ࡱ> G wbjbjَ  :]""""*****>,**~~~~~)))))))$]+Q-)1)""~~ ~"R~~)@@""""){(t")~j\ֲ**)IMUNOKEMIJSKE TEHNIKE Imunokemijske tehnike koje se primjenjuju za otkrivanje, razlikovanje i mjerenje koncentracije razli itih antigena ( Ag ) i protutijela (At ) osnivaju se na reakciji izmeu antigena i protutijela. Zbog izuzetne specifi nosti ove reakcije, imunokemijske tehnike se odlikuju: osjetljivoau, reproducibilnoau i jednostavnoau. U veini imunokemijskih tehnika, koje se primjenjuju u klini kim laboratorijima, protutijela su radni reagens za kvalitativnu ili kvantitativnu analizu antigena u ispitivanom uzorku. S obzirom na na in otkrivanja reakcije izmeu antigena i protutijela, imunokemijske tehnike mogue je podijeliti u dvije skupine: Neobilje~ene ili izravne tehnike, u kojima se mjerenja izvode u zoni ekvivalencije reaktanata kada dolazi do stvaranja netopivih imunoprecipitata. Njih mo~emo dalje podijeliti na: Kvalitativne: imunoelektroforeza protusmjerna imunoelektroforeza imunofiksacija imunoselekcija Kvantitativne: radijalna imunodifuzija imunoturbidimetrija imunonefelometrija Obilje~ene tehnike, kod kojih se mjeri primarna reakcija izmeu Ag i At uz koriatenje obilje~enih reaktanata. Pripadaju najva~nijim analiti kim tehnikama u klini koj kemiji. Pronalazak ovih tehnika zna io je golem napredak u daljnjem prou avanju fiziologije i patologije ovjeka. Pridonio je zna ajnom razvoju raznih grana medicine, a posebno endokrinologije. Ovim tehnikama omogueno je mjerenje tvari koje se u organizmu nalaze u vrlo malim koli inama (do 10-14 mol/L). One se takoer mogu podijeliti na: Kvalitativne: imunoblot tehnike gdje se proteini razdvoje elektroforezom u agaroznom gelu. Razdvojeni se proteini prenesu na vrsti nosa  ( nitrocelulozni ili najlonski filtar ), te izravnim nanoaenjem odgovarajueg antiseruma na nosa  izvodi reakcija imunoprecipitacije. Kvantitativne: kod kojih se antigeni ili protutijela mogu obilje~iti radioizotopom, radioimunoloake tehnike (RIA) enzimom, inhibitorom, kofaktorom ili supstratom enzima, enzimoimunokemijske tehnike, (ELISA) luminiscentnim spojem, luminoimunokemijske tehnike (LIA) fluorescentnim spojem, fluoroimunokemijske tehnike (FIA) te mnogim drugim obilje~iva ima. Vezanje antigena i protutijela dogaa se preko nespecifi nih vodikovih, Coulombovih, hidrofobnih i Van der Waalsovih veza. Jakost tih veza vrlo je mala i postaje znatno vea tek nakon bliskog pristajanja antigena uz protutijelo. Jakost ili energija vezanja antigena i protutijela odreena je dvama pojmovima: afinitet i avidnost. Afinitet ozna ava jakost vezanja jednoga spojnog mjesta protutijela i jednog epitopa odnosno determinante antigena, dok avidnost ozna ava jakost vezanja protutijelai cijele molekule antigena, odnosno zbroj afiniteta svih spojnih mjesta na protutijelu. Afinitet je stoga svojstvo antigena, a avidnost je svojsvo protutijela. Stvaranje protutijela mogu uzrokovati razli ite kemijske tvari kao ato su bjelan evine, polipeptidi, nukleinske kiseline, lipidi, aminokiseline, steroidi i druge molekule. Dok bjelan evine, polipeptidi, nukleinske kiseline i lipidi ozna uju prirodne imunogene, male kemijske molekule  hapteni, kao ato su primjerice steroidi, lijekovi i aminokiseline, postaju imunogene tek nakon povezivanja s nosa em, naj eae bjelan evinom ( goveim ili ljudskim albuminom ). Stoga svaka kemijska tvar koja je sposobna uzrokovati stvaranje protutijela, kad se unese u ~ivotinju domaina, nosi naziv imunogen. U veini imunokemijskih tehnika koriste se poliklonska protutijela kao radni reagens. Ona tvore heterogenu smjesu protutijela s razli