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CHNG III: CC K THUT HUYT THANH

Cc k thut huyt thanh l k thut s dng khng th pht hin protein khng nguyn ca vi-rt, vi khun hoc nhng p ng ca vt ch vi vi-rt, vi khun hay k sinh trng trong mu huyt thanh. Trng tm ca cc k thut huyt thanh l xc nh s tip xc ca vt ch vi mm bnh, tuy nhin, cc k thut ny cung cp t thng tin v tnh trng nhim bnh ca vt ch hoc ca c n thy sn nui.

III.1. PHNG PHP KT TA MIN DCH III.1.1. Nguyn l Khi cho khng th c hiu phn ng vi khng nguyn ha tan liu lng chun s xut hin c kt ta nhn thy c bng mt thng. Phn ng ny c dng ph bin pht hin khng nguyn khi c sn khng th c hiu hoc pht hin khng th khi c khng nguyn ha tan c hiu. Phn ng kt ta b c ch khi c qu tha khng nguyn hoc khng th. S kt ta ti u khi c nng khng nguyn ph hp vi khng th. Nguyn l chung: kt ta min dch ch c th s dng c khi cho khng nguyn ha tan phn ng vi khng th cng ha tan. Hin tng kt ta min dch xy ra in vitro. Hin tng ta nhng phc hp phn t do lin kt khng nguyn-khng th l do hnh thnh mt mng li ba chiu cc phn t khng nguyn ni li vi nhau bi cc khng th (Marrack, 1934) (hnh 3.1).

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Hnh 3.1. Hin tng ta vi s hnh thnh mng li cn bng Vic hnh thnh mng li kt ta phi c nhiu iu kin nh sau: Khng th phi c t nht hai ha tr. Khng nguyn a ha tr Khng nguyn phi ha tan v thnh phn ca mi trng nh lc ion hay pH c vai tr nht nh trong vic lm xy ra hin tng kt ta. Cc khng th IgG ch c cha 3% glucid l nhng cht gy ta tt nht, cn khng th IgM cha ti 10% nn d ha tan v t kt ta hn. Cc phc hp khng nguyn-khng th cng to th cng d kt ta

III.1.2. ng dng K thut min dch khuch tn l k thut n gin gip pht hin khng nguyn trong mu huyt thanh hay huyt tng bng cch nh tnh hay nh lng.

III.1.3. Mu phn tch Mu huyt thanh hay huyt tng.

III.1.4. Cc dng khuch tn min dch III.1.4.1. Kt ta trong mi trng lng Phng php Heideiberger v Kendall Phng php ny cho php nh lng mt phn ng min dch, n nay vn cn c dng gii thch hin tng ta ti sao khi xut hin khi khng, l do thay i t l nng tng i gia khng th v khng nguyn. Trong mt lot ng th nghim, cho u cng mt lng khng th khng thay i. Sau cho vo khng nguyn vi nng tng dn. Kt qu l hin tng kt ta s tng dn t ng u v sau gim i (hnh 3.2). Nu em ly tm th c th ly ta v nh lng nitrogen ca ta trong tng ng. Nu khng nguyn khng phi l protein th lng nitrogen ta t l vi lng khng th b ta. Nu ly ng c ta nhiu nht th c th xc nh c lng khng th c trong huyt thanh bi v trong ng y hn 95% khng th b ta. Hn na, nu bit c lng khng nguyn cho vo mi ng v trng lng phn t ring ca khng nguyn v khng th th ngi ta c th tnh ra cng thc ca phc hp Sau khi ly tm tch ta, c th ly nc mt ri nu cho thm khng nguyn vo nhng ng u v khng th vo nhng ng cui th vn c c ta thm. iu ny cho php xc nh c 3 khu vc: 35

Trong nhng ng u l khu vc tha khng th, ni m do thiu khng nguyn nn ngn khng cho kt ta ht khng th. Trong nhng ng cui l khu vc tha khng nguyn nn cng khng cho php hnh thnh mng li 3 chiu. Nhng ng gn ni ta ti a tng ng vi khu vc tng ng khng nguyn v khng th c t l nng ti u cho php ta gn ht cc phn t khng th v khng nguyn.

