saatavilla olevat immunohistokemialliset vasta-aineet myös poroilla. Porojen keuhkokudosnäytteet oli formaliinifiksoitu ja valettu paraffiiniin. Kokeessa käytetyt näytteet olivat porojen keuhkokudosta. Värjäysmenetelmäksi valittiin avidiini-biotiini-kompleksi (ABC) -menetelmä sen sensitiivisyyden vuoksi. Menetelmä perustuu siihen, että avidiini kykenee sitoutumaan biotiinin neljään molekyyliin non-immunologisesti. Menetelmässä käytetään kolmea eri reagenssia. Ensimmäinen reagenssi on primaarivasta-aine, joka on spesifinen kulloinkin paikallistettavalle antigeenille. Toisena reagenssina on sekundaarivasta-aine. Sekundaarivasta-aine on konjugoitu biotiiniin ja kykenee sitoutumaan primaarivasta-aineeseen. Kolmas reagenssi on peroksidaasikonjugoidun biotiinin ja avidiinin kompleksi. Avidiinimolekyylin vapaat kohdat sitoutuvat sekundaarivasta-aineen biotiiniin. Peroksidaasientsyymi saadaan näkyviin sopivalla väriaineella ja samalla selviää myös alkuperäisen antigeenin sijainti kudoksessa. Tulosten perusteella useimmat kaupallisessa käytössä olevista immunokemiallisista vasta-aineista sopivat myös poron keuhkokudoksen sisältämien antigeenien paikallistamiseen. Positiivinen tulos saatiin aikaan von Willebrandin faktorin (endoteeli), CD 3:n (T-lymfosyytit), korkean molekyylipainon sytokeratiinin (värekarvaepiteeli), sytokeratiini 8:n (värekarvaepiteeli, rusto), sytokeratiini 1:n (basaalisolut), vimentiinin (endoteeli, basaalisolut, rusto, lymfosyytit, sileälihas), -sileälihasaktiinin (sileälihas), sytokeratiini 7:n (Clara-solut, värekarvaepiteeli) ja desmiinin (sileälihas) vasta-aineilla. S-100-vasta-aineella saatiin vain yksi positiivinen reaktio (rusto). Sytokeratiini 18:n, sytokeratiini 19:n ja makrofagivasta-aineilla ei saatu reaktioita.