用体外免疫的人淋巴细胞建立抗破伤风类毒素的杂交瘤克隆

我们应用破伤风类毒素体外免疫的人扁桃体细胞和人-鼠异源骨髓瘤RF 系细胞进行融合,从中筛选到一株杂交瘤细胞89112—50,并通过克隆化筛选获得了两个亚克隆,其分泌的抗体是抗原特异的,和三种抗原(OVA、TC-S、FYG)都不交叉,分泌抗体的功能也比较稳定,在培养瓶内连续扩增传代13次后,仍维持相当高的抗体分泌能力,在常规传代培养过程中所收集的培养物上清液中抗体的含量平均为69.6μg/ml。     

破伤风类毒素的发展历史、现状和新型破伤风疫苗的研究进展

简要回顾了破伤风类毒素主要的发展历程;概述了目前国内外精制破伤风类毒素的两种生产工艺各自的优缺点,并且肯定了吸附精制破伤风类毒素几十年实际应用的效果,同时指出了在大规模人群接种时可能出现为数不多的各种不良反应,以及引起不良反应的可能的原因。对国内外破伤风疫苗研究进展和主要发展方向,如亚单位疫苗和新型破伤风类毒素的研究概况和前景也作了介绍。     

破伤风类毒素不良反应述评

本文简略回顾了破伤风类毒素接种后不良反应的历史,介绍了反应（主要是超敏反应）的类型,讨论了发生反应的原因,提出了减少反应的措施。     

一种提高破伤风类毒素马匹免疫成功率的方法

采用抗原加倍方法用于成率率低于705群的巴匹免疫,结果显示,进一步免疫成功的马匹明显多于常规免疫（P＜0．05）,免疫效价和死亡率无明显变化（P＞0．05）。     

双佐剂包被的破伤风类毒素马匹免疫试验

为提高破伤风免疫马匹的血浆抗体效价,应用不同佐剂配制TT抗原,进行马匹超免疫比较研究;采用FIA和植物油双佐剂包被与单佐剂包被的TT抗原,注射马匹进行超免疫,比较三组血浆的效价;结果显示,双佐剂抗原较单佐剂的免疫效果好,但可能对马匹刺激较强,有待调整注射剂量和免疫程序。     

聚合破伤风类毒素抗原特性的研究

由戊二醛脱毒的聚合破伤风类毒素,经高压液相层析及聚丙烯酰胺凝胶电泳分析,类毒素中多聚体含量占８１．９％以上,多聚体分子量为８００ｋＤ,常规破类多聚体仅占有２．２４％。聚合破类免疫豚鼠后,平均心血抗体单位达２ＩＵ／ｍｌ,常规破类仅为０．７５ＩＵ／ｍｌ（Ｔ＝１３．１５,Ｐ＜０．００１）,聚合破类免疫马匹后所诱发的抗体水平较常规抗原的高。     

抗破伤风类毒素单抗可变区基因的克隆及在大肠杆菌中表达的三种工程抗体

The heavy and light chain variable region genes of anti-tetanus toxoid (TT) antibody and the heavy chain Fd genes were amplified and cloned through RT-PCR from mouse hybridoma cells. The sequences of VH and VK were determined. Fd gene fragments were expressed in E. coli. The ELISA results indicated that the expressed Fd showed antigen binding activity but was nonspecific. Furthermore, through SOE and PCR techniques, the VH and VK gene fragments together with ScFv linker were assembled into single chain antibody (ScFv) gene fragment. While together with human heavy chain CH 1 gene fragment and Fab linker, they were assembled into chimeric Fab gene fragment. The two assembled gene fragments were separately inserted into phagemid pHEN 1, which was a fd-based vector containing gene 3 encoding the minor coat protein. In presence of helper phage M 13-VCS the anti-TT phage-ScFv or phage-Fab were displayed on the surface of phage particles respectively. Results from phage-ELISA indicated that both phage antibodies were TT-specific.     

