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

应用具有高中和活性的抗破伤风人单克隆抗体代替现有多克隆破伤风抗毒素制剂的研究。

基本信息

批准号：
07557212
负责人：
MATSUDA Morihiro
金额：
$ 1.6万
依托单位：
Osaka Unviresity
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1996
项目状态：
已结题

项目摘要

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.

1.对来自产生抗破伤风人单克隆抗体（MAb）的杂交瘤G2、G6主细胞库的细胞进行了体内和体外安全性检测：体内试验：（1）卵接种试验，（i）羊膜，（ii）绒毛尿囊，（iii）卵黄囊，（iv）尿囊接种试验，和（2）动物试验，（i）乳鼠，（ii）成年鼠，（iii）用Hy 606培养基第3代和第4代培养物的培养上清液进行豚鼠接种试验，未检测到胎儿或动物死亡，也未检测到异常的临床体征和症状，未检测到诱导血凝的因素，也未检测到凝集素吸附。在体外，（3）（i）MRC-5、（ii）Vero、（iii）MDCK细胞和（iv）通过用杂交瘤培养物的超声提取物处理进行的共培养试验均未显示病毒污染或逆转录酶（RT）活性。（4）逆转录病毒诱导试验显示G_2和G_6细胞系RT活性均为阳性，G_2细胞系电镜下可见A型颗粒，S+ L-灶试验阳性。2.通过使用纯化的毒素蛋白酶及其氨基酸测序和免疫印迹，确定G2、G6和G4单抗在毒素分子中的表位定位。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剂，利用上述杂交瘤细胞株，采用混合寡核苷酸引物和PCR技术，对G6、G2和G4单克隆抗体的重链和轻链可变区基因进行了分子克隆和测序，为制备人用重组破伤风抗毒素提供了基础。