Hnh 3.2. Hin tng ta trong mi trng lng

nh lng bng o c Dng mt tia sng mnh n sc cho i qua ng nghim trong c trn khng nguyn vi khng huyt thanh tng ng. Tia sng s cng b khuch x mnh nu ta cng nhiu. Vic nh lng c tin hnh ti vng c tha khng th v nh vic c cn quang ri so snh vi ng chun. Vic s dng tia laser cho php vic nh lng c gi tr cao. K thut ny c rt nhiu ng dng trong vic nh lng cc khng nguyn khc nhau m nng trong mt mi trng sinh hc trong khong mg/l nh IgG, IgA, IgMv trong vic tm cc khng khng th.

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III.1.4.2. Ta trong mi trng gel Nguyn l Khng th cng nh khng nguyn c th khuch tn trong thch (gel) v khi gp nhau th gy ta, ta y khng di chuyn v hin ln trng thy bng mt thng hoc nhum. Min dch khuch tn kp (k thut Ouchterlony) Trong mt lp thch khng dy, c thng sut nhng l cch u nhau, khong cch ny ph thuc vo h s khuch tn ca tng cht phn ng. Ri nh vo trong mi l i din vi nhau mt dung dch khng nguyn v khng th. Thi gian xut hin cc ng cung ta c th l hai ngy n mt tun.

Hnh 3.3. Khuch tn kp (k thut Ouchterlony)

K thut ny d t nhy nhng cho php phn tch cc h thng khng nguyn- khng th. Mun so snh gia hai khng nguyn hay pht hin s c mt ca nhiu khng th trong mt huyt thanh th c th b tr hai l khng nguyn trc mt l c cha huyt thanh. Kt qu c th: (i) Phn ng ging ht khi hai khng nguyn y ht nhau hay c epitop tng t th cc ng kt ta vi khng th s ni lin nhau; (ii) Phn ng khng y ht nu hai khng nguyn khc nhau th s kt hp ring vi hai khng th v hai ng kt ta s ct cho nhau hoc (iii) Phn ng ging mt phn khi hai khng nguyn c cng mt epitop v mt epitop ring khc nhau th cc khng th tng ng s cho mt ng ta chung lin vi nhau v mt ng ta ph gn vi ng ta trc i vi khng nguyn th hai (hnh 3.3). Ngc li, c th dng k thut ny so snh vi nhau hai khng huyt thanh gy ta xem chng c nhn bit cng mt khng nguyn hay khng. K thut ny rt hay c dng pht hin cc khng th chng nhn ca mt ngi so vi mt huyt thanh chun. 37

Min dch khuch tn vng (k thut Mancini) K thut ny dng nh lng khng nguyn. Khng th c hiu c ha u vo trong thch trc khi dy thch ln trn knh hay hp petri. Sau , c cc l v cho vo dung dch khng nguyn c pha long khc nhau. Khng nguyn s khuch tn v gp khng th ngay trong thch v to nn nhng vng kt ta m b mt ca n s t l thun vi nng khng nguyn. Vic nh lng c suy ra t mt ng biu din chun lp ra t nhng nng bit ca khng nguyn. III.1.4.3. Min dch khuch tn in Min dch khuch tn in n (k thut Laurell) Xut pht t nguyn l ca k thut khuch tn vng, k thut ny ch khc ch l s di chuyn ca cc khng nguyn nh lng c thc hin trong in trng. Do vng kt ta c hnh tn la (cn gi l in di tn la) (hnh 3.4). Chiu di ca hnh tn la t l vi nng khng nguyn. K thut ny nhy hn k thut Mancini nhng dng nhiu cht phn ng hn.

Hnh 3.4. Khuch tn in: 1,2,3 mu chun; x: mu

Min dch in di (k thut Grabar v Williams) Khi dung dch nh phn tch c cha nhiu khng nguyn th khuch tn kp khng xc nh mi ng kt ta. Min dch in di b sung thiu st y bng cch tch trc cc khng nguyn theo tc di chuyn ca chng trong in trng ri mi gy ta bng khng huyt thanh c hiu ri trong mt rnh o sn dc theo chiu in di. Cc ng ta xut hin theo hnh thc nhng cung hai bn rnh. K thut ny ch yu c dng nh tnh, cho php nh gi c hin tng ta hay khng so vi mt i chng. y l mt k thut tt xc nh mt Ig n dng do to ra mt cung ta dy hn, cong v gn rnh hn ng thi ni lin vi cung ta ca Ig huyt thanh cng lp. III.1.4.4. Min dch khuch tn: in di vi min dch khuch tn in situ Trong k thut ny dch sinh hc nh nghin cu c cho chy in di trc, sau cho tc dng ca khng th c hiu ngay trn lp thch hnh thnh kt ta khng nguyn- khng th. Nh vy, cc thnh phn ca hn hp phc tp nh huyt thanh c tch ra bn in di trong thch. Sau , cc phn t khc nhau nh nghin cu s c ta mt cch chn lc bng cc khng huyt thanh c hiu vi cc ptotein di chuyn 38