人破伤风类毒素免疫血浆筛选试验中两种抗体检测方法的比较

应用酶联免疫法(ELISA)和间接血凝法(IHA)对1014份破伤风类毒素全程免疫后人血浆进行抗体水平检测,比较两者的收浆率、收浆符合率以及与动物实验的相关性。结果表明两者收浆率均达80%。ELISA法与IHA法的合格浆符合率达92%,与动物实验的相关性更好。ELISA法操作简便快速,结果判读客观明确,优于传统的IHA法,可作为人破伤风类毒素免疫血浆筛选的常规方法。     

14型肺炎球菌荚膜多糖-破伤风类毒素结合疫苗的研制

将14型肺炎球菌的荚膜多糖(PS)与破伤风类毒素(TT)通过化学方法结合,制备成多糖-蛋白结合疫苗(PS14-TT)。用该结合疫苗免疫小鼠,在小鼠体内产生了高滴度的PS-IgG抗体和TT-IgG抗体,且再次注射后有加强应答效应,表明制备的结合疫苗保留了完好的抗原性,具有胸腺依赖性抗原的特性。     

Effective production of anti-tetanus toxoid and anti-HBsAg human monoclonal antibodies by serum-free culture of hybridomas

Two hybridoma systems, mouse·human-human (m·h-h) heterohybridoma and human-human (h-h) hybridoma, have been established, and hybridomas secreting anti-tetanus toxoid and anti-HBsAg human monoclonal antibodies (MoAbs), both having a neutralizing activity have been obtained. Cell-line improvement was shown to be an efficient method for improving the productivity in a cell culture process. Two kinds of serum-free media, GFS (a serum substitute)-containing media and polyethylene glycol (PEG)-containing media, have been established to produce human MoAbs. m·h-h Heterohybridomas could be cultivated for a long period by perfusion culture in an agitation vessel, but h-h hybridomas could not. We found that h-h hybridomas show growth-associated antibody production kinetics and established two kinds of long-term cultivation systems: continuous perfusion culture and semicontinuous immobilized perfusion culture. We also scaled up batch culture and short-term perfusion culture to 200-L and 50-L fermentors, respectively. Processes for large-scale purification from the culture supernatants of both GFS- and PEG-containing serum-free media have also been developed.     

The induction and suppression of in vitro IgG anti-tetanus toxoid antibody synthesis by human lymphocytes stimulated with tetanus toxoid in the absence of in vivo booster immunizations

This report presents a new approach that by-passes booster immunizations with tetanus toxoid (TT) before in vitro studies of antibody (Ab) production. The methodology for optimal TT-induced synthesis of specific IgG anti-tetanus toxoid Ab (IgG anti-TT) by peripheral blood mononuclear cells (PBMC) from randomly selected TT immune individuals without recent booster immunizations is described. PBMC from most normal immune subjects could be repeatedly induced to produce in vitro IgG anti-TT; PBMC from subjects with high TT titers are not required for this new approach. This approach uses high cell concentrations in multiple replicate microcultures and TT washout to obtain optimal IgG anti-TT synthesis. Washed cultures produced more Ab than nonwashed cultures (p less than or equal to 0.005). The readdition of TT (2.5 to 250 ng/ml) to the culture media after washout of TT on day 4 suppressed specific Ab formation, whereas diphtheria toxoid added at comparable doses did not inhibit specific Ab formation. Suppression of antibody synthesis mediated by T cells could be induced by TT per se, and was not due to binding of synthesized Ab to TT in the latter 8 days of culture. In addition, suppression could not be induced in the first 4 days of culture by IgG anti-TT, IgG, or IgM. This approach permits the analysis of antigen-specific regulatory circuits in the steady and activated immune states, and the evaluation of in vivo and in vitro effects of biologic response modifiers on specific Ab production.     

Feasibility studies of large scale production of human anti-tetanus toxoid monoclonal antibodies

The feasibility of large scale production of human anti-tetanus toxoid monoclonal antibody for therapeutic use was evaluated using a human heterohybridoma. The effects of duration of subculture, transition from static to agitated culture conditions and the level of serum concentration were studied. The level of antibody secreted by the clone decreased with increasing length of subculture and decreasing serum concentration. The clone exhibited heterogeneity in expression of surface IgG after 2 or 7 weeks of subculture in static culture conditions irrespective of the serum concentration. However, a prolonged duration of subculture (9 weeks) in 3% serum medium had an effect on the expression of surface IgG both in static and agitated culture conditions. With respect to total (surface and intracellular) IgG, two distinct cell populations were observed. On long term subculture (9 weeks) in low serum medium (3% FCS), there was a decrease in the population which was the high synthesizer. In addition, when these cells were cultivated in agitated spinner flasks, a defect in secretion of antibodies was observed. Thus a general fall in the amount of antibody in the supernatant of agitated cultures was due to decrease in antibody synthesis as well as the defect in secretion of antibodies.     

