破伤风类毒素和白喉类毒素原液的质量分析

目的  分析破伤风类毒素（tetanus toxoid,TT）和白喉类毒素（diphtheria toxoid,DT）原液的细菌内毒素含量、单体与多聚体之比以及游离氨基数。方法  取TT和DT原液各10批,用凝胶法、高效液相色谱法及三硝基苯磺酸法分别检测原液的细菌内毒素、单体和多聚体以及游离氨基。 结果  各10批TT、DT原液的细菌内毒素含量均值分别为1.14和0.35 EU/Lf。TT原液的单体平均含量为72.1%,是多聚体的3倍;DT原液的单体平均含量为88.9%,是多聚体的8倍。脱毒后,每个TT蛋白分子含游离氨基数均值为43.5,比破伤风毒素减少约60%;每个DT蛋白分子含游离氨基数均值为9.9,比白喉毒素减少约75%。10批TT、DT的单体与多聚体含量之比以及游离氨基数的批间一致性较好（变异系数：1.59% ~16.26%）。 结论   细菌内毒素含量、单体与多聚体之比以及游离氨基数检测指标可用于对TT、DT生产过程的控制以及批间一致性的监控。     

检测破伤风类毒素的酶联免疫吸附法的建立及应用

目的  建立定量检测白喉-破伤风-无细胞百日咳疫苗生产过程中破伤风类毒素（tetanus toxoid,TT）的方法。方法  TT免疫家兔以制备高效价血清抗TT抗体。辛酸-硫酸铵沉淀法纯化抗TT多克隆抗体并进行辣根过氧化物酶标记,建立双抗体夹心ELISA法。结果  建立的ELISA法与丝状血凝素、百日咳毒素及白喉类毒素无交叉反应,特异性较好。该ELISA法在0.5~16.00 Lf/L TT测量区间有最佳线性,决定系数＞0.99。实验内和实验间检测14.0、12.0、6.0、3.0和1.5 Lf/L TT,变异系数为4.7%~9.8%,回收率为92.7%~109.0%,精密度和准确度均符合常规质控要求。该法的定量下限为1.5 Lf/L TT。结论  建立的ELISA法可有效检测破伤风疫苗纯化过程中的TT含量,为破伤风疫苗质量控制奠定了基础。     

Interaction of tetanus toxin and toxoid with cultured neuroblastoma cells

Summary The primary interaction of tetanus toxin and toxoid with mouse neuroblastoma cells (C 1300, clone NB2A) in tissue culture was studied using direct immunofluorescence. Experiments were done in standard routine cultures and also those influenced by chemical modulators.There is a difference in the characteristic binding response between the growth culture cells (grown in presence of fetal calf serum) and differentiating culture cells (grown in absence of serum). Exposure to the toxin gives no visible effect on the cell division or viability in growth cultures; whereas in differentiating cells the processes are shortened and the adherence to the glass is diminished without involving significant cell death. The toxoid did not bind at all under the same experimental conditions.Since there was no biological effect in growth cultures we have called this binding ineffective, and in the case of the differentiating cells, effective binding. Stimulation of pinocytosis increases the uptake of toxin in both cultures. Presence of some surface bound toxin still remaining on the differentiating cells indicates the possibility of another sort of mechanism for internalization. Pre-treatment of the cells with neuraminidase of -galactosidase to alter the membrane gangliosides eliminates binding in growth cultures but not in differentiating cultures.From these results we suggest that even though the toxin may well bind to gangliosides, at least in the differentiating cultures they are not solely responsible for the fixation. The morphologically observed effective binding is probably that not related to gangliosides.This work was supported by a Medical Research Grant from World Health Organization to J.M.Z. A communication was presented at the Conference of the Swiss Society for Microbiology, Geneva, June 17–19, 1976     

