NGHIEN CLFU SAN
X U A TKHANG HUYiT THANH ROTA DUNG CHO KI^M DINH VAC XIN
Le Thj Luan, Ngo Thu Huong
Trung tam Ngltiin cuu, San xuat Vac xin va Sinh pham y te. Ha Noi
TOM TAT
Trung tam nghien cii'u san xuat vac xin va sinh pham y te (POLYVAC) da san xuat du'O'c khang huyet thanh rota dat tieu chuan co so. Tat ca cac khang huylt thanh san xuat deu vo triing. Khang huyet thanh san xuat tif 4 Chung (WA, DS1, RRV, G1P8) deu c6 hieu gia rat cao, thap nhSt la chung G1P8 (6.400). Hieu gia khang huyet thanh rota san xuat tif chung hoang dai (WA, DS1, RRV) cao han chung giam doc lye rat nhieu, hieu gia huyet thanh chung G1P8 giam dpclyc chf bSng 1/4 den 1/16 chung hoang dai. Huyet thanh san xuat tir cac chung Quae ieng dung tot trong Xhu nghiem nhan dang, chuan dp hieu gia vlic xin rota va thir nghiem nghien ciru li§n quan den virut rota (ELISA) thay th6 du-gc khang huyet thanh nhap ngoai TCH khoa: khang huyet thanh rota, khang nguyen virut rota, tho, ta dircc, hieu gia
I. DAT VAN DE
Trung tam nghien cuu, san xuat vacxin va sinh pham y te hien dang nghien ciru san xuat vac xin rota phong tieu chay cho tre em.
vac xin rota dang dugc thu nghiem lam sang giai doan cuoi va dat ket qua tot. Trong thoi gian toi trung tam se tien hanh san xuat vac xin do do kiem djnh chat lugng vac xin la van de can dugc quan tam. Mot trong nhirng nguyen vat lieu co ban dugc su dung de danh gia tinh an toan ciia vac xin trong phong thi nghiem la huyet thanh khang virut rota. Khang huyet thanh nay sir dung cho thu' nghiem nhan dang, chuan do hieu gia bang phuong phap mien djch huynh quang [1,2,3,5,6]... mat khac con sir dung cho cac thir nghiem nghien ciiu lien quan din virut rota (ELISA) [4]. Hien nay khang huyet thanh rota dugc cung cap tir cac nha san xuat cua My voi gia thanh rat cao. De tiet kiem chi phi cung nhu chii dgng trong cong viec, Trung tam da tien hanh san xuat khang
huyet thanh rota tren tho. Trong nghien ciru nay chiing toi dua ra quy trinh san xuat khang huyet thanh rota dat tieu chuan co so su dung cho kiem djnh vac xin va danh gia chat lugng huyet thanh ve tinh dac hieu, hieu gia so voi huyet thanh nhap ngoai.
II. PHU'aNG PHAP NGHIEN ClTU Chung virut rota WA, DSl., RRV do Trung tam kiem soat va phong chong benh tat Hoa Ky (CDC) cung cSp, chiing G1P8 POLY- VAC phan lap va cay truyen giam doc luc.
Virut dugc nhan len tren te bao Ma (doi voi cac chiing WA, DSl., RRV ) hoac te bao Vero (doi voi chiing G1P8) kin mot lop, hon dich virut dugc dong tan, ly tam, lay nuoc noi sau do tinh sach qua duofng Sacahrose 40%, lay can, hoa can , ly tam 15.000 vong /phiit/40 phiit, lay nuoc noi, pha Ioang sao cho hieu gia
10'"ffii/lml.
Tho dugc mua tii' trai tho Ba Vi trgng Tac gia: Le Thj Luan
Dja chi: 135 L6 due, Hai Ba Trung, Ha Ngi Dien thoai: 04.3.9713048; 0904001727 Email: [email protected]
28 Tap chi Y hoc dir phong. Tap XXI, so 3 (121)
lugng tir 1,5 kg den 2,5 kg, khoe manh, dugc dugc toan phan (PCA) hoac ta dugc khong toan each ly it nhat 10 ngay truoc khi su dung, chgn phan (FIA) pha chi h6n dich khang nguyen.
