Ngy nay, phng trnh cc cn bnh him ngho, pht hin cc yu t l trong thc phm cy trng, ngi ta c th s dng nhiu phng php khc nhau nh phng php vt l, phng php gii phu bnh sinh thit v phng php ha sinh, hoc kt hp cc phng php ny. Mi phng php c u v nhc im ring. Tuy nhin, phng php n gin, gi thnh xt nghim r v tng i chnh xc, c ph bin hin nay l ELISA (Enzyme Linked Immunosorbent Assay) - xt nghim hp th min dch lin kt vi enzyme. H Ni, 14/4/2012 I. Gii thiu phng php ELISA

1. Lch s pht trin Vo nm 1960, Rosalyn Sussman Yalow v Solomon Berson Prior m t phng php min dch c gn cht phng x (radioimmunoassay) dng xc nh khng nguyn, khng th . Trong phng php ny, khng th hay khng nguyn c gn cht nh du phng x v s hin din ca chng c xc nh thng qua tn hiu phng x. Tuy vy, cc cht phng x li tim n mi nguy him v vy ngi ta ngh n mt phng php mi trong tn hiu c pht ra khng phi nh phng x. Khi ngi ta bit rng mt s loi enzyme nh cc peroxidase phn ng vi mt s c cht nht nh nh Tetramethylbenzidine hay 3-ethylbenzthiazoline-6-sulphonic acid s pht mu. Mu sinh ra t cc phn ng ny c th c s dng lm tn hiu nhn bit (tn hiu ch th). Tuy nhin, tn hiu ny c s dng nh cht phng x trong radioimmunoassay, tn hiu mu pht ra phi ng ngha vi s c mt ca khng th hay khng nguyn. Chnh v vy, gii php c a ra l enzyme s c gn vi khng nguyn hay khng th. K thut gn kt enzyme vi khng nguyn hay khng th c pht trin bi Stratis Avrameas v G.B. Pierce . Tip sau , vo nm 1966, Wide v Porath pht trin phng php gn khng nguyn v khng th trn b mt rn (b mt ca cc vi phim hay cc a) nh m khng nguyn hay khng th khng c gn kt s d dng c ra tri. Peter Perlmann v Eva Engvall (Thy in) cng vi nhm Anton Schuurs v Bauke van

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Weemen (H Lan) cng b phng php c t tn ELISA (EIA) da trn tt c nhng bc pht trin ni trn. Phng php chnh thc ra i vo nm 1971. 2. Khi nim v ELISA nh ngha: ELISA (Enzyme-Linked ImmunoSorbent Assay) hay EIA (Enzyme ImmunoAssay) l mt k thut sinh ha pht hin khng th hay khng nguyn trong mu xt nghim. *Ch thch: Khng nguyn (antigen) l nhng cht c kh nng huy ng h min dch v gy mt phn ng min dch. Khng nguyn bao gm protein l, acid nucleic, mt s lipide v polysaccharide. Cha bit gn trn b mt. Khng th (antibody) to thnh phn gramma globulin trong cc protein mu. Khng th l nhng protein ha tan do cc t bo B hay tng bo tit ra p ng vi mt khng nguyn v chng c th gn c hiu vi khng nguyn . K thut ELISA gm ba thnh phn tham gia phn ng l: khng nguyn, khng th v cht to mu; thc hin qua hai bc: - Phn ng min dch hc: L s kt hp gia khng nguyn v khng th. - Phn ng ha hc: Thng qua hot tnh xc tc ca enzyme lm gii phng oxy nguyn t [O] t H2O2 oxy ha c cht ch th mu, do lm thay i mu ca hn hp trong dung dch th nghim. Enzyme thng c s dng l : + Peroxidase + Beta galactoxidase + Alkaline phosphatase 3. u im v hn ch.