ngay trn b mt ca thch. Khng th ny nhanh chng thm vo bn trong gel ca thch v kt ta ti ch khng nguyn tng ng. Nhng phn t khng b kt ta th s c ra cho n ht. T , d dng nhum cc phc hp khng nguyn- khng th v s cho ra mt hnh nh chnh xc ca tnh trng phn b khng nguyn sau in di. K thut ny rt nhanh v nhy hn k thut in di chun on nhng bt thng n dng ca Ig. N c li hn l khng nguyn khng c thi gian khuch tn trc khi b kt ta bi khng th. Tnh thun nht ca mt thnh phn n dng no nu c mt lng rt thp th cng c bo tn trong k thut ny. Sau cng vic gii thch nhng hnh nh thu c cng d dng hn trong k thut min dch in di.

III.1.5. u v nhc im ca phng php III.1.5.1. u i m: K thut n gin III.1.5.2. Nhc im: Khng nguyn mun pht hin phi dng ha tan th mi c th khuch tn c. Mt khc phng php ny c tnh nhy khng cao.

III.2. PHNG PHP NGNG KT MIN DCH III.2.1. Nguyn l Hin tng ngng kt l do c ch hnh thnh mt mng li gia khng nguyn v khng th cho php mt lng cc ht y sp li vi nhau hnh thnh m ngng kt to mt thng c th nhn thy c. Mng li ch c th xut hin vi cc khng th c t nht l hai ha tr. IgM vi n 10 v tr kt hp nn c kh nng ngng kt mnh hn IgG. Ni chung cc khng nguyn hu hnh bao gi cng a ha tr cho nn khng b hn ch. phn ng ngng kt min dch i hi khng nguyn hu hnh. l khng nguyn c kch thc ln nh hng cu v t bo vi sinh vt. Khng nguyn hu hnh c epitop b mt c th lin kt cho vi cc khng th to thnh tng cm c th nhn thy c bng mt thng. Khng th trong phn ng ny gi l khng th gy ngng kt. Phn ng ngng kt nhy hn phn ng kt ta nn c dng nh tnh v bn nh lng khng th trong huyt thanh. C s l ha ca phn ng ngng kt: mt hn hp bao gm nhng tiu phn l lng trong mt dung mi. Cc tiu phn gi c trng thi.

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III.2.2. Xp loi cc phn ng ngng kt Ngi ta phn bit thnh ngng kt ch ng hay trc tip trong ht hu hnh c mang sn nhng nhm quyt nh khng nguyn c hiu nh hng cu, bch cu, tiu cu, tinh trng, vi khun v ngng kt th ng khi cc ht ch l cht tr lm gi cho cc quyt nh khng nguyn ha tan c gn mt cch nhn to ln b mt ca n, trong thng dng l hng cu x l bng formol. III.2.2.1. Ngng kt trc tip: Cho cc ht hu hnh c mang sn cc nhm quyt nh khng nguyn tip xc vi khng th cc nng pha long khc nhau. Cc khng th kt hp vi khng nguyn gn trn cc ht s dn n hnh thnh mng li v ngng kt. III.2.2.2. Ngng kt gin tip: Khng nguyn ha tan c c nh trn nhng ht khc nhau ty theo typ phn ng, sau hn hp ht s c cho tip xc vi huyt thanh trong nhng iu kin nh ngng kt trc tip. III.2.2.3. Ngng kt nhn to: Khi khng th khng pht huy c tc dng trn hai v tr trn, ngha l hin tng kt hp gia khng nguyn v khng th c xy ra m vn c hin tng ngng kt. Coombs cho thm khng th anti-globulin to mt cu ni gia globulin min dch c sn trn ht theo hai hnh thc sau: test coombs trc tip th trn hng cu c gn sn mt cch t nhin cc khng th khng gy ngng kt, khi cho thm khng th anti-globulin vo th s gy ngng kt; Test Coombs gin tip l mt test huyt thanh cho php tm xem trong mt huyt thanh c hay khng c khng th chng hng cu nhng khng gy ngng kt. u tin, huyt thanh vi mt loi hng cu bit r nhm. Sau , ra sch th trn hng cu ch cn bm khng th trong huyt thanh cho nn khi thm khng th antiglubolin v nu c ngng kt th c ngha l trong y c khng th nh tm (hnh 3.5).