Igc tho CO khang the truoc tiem khong qua 4 Tiem tho, lay mau kiem tra dap irng miln djch don vj voi khang nguyen dac hieu. Su dung ta theo quy trinh ciia CDC (Hoa Ky):
stt
1
2
3
4
5
6
7 Ngay
0
14
28
42
56
70
84
Liy mau
Mo
L3y mau tai kiem tra hieu gia khang the truoc tiem
Ml
Lay mau tai M2 Lay mau tai
M3 Lay mau tai
M4 Lly mau tai M5 L§y mau tai
M6
(Lay mau dpng mach canh)
Cach tiem
Tiem 0,2ml virut rota + 0,2ml ta dugc FCA; Tiem trong da nhiiu vi tri, 0,1 ml/1 vi tri;
Tiem 1ml virut rota + 1ml ta dugc FIA; Tiem vao eg diii 2 ben, m6i ben 0,5 ml;
Tiem 1ml virut rota + 1ml ta dirge FIA; Tiem vao eg diii 2 ben, m6i ben 0,5 ml;
Tiem 1ml virut rota + 1ml ta du'gc FIA; Tiem vao eg diii 2 ben, moi ben 0,5 ml;
Tiem 1ml virut rota + 1ml ta dugc FIA; Tiem vao eg diii 2 ben, moi ben 0,5 ml;
Tiem 1ml virut rota + 1ml ta dugc FIA; Tiem vao eg diii 2 ben, m5i ben 0,5 ml;
_ _ _ _
Mau dugc ly tam 3.000 vong/phiit/lO phut, lay huylt thanh, bao quan huyet thanh rieng re cua tirng tho tai nhiet do -20''C cho toi khi sir dung.
Xac djnh hieu gia khang huyet thanh bang phuong phap ELIZA (CDC, Hoa Ky).
Kilm tra vo khuSn huylt thanh bang phuong phap nuoi cay tren moi truong Thio- glycolate va Soybean casein (POLYVAC).
Su dung khang huylt thanh rota trong cac thir nghiem kilm djnh xac djnh tinh dac hieu, chuan do hieu gia bang phuong phap
mien djch huynh quang (CDC; Hoa Ky) III. KET QUA VA BAN LUAN 3.1. Chon loc tho
Lay mau 25 con tho chiing toi kiem tra khang huyet thanh rota. Ket qua 8 trong tong so 25 con CO hieu gia khang the <4 dugc sir dung de san xuat khang huyet thanh. Chiing toi sir dung 4 chiing virut rota de gay mien djch, tiem 2 tho/chiing; Wa (tho 6 va 18), DSl (tho 8 va 22), RRV (tho 13 va 20), G1P8 (tho 24 va 25).
Tap chi Y hpc du phong. Tap XXI, so 3 (121) 29
Bang 1. Chpn loc tho
Sdtho
01 02 03 04 05 06 07 08 09 10 11 12 13
Can n^ng tripoc tiem(kg)
1.7
1.7
1.5
Can nang sau tiem
(kg)
2.2
2.8
2.3
Khang huyit thanh (Mo)
256 64 64 16 16
<4 16
<4 16384
16 4 16384
<4
Sdtho
14 15 16 17 18 19 20 21 22 23 24 25
Can nang trifo'c tiem(kg)
2.0
1.8
2.0
1.6 1.9
Can nang sau tiem
(kg)
2.8
2.5
2.5
2.7 2.8
Khang huylt thanh (Mo)
4096 16 16 4
<4 4
<4 64
<4 16
<4
<4
Bang 1 cho thay can nang tho truoc tiem phat triln binh thuong.
dap ung miln djch tir 1,5 kg din 2,0 kg; can 3.2. Ket qua dap urng mien dich
nang tho o thoi diem lay toan bg mau tir 2,2kg Kit qua hieu gia khang huylt thanh thu den 2,8 kg. Dieu do chung to sau khi tiem dugc trong qua trinh gay dap ung mien djch the khang nguyen ca 08 con tho diu khoe manh, , hien trong bang 2.