Inhibition of synaptosomal choline uptake by tetanus and botulinum a toxin

Summary Tetanus toxin and, to a lesser degree, botulinum A toxin inhibit partially and noncompetitively the uptake of [³H]choline into a crude synaptosomal fraction from rat brain cortex. Botulinum toxin acts by its neurotoxin content. The effect is not due to nonspecific synaptosomal damage by the toxins as shown by the lactate dehydrogenase occlusion test, by the absence of swelling and by the preservation of choline stores. The ratio between [³H]acetylcholine and [³H]choline was decreased by both toxins.Inhibition by either toxin depends strongly on the temperature and duration of incubation, and is preceded by an initial latency period. The effect of tetanus toxin, once manifest, is largely resistant against antitoxin. It is not significantly diminished by pretreatment of the synaptosomes with V. cholerae neuraminidase.Fixation of ¹²⁵I-tetanus toxin proceeds fast, is largely independent of temperature and is diminished by pretreatment of the synaptosomes with neuraminidase. Thus only some of the fixation sites, and not the long-chain gangliosides, are required for the effects of tetanus toxin. A slow, temperature-sensitive process links the fixation with the action.In contrast to rat synaptosomes the chicken preparation is more sensitive to botulinum A than to tetanus toxin, which reflects the differences in sensitivity between live birds and rodents.Our data underline the similarities between the effects of tetanus and those of botulinum A toxin. Their dependence on time and temperature, the time dependence of efficacy of antitoxin, and the concordance in species specificity indicate that the in vitro system mirros some crucial features of poisoning of isolated organs and live animals.with the technical assistance of Eva BolzThe essentials of this communication, which is part of the Thesis of I. Heller, have been presented at the Joint Meeting of the Scandinavian and German Pharmacological Societies, Lübeck, FRG, September 1980 (Bigalke et al. 1980), as well as at the Spring Meeting 1980 of the Gesellschaft für Biologische Chemie, Münster, FRG, March 1980 (Habermann and Bigalke 1980)     

Suppression of 3H-acetylcholine release from primary nerve cell cultures by tetanus and botulinum-A toxin

Summary Primary nerve cell cultures derived from embryonic rat central nervous system form [³H]ACh from exogenous [³H]Ch, and release it upon potassium depolarization.Pretreatment of the cultures with botulinum-A toxin or tetanus toxin diminishes the cellular accumulation of [³H]ACh. Poisoning the cultures during the period of [³H]Ch uptake fails to lower [³H]ACh formation.Dependent on dosage, both toxins suppress the release of [³H]ACh upon potassium depolarization.Heat-denaturated toxins as well as tetanus toxin preincubated with tetanus antitoxin were without effect.Abbreviations used BTA Botulinum-A toxin - TET Tetanus toxin - ACh Acetylcholine - Ch Choline - [³H]Ch [³H-Methyl]-choline - [³H]ACh Acetyl [³H-Methyl]-choline - DMEM Dulbecco's modified Eagle's medium - ChAc (EC 2.3.1.6.) Choline acetyl transferase - [¹⁴C]ACh [1-¹⁴C]Acetylcholine - PBS Phosphate buffered saline This work will be presented as part of the thesis of H. B. at the Justus Liebig-Universität GiessenThis work was supported by DFG via Sonderforschungsbereich 47Part of this work was presented at the sixth International Meeting of the International Society for Neurochemistry in Copenhangen, Denmark, 1977.     

Induction of high antitoxin titers against tetanus toxoid in rabbits by intranasal immunization with dextran microspheres

Poor absorption of protein antigens through the mucosal membranes necessitates the use of mucoadhesive delivery systems. Regarding the advantages of mucosal immunization and also the penetration enhancement potential of dextran microspheres, in this study the adjuvant potential of these microspheres was compared with CpG-ODN. Cross-linked dextran microspheres (CDMs) were loaded with tetanus toxoid (TT). In vitro release studies were performed in a model, simulating the nasal cavity. The immunoreactivity of encapsulated TT was assayed by ELISA. Membrane toxicity and local irritating potential of CDM was examined by erythrocyte hemolysis and nasal administration to human nose, respectively. The various formulations were nasally administered to rabbits (n=4). Alum-adsorbed TT (AATT) was injected as the positive control. The serum IgG and nasal lavage sIgA titers were determined by ELISA method. Serum antitoxin titers were determined by toxin neutralization (TN) bioassay method. Mean diameter of CDM was 128.1+/-25.8 microm. Mean encapsulation efficiency was 20.3+/-3.2% (n=3). Antigenicity of encapsulated TT was 90.5+/-1.8% (n=3) that of original TT. Hemolysis studies showed no membrane disruption by CDM and none of the human subjects reported nasal irritation. Among the nasally immunized animals, the highest antitoxin titers was seen in the group immunized with CDM+TT (P<0.0001). The serum IgG titers of the CDM+TT group was higher than the TT solution group (P<0.05). The adjuvant potentials of CDM and CpG-ODN in inducing IgG titers was not significantly different (P>0.05). The lowest sIgA titers in the bronchial lavage were seen in the group of animals received AATT parenterally. Considering the proper release characteristics, desirable preservation of the antigen activity of TT, good mucoadhesion properties and also safety of CDM+TT, these microspheres could be regarded as an efficient mucosal adjuvant and antigen delivery system. These microspheres could induce very high antitoxin titers following nasal administration, while the CpG-ODN could not induce such titers. The antitoxin titers induced by CDM+TT was 175 times higher than the protective levels.     

