HASHIZUME Shuichi Morinaga Inst.Biol.Sci., Director (Scientist), 所長(研究者)
KATAHIRA Jun Osaka University, Res.Inst.Microb.Dis., Bact.Toxinol., Res.Assoc., 微生物病研究所, 助手 (30263312)
HORIGUCHI Yasuhiko Osaka University, Res.Inst.Microb.Dis., Bact.Toxinol., Res.Assoc., 微生物病研究所, 助手 (00183939)
SUGIMOTO Nakaba Osaka University, Res.Inst.Microb.Dis., Bact.Toxinol., Assoc.Prof., 微生物病研究所, 助教授 (20142317)
|Budget Amount *help
¥2,500,000 (Direct Cost : ¥2,500,000)
Fiscal Year 1996 : ¥2,500,000 (Direct Cost : ¥2,500,000)
1.Cells from master cell bank of hybridomas G2, G6 producing anti-tetanus human monoclonal antibodies (MAbs) were tested for their safety, in vivo and in vitro : in vivo, (1) egg inoculation tests, (i) amniotic, (ii) chorioallantoic, (iii) egg yolk sac, (iv) allantoic inoculation tests, and (2)animal tests, (i) sucking mice, (ii) adult mice, (iii) guinea pig inoculation test by using culture supernatants of 3rd and 4th passage culture in Hy606 medium, no death of fetus or animals, nor abnormal clinical signs and symptoms, no factors inducing hemagglutination, heuradsorption were detected. In vitro, (3) (i) MRC-5, (ii) Vero, (iii) MDCK cells and (iv) their co-cultivation tests by treatment with sonic extract of the hybridoma cultures did not show the contamination of viruses nor reverse transcriptase (RT) activity. However, (4) retrovirus inducing test showed positive RT activity in both G2 and G6 lines and for G2 the presence of A type particles electronmicroscopically and positive S+L-focus assay were shown. 2.Epitope localization in the toxin molecule for G2, G6 and G4 MAbs were determined by using purified protease digests of the toxin and their amino acid sequencing and immunoblotting. 3.On the basis of the above safety tests, to develop more safe, economical preparation of MAbs, by making the best use of the above hybridomas, molecular cloning and sequencing of the variable regions genes of heavy and light chains of G6, G2 and G4 MAbs were performed using mixture of oligonucleotide primers and PCR,providing the basis for preparation recombinant tetanus antitoxins having high neutralizing activity for human use.