itim specifi nostima. Proizvode ih razli ite stani ne linije plazma stanica kao odgovor na razli ite epitope imunogena. Nasuprot tomu, monoklonska su protutijela homologna i potpuno specifi na. Njih proizvode stanice koje su podrijetlom iz jedne jedine hibridne stanice nastale spajanjem B limfocita imunizirane ~ivotinje i mijelomske plazma stanice. Budui da monoklonska protutijela ulaze u reakciju samo s jednim jedinim epitopom imunogena, nisu pogodna za precipitacijske i aglutinacijske tehnike, zbog nemogunosti kri~nog povezivanja antigena i protutijela. No, vrlo su pogodna u tzv. obilje~enim tehnikama kao ato su radioimunokemijska tehnika, heterogena enzimimunokemijska tehnika i imunofluorometrija. Reakcija izmeu antigena i protutijela Vezanje antigena i protutijela ravnote~na je reakcija koja se zbiva u tri faze, sl.1. U prvoj fazi ili primarnoj reakciji dolazi do vezanja viaevalentnog antigena i dvovalentnog protutijela u kompleks antigen-protutijelo u razdoblju od nekoliko sekundi. Nakon nastanka primarnog kompleksa antigen-protutijelo slijedi rast kompleksa koji mo~e potrajati od nekoliko minuta do nekoliko dana. U treoj fazi, ovisno o relativnim koncentracijama antigena i protutijela, kompleksi se mogu kri~no povezivati pri emu nastanu veliki trodimenzionalni kompleksi koji precipitiraju iz otopine. Kad se uspostavi ravnote~no stanje izmeu svih nastalih kompleksa, prestaje njihov daljnji rast.  1. faza 2. faza 3. faza Slika 1.: Reakcija vezanja antigena i protutijela Brzina tih reakcija ovisna je o vrsti antigena i protutijela, afinitetu vezanja protutijela, kao i o koncentraciji elektrolita, pH i temperaturi. Razli iti linearni polimeri kao polietilenglikol, dekstran, preina ena celuloza, propilenglikol, polivinilni alkohol, imaju svojstvo da smanje topljivost bjelan evina, ato se primjenjuje u nekim imunokemijskim tehnikama za poveanje precipitacije kompleksa antigen-protutijelo. Reakcija izmeu antigena i protutijela prikazana je Heidelberger-Kendallovom krivuljom na sl.2. Ona ozna uje rezultate mjerenja koli ine precipitata nastalih dodatkom razli itih koli ina antigena stalnoj koli ini protutijela.   Slika 2. Heidelberger-Kendallova krivulja Odnos izmeu antigena i protutijela je razli it u svakoj zoni precipitacijske krivulje. U zoni suviaka protutijela (1. faza), odnos je izmeu antigena i protutijela manji od 1. Budui da je broj veznih mjesta protutijela velik, gotovo sva mjesta za vezanje na antigenu zasiena su prije nego ato mo~e doi do kri~nog povezivanja izmeu antigena. Stoga nastaju mali topljivi kompleksi sastava AgAt2. Daljnjim dodatkom antigena, pri emu je protutijelo prisutno u umjerenom suviaku, vjerojatnost je kri~nog povezivanja antigena s protutijelom vea pa nastaju veliki netopljivi kompleksi. Veli ina kompleksa raste do zone ekvivalencije u kojoj je sav antigen vezan s protutijelom. Odnos izmeu antigena i protutijela iznosi 1 (2. faza). U zoni suviaka antigena odnos izmeu antigena i protutijela raste pri emu se veli ine kompleksa smanjuju. Kad je suviaak antigena velik, nastaju mali topljivi kompleksi sastava Ag2At (3. faza). Precipitacija u zoni ekvivalencije tvori osnovu za otkrivanje i mjerenje koncentracije antigena u veini izravnih imunokemijskih tehnika. Neobilje~ene kvalitativne imunokemijske tehnike Imunoelektroforeza je spoj zonske elektroforeze i reakcije izmeu antigena i protutijela i omoguuje razdvajanje razli itih antigena tj. proteina u jednoj bioloakoj tekuini. Pomou imunoelektroforeze mogu se pojedini proteini definirati prema njihovoj pokretljivosti u elektri nom polju kao i njihovoj