Studies on the application of anti-tetanus human monoclonal antibodies having high neutralizing activity to use in place of current polyclonal tetanus antitoxin preparations.

1.从产生破伤风人单克隆抗体（MAbs）的杂交瘤G2、G6主细胞库中提取细胞，进行体内外安全性试验。体内(1)鸡蛋接种试验，(1)羊膜接种试验，(2)绒毛膜尿囊接种试验，(3)卵黄囊接种试验，(4)尿囊接种试验，(1)吸吮小鼠，(2)成年小鼠，(3)Hy606培养基中第3代、第4代培养上清豚鼠接种试验，未发现胎儿和动物死亡，未发现异常临床体征和症状，未发现诱导血凝、启发吸附的因素。在体外，(3)(i) MRC-5, (ii) Vero, (iii) MDCK细胞和（iv）杂交瘤培养物的超声提取物处理的共培养试验未显示病毒污染和逆转录酶（RT）活性。然而，(4)逆转录病毒诱导试验显示G2和G6系RT活性均为阳性，G2在电镜和S+ l聚焦试验中均表现出A型颗粒的存在。2.G2、G6和G4单克隆抗体的抗原表位定位采用纯化的蛋白酶酶切毒素及其氨基酸测序和免疫印迹法。3.在上述安全性试验的基础上，为开发更安全、经济的单克隆抗体制备方法，充分利用上述杂合瘤，采用寡核苷酸引物与PCR的混合方法对G6、G2和G4单克隆抗体重链和轻链可变区基因进行分子克隆和测序，为制备具有高中和活性的重组破伤风抗毒素提供基础。

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.