Hnh 3.5. Phng php test coombs trc tip v gin tip

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III.2.3. ng dng Pht hin khng nguyn v nh gi s tng tc gia khng th vi mt khng nguyn dng ht. C 3 dng thng gp: c ch s ngng kt hng cu (Hemagglutination inhibition-HI): nh gi s tng tc gia khng th vi vi-rt c cha protein ngng kt hng cu. Ngng kt vi khun (Bacterial agglutination): xc nh khng th huyt thanh c to ra chng li s nhim khun. c ch ngng kt (Agglutination inhibition): pht hin mt lng nh khng th chng li mt tc nhn gy bnh no .

III.2.4. Mu phn tch Mu huyt tng hay huyt thanh

III.2.5. u v nhc im ca phng php III.2.5.1. u i m: K thut kh n gin III.2.5.2. Nhc im: Khng nguyn ca mm bnh mun pht hin phi dng ht. Mt khc phng php ny c tnh nhy khng cao.

III.3. K THUT MIN DCH HUNH QUANG III.3.1. Nguyn l Trong k thut ny khng th c nh du bng thuc nhum hunh quang. K thut da trn tnh cht ca thuc nhum khi c kch thch bi bc x c bc sng c hiu s pht sng. Chng hn fluorescein pht hunh quang mu vng lc, cn rodamin pht quang mu da cam. Khng th gn thuc nhum hunh quang gi l khng th nh du hoc khng th hunh quang.

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III.3.2. Phng php III.3.2.1. K thut min dch hunh quang trc tip K thut ch c hai thnh phn: Khng nguyn v khng th, to ra hai lp ca phn ng. Mt trong hai thnh phn c gn trc tip vi thuc nhum hunh quang. u tin ngi ta c nh vi khun trn lam knh sau ph khng th hunh quang ln khng th gn vi t bo vi khun. Ra loi b khng th tha ri quan st di knh hin vi hunh quang. Ch c cc vi khun c hiu vi khng th hunh quang mi c quan st (hnh 3.6).

Hnh 3.6. Khng th hunh quang v k thut min dch hunh quang trc tip

III.3.2.2. K thut min dch hung quang gin tip K thut ny ngoi hai thnh phn chnh l khng nguyn v khng th c hiu to ra hai lp ca phn ng cn c mt thnh phn na c gn vi thuc nhum hunh quang, to lp th ba l khng- khng th (hnh 3.7).

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III.3.3. ng dng K thut min dch hunh quang c s dng pht hin khng nguyn ca mt mm bnh chuyn bit trong m hay t bo b nhim bnh. Phng php kh tng t nh k thut IHC, cho php vic pht hin khng nguyn qua s kt hp c hiu vi khng th c nh du hunh quang. III.3.4. Mu phn tch Mu phn tch c th l mu m, mu t bo, huyt thanh. Cc mu ny c th c lu tr bng cch ng lnh, lm kh hay c nh trong dung dch c nh thch hp.

III.3.5. u v nhc im ca phng php III.3.5.1. u im: C tnh nhy cao v thao tc nhanh pht hin v nh dng mm bnh trong mu knh pht, m hay t bo. III.3.5.2. Nhc im: i hi phi s dng khng th hunh quang hoc khng huyt thanh. Vic c kt qu i hi k nng v kinh nghim ca ngi thc hin vic chn on phn bit nhng trng hp dng tnh gi v m tnh gi. Phn ng cho ca khng th vi khng nguyn kh c th xy cho nn khng th s dng phi c tnh chuyn bit cao.

