8122 2010 BdYTf BAO CAO idl QUA NGElfiN Ctfl] fli T.4l Clp BO nUESM ecu rjNG DtJNG QUI TRINH SAN XUXT KHANG HIJYf F THANH Sdl, BAILE^, ROTA StJTOWG SQ^M BBStH vAC SIN Cbunhttind^ai: PGS.TS.L«TMl«te Cff quan (Td ch6c) did hi S« Oil: Trung tftm Ngbi«n c«iu Sin %.u& Vic siii v^ Sinfe phlas Y t* Nam - 2010 BOYTE —SOQGS— BAO CAO KET QUA NGHIEN ClTU DE TAI CAP BO Ten de tai: NGHIEN Ctru tTNG DVNG QUI TRINH SAN XUAT KHANG HUYET THANH SOU, BAI LIfT, ROTA SlJ DVNG KIEM SpjH VAC XIN Chu nhifm de tii: PGS.TS.Lg Thi Luan Ctf quan chu tri de tai: Trung tam Nghien cuu San xuat V^c xin va Sinh pham Y te Cap quan ly: B9 Y te Ma so de tai (neu c6): Tlioi gian tliirc hi^n: ttr thang 7 nam 2007 den thang 12 nam 2009 Tong Idnh plii tliuc liif n de tai: 910 trif u dong TrODg do: Idnh phi SNKH : 598 trifu dong Nguon khac (neu co); Nam 2010 p(/0 ( 2MO Boo cdo kit qua nghien cuu Se tai cap Bo 1. Ten de tai: NGHIEN ctru tTNG DVNG QUI TRINH SAN XUAT KHANG HUYET THANH sen, BAI LIET, ROTA SU" DVNG KIEM DpVH VAC XIN 2. Chu nhifm de tai: PGS.TS.Le Thj Luan 3. Cor quan chu tri de tai: Trung tam Nghien ciru, San xuat Vac xin va Sinh pham Y te 4. Cv quan quan ly de tai: B9 Y te 5. Thu- Ity de tai: 6. Pho chu nhif m de tai ho^c ban chii nhi|m de tai (neu c6): 7. Danh sach nhimg nguM thuc hien chinh: Nguyin DangHiSn Nguyen Thj Quy Le Trung Diing Nguyin Thiiy Huong Nguyen Thi Mai Huong TrSn Bich Hanh Dang Mai Dung Nguyin NO Anh Thu Ngo Thu Huong 8. Cac dl tai nhanh (de mi^c) ciia de tai (neu c6) 9. ThM gian th^c hi|n de tai: tu thang 7 nam 2007 dSn thang 12 nam 2009 Cac chir viet tat BS (Bovin serum) PBS (Photphat buffer solution) P-S (Penixilin - Streptomycin) MEM (Minimum Eagle Medium) FBS (Fetal Bovine Serum) : Huyet thanh be : Dung dich dem muoi phot phat : Khang sinh Penixilin va Streptomycin : Moi truong Eagle toi thieu : Huyet thanh be bao thai DMEM (Dubeco Minimum Essencial Medium): Moi truong ca ban t6i thi6u CPE (Cytopathic effects) : Huy hoai tk bao FFU(fluorescence forcus unit) : Bon vi mien dich huynh quang CCID50(cell culture infectious dose 50) : Lieu gdy nhilm 50% tl bao IgG (Immuno Globulin 0) IgA (Immuno Globulin A) IgM (Immuno Globulin M) IgE (Immuno Globulin E) IgD (Immuno Globulin D) MAI 04 Vero Hep2 Da FIA (Freund's Incomplete Adjuvan) FCA (Freund's Complete Adjuvan) WHO (World Health Organization) : Khang the IgG : Khang thi IgA : Khang the IgA : Khang thi IgA : Khang the IgA : Te bao than bao thai khi Rhesus : T8 bslo than khi xanh Chau Phi ; Te bao ung thu thanh quan nguai : Dalton : Ta dirge khong hoan toan : Ta dugc hoan toan : To chuc Y te The gi(5i Bao cao torn tat de tai Bit van d€ Trung tam Nghien ciiu San xult Vic xin vi Sinh phSm Y te hay Trung tam NghiSn cuu San xult