IgG anti-tetanus toxoid antibody production induced by Epstein-Barr virus from B cells of human marrow transplant recipients

This investigation shows that Epstein-Barr virus (EBV)-activated human B cells from marrow transplant recipients can produce in vitro IgG anti-tetanus toxoid antibody (anti-TT) without booster immunizations with tetanus toxoid (TT). Purified B cells (E-rosette negative) from 8 normal subjects, 6 healthy long-term marrow graft recipients, and 15 long-term marrow graft recipients with chronic graft-vs-host disease (GVHD), were stimulated for 12 days with EBV to induce anti-TT production in culture supernatants. The amount of anti-TT in culture supernatants was quantitated using a enzyme-linked immunosorbent assay. B cells from all 8 normal controls produced in vitro IgG anti-TT after EBV stimulation. Five of 6 healthy recipients had B cells that produced anti-TT after EBV stimulation. Four of 15 recipients with chronic GVHD had B cells capable of producing anti-TT after EBV stimulation. The number of cultures making anti-TT responses was less in those with chronic GVHD than in those without chronic GVHD or normal individuals (P less than 0.001). B cells from patients with chronic GVHD had fewer responses exceeding the overall median of 0.7 ng/ml when compared with the other two groups (P less than 0.03). These data show that B cells of donor origin can produce in vitro IgG anti-TT antibody to tetanus toxoid antigen in a T-independent fashion.     

Influence of prednisolone on cells erythroid series in the bone marrow of rabbits immunized with diphtheria toxoid]

The bone marrow reaction to prednisolone showed significant changes after the administration of the antigen: there proved to be a significant reduction of the erythrocyte count, hemoglobin content and of hematocrite. The mean erythrocyte volume increased. Along with this there was a reduction of the number of basophilic and polychromatophilic erythroblasts and an increase in number of proerythroblasts. The degree of depression of the red series of the bone marrow (induced by prednisolone) was much less in case of combined administration of prednisolone and diphtheria toxoid.     

Differential synthesis of IgM and IgA anti-tetanus toxoid antibody in vitro following in vivo booster immunization of humans.

The in vitro syntheses of IgM and IgG anti-tetanus toxoid antibody by human peripheral blood leukocytes were compared prior to and at various intervals following in vivo booster immunization with soluble tetanus toxoid. Prior to booster immunization, the in vitro synthesis of IgG anti-tetanus toxoid antibody by combinations of B cells and irradiated T lymphocytes was negligible following pokeweed mitogen stimulation. Within 2 weeks after booster immunization, the quantity of IgG anti-tetanus toxoid antibody synthesized in vitro increased 5- to 20-fold. There was no comparable increase in total IgG synthesis. In contrast to the synthesis of IgG antibody, in vitro synthesis of IgM anti-tetanus toxoid antibody occurred prior to booster immunization and did not increase significantly following booster immunization. This dichotomy in anti-tetanus antibody production was further demonstrated in an individual with common variable hypogammaglobulinemia whose lymphocytes synthesized normal quantities of total IgG, IgM, and IgM anti-tetanus toxoid antibody in vitro, but failed to synthesize IgG anti-tetanus antibody following in vivo booster immunization.     

Characterization of mouse-human hybridoma as a useful fusion partner for the establishment of mouse-human-human hybridoma secreting anti-tetanus toxoid human monoclonal antibody of IgM or IgG class

We succeeded in establishing a mouse-human (M-H) heterohybridoma clone which provides parental cells useful for human monoclonal antibody (hMoAb) production. Electron micrographs show that the M-H hybridoma cells retain characteristics of murine origin with regard to chromatin patterns, small granules, granular endoplasmic reticulum and Golgi apparatus. The human DNA incorporated into the M-H hybridoma is estimated to be about 1% of the total human chromosomal DNA. Mouse-human-human (M-H-H) hybridomas obtained by hybridization of the M-H hybridoma cells with Epstein-Barr virus(EBV)-transfected human B cells secrete immunoglobulin (Ig) in amounts comparable to those of murine hybridomas. Also the M-H-H hybridomas grow in nude mice and are capable of producing ascites containing large quantities of Ig. The Ig class switching takes place in the M-H-H hybridomas at a much higher frequency than in the original EBV transformant and the M-H hybridoma. Cells secreting specific monoclonal antibody of different Ig classes could be separated and concentrated by the use of fluorescence activated cell sorter (FACS).