30 Tap chi Y hoc du phong. Tap XXI, so 3 (121)
Bang 2. Hi^u gia khang huylt thanh IgG tho dac hi$u chung virut rota
Hieu gia khang huyet thanh Tho s6
6-Wa 18-Wa 8-DSl
22-DS1 13-RRV 20-RRV 24-G1P8 25-G1P8
MO
<4
<4
<4
<4
<4
<4
<4
<4
M1 102.400
25.600 6.400 1.600 1.600 6.400 2.560 25.600
M2 1.638.400
1.638.400 409.600 409.600 409.600 1.638.400 2.560 25.600
MS 1.638.400 6.553.600 409.600 409.600 409.600 1.638.400 25.600 102.400
M4 1.638.400 1.638.400 1.638.400 409.600 6.553.600 1.638.400 25.600 25.600
M5 1.638.400 6.553.600 409.600 409.600 1.638.400 1.638.400
102.400 102.400
M6 1.638.400 1.638.400 409.600 409.600 1.638.400 1.638.400 102.400 102.400
Bang 2 cho thSy k i t qua hieu gia IgG d6i nay, chung toi nhan thay cac chiing hoang dai voi cac chung Wa, D S l , RRV rk cao. Tho 6 va WA, D S l , RRV gay dap irng m i l n djch cao tho 20 sau gay dap ung m i l n djch 15n 1 hieu hon rat n h i l u so voi chiing giam doc lire G l P8 gia da tang len tuong ung la 102.400 va 6.400, va qui trinh gay dap ung m i l n djch d6i voi cac sau l i l u 2 tang len 1.638.400 va 6n djnh cho toi chiing hoang dai chi nen thuc hien trong 4 lieu sau lieu 6. Tho 8 sau l i l u 4 cao nhit, sau do lai d l dat dugc hieu gia khang t h i cao nhat. Danh giam nhu lieu 2,3; Tho 13 hieu gia cao nhat sau gia tiing chiing dap ung m i l n djch chiing toi l i l u 4, hieu gia cao toi 6.553.600; Tho 18 sau thSy chiing Wa va RRV cho hieu gia cao nhat lieu 3 va 5 cao nhat dat toi 6.553.600. Hieu gia va dong d I u ca 02 tho sii' dung. Chiing DSl khang the doi voi chung G1P8 thap hon nhilu cho hieu gia thap nhat chi dat 'A so voi cac chi dat 102.400 sau l i l u 5 va 6. V o i k i t qua chiing khac.
Tap chi Y hoc du phong. Tap XXI. so 3 (121) 31
3.3.'Danh gia ch§t lu-ong khang huyet thanh 3.3.1. Tieu chuan ca s& cho khang huyet thanh
Bang 3. Tieu chuan khang huylt thanh rota
Tieu chi danh gia Vo triing
Bao quan
Hieu gia khang huylt thanh
Tinh dac hieu khang huylt thanh
Wa DSl RRV G1P8 Wa DSl RRV G1P8
Tieu chuan Hoa Ky Vo triing
-200 0
5.000 5.000
Thong qua thir nghiem nhan dang bSng ky thuat miln djch huynh quang
Tieu chuan co so Vo triing
-200 C
> 1.000.000
>100.000
> 1.000.000
> 6.400
Thong qua \h\jf nghiem nhan dang bang ky thuat miln djch huynh quang
3.3.2. Danh gia chat luffng ciia khang huyet thanh
Bang 4. Chit lu'gng cua khang huylt thanh
Tho
6-Wa 18-Wa 8-DSl 22-DS1 13-RRV 20-RRV 24-G1P8 25-G1P8 Tong
The Tich (ml)
45,0 39,0 51,0 39,0 60,0 48,0 36,0 25,5 353,5
Nhift do bao
quan
-200C -200C -200C -2000 -2000 -2000 -2000 -2000
Vo trung Nhan dang
+ + + + + + + +
Ty le pha Ioang KHT ThCr nghiem hieu gia POLYVAC
1:3.000 1:3.000 1:1.000 1:1.000 1: 3.000 1: 3.000
CDS Hoa Ky
1:200 1:200
32 Tap chi Y hpc du phong, Tap XXI, so 3 (121)
Bang 4 cho thSy tong so huylt thanh thu dugc la 353,5 ml dugc bao quan -20"C Tat ca huyet thanh san xuat ra deu khong bj nhilm vi khuan va nam. Chiing toi da su dung cac huylt thanh nay cho thir nghiem nhan dang va chuin do hieu gia khang nguyen rota bang phuong phap miln djch huynh quang. Voi ty le pha Ioang 1:3000; 1:1000 ; 1:3000; tuong irng voi khang huyet thanh san xuat tir cac chiing WA, DSl, RRV cho ket qua hieu gia khang nguyen rota ciia 3 loat vac xin Rotavin Ml do POLY- VAC san xuat tuong duong khi chiing toi sir
dung khang thi CDS ciia Hoa Ky pha Ioang 1:200. Nhu vay hieu gia khang huyet thanh ma chiing toi san xuat tir cac chung hoang dai tuong doi cao, gap 5-15 lan so voi khang the nhap ngoai.