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K thut ny kh nhy v n gin, cho php ta xc nh khng nguyn hoc khng th mt nng rt thp (khong 0,1 ng/ml). So vi k thut min dch phng x (RIARadio Immuno Assay) th k thut ny r tin v an ton hn m vn m bo chnh xc nh nhau. ELISA c dng xc nh nhiu tc nhn gy bnh nh virus, vi khun, nm, k sinh. u im: +D thc hin + C th tin hnh ng thi nhiu mu + C th pht hin khng th IgM, IgG, IgA v IgE Hn ch: + i hi thi gian (c kt qu sau 5h) + Thit b mc tin II. Phn loi

1. Direct ELISA (ELISA trc tip) y l dng n gin nht ca phng php ELISA. Trong , khng nguyn cn pht hin s c gn trc tip ln b mt gi th v s c pht hin bng mt khng th duy nht (khng th ny c gn enzyme). Quy trnh thc hin:

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u nhc im: u im: + n gin nht, Nhanh chng bi v ch c mt khng th v tri qua t bc hn. + Phn ng ca khng th th cp c loi b. Nhc im: + c hiu b gii hn v thng th khng nguyn c t nht l 2 epitope (trnh din khng nguyn) m phng php ny ch s dng 1 khng th gn vo mt epitope. + Phn ng min dch ca khng th chnh c th nh hng bt li bng cch tc dng vi cc enzyme. + Phi nh du cho tng khng th chuyn bit vi tng i tng. + Khng c s linh hot trong la chn khng th chnh t mt th nghim khc + Khuch i tn hiu ti thiu.

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p dng: - Pht hin nhim HIV tr s sinh c sinh ra t m nhim HIV. - Pht hin sm giai on mi nhim HIV (giai on ca s) hoc khi kt qu pht hin KT khng r rng. - Theo di din tin bnh, nh gi hiu qu iu tr thuc khng virt v trong cc mc ch nghin cu khc. 2. Indirect ELISA (ELISA gin tip) Phng php ny khc Direct ELISA ch khng th bt khng nguyn khng c gn enzyme m n l mc tiu gn c hiu ca mt khng th khc (khng th ny mi l khng th c gn vi enzyme). Quy trnh thc hin:

- Chuyn khng nguyn bit ln b mt cng ( c gi chung l cc a). Khng nguyn s c c nh trn b mt. Nng ca cc mu khng nguyn ny dng thit lp ng chun cho vic tnh nng ca khng nguyn trong cc mu cha bit. - Chuyn cc mu khng nguyn cha bit vo cc ging khc. Khng nguyn cha bit

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c ha tan trong cng mt loi dung dch m ging cc mu KN chun. - Thm dung dch protein khng tng tc (non-interacting protein) nh albumin huyt thanh b (bovine serum albumin) hay casein vo tt c cc mu (k c mu chun). Bc ny c gi l "blockin" do protein huyt thanh c tc dng ngn cn s hp ph ca cc protein khc ln b mt ca a. - Ra b mt a sau chuyn khng th (bit trc) vo tt c cc ging ca a. Khng th s kt hp vi cc khng nguyn c c nh m khng kt hp vi protein ca huyt thanh. - Thm khng th th cp (secondary antibody), khng th th cp s kt hp vi bt k mt khng th cn d (bc ny c th c b qua nu khng th dng pht hin khng nguyn gn vi enzyme). - Ra a, cc khng nguyn gn enzyme cn d s c loi b. - Thm c cht. Enzyme s lm bin i c cht lm sn sinh tn hiu hunh quang hay tn hiu in ha hc (enzyme c tc dng nh yu t khuych i). u nhc im: u im: + Bc c nh khng nguyn khng c tnh c hiu nn bt k protein no cng gn vi b mt a v vy khng th (c nng thp) phi cnh tranh vi cc protein khc trong huyt thanh khi gn kt vi b mt ca a. Sandwich ELISA hn ch c nhc im ny. + Khng th gn enzyme c th s dng nh du cho nhiu loi khng nguyn nn tin li v kinh t hn, d dng thng mi ha. + Linh hot bi v cc khng th nhiu chnh c th c thc hin trong mt loi v cng mt khng th th cp c th c s dng pht hin. + nhy sng c tng ln bi v mi khng th chnh cha cc epitope mt s c th c rng buc bi cc khng th th cp , cho php khuch i tn hiu. Nhc im: + c hiu ca tng khng huyt thanh l khc nhau. iu ny dn n kt qu khc nhau gia cc th nghim v do cn phi th nghim vi nhiu khng huyt thanh khc nhau kt qu c th tin tng c.