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Hnh 3.7. K thut min dch hunh quang gin tip

III.4. K THUT MIN DCH LIN KT ENZYM III.4.1. Nguyn l K thut min dch lin kt enzym (ELISA-Enzyme- Linked Immunosorbent Assay) l k thut kh nhy v n gin, cho php xc nh khng nguyn hoc khng th nng rt thp (khong 0,1 ng/ml). So vi k thut phng x th k thut ny rt r tin v an ton hn m vn chnh xc nh nhau. Nguyn tc ca k thut ELISA ging nh k thut min dch hunh quang, nhng thay v khng th gn vi thuc nhum hunh quang th ngi ta gn khng th vi mt enzym. Khi cho thm c cht thch hp vo phn ng, enzym s thy phn c cht ny thnh mt cht c mu. S xut hin mu chng t xy ra phn ng c hiu gia khng nguyn vi khng th, thng qua cng mu ngi ta bit c nng ca khng nguyn cn pht hin. K thut ELISA c chia lm hai dng l ELISA trc tip v ELISA gin tip. Cc enzym thng dng trong k thut ELISA l -galactoxidaza, glucooxidaza, peroxidazza 44

v phophadaza kim. ELISA dng xc nh nhiu tc nhn gy bnh nh vi-rt, vi khun, nm, k sinh trng.

III.4.2. ng dng ELISA c s dng nh gi s tip xc ca mu bnh phm vi nhng tc nhn gy bnh khc nhau nh vi khun, vi-rt, k sinh trng. Khng nguyn c lm tinh sch v hm lng khng th s dng cng nh hm lng khng nguyn c th xc nh c.

III.4.3. Mu phn tch Huyt thanh hay huyt tng

III.4.4. Phng php Hm lng khng th s dng kt hp vi khng nguyn ca mm bnh c xc nh bng biu hm lng khng th chun. Kt qu c c bng hm lng khng th. III.4.4.1. K thut ELISA gin tip Thng c dng nh tnh hoc nh lng khng nguyn. Nh huyt thanh c khng th (v d antitoxin) vo ging bng nha cho khng th bm vo thnh ging. Nh tip dch khng nguyn cn xt nghim (v d toxin). Nu l khng nguyn c hiu vi khng th th s gn vi khng th. Thm cng hp gn enzym vo ging cho khng th ca cng gn vi khng nguyn m trc gn vi khng th u tin, to nn mt bnh m kp ch l khng th- khng nguyn- khng th gn enzym (hnh 3.8a). B sung c cht ca enzym. Enzym s thy phn c cht ny lm thay i mu ca dung dch. Tc thy phn ca enzym s t l thun vi lng khng th gn enzim, cng c ngha l t l thun vi khng nguyn cn xt nghim. S thay i mu dung dch c th nhn thy c bng mt thng hoc o c bng quang ph k.

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Hnh 3.8. K thut ELISA: (a) ELISA gin tip; (b) ELISA trc tip (KT: khng th; KN: khng nguyn; S: c cht)

III.4.4.2. K thut ELISA trc tip Thng c dng nh tnh hoc nh lng khng th. Nh dch khng nguyn cho hp th ln thnh ging. Nh tip khng huyt thanh (cha khng th) cn xt nghim ri . Nu trong huyt thanh c cha khng th c hiu th s gn vi n. Thm cng hp khng- khng th gn enzym. Khng th ny c to thnh khng ch khng th ngi. Thm c cht ca enzym. Tc thy phn c cht gn lin vi s thay i mu ca dung dch v t l thun vi lng khng th cn xc nh trong mu. S thay i mu c th quan st bng mt thng hay quang ph k (hnh 3.8b).

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III.4.5. u v nhc im ca phng php III.4.5.1. u im: Phng php ELISA l phng php kh nhy v d thc hin. C th xt nghim cng mt lc nhiu mu bnh phm, thi gian thc hin ngn nn c xem nh l phng php vng trong sng lc/pht hin bnh. III.4.5.2. Nhc im: Cn phi c khng th n dng hay a dng ph hp vi khng nguyn ca mm bnh cn xt nghim.

III.5. TI LIU THAM KHO CHNG III 1. Kuby, J. (1997). Antigen-Antibody Interactions. In: Immunology. 3rd edition. 2. V Triu An v Jean, C.H. (2001). Min dch hc. 3. Nguyn Ln Dng. (2000). Vi sinh vt hc. 4. Nguyn Ngc Lnh. (1997). Min dch hc. 5. Madigan, M.T., Martinko, J.M. and Parker, J. (2002). Biology of Microorganisms. 10th edition. 6. Ngc Lin. (1999). Min dch hc c s.

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