Vac xin Sabin dugc thanh lap tii nam 1994 c6 nhiem vu san xuat v4 kiem dinh vac xin. Hien nay Trung tam dang san xuat va kiem dinh vac xin Bai liet, vac xin Soi; nghien cuu, san xuat vac xin Rota phong tieu chay cho tre em. He thong kiem dinh cua Trung Tam duoc dao tao co ban va chuyen sau phuc vu cong tac kiem djnh chat lucjmg vac xin. Mot trong nhung nguyen vat lieu co ban sit dung danh gia an to4n cua vac xin bin thanh pham trong phong thi nghiem la huySt thanh khang Soi, khang virut Bai lift, vk khang virut Rota. Khang thi nay s\jt dung cho thii nghiem tim tdc nhan ngoai lai trong h5n djch vac xin san xuat. D5ng thoi khang the nay ciing sii dung de chuan dO hieu gia vie xin Rota va vie xin Bai liet thanh phAm. Nhiing san pham nay truac dJy dugc cung cap hoac mua tir cac nha san xuat Nhat, My. Tuy nhien de chu dgng trong cong vi?c, Trung tam thuc hien de tai "Nghien ciiu ling dung quy trinh san xudt khdng huySt thanh Soi, Bai liet. Rota sii dung kiSm dinh vie xin" vdi nhiing muc dich sau: 1. LTng dijng vk hoan thien qui trinh san xuat huySt thanh dac hiSu vurut Soi, Bai lift. Rota sii dymg trong kifm djnh vac xin; 2. San xuit dugc huySt thanh khang vurut Soi. Bai liet. Rota dat tieu chuin co sd; 3. Danh gia chat lugng huyet thanh ve tinh dac hieu, hieu gia so vdi huySt thanh nhap ngoai Nguyen v|t lien va phinrag phap tiln hanh De CO dugc khang the, trade het de tai pliai san xuat khang nguyen, phuong phap san xuat khang nguyen Soi, Bai liet. Rota la sii dung chung virut da dugc cung cap tir cac phong thi nghiem chuan quoc tS hojc chung san xuat vac xin nhan len tren nuoi cay te bao thich hop vdi the tich va hieu gia du de sau khi tinh sach gay dap ling miln djch t6t. Sau khi nhan virat tren te bao, h5n dich virut dugc dong tan, ly tam, lay nude noi sau do tinh sach qua dudng Sacahrose 40%, lay can, hoa can , ly tam 15.000 vdng /phut/40 phiit, lay nude noi, dieu chinh sao cho hieu gia theo lieu gay nhilm thich hgp ciia timg loji virut: 10*'° CCID 50/0,5ml cho virat Soi; 10''° CCID 50/0,lml cho virat B?ii liet va lO''" ffii/lml cho virat Rota. Thd dugc mua tii trai thd Ba vi trgng lugng tir 1,5 kg din 2,5 kg, khoe manh, dugc cich ly it nhat 10 ngay trade khi sii d\mg, khang thi khong qua 4 don vi vdi khang nguygn die hieu. Don vj khang thi d day dugc hieu la hi?u gia cua khang huylt thanh vi dij: Hifu gia khang huyet thanh khi chuin df cho ket qua 4.000 va dugc coi la 4.000 don vi khang huyet thanh. Sii d\mg ta chit hoan chinh (FCA) hoac ta chit khong hoan chinh (FIA) pha chl h5n dich khang nguySn, tiiy loai khang nguyen c6 Hch v4 lilu ti6m khac nhau. Doi vdi virat Sdi cd 2 phuong phap tao khang thi, do la phuong phap cua vien Kitasato Nh|t Ban va phuong phap cua