VAcdNCiNGHfi 2010 " TAI D6C L.4P C ^ NHA NUOC g ^ i'\ i~^ A r% 'Ti'\^ri if E-l!o o ^ ^ i; oo O O - > - O O ^ < < < < Z Z Z Z Q a > > ir, iTi zn iri ^/i K « i« ts "« o o o o < < < < &&£ & tu cy 51) ^ ^ U CJ S cu -S 3 3 K I I t3 fiv e> CJ ^; ^: t: ^: in « i Phu lyc 24: QUY TRINH SAN X U A T H U Y ^ T THANH KHANG TA BAC HIEU d QUY MO PHONG THf NGHlfM Pham vi ap dyng Quy trinh du0c xay dimg de s4n xuat huyet tho dJc hi^u khang ta dac hi?u co the ap dung cho cac co so nghign cOru, cac tnrcmg djii hgc va cac cong ty sinh pham de san xu^t huyet khdng tA phuc vy cho cong tac nghien cuu va chan doan benh ta tai Viet Nam Nguyen tac S i> dung te bao V.cholerae 01 va 0139 da bat hoat bang formalin 0,05% gay mien dich cho tho de tao khang the dac hi?u khang lai khang nguyen O ctia V.cholerae 01 va 0139 Chung VK + Chiing V.cholerae Ol: gom ca typ huyet Ogawa va Inaba + Chung V.cholerae 0139 + Dan miu chiing VK de kiem tra huySt khang ta Dong vat thi nghifm: Tho cai to, chua de (tho non) can nSng 1,5-1,8kg Cac bvorc tien hanh Buac 1: Xac djnh cac chung gay miin djch t^o huyet Idiang ta - Kiem tra tinh chat khuan l?c, sinh hoa va ngung kit vdi huylt dac hieu cua cac chung V.cholerae 01 va 0139 - Phat hien gen dJc hieu va gen doc t6 ctxK cua cac chung gay mien dich bang ky thult PCR 90 • '•'• ^ Chuan bj huyen djch l(hang nguyen cay chiing tren moi tnrcmg thach TCBS, li 37°C/18 - 24 gio hiy khuan lac ciy tang sinh canh thang LB, u 37''C/6 - gio Cay 0,5 ml canh khuin sang cac binh chiia 50ml LB li 37°C/6 - giot (OD = 0,5 - 0,7) - Ly tam 6ng 5000 v.p./5 phut lly can VK Riia te bao VK bSng nuoc muoi sinh ly Xac dinh nong dp VK bing phirong phap pha loang VK va cly dim - Bat hoat VK bing formalin 0,05% w/v - Giu o nhiet dp C Sti dung dl gay mien dich vong tuin Bu*c 3: Quy trinh gSy mien djch cho tho - Chia tho nhom dl gay miln dich tao cac loai huylt khang ta rieng biet - Tho dugc gay miln dich lam dot mli dpt each tuin bing each tiem bip lugng 0,3ml huyIn dich co 10'° VK Dpt huyIn dich VK dugc tron them vori 0,3ml complet ajuvant, dpt va huyIn dich VK dugc tron them voi 0,3ml incomplet ajuvant - Lugng VK can thiet de gay mien dich: + Tao huyet idiang V.cholerae da gia, huyen dich VK co x lO' VK V.choleraeae typ Ogawa va x lO' VK V.choleraeae typ Inaba + Tao huylt khang V.cholerae 0139, huyen dich VK co lO'" VK V.choleraeae 0\39 + Tao huylt don gia khang V.cholerae Ol typ huylt Ogawa, huyIn dich VK co lO'" VK V.choleraeae typ huylt Ogawa + Tao huylt don gia khdng V.cholerae Ol typ huyet Inaba, huyIn djch VK co lO'" VK V.choleraeae typ huylt Inaba - Theo doi tinh trang can ndng, bpnh t?it ctia tho suot qui trinh gay mien dich Buo'c 4: Thu thap cdc mau huyet sau gSy miln djch - Thu thap cdc mlu huyet tho vao cdc ngdy tru6c tiln hdnh cdc dpt gay mien djch - Lay ml mdu tmh mach tai tho, chdt lay huylt thanh, gift - 20°C Biro'c 5: Kiem tra hi|u gid khang the gay miin djch - Chuan bi huyen dich VK V.cholerae 01 typ huylt Ogawa, Inaba va 0139 nuoc muoi sinh ly (lO'vk/ml), cdc VK dugc thu hoach vo khuan tren moi truong canh LB da nuoi cay 6-8 gio - Pha loang huyet bac nuoc muoi sinh ly: n6ng dp 1/2; 1/4, 1/8; 1/16; 1/32; 1/64 vd 1/128 - Nho 50)il ciia m6i dam dp pha loang huylt len be m?t phiin kinh - Nho 50nl dung dich treo khang nguyen vao tung d§m dp huylt pha loang trpn ky va dgc ket qua 30-60 giay - Cdc mau chiing am gom huyet tho chua gay miln dich vd nu6c muoi sinh ly Buffc 6: Thu hoach huylt Tuan thii (ngay thii 42) kl tir dpt miln djch diu tien tho dugc lly mau va thu hoach khdng huylt theo quy trinh duoi ddy: - Hai truoc lay mdu chi cho tho u6ng nuorc (khong an) nhim han che phin protein khde ciia huylt