specifi noj reakciji s homolognim protutijelima. Razli itim tehnikama bojenja mogu se razlikovati proteini, lipoproteini i glikoproteini, a specifi nim reakcijama mogu se dokazati i enzimi. Imunoelektroforeza ima posebnu va~nost u definiranju razreda i tipa monoklonskog imunoglobulina kod monoklonskih gamapatija. Kako je monoklonski imunoglobulin proizvod jednoga klona plazma stanica, njegova osnovna karakteristika je kemijsko-fizi ka i imunoloaka homogenost koja se na elferogramu vidi kao uska homogena proteinska vrpca, a na imunoelferogramu kao precipitacijska linija promijenjenog oblika, polo~aja i intenziteta (sl.3.)  serum-monospeci ini antiserumi likvor-polispecifi ni antiserum Slika 3.: Imunoelektroforeza Nakon elektroforetskog razdvajanja proteina u agar gelu, izvodi se imunokemijska reakcija precipitacije pojedinih proteina pomou monospecifi nih ili polispecifi nih antiseruma. Kako se ova metoda koristi prvenstveno za odreivanje razreda i tipa monoklonskih proteina , imunokemijska precipitacija izvodi se pomou specifi nih antiseruma na teake lance imunoglobulina  (G),  (A),  (M), vrlo rijetko  (D) i  (E), kao i na vezane i slobodne lake lance imunoglobulina kapa i lambda tipa. Protusmjerna imunoelektroforeza je vrlo sli na dvostrukoj imunodifuziji, ali znatno br~a jer pasivna difuzija je zamijenjena djelovanjem elektri nog polja. Ako se odaberu elektroforetski uvjeti pri kojima antigen ima pokretljivost prema anodi, a protutijelo prema katodi, pod u inkom elektri nog polja dolazi do njihova kri~anja u gelu agara. Kad su koncentracije antigena i protutijela optimalne, na mjestu susreta nastaje netopljiva precipitacijska linija koja viae ne putuje. Rabi se za razlu ivanje uzoraka po na elu ima-nema. Imunofiksacija je svakako tehnika izbora za odreivanje razreda i tipa monoklonskog imunoglobulina. Izvodi se u gelu agaroze. Nakon elektroforetskog razdvajanja proteina u ispitivanom uzorku (serumu, mokrai, likvoru) u aest jednakih, paralelnih kanala, izravno se nanese u prvi (referentni) kanal otopina za fiksiranje proteina te redom na ostale kanale monospecifi ni antiserumi na teake lance  (G),  (A),  (M) i vezane lagane lance kapa i lambda tipa. Monoklonske slobodne lagane lance imunoglobulina ( Bence Jones protein ) dokazujemo monospecifi nim antiserumom na slobodne lagane lance kapa i lambda tipa. Ako je prisutan monoklonski imunoglobulin, nakon bojenja pojaviti e se uska homogena proteinska vrpca u kanalu razreda i tipa odreenog imunoglobulina ( sl. 4.). Kod poliklonski sintetiziranih imunoglobulina nastat e airoke difuzne vrpce. Polo~aj obojene i imunofiksirane proteinske vrpce usporeuje se s polo~ajem vrpce u referentnom kanalu.  serum Slika 4.: Imunofiksacija Imunofiksacijom nativnog uzorka likvora , bilo da se proteini u likvoru razdvoje elektroforetski na acetat celuloznom nosa u ili izoelektri nim fokusiranjem na poliakrilamidnom gelu (PAG), dokazuju se oligoklonski imunoglobulini (sl.5.). Nalaz oligoklonskih imunoglobulina upuuje na sigurnu intratekalnu sintezu imunoglobulina odnosno ukazuje na lokaliziranu imunoloaku reakciju unutar centralnog nervnog sistema.  