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+ Phn ng c th xy ra vi khng th th cp, dn n tn hiu khng c hiu. + Mt bc bnh thm l cn thit trong th tc.

3. Sandwich ELISA y l mt dng ELISA c s dng ph bin nht trong thc tin do n cho phn ng mnh v nhy. c gi l sandwich l do kt qu th nghim c nh gi thng qua s kt hp ca hai loi khng th l khng th bt (capture antibodies) v khng th pht hin (detection antibodies). K thut ny cng c phn lm hai dng l Direct sandwich ELISA (DAS-ELISA - Double antibody sandwich) v Indirect sandwich ELISA (TAS-ELISA Triple antibody sandwich). 3.1 Sandwich ELISA trc tip

- u im: C th pht hin s khc bit nh gia cc khng nguyn nu s dng khng th bt v khng th pht hin khc nhau. - V phng php ny c u im hn hn nhng phng php khc m chng ti chn phng php ny chn on bnh virus ang nghin cu.

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Ch : nu s dng khng th bt v khng th pht hin ging nhau c th dn n vn nu c s gii hn v tr kt hp sn c pht hin. Mi quan h v kch thc v v tr khng gian ca cc epitope cng c nh hng n th nghim. 3.2 Sandwich ELISA gin tip

Chuyn bit hn Direct sanwich ELISA do antispecies khng th c gn enzyme khng phn ng vi khng th bt khng nguyn. 4. ELISA cnh tranh Quy trnh thc hin: + khng th khng c nh du vi khng nguyn. + a hn hp ny vo cc ging ca vi phim c cha khng nguyn. + Ra a, khng th khng c gn kt s b ra tri. Lng khng nguyn cng ln, lng khng th gn thnh cng vi khng nguyn trong a cng thp do cnh tranh

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+ Thm khng th th cp (khng th ca khng th bc 1). Khng th th cp gn vi enzym. + Thm c cht. Lng enzym d s gii phng tn hiu hunh quang hay tn hiu mu. Trong phng php ny, hm lng khng nguyn gc cng cao, tn hiu sn sinh cng yu. III. Cc yu t nh hng n phng php

1. nh hng n nhy S lng khng th th nht c gn vo y ging i lc ca khng th th nht i vi khng nguyn i lc ca khng th th hai i vi khng nguyn

2. nh hng n kt qu

Nu cc i chng m cho kt qu dng tnh th c th do s nhim t cht to mu hoc t khng th c nh du hoc chnh cc i chng b nhim. Nu mu khng xut hin i vi i chng dng hoc i vi mu th phi kim tra li tt c ho cht bao gm: hn s dng, nng , iu kin bo qun Nu mu xut hin qu thp i vi i chng dng v c mu kim tra th phi kim tra li khng th c gn enzyme v nng ca cht to mu. Nu c to mu i vi mu nhng khng to mu vi i chng dng th c th kim tra li ngun gc i chng, hn s dng v iu kin bo qun. Khi chy li mt th nghim trong iu kin ang gp s c th ch nn thay i mt yu t th nghim. Mt s thit b s dng trong phng php ELISA Khay nha c cc ging. My quang ph vi cc bc sng khc nhau. Micropipette. My ra khay nha.