Trung tam phong chdng va kiem soat b$nh tat CDC-Hoa k^. hai phuong phap niy khac nhau ve chuin bi khang nguyen, va lich tiem cv thi nhu sau: Phuong phap san xuat khang huylt thanh Sdi khong qua tinh sach vk phuong phap gay dap ling miln dich trSn tho (Phuong phap vien Kitasato-Nhat ban) Thuc hifn 5 lin gay dap ling miln dich theo lich ngay: 0 : 7 : 14: 28 : 42. Lay mau (5ml) trade khi tiem lin l(Mo), 5ml trudc khi gay mien dich mQi 4 (Ml)va liy toto b$ mau vko ngay thii 49 sau tiem Ian 1(M2), nhu vjy se co 3 mau huylt thanh kiem tra hif u gia. Lilu lugng tiem moi Ian nhu sau: Tiem lin 1 ngay 0: Tiem 2,5ml hdn dich virut + 2,5 ml ta chit FCA; Tiem trong da nhilu vj tri, 0,1 ml/1 vi tri; Tiem Ian 2 ngay 7: Tiem 5 ml virat Sdi khong cd ta chat; Tiem vao dudi da diii 2 bta, m6i bSn 2,5 ml; Tiem lin 3 ngay 14: Tiem 5 ml virat Sdi khong cd ta chat; Tiem vao dudi da dCii 2 bgn, m5i ben 2,5 ml; Tiem Ian 4 ngky 28: Tiem 10 ml virut Sdi khong cd ta chat; Tiem vao dudi da diii 2 ben, mdi ben 5 ml; Tiem Ian 5 ngay 42: Tiem 10 ml virat Sdi khong cd ta chat; Tiem vao tinh mach tai 2 ben, m5i ben 5 ml. Phnvng phap san xuat khang huyet thanh Bai liet. Rota theo phuong phap gay d4p ung miln djch cua Trung tam Phong chong va kiem soat benh tat CDC-Hoa Ky Thd dugc each ly tir 7-10 ngay, gay dap img mien dich 6 Ian tiem each nhau 2 tuin vko ngay: 0:14:28:42:56:70; 84 liy mau tai 5ml trade khi tiem lin 1 va ngay trade khi tiem cao Ian sau, lay mau toan bg cu6i cilng vao ngay 84 sau lin tifm 1. Nhu vay se cd 7 mlu huylt thanh (MO, Ml, M2, M3, M4, M5, M6) hoac se co 6 miu mau khi Hch liy mau 0:28:42:56:70:84 (Mo, Ml, M2, M3, M4, M5) Lich va lilu tiem cu thi: Tiem lin 1 ngay 0: Tiem 0,2nil virat B^i li?t + 0,2ml ta dugc FCA; Tiem trong da nhilu vi tri, 0,1 ml/1 vi tri; Tiem Ian 2 ngay 14: Tiem 1ml virat Bai liet + 1ml ta dugc FCA; Tiem vao co dii 2 bfn, mSi ben 0,5 ml; Tiem Ian 3 ngay 28: Tiem 1ml virat B?i lift + 1ml ta dugc FCA; TiSm vao co diii 2 ben, mdi bto 0,5 ml; Tiem lin 4 ngay 42: Tiem 1ml virat Bai lift + 1ml ta dugc FCA; Tiem vao co diii 2 ben, mdi bfn 0,5 ml; Tiem lin 5 ngay 56: Tiem 1ml virat Bai lift + 1ml ta dugc FCA; Tiem vao co dCii 2 ben, mSi ben 0,5 ml; Tiem ISn 6 ngay 70: Tiem 1ml virat Bai lift + 1ml ta dugc FCA; Tiem vao eg diii 2 ben, mdi ben 0,5 ml; Sau mSi lin tiem, lay mau kilm tra diin bien dap ling khang the va chon ra qui trinh thich hgp cho hieu gia khang the cao nhat. Sii dung phuong phap trang hoa vi lugng tren te bao vero xac dinh khang huylt thanh Sdi va phuong phap tnmg hoa vi lugng tren te bao Hep2 xac dinh khang huylt thanh Bai lift. Xac dinh khang huylt thanh Rota bang phuong phap ELISA. Dong thdi sii dung phuong phap trung hoa de ling dung khang the