D Nhd khang nguySn
n Nhd huyit thah thd
3.1.1. Qui trinh sdn xuat khdng huyit thanh Sd
Qui trinh tdm tdt
Virut Sdi chiing Edmonston
Ham luong khdng nguyen: 10^'°CCID50/0,5inl
Thd
Huyet thanh
Kidm djnh: Hieu gia, vd trung
Qui trinh chi tidt
Buac Ị Sdn xudt khdng nguyen Sai
Virus Edmonston Verỏ dugc sdn xudt tren te bdo vero tao virut dugc ggi la Edmonston Vero 7/P2. Hon d(ch virut nay sau khi gat dugc ly tam bd t^ bdo va sau do dugc Igc vdi mang igc 0,65. Hon djch dugc thir vd triing vd kiem tra hieu gid, hỉu gid sir dung cho gay đp img mien dich idlỐ''CCID50/0,5ml
Buac 2. Chpn Igc vd cdch ly thọ Thd mua ve dugc cdch ly trong 7-10 ngdy, thd dugc kidm tra v^ can n$ng, b|nh ngodi da, huyet thanh Sdị Tieu chudn tho sic dung: Khdng huyit thanh Sdi
trudc khi thii nghiSm <4 don vi; Khde manh, khdng bi bat ky benh ngoai da nao; Can ndng: 2,0kg den 2,5 kg; Thd dugc each ly tir 7-10 ngdy
Bdng 3.1. Qui trinh gay dap img miln djch t^o khdng huyet thanh Sdi tren thd Phuong phdp vien Kitasato-Nhat ban
Stt 1 2 Ngiy 0 07 Lay miu Mo Cich tiem
Tifm 2,5 ml hdn dich virut + 2,5 ml ti chat FCA
Tifm tiong da nhieu vi tri, 0,1 ml/1 vj tri
Tiem 5 ml hdn dich virut khdng ti chit Tiem vio dudi da dCii 2 ben, mdi ben
3 4 5 6 14 28 42 49 Ml Lay mau giiia ky M2 Lay miu loan bp 2,5 ml
Tiem 5 ml hdn dich virut khdng ti chat Tiem vao dudi da diii 2 ben, mdi ben 2,5 ml
Tiem 10 ml hon dich virut khdng ti chat
Tiem vao dudi da diii 2 ben, mdi ben 5 ml
Tiem 10 ml hdn dich virut khdng ti chit
Tiem vio tinh mach tai
Gay mien dich mui 1, tiem 2,5 ml khing nguyen + 2,5 ml ti dupe (ti dupe hoin toin), tiem trong da vimg lung. Gay mien dich mui 2 sau tiem mui 1 li 7 ngiy, tiem 5 ml khing nguyen, tiem dudi da diiị Giy mien dich mui 3 sau tiem mui 2 li 7 ngiy, tiem 5 ml khing nguyen, tiem dudi da diiị Giy mien djch mui 4 sau tiem mui 3 li 14 ngiy, tiem 10 ml khing nguyen, tiem dudi da diiị Gay mien dich miii 5 sau tiem mui 4 li 14 ngay, tiem 10 ml khing nguyen, tiem tmh mach taị
Budc 3. Ldy mdu
Liy mau kilm tra huyet thanh Sdi 3 lan, lan 1 trudc khi giy miln dich mui 1; Lin 2 liy mau tai hirdc khi giy miln dich mui 4 dl kilm tra khi nang tao huylt thanh giiia ky; Lin 3 liy miu toin bp sau 7 ngiy gay miln dich mui 5 (ngiy tilir 49 kl tir ngiy gay miln dich mui 1)
T^t ngiy thii 49 liy toin bp miu thd vao chai det, bio quan 4°C qua dem , tich huyet thanh, ly tim 3000 vdng / phut trong 20 phut, liy huylt thanh, sau do bio quin - 30°C cho tdi khi sir dung.
Budc 4. Kiem dinh huyit thanh
Kiem tra vd khuan
Xic dinh hifu gii huylt thanh bing phuong phip tiling hoi tien tl bio vero mpt ldp. Huylt tiianh dupe bat boat 56°C ti-ong 30 phut, sau đ dupe pha bang bac 2 vi dupe tnmg hda vdi 100 don vj khing nguyen Sdi, hdn dich trung hoa dupe giy nhilm trfn tl bio vero 1 Idp, sau 2 tuin dpc kit quị Hifu gii khing huylt thanh la ndng dp huylt thanh pha loang nhit ma tai đ tao ra phin img trung hoi (tl bao binh thudng khdng bj buy hoai)
3.1.2. Qui trinh sin xuat khing huyit thanh Bgi lift
Him lirang khing nguySn: 10'-°CCIDS0/0,lml
Thd
Huyet thanh
1
Kiem dinh: Hi|u gia, vd trung
Qui trinh chi tidt
Buac 1. Sdn xudt khdng nguyen
Sdn xuat khdng nguyin bai li$t: Nhdn virut bai liet typ I, hodc typ II hodc typIII tren te bao
vero mgt (dp vdi nong dO 1 CCID50 trong 300 td bdo vdi mdi trudng gay nhiem DMEM tgi nhiet dg 37 C trong 72-96 gid, gat hdn dich virut khi huy hoai te bdo >90%.