IV. -

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V.

ng dng 1. Trong thc phm

-Phng php Elisa c th pht hin v nh lng vi sinh trong thc phm trong thi gian vi gi sau khi tng sinh. -Pht hin yu t gy d ng thc phm nh sa, u phng, qu c ch, hnh nhn v trng -Pht hin c t trong to. -Pht hin vi khun E.coli, Salmonella, Staphylococcus aureus,sn l gan trong thc phm. -Pht hin cht chloramphenicol (cht khng c php c trong tm, c v cc sn phm thu sn khc) -Kim tra d lng khng sinh trong thc phm,tn d thuc dit c,thuc tr su 2. Trong nng nghip -Chun on bnh Tristeza(tc nhn gy bnh ho r) trn cy cam qut. -Gim nh s hin din ca BBTV(Banana Bunchy Top Virus) gy bnh chn t chui. -Pht hin khng th chng Mycoplasma hyopnewmonia(MH) heo. -Gim nh bnh ln xon l trn la bng phng php DAS ELISA ci tin 3. Trong y hc -Chun an v iu tr bnh vim gan siu vi B v C,bnh ung th. -ng dng pht hin bnh A. cantonensis (bnh vim mng no) do loi giun k sinh phi chut gy ra. - Xc nh t l nhim k sinh trng st rt min Trung-Ty Nguyn. ng dng ca ELISA trong xt nghim HIV

VI.

1. V HIV/AIDS

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AIDS(Acquired Immuno-Deficiency Syndrome) l hi chng suy gim min dch mc phi gy ra bi HIV (Human Immuno-Deficiency virus) AIDS xy ra do s tn cng HIV vo t bo ca h min dch. Trc ht l lm suy gim s lng v ri lon chc nng ca t bo min dch dn dn hng lot ri lon khc ca h min dch, gy ra suy gim min dch nghim trng, to iu kin cho nhim trng c hi pht trin, cui cng l t vong. Cu trc HIV: HIV c c im chung ca h retroviridae. Ht virus hon chnh (virion) c cu trc gm 3 lp. - Lp v ngoi (v peplon): lp ny l 1 mng lipid kp c khng nguyn cho vi mng nguyn sinh cht t bo. Gn ln mng ny l cc nh, l cc phn t Glycoprotein c trng lng phn t 160 kilodalton (gp160). N gm c 2 phn: + Glycoprotein mng ngoi :c trng lng phn t l 120 kilodalton (gp120). GP120 l khng nguyn bin i nht, gy kh khn cho phn ng bo v c th v ch vaccin phng bnh. +Glycoprotein xuyn mng: c trng lng phn t 41 kilodalton. - V trong (v capsid): v ny gm 2 lp protein: + Lp ngoi hnh cu, cu to bi protein c trng lng phn t 18 kilodalton (p18). + Lp trong hnh tr, cu to bi cc phn t c trng lng phn t l 24 kilodalton (p24). y l khng nguyn rt quang trng chn on nhim HIV/AIDS - Li: L nhng thnh phn bn trong ca v capsid, bao gm: + Hai phn t ARN n, l b gen di truyn HIV(genom).Genom ca HIV cha 3 gen cu trc: Gag (group specific antigen) l cc gen m ho cho cc khng nguyn c hiu ca capsid cu virus; Pol (polymerase) m ho cho cc Enzym: reverve transcriptase (RT:Enzym sao m ngc), protease v endonuclease (cn gi khng nguyn integrase); v EnV (envelop) m ho cho glycoprotein lp v peplon ca HIV. 2. Nguyn l ca phng php ELISA pht hin khng th khng HIV