trong thii nghif m tim tac nhan ngoai lai trong hdn dich virat vacxin khi trang hoa hoan toan me gjt don. Tim ra lugng khang huylt thanh trang hoa hoan toan me gat don va ap ung trong kiem dinh vie xin; Khang huyet thanh san xuat ra con dugc sii dmg vao thu nghiem xac dinh nhan dang virut vacxin va xac dinh hieu gia virat vacxin bang each tim don vi khang thi ap dung cho thu nghiem nay. Ket qua 1. Qui trinh san xu^t khang huylt thanh l.I. Qui trinh san xuat khang huyet thanh Soi Bu&c 1. San xudt khang nguyen S&i Viras Edmonston Vero7 dugc san xuat tren te bao vero tao virat dugc ggi la Edmonston Vero 7/P2. H5n dich virat nay sau khi gat dugc ly tam bd te bao va sau do dugc Igc vdi mang Igc 0,65. Hdn dich dugc thii v6 triing va kiem tra hieu gia, hifu gia SU dung cho gay dap ling miln dich la 10'-°CCID 50/0,5ml Buac 2. Chon Igc va each ly tho. Thd mua vl dugc each ly trong 7-10 ngay, thd dugc kiem tra v6 can njng, benh ngoai da, huylt thanh Sdi. Tieu chuan tho sii dung: Khang huyet thanh Sdi trade klii thii nghifm <4 don vi; Khde manh, khong M bit ky bfnh ngoai da nao; Can njng: 2,0kg din 2,5 kg; Thd dugc each ly tir 7-10 ngay. Qui trinh gay dap img mien dich tao ichdng huyet thanh S&i tren tho, /"huong phap vifn Kitasato-Nh$t ban Stt 1 2 3 4 5 6 NgJy 0 07 14 28 42 49 Lay mau Mo Ml Lay mau gitia ky M2 Lay mau toan bo Cach tiSm Tiem 2,5ml hdn dich virat + 2,5 ml ta chit FCA; TiSm trong da nhilu vi tri, 0,1 ml/1 vi tri Tifm 5 ml virat Sdi khong cd ta chat; Tiem vao dudi da diii 2 ben, moi ben 2,5 ml Tiem 5 ml virat Sdi khong cd ta chit; Tiem vao dudi da diii 2 bSn, moi b6n 2,5 ml Tiem 10 ml virat Sdi khong cd ta chat; Tiem vao dudi da diii 2 ben, moi ben 5 ml; Tiem 10 ml virut Sdi khong co ta chat; Tiem vao tinh mach tai 2 ben, moi ben 5 ml Budc3.Lay mau Lay mau kiem tra huyet thanh Sdi 3 Ian, lin 1 trade khi gay miln dich mili 1; Lan 2 lay mau tai trade khi gay mien dich miii 4 de kiem tra kha nang tao huyet thanh giila ky; Lan 3 lay mau toan bg sau 7 ngay gay miln dich miii 5 (ngay thii 49 ke tit ngay gay miln dich miii 1) Tai ngay thii 49 liy toan bg mau thd vao chai det, bao quan 4*'C qua dSm , tach huylt thanh, ly tam 3000 vong / phiit trong 20 phiit, liy huylt thanh, sau dd bao quan - 30°C cho tdi khi sii dung. Budc 4. Kilm dinh huylt thanh Kif m tra v6 khuin xac dinh hieu gia huyft thanh bjng phuong phap trung hoa tren te bao vero mdt Idp. Huyet thanh dugc bat boat 56''C trong 30 phiit, sau do dugc pha loang bac 2 va dugc trang hda vdi 100 dgn vi khang nguyen Sdi, hon dich trung hoa dugc gay nhiim tren te bao vero 1 lop, sau 2 tuin doc ket qua. Hifu gia khang huylt thanh la nong do huyet thanh pha loang nhat ma tai dd tao ra phan ling trang hoa (tl bao binh thudng