Kit qua tir cac thir nghiem nhan dang cho thay khang huyet thanh ma chiing toi san xuat dac hieu voi khang nguyen virut rota, cho hinh anh nhan dang dep, ro khi pha Ioang 6 cac nong do tuong u'ng voi nong do thuc hien chuan do hieu gia.
T l bao nhiem virut Rota (nong do 10'^) T l bao doi chu'ng Hinh 1. Hinh anh virut rota diroi kinh hiln vi huynh quang
Chiing toi da sir dung khang huylt thanh nguyen virut rota o nong do pha Ioang 10.000 san xuat til- chiing WA tinh sach qua cot ai luc lin cho kit qua tuong duong voi Kit phat hien Protein A -sepharose va khang thi nay dugc rota (Prospect Kit) cua Uc khang huyet thanh diing trong phan ling ELISA phat hien khang wa pha Ioang 1:10.000.
Tap chi Y hpc dir phong. Tap XXI, so 3 (121) 33
POLYVAC Prospect Kit
Hinh 2. Phan u'ng ELISA phat hien khang nguyen rota sii' dung
Voi kit qua hieu gia khang huyet thanh thu dugc 6 tren chung toi thay tat ca cac khang huylt thanh dIu dat tieu chuan co so de ra.
IV. KET LUAN
Trung tam nghien ciru san xuat vac xin va sinh pham y te da san xuat dugc khang huyet thanh rota dat tieu chuan co so. Hieu gia khang huyet thanh rota san xuat tir 4 chiing (WA, DS1, RRV, Gl P8) deu cho hieu gia rat cao, thap nhat la chiing G1P8 (6.400). Hieu gia khang huylt thanh rota san xuat tir chiing hoang dai (WA, DSl, RRV) cao hon chiing giam doc luc rat nhieu, hieu gia huyet thanh chung G1P8 giam doc lire chi bang 1/4 den 1/16 chung hoang dai.
Huyet thanh san xuat tir cac chiing dugc irng dung tot trong thir nghiem nhan dang, chuan dp hieu gia vac xin rota va thir nghiem nghien ciru lien quan den virut rota (ELISA) thay the dugc khang huyet thanh nhap ngoai.
TAI LIEU THAM KHAO
1. Le Thj Luan, Nguyen Dang Hien. Virut rota. Dac tinh va bien phap phong ngira.
Nha xuat ban y hgc 2007;
2. Nguyin Thj Tuyen. Rotavirut. Bai giang Vi sinh vat Y hgc, NXB Y hgc 1993. tr. 3 - 26.
Hendriksen CFM. Laboratory animals in vaccine production and control. Replace- ment, reduction and refinement. Dordrecht, Kluwer Academic Publisher, 1998.
4. Herrmann, J.E., Blacklow, N.R., Perron, D.M., Cukor, G., Krause, P.J., Hyams, J.S..
Barrett, H.J., Ogra, P.L. '"Monoclonal Anti- body Enzyme Immunoassay Jar detection oj Rota virus in stool specimens , J Infect Dis.
152, 1985, p. 830 - 832.
5. Taniguchi K., Urasawa S., Urasawa T ''Preparation and characterization of neu- tralizing monoclonal antibodies with differ- ent reactivity patterns to human Rota virus- es- J Gen Virol, 66, 1985, p. 1045-1053.
6. WHO. Manual of laboratory methods.
WHO/VSQ/97.4
34
Tap chi Y hpc du phong. Tap XXI, so 3 (121)RESEARCH ON PRODUTION OF ROTAVIRUS ANTISERA USING FOR VACCINE QUALITY CONTROL
Le Thi Luan, Ngo Thu Huong
Center for Research and Production of Vaccines and Biologicals(POLYVAC) Center for Research and Production of
Vaccines and Biologicals (POLYVAC) has pro- duced rotavirus antisera satified the in-house standard. The produced antisera were sterile, rhe titre of antisera from rabbits injected by 4 rotavirus strains of WA, DS 1, RRV, G1P8 were such high, in which the lowest one was the strain G1P8 (6.400). The titre of rotavirus anti-
sera from rabbits injected by wild strains (WA, DSl, RRV) were significantly higher than at^
tenuated strain G1P8 (4-16 times higher). Pro- duced antisera have been successfully applied in rotavirus identification, titre and ELISA; and this product could be used instead of imported antisera.
Tap chi Y hpc du phong. Tap XXI, so 3 (121)