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Elisa gin tip Khng th khng HIV c trong mu bnh nhn kt hp c hiu vi khng nguyn virt HIV c c nh sn trn gi (cc ging phn ng trn phin nha). Phc hp khng nguyn -khng th ny c nhn bit c hiu bi mt cng hp l mt khng th khng immunoglobuline ngi (Ig) c gn enzyme v s cho phn ng hin mu vi mt c cht thch hp. Gi tr mt quang ca phn ng mu (OD: optical density) t l thun vi lng khng th khng HIV hin din trong mu th. Elisa cnh tranh Nguyn tc ca k thut da trn s cnh tranh gia khng th khng HIV c trong mu th vi cng hp l khng th khng HIV gn vi enzyme c gn ln cc khng nguyn c nh trn ging. Gi tr mt quang (OD) t l nghch vi lng khng th trong mu th. Sanwich elisa Khng th khng HIV trong mu th s kt hp c hiu vi khng nguyn c nh trn ging v c pht hin bi cng hp l cc khng nguyn virt gn enzyme. Gi tr mt quang ca phn ng mu (OD) t l thun vi lng khng th khng HIV hin din trong mu th. Cc th h sinh phm Sinh phm th h th nht s dng khng nguyn l vi rt ton phn c ly gii v tinh ch t cc t bo gy nhim vi HIV. Cc sinh phm ny c nhy v c hiu hn ch. Sinh phm th h th hai dng cc khng nguyn l protein ti t hp hoc protein tng hp. Sinh phm c nhy v c hiu cao hn. Sinh phm th h ba x dng cc khng nguyn ti t hp v cc proteine tng hp nhng khng th c pht hin theo nguyn tc ELISA sandwich. nhy v c hiu c ci thin r rt. Sinh phm th h th t pht hin ng thi khng nguyn v khng th c trong mu th. Sinh phm ny cho php pht hin nhim HIV trc khi c s chuyn i huyt thanh v l sinh phm c khuyn co nn la chn cho kim tra an ton trong truyn mu. Cc sinh phm ELISA ngy cng c hon thin c th pht hin sm v chnh xc

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khng th khng HIV, rt ngn thi gian ca s huyt thanh. Cc sinh phm dng cc khng nguyn c cc epitop to ra cc khng th sm v c cc loi khng nguyn cho php pht hin khng th khng virt HIV 1 & 2, cc th type virt v cc virt HIV nhm O. 3. Ch phm HIV UNIFORM II Plus O pht hin nhim HIV 3.1. Nguyn tc

L phn ng ELISA Sandwich mt bc. - Khng nguyn gn trn ging l cc protein v gp160 ca HIV-1 v ANT70 (nhm O); gp36 ca HIV2 - Nu trong mu th c khng th khng HIV, khng th ny s gn c hiu vi khng nguyn c nh trn ging. ng thi khi cu cng hp l khng nguyn HIV gn men cng c sn trong mi ging s to phc hp kp ch: Khng nguyn - Khng th - Cng hp. - Phc hp Khng nguyn - Khng th - Cng hp s c pht hin bng phn ng hin mu vi c cht 3.2. Quy trnh thc hin

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4. u im v hn ch u im:

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+ Cho php thc hin ng thi nhiu mu. C th dng my t ng gim bt thao tc cho ngi lm xt nghim, trnh sai st v ly nhim. + c kt qu bng my khng ph thuc vo ch quan ca ngi lm xt nghim. + C th lu cc bng kt qu v cc thng s k thut thun li cho vic kim tra nh gi cht lng xt nghim. + Gi thnh xt nghim tng i r. Hn ch:

+ Cn c s u t cho trang thit b ban u v bo dng my mc. + Cc sinh phm phi bo qun nhit lnh (4-80C) + Thi gian thc hin xt nghim 2-3 gi. + Khng thch hp cho nhng ni s lng mu t do phi lm nhiu chng. 5. Cc kt qu bt thng Mt kt qu dng tnh khng nht thit ngha l ngi b nhim HIV v c mt s bnh l c th gy dng tnh gi(nh bnh l hch lymph, giang mai, lupus). Khi kt qu ELISA dng tnh, th nghim xc nh Western blot lun lun phi c thc hin sau . Western blot dng tnh nhn chung c cho l chc chn c nhim HIV. Kt qu m tnh khng th loi tr nhim HIV, v cn mt khong thi gian cho t lc nhim HIV n khi xut hin nng khng th khng HIV c th o c(do c gi l giai on ca s). V vy, nu mt ngi b ngi ng nhim HIV cp hay nguyn pht ngha l c giai on ca s, th kt qu ELISA v Western blot m tnh khng th loi tr chn on. Nn cn thc hin thm nhng xt nghim b sung nh xt nghim tm khng nguyn p24 hay dung np virus HIV. Hoc phi xt nghim li sau 3 6 thng.