khdng bi huy hoai) 1.2. Qui trinh san xuat khdng huylt thanh Bai lift Bird^c 1. S&n xuat kh^ng nguyen San xudt Ididng nguyen bgi lift: Nhan virut bai lift typ 1, ho5c typ II hoSc typlll tr6n tl bao vero mpt Idp vdi nong d0 1 CCID50 trong 300 te bao vdi moi trucrag gay nhiem DMEM t^ii nhift d0 37°C trong 72-96 gid, gSt hon dich virut khi huy hoai tl bao >90%. Tinh sgch khdng nguyen: HSn dich virut sau khi dugc dong tan 3 lan, ly tam 3000 vdng/phut trong 30 phut, Idy nude noi. Can dugc sieu am va ly tam lay nude noi. Nude no: hpn lai va dugc loc vdi co Igc lan lugt 0,65 pm, 0,45 ^m, 0,2 |xm. Hon dich virut dugc sieu Ipc vdi c8 dac 100-200 lin va dugc sieu li tam 36.000 v6ng/phuf3 gid tai 4''c, lay can, hoa loang c^ bing EMS, pha loang sao cho nong do virut lO'' CCED50/0,lml. Hon dich virut niy dugc chia nho va bao quan tai -80°C cho tdi khi sii dung. Chi dong tan 01 lin trudc khi sii d\ing. Budc 2. Chpn loc vd cdch ly tho. Tho mua ve dugc each ly trong 7-10 ngay, tho dugc kiem tra vl cSn n^g, bfnh ngoai da, huyet thanh Sdi. Tieu chuan tho sic dung: Khdng huyit thanh B?i Uft trade khi thu nghiem <4 don vi; Khoe manh, khong bi bat ky benh ngoai da nao; can n^g: 2,0kg din 2,5 kg; Thd dugc each ly tii 7-10 ngay. Qui trinh gay dap img miin dich tao khdng huyit thanh Bai liet tren tho- Qui trinh Trung torn Phong chong vd Kiim sodt benh tat CDC-Hoa Ky Stt 1 2 3 4 5 6 7 Ngay 0 14 28 42 56 70 84 Lay mau Mo Lay mau tai Ml Lay mau tai M2 Liy mau tai M3 Lay man tai M4 Liy mau tai M5 Liy mau tai M6 (Liy mau dgng mach catih) each tiem Tiem 0,2ml virat Bai hft + 0,2nil ta dugc FCA; TiSm trong da nliieu vi tri, 0,1 ml/1 vi tri; Tiem 1ml virat Bai lift + 1ml ta dugc FIA; Tiem vao co dui 2 bSn, m§i ben 0,5 ml; Tiem 1ml virat Bai lift + 1ml ta dugc FIA; Tiem vao co diii 2 bSn, mSi ben 0,5 ml; Tiem 1ml virat Bai lift + 1ml ta dugc FIA; Tiem vao co diii 2 ben, mdi ben 0,5 ml; Tiem 1ml vurut Bai lift + 1ml ta dugc FIA; Tiem vko co dui 2 ben, mdi ben 0,5 ml; Tiem 1ml virat Bai lift + 1ml ta dugc FIA; Tifm vko co dtii 2 bfn, moi bSn 0,5 ml; Bu&c 3. Lay mau vd thu huyet thanh Liy mau 7 lin, lin thii nhit trade khi gay dap ling miln dich lan 1, cac lan tiep theo liy mku ngay trade mdi lin gay dap irng mien dich tiep theo; lan cudi cung lay toan bg mau vao ngay thii 84. Mau dugc ly tSm 3.000 vdng/phut/10 phiit, liy huyet thanh, bao quan huylt thanh ri6ng rg cua timg thd tai nhift dp -20°C cho tdi khi su diing Bit&c 4. Kiim dinh huyet thanh Kiem tra v6 khuin X4c dinh hifu gia huylt thanh bing phuong phap trung hoa trfn tl bao Hep2 mOt Idp. Huylt thanh dugc bit boat 56°C trong 30 phiit, sau dd dugc pha loang bac 2 [...]