Tinh sgch khdng nguyin: Hon djch virut sau khi dugc đng tan 3 \hn, ly tam 3000
vdng/phut trong 30 phut, ldy nude ndị Cdn dugc sieu dm vd ly tam lay nude noị Nuoc ndi hgn lai vd dugc Igc vdi cd Igc lan lugt 0,65 |im, 0,45 ^m, 0,2 |im. Hdn dich virut dugc sieu loc vdi cd đc 100-200 lan va dugc sieu li tdm 36.000 vdng/phut/3 gid tai 4'^C, lay c^n, hda loang c|in bdng BMS, pha loang sao cho ndng do virut 10^'°CClD50/0,lml. Hdn dich virut ndy dugc chia nhd vd bdo qudn tai -80°C cho tdi khi sir dung. Chi đng tan 01 lan trudc khi su dung.
Buac 2. Chpn IQC vd cdch ly thọ Thd mua ve dugc cdch ly trong 7-10 ngay, thd dugc kidm tra ve cdn ndng, benh ngodi da, huyet thanh Sdị Tieu chudn tho sic diing: Khdng huyit thanh Bai
liet trudc khi thOr nghiem <4 don vi; Khde manh, khdng bi bat ky b?nh ngodi da nao; Can ndng: 2,0kg den 2,5 kg; Thd dugc cdch ly tu 7-10 ngay
Bang 3.2.Qui trinh gay dap ung miln dich tao khang huyet thanh Bai lỉt tren thd
Stt 1 2 3 4 5 6 7 Ngiy 0 14 28 42 56 70 84 Mo Liy miu tai
Ml Lay mau tai
M2 Lay miu tai
M3 Lay miu tai
M4 Lay mau tai
M5 Lay mau tai
M6 (Liy miu dpng
mach cinh)
Tiem 0,2ml hdn dich virut + 0,2m ti chit FCA, tiem trong da nhifu vi tri, 0,1 ml/1 vi tri
Tiem I ml hdn dich virut + I ml ti chat FIA Tifm vio co diii 2 ben, mdi ben 2 vi tri, mdi vi tri 0,5 ml
Tifm 1 ml hdn dich virut + 1 ml t i chit FIA Tiem vio co did 2 bfn, mdi ben 2 vj tri, mdi vi tri 0,5 ml
Tifm 1 ml hon dich virut + 1 ml ta chit FIA Tiem vio co diii 2 ben, mdi ben 2 vj tri, mdi vi tri 0,5 ml
Tifm I ml hon dich virut -(-1 ml ti chit FIA Tiem vao co diii 2 ben, mdi bfn 2 vi tri, mdi vi tri 0,5 ml
Tiem 1 ml hdn dich virut + 1 ml ti chat FLA Tifm vio ca did 2 ben, mdi bfn 2 vi tri, mdi vi tri 0,5 ml
Tidm 6 l4n, l4n thii nhdt tiem trong da lung, nhieu vi tri su dung td chat hodn chinh. Tiem nhttng lan sau, tiem vdo ca dui 2 ben, sir dung ta chat khdng hodn chinh
Buac 3. Ldy mdu vd thu huyet thanh
Lay mdu 7 lan, lan thii nhdt trudc khi gdy dap iing mien dich lan 1, cdc lan tiep theo lay mdu ngay trudc mdi Idn gdy dap ung midn dich tidp theo; Idn cudi cimg liy toan bg mau vdo ngdy thir 84.
Mdu dugc ly tdm 3.000 vdng/phiit/lO phut, ldy huyet thanh, bao quan huyet thanh rieng re ciia tung thd tai nhiet do -20'*C cho toi khi sii dung
Bu&c 4. Kiem dinh huyit thanh
Ki^m tra vd khudn
Xdc djnh hi^u gid huy^t thanh bdng phuong phap trung hod trdn td bao Hep2 mdt ldp. Huydt thanh dugc bat boat 56°C trong 30 phiit, sau do dugc pha loang bac 2 vd dugc trung hda voi 100 don vj khdng nguyen Bai lỉt, hon dich trung hod dugc gdy nhiem tren te bdo Hep2, sau 5-7 ngdy dgc ket qud. Hỉu gid khdng huyet thanh Id ndng do huyet thanh pha loang nhat md tai đ tao ra phdn ling trung hod (td bdo bhih thudng khdng bi huy hoai)
3.1.3, Qui trinh sdn xuat khdng huyet thanh Rota
Qui trinh tdm tdt
Virut Rota G1P8, Wa, DSl, RRV, RRV mix
Hdm lupng khdng nguyen: lÓ^CCIDSO/lml
Huylt thanh
Kiem djnh: Hi^u gia, vd trOng Qui trinh chi tiet
Budc 1. Sdn xuat khdng nguyin
Ti bdo MA 104 mgt Idrp hodc td bdo vero mgt ldp, d?p dugc sir dyng cho su nhan len cua
virut Rotạ Te bdo dugc rira 2 lan vdi mdi truorng DMEM dm, sau đ dugc dat tai 37°C it nhat 3 gid.