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Kt lunELISA l mt phng php c nhiu u im ni tri so vi cc phng php khc, cho php thc hin ng thi nhiu mu, dng my t ng gim bt thao tc cho ngi lm xt nghim, trnh sai st v ly nhim, kt qu c bng my khng ph thuc vo ch quan ca ngi lm xt nghim nn kt qu o c c chnh xc cao. Hn na gi thnh xt nghim r, thi gian thu c kt qu khng qu lu. Vy nn phng php ny nn c s dng ph bin rng ri nng cao kt qu iu tr bnh, nng sut cy trng cng nh cht lng thc phm ca x hi. c bit, ELISA gip xt nghim HIV/AIDS cn bnh th k ca con ngi. Trong qu trnh lm tiu lun ny, hn vn cn nhiu thiu st do hn ch v kin thc cng nh thi gian, v vy, rt mong c gio v cc bn gip tiu lun c hon thin hn. Xin chn thnh cm n!

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Ti liu tham khoVi sinh vt hc, Nguyn Ln Dng, NXB Gio dc, 1997. Molecular Biotechnology, Glick BR and Jackj P, 1994. Genetic Engineering and Biotechnology, Chopra BL and Anwan N, 1990. Hng dn quc gia v xt nghim HIV, B Y T, 2009. www.sinhhocvietnam.com www.genprice.com

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Mc LcI. 1. 2. 3. II. 1. 2. 3. Gii thiu phng php ELISA ........................................................................................................ 1 Lch s pht trin ............................................................................................................................ 1 Khi nim v ELISA ....................................................................................................................... 2 u im v hn ch. ....................................................................................................................... 2 Phn loi .......................................................................................................................................... 3 Direct ELISA (ELISA trc tip).................................................................................................... 3 Indirect ELISA (ELISA gin tip) ................................................................................................ 5 Sandwich ELISA ............................................................................................................................. 7 3.1 Sandwich ELISA trc tip............................................................................................................... 7 3.2 Sandwich ELISA gin tip.............................................................................................................. 8 4. III. 1. 2. IV. V. 1. 2. 3. VI. 1. 2. 3. ELISA cnh tranh ........................................................................................................................... 8 Cc yu t nh hng n phng php ...................................................................................... 9 nh hng n nhy ................................................................................................................. 9 nh hng n kt qu .................................................................................................................. 9 Mt s thit b s dng trong phng php ELISA .................................................................... 9 ng dng ........................................................................................................................................... 10 Trong thc phm .......................................................................................................................... 10 Trong nng nghip ........................................................................................................................ 10 Trong y hc .................................................................................................................................... 10 ng dng ca ELISA trong xt nghim HIV ............................................................................. 10 V HIV/AIDS................................................................................................................................. 10 Nguyn l ca phng php ELISA pht hin khng th khng HIV .................................... 11 Ch phm HIV UNIFORM II Plus O pht hin nhim HIV .................................................... 13 3.1. 3.2. 4. 5. Nguyn tc ............................................................................................................................. 13 Quy trnh thc hin............................................................................................................... 13

u im v hn ch ...................................................................................................................... 14 Cc kt qu bt thng ................................................................................................................ 15

Kt lun ....................................................................................................................................................... 16 Ti liu tham kho ...................................................................................................................................... 17

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