... ngiy: 0:1 4:2 8:4 2 Lich va lilu tiem cu the: Tiflra lin I ngiy 0: Tiem 0,2ml virut Rota + 0,2m! ta dugrc FCA; Tiem trong da n h i k vi tri, 0,1 mJ/1 vj tri; Ti6m lin 2 ngiy 1 4: TiSm 1ml vinit Rota + 1ml t i dirijc FCA; Tiem v4o c a diii 2 ben, m5i b8n 0,5 ml; TiSm lin 3 ngiy 2 8: TiSm 1ml virut Rota + 1ml t i dtrpc FCA; TiSm vio co diii 2 bdn, m6i ben 0,5 ml; Tifim lin 4 ngiy 4 2: TiSm 1ml virut Rota. .. huyet thanh thu dirge Bing 3.31 Dinh gii chit luiTng huylt thanh khing Wa qua kilm tra hi?u gii vie xin Rotavin-Ml 98 99 100 101 Bii^3.32 Danh gji chit lugng huylt thanh khang DSl qua ki6m tra hieu gii vie xin Rotavin-Ml 101 Bing 3.33 Dinh gii chit lu(?ng huylt thanh khang RRV qua kilm tra hieu gia vac xin Rotavin-Ml 102 Bing 3.34 Dinh gii chit lugng huylt thanh khang RRVmix qua kiem tra hieu gia vie xin. .. Ueu ca ban sir dung danh gia an toan ciia vac xin ban thanh phim trong phong thi ngliiem la huyet thanh khang Soi, khang virut Rota, va khang virut Rota Khang huylt thanh nay sur dung cho thu nghiem tim tac nhan ngoai lai trong h6n dich vac xin san xuit D6ng thoi khang huylt thanh nay cung sir dyng dk chuin dp hieu gid vie xin Rota va vie xin Bai liet thanh phim Nhting san phim nay truoc day du^c cung... vao ngay: 0:1 4:2 8:4 2:5 6:7 0; 84 Lich va Ulu tiem cu thi: TiSm lin i ngiy 0: TiSm 0,2ml virut Bai liet + 0,2ml ta dupe FCA; Tiem trong da nhiSu vi tri, 0,1 mi/1 vi tri; TiSm lan 2 ngiy 1 4: TiSm Iml virut Bai liet + 1ml ta tlupc FCA; Tiem v ^ CO diii 2 ben, mdi ben 0,5 ml; TiSm lin 3 ngiiy 2 8: TiSm 1ml virut Bai list + Iml ta dugc FCA; TiSm vio co diii 2 bSn, mdi bSn 0,5 ml; TiSm lin 4 ngSy 4 2: TiSm 1ml... 25.600 25.600 6.400 1.10.000 1.10.000 1:2 .500 1:1 0.000 1:5 .000 1:1 0.000 1:2 .500 1:2 .500 1:5 .000 1.638.400 1.638.400 1.30.000 1.30.000 409.600 409.600 27 27 20 27 20 25 30 27 32 1.10.000 1.10.000 235 30 26 56 34 26 60 1.638.400 1.638.400 1: 3.000 1: 3.000 1.30.000 1.30.000 1.638.400 409.600 40 32 1; 3.000 1: 1.000 1.30.000 1.10.000 72 24 17 41 974 Tong sd khang huyet thanh sdn xuat dugc 741,5ml trong dd... hifu gia vie xin thanh phim huyet >8.000 cho thu nghifm xac dinh Typ II >16.000 thanh tac nhan ngoai lai bai 16.000 cho thir nghifm xac dinh Typ III >16.000 tac nhan ngoai lai . cuu San xult Vac xin Sabin dugc thanh lap tii nam 1994 c6 nhiem vu san xuat v4 kiem dinh vac xin. Hien nay Trung tam dang san xuat va kiem dinh vac xin Bai liet, vac xin Soi; nghien. tdc nhan ngoai lai trong h5n djch vac xin san xuat. D5ng thoi khang the nay ciing sii dung de chuan dO hieu gia vie xin Rota va vie xin Bai liet thanh phAm. Nhiing san pham nay truac. dung quy trinh san xudt khdng huySt thanh Soi, Bai liet. Rota sii dung kiSm dinh vie xin& quot; vdi nhiing muc dich sau: 1. LTng dijng vk hoan thien qui trinh san xuat huySt thanh