thanh, lipit, •92 Ldy mdu dpng mach co vao cdc Ing Falcon 50ml vo tning, de 4°C/3 gio Ly tam mau 2500 v.p./5 phiit Chdt huylt Tilp tuc ly tam huylt 10.000 v.p /20 phiit dl loai bo can Bat hoat bl thi nhiet dp 56°C vong gio Voi huylt da gia khdng V.cholerae Ol vd 0139 cho them NaNs vao huylt v6i nong dp culi la 0,1% - Bong ong 1ml giir o nhiet dp-20''C Btforc 7: Hap phu cheo tao huyet khdng V cholerae doTi gia • Huyet do-n gia khdng Ogawa: - Chuan bi khdng nguyen: nuoi cly chiing V cholerae 01 typ huylt Inaba moi trudng canh thang LB, ti 37°C/20 gio Gat tl bdo VK bang cdch ly tam 3000 v.p./15 phut Rtia tl bdo bing nuoc muli sinh ly 9%o - Pha loang huyet ctia tho gay miln dich bang chting V.choleraeae Ol typ huylt Ogawa vl cimg mot hieu gia 1/30 bing NaCl 9%o - Hap phu huyet thanh: trpn 1ml huyet vol lg te bdo VK U hon dich 42°C /I tieng be li nhiet De hon dich o 4''C 20 gio Ly tam (3000 v.p ^ 15 phiit) thu nuctc noi Lpc vo trimg nuoc nii bing mdng lpc vo triing 0,45 (im - Kilm tra khdng huylt sau hip phu bang phdn ting ngung ket tren phiin kinh voi V.cholerae 01 typ huylt Ogawa va Inaba • Huyet don gia khang Inaba Lam gilng nhu voi huylt don gia Ogawa nhung khdng nguyen su dimg la chiing Vcholerae Ol typ huylt Ogawa vd huylt cua tho gay mien djch bing chung V.choleraeae 01 typ huyet Inaba Bu^C 8: Danh gia d§ nh?y va d?c hieu cua huyet khang ta - Sii dung dan mdu chuan de ddnh gid dp nh?y va dg d|c hifu ciia huylt khdng ta dac hi?u bing phan img ngung kit phiin kinh - Ddnh gid huylt khdng td tren cdc mlu lam sdng Buoc 9: Tao huyet khang td thdnh phim thdnh phim - Pha huylt khang td ddc hi?u tir huylt d^m d|c - Kilm tra khd ndng ngung kit tryc tiep vdi VK td trSn phien kinh - Dong lp 2ml, dan nhan Bdo quan huyet thdnh pham d 4''C 94 Phu lyc 25: HUOfNG D A N S C DUNG HUYET THANH K H A N G TA Myc dich sir dyng: Huyet khdng td dac hieu diing dl chin dodn xdc dinh VK td bdng phan ling ngung kit true tilp tren lam kinh, iing dung dl chin dodn VK ta phong xet nghiem Nguyen ly ky thuat: Huyet khdng ta dac hieu dua tren nguyen ky kit hgp khang nguyen khdng the: Thanh phan ciia huylt co chiia khang the da dong dac hieu voi khdng nguyen O cua V.cholerae, mli loai duoc chiia cac lp rieng Thanh phan huyet khang td: Bp huyet khdng td gIm lp, mli lp co 2ml huylt mSi loai: - Huyet da gid khdng V.cholerae Ol - Huyet da gid khdng V cholerae 0139 - Huyet don gid khdng Ogawa - Huyet don gid khdng Inaba Cach SV dyng: Buoc 1: Nho mot gipt huyet len lam kinh Buoc 2: Ldy khudn l£ic nghi ngo trpn deu vcti gipt khang huyet Budc 3: Quan sdt phdn irng ngung ket vong 30-60 giay duoi nen den bdng mat thudng hoac bang kinh lup Thai han sa dyng va cac luu y can thiet: - Thdi gian su dung 24 thdng kl tir ngdy sdn xuit 95 Bdo quan d 4°C Huylt co chira 0,1% sodium azide co the phan ting vdi chi hoSc ding tao hdn hgp kim lo?i + azide co khd ndng gay no De phong trdnh azide tilp xuc vdi cdc dudng ong din nudc, lo^ii b6 cac hda chit cdn sii dyng binh chiia bdng nhya ho$c thUy tinh 9« Phy lyc 26: TIEU C H U A N CO S d CUA HUYET THANH K H A N G T A ^ Huyet khang ta dac hieu phai dat tieu chuln ciia TCYTTG vl dp nhay va dp dac hieu theo dan mdu kilm tra dl sir dung chin doan V.cholerae Ol va 0139 bing phdn img ngung kit true tilp tren phiin kinh ^ De sti dung > Dp dac hieu cao 100% > Dp nhay cao 98% > Trong dieu kien bao quan chuln (4°C), huylt khang td cd han su dung 24 thdng Phy luc 27: KET QUA KIEM DJNH CUA iViOT S hOJ^T HVVtT THANH K H A N G T A DA S A N X U A T I.N lill'M OINII (iVin (.IA VAC VIN VA.SINH PHAM V Tf: Hl.> \Tf, Vl^T N.AM iiariMBBM- ;Mai jiii,o I'Hii'^i; 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