Virut dugc hôt hda vdi trypsin nong dg 15^1/ml tgi nhi^t dg 37°C trong I gid, sau do hip phy virut !en te bdo tai nhi^t dg 37*'C trong 1 gid
Bd het mdi trudng gay nhiem, riia te bao 01 lan bdng mdi trudng DMEM im khdng co
trypsin, sau đ cho thdm mdi trudng cd trypsin vdi ndng d^ 10-20ng/ml. D|t chai ti bdo da gdy
nhiem virut tai nhỉt dg 37''C trong 3 ngdỵ
Gdt hon dich virut, đng tan 3 lan, ly tam 3.000 vdng/phut/30 phut bd cdn.
Nude noi thu dugc sieu ly tdm 25.000 vdng/phut/2 gid t£ii 4'^C vdi nong do dudng 40%. Bd nude noi, thu can vd hda can vdi TNC, sau đ pha loang khang nguyen thu dugc sau cho lugng vimt khodng 10''-10^ ffii/ml
Bu&c 2. Chpn Igc vd cdch ly tho
Thd mua vd dugc cdch ly trong 7-10 ngdy, thd dugc kiem tra ve can nang, benh ngodi da,
huyet thanh Sdị Tieu chudn thd sir dung: Khdng huyet thanh Rota trudc khi thir nghiem <4 don vi;
Khde m^nh, khdng bj bat ky b?nh ngodi da nao; Can ndng: 2,0kg den 2,5 kg; Thd dugc cdch ly tir 7-10 ngdy
Bang 3.3.Qui trinh gdy dap img midn dich tao khang huyet thanh Rota tren thd
Qui trinh Trung tdm Phong chdng va Kiim sodt benh tdt CDC-Hoa Ky
Stt 1 2 3 4 5 6 7 Ngay 0 14 28 42 56 70 84 Lay mau Mo Lay miu tai
MI Lay mau tai
M2 Lay miu tai
M3 Lay mau tai
M4 Lay mau tai
M5 Lay mau tai
M6 (Liy miu d^ng
m^ch canh)
Cich tifm
Tifm 0,2ml hon dich virut + 0,2m ta chat FCA, tifm ti:ong da nhifu vi til, 0,1 ml/l vi til
Tifm 1 ml hdn dich virut + 1 ml ta chit FL^ Tifm vao ca diii 2 bfn, mdi bfn 2 vi tri, mdi vi tri 0,5 ml
Tiem 1 ml hdn dich virut + 1 ml ta chit FLA Tifm vio ca dui 2 bfn, mdi bfn 2 vi tri, mdi vi til 0,5 ml
Tiem 1 ml hdn dich vuut + 1 ml ta chit FIA Tifm vio ca dui 2 bfn, mdi bfn 2 vi tri, mdi vi til 0,5 ml
Tifm 1 ml hdn dich vuut + 1 ml ta chit FIA Tifm vao co diii 2 bfn, mdi ben 2 vi tri, mdi vi tri 0,5 ml
Tifm 1 ml hdn dich virut + 1 ml ta chit FIA Tiem vao co diii 2 bfn, mdi bfn 2 vi tri, mdi vi tii 0,5 ml
Tiem 6 Idn, Idn thii nhdt tiem trong da lung, nhieu vj tri su dung td dugc todn phdn. Tidm nhihig lan sau, tiem vao ca diii 2 ben, su dung td dugc khdng todn phin
Bvd^c 3. Lay mau vd thu huyet thanh
Lay mdu 7 lan, lan thii nhdt truoc khi gay đp ting mien dich lan 1, cdc lan tiep theo liy mdu ngay trudc mdi lan gay dap iing mien dich tiep theo; Idn cudi cimg ldy todn bg mdu vdo ngay thii 84.
Mdu dugc ly tdm 3.000 vdng/phiit/lO phiit, lay huyet thanh, bdo quan huyet thanh rieng re cua tiing thd tai nhiet dg -20°C cho tdi khi sir dung.
Bvd'c 4. Kiem dinh khang huyet thanh Kiem tra vd khudn
Xdc djnh hi^u gid khdng huyet thanh bdng phuong phdp ELISẠ huyet thanh dugc pha lodng b^c 4 . Hỉu gid huyet thanh Id ndng do pha loang nhat md tai do xdy ra phdn iing khang nguyen khdng thẹ