Slika 5.: Odreivanje oligoklonskih IgG metodom izoelektri nog fokusiranja na PAG-u i direktnom imunofiksacijom na gelu uz bojenje srebrom Nakon ato se proteini, putujui u elektri nom polju uguste u uskom podru ju svoje izoelektri ne to ke (pI), nanose se specifi ni antiserumi izravno na gel uz bojenje proteina ili srebrom ili Coomassie-Blue bojom. Imunoselekcija je imunokemijska tehnika kojom se mogu dokazati monoklonski sintetitzirani teaki lanci. U gelu agara, u koji su prethodno dodani antiserumi na vezane lake lance kapa i lambda tipa, elektroforetski se razdvoje proteini u serumu. Pri tom dolazi do vezanja kompletnih molekula imunoglobulina, dok eventualno prisutni slobodni teaki lanci ostaju nevezani. U slijedeem koraku izvodi se imunokemijska reakcija precipitacije slobodnih teakih lanaca sa specifi nim antiserumom na  (G),  (A) ili  (M) teake lance. Sve bolesti teakih lanaca povezane su s istodobnim postojanjem jedne od malignih limfoproliferativnih bolesti. To su naj eae maligni limfomi i kroni na limfocitna neoplazma. Neobilje~ene kvantitativne imunokemijske tehnike Radijalna imunodifuzija osniva se na pasivnoj difuziji antigena u agarozni gel koji sadr~i jednoli no rasporeena protutijela. Pri prvom dodiru s protutijelima, antigen je u velikom suviaku pa nastaju kompleksi Ag-At. Daljnje difundiranje antigena uzrokuje pad koncentracije antigena, dok koncentracija raspolo~ivih protutijela raste. Kad se postigne optimalan koncentracijski odnos izmeu antigena i protutijela, nastaje netopljiv i nepokretan precipitacijski prsten koji se mo~e mjeriti (sl.6.). Povraina precipitacijskog prstena razmjerna je koncentraciji antigena. Usporeivanjem s povrainama precipitacijskih prstena poznatih koncentracija antigena mo~e se mjeriti nepoznata koncentracija antigena u ispitivanom uzorku. Ovom tehnikom odreuju se koncentracije imunoglobulina, lipoproteina, hemoglobina kao i mnogih drugih specifi nih proteina.  Slika 6.: Radijalna imunodifuzija Imunoturbidimetrija i imunonefelometrija su imunokemijske tehnike koje se izvode u tekuem mediju. Prva reakcija izmeu antigena i protutijela bila je provedena u otopini. Dodatkom otopine protutijela u otopinu koja sadr~i antigen nastaje zamuenje zbog nastalih kompleksa Ag-At. Mjerenje reakcije izmeu antigena i protutijela, u zoni ekvivalencije kad je postignuta maksimalna koncentracija kompleksa Ag-At, naziva se metoda krajnje to ke ( End-point method). Svaki uzorak antigena pomijeaa se sa stalnom koncentracijom protutijela u prisutnosti polietilenglikola 6000 koji poja ava reakciju vezanja, pomi e to ku ekvivalencije prema viaim koncentracijama antigena i odr~ava homogenost kompleksa Ag-At koji lebde u otopini. Nakon inkubacije mjeri se kod imunoturbidimetrije apsorpcija svjetla ili kod imunonefelometrije rasap svjetla. Standardna krivulja apsorpcije ili rasapa svjetlosti dobije se mjerenjem poznatih koncentracija antigena. Kod ovih tehnika mjeriti se mo~e i brzina stvaranja kompleksa Ag-At, i to unutar prvih nekoliko minuta reakcije izmeu antigena i protutijela, jer se tada dobiju najvee promene intenzteta apsorpcije ili rasapa svjetlosti s obzirom na vrijeme ( Fixed-time method). Osjetljivost imunonefelometrije je neato vea od imunoturbidimetrije i iznosi 1-10 g/mL. Osim za odreivanje koncentracije raznih specifi nih proteina obje metode se primjenjuju i za mjerenje koncentracije haptena, malih kemijski odreenih molekula kao ato su na pr. lijekovi.  .0V0 t @ Z BZ.4xp5:\F!(....9......3333477;;"==DDDDDEEEFHHHI I I2I4I5CJCJH*jCJUmH5 jUmHCJCJH*5CJCJU.0V*. 0 @ x" j    j  h .0V*. 0 @ x"\"*F!#(((...........`/,22333333p444 ="===DDDDEEEF F FII N NUUUUUUUUVVV,W.W0WpZ[[]\"*F!#(((...........`/,2233h j 33333p444 ="===DDDDEEEF F FII N NUUq4IBIDII"J N*N&Q(Q4Q6QBQDQR RRR R"RUUUVpZvZ[\j^^:b>bFbLbZb`bfbjbc~djjjkkJkNkkruvvvvw jUmHjCJUmHCJ5CJ54UUUUUUUVVV,W.W0WpZ[[8\\\f^j^bccccLdNdj[8\\\f^j^bccccLdNdjjjjkkJkLkNkruvvvvvvvvww jjjjkkJkLkNkruvvvvvvvvww. A!"m#$%Fќ(~#SY OiJFIF,,LEAD Technologies Inc. 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