伤寒Vi多糖、白喉、破伤风联合疫苗初探

目的   制备成人及青少年用吸附伤寒Vi多糖、白喉、破伤风联合疫苗,观察伤寒Vi多糖含量、白喉及破伤风效价.方法   按设计的配方,将白喉、破伤风类毒素加入佐剂中吸附后,再加入伤寒Vi多糖,要求配制的吸附伤寒Vi多糖、白喉、破伤风联合疫苗每人用剂量0.5ml伤寒Vi多糖含量≥30μg,白喉类毒素效价≥2IU,破伤风类毒素效价≥40IU.结果    三组配方试验样品每人用剂量0.5ml伤寒Vi多糖含量均低于30μg,白喉类毒素效价3.127IU～5.911IU,破伤风类毒素效价7.784IU～23.976IU.除白喉类毒素效价达到要求外,伤寒Vi多糖含量及破伤风类毒素效价未达到要求.结论   初探试验表明,试验的联合疫苗存在抗原干扰,伤寒Vi多糖含量及破伤风类毒素效价下降.     

Reductive cleavage of tetanus toxin and botulinum neurotoxin A by the thioredoxin system from brain

Summary Inhibition of neurotransmitter release by tetanus toxin and botulinum neurotoxin A can be mimicked by intracellular application of the corresponding toxin light chains. The aim of this study was to determine whether the two-chain toxins are reduced by brain preparations to yield free light chains which would represent the ultimate toxins.The interchain disulfide of two-chain tetanus toxin was cleaved by rat cortex homogenate fortified with NADPH. Reduction was promoted further by addition of thioredoxin. Thioredoxin reductase was demonstrated in and purified from porcine brain cortex. The thioredoxin system which consisted of purified enzyme, thioredoxin and NADPH reduced both toxins. The resulting light chains appeared homogeneous in SDS gel electrophoresis. The complementary heavy chain of tetanus but not of botulinum toxin migrated in two bands, the faster one with the velocity of heavy chain obtained by chemical reduction. The major, slower form was converted into the faster by chemical but not by enzymatic reduction. Tetanus toxin, whether in its single-chain or two-chain version also occurred in two forms which differed by their electrophoretic mobility. The two forms of single-chain toxin were interconverted by chemical reduction or oxidation but not by the thioredoxin system.It is concluded that a) a thioredoxin system in brain tissue reduces the interchain disulfide of two-chain tetanus toxin and botulinum neurotoxin A, b) tetanus toxin but not botulinum neurotoxin A consists of two electrophoretically distinct forms which differ by the thiol-disulfide status of their heavy chains, c) the disulfide loop within the heavy chain of tetanus toxin is resistant to the thioredoxin system. Send offprint requests to E. Habermann at the above address     

2型肺炎链球菌多糖-破伤风类毒素结合疫苗的制备及免疫原性研究

目的 制备2型肺炎链球菌多糖(Streptococcus pneumoniae type 2 polysaccharide,SPNPS2)-破伤风类毒素(tetanus toxoid,TT)结合疫苗(SPNPS2-TT),并研究其免疫原性.方法 采用溴化氰活化SPNPS2,己二酰肼作连接剂,碳二亚胺为耦联剂,将活化的SPNPS2与TT耦联制成SPNPS2-TT.以SPNPS2-TT或单纯SPNPS2免疫Balb/c小鼠,用ELISA检测小鼠血清抗SPNPS2 IgG水平.结果 SPNPS2-TT的各项指标均达到质控标准.SPNPS2-TT免疫小鼠产生的特异件抗SPNPS2 IgG水平显著高于单纯SPNPS2免疫小鼠,且SPNPS2-TT诱生了免疫记忆应答.结论 采用此项技术制备SPNPS2-TT疫苗是可行的.     

Interaction of botulinum type A,B and E derivative toxins with synaptosomes of rat brain

Summary Clostridium botulinum ¹²⁵I-labelled derivative toxin immediately bound to rat synaptosomes. Of the two fragments of type B derivative toxin, the large-molecular-weight fragment (fragment I) inhibited the binding of labelled type B derivative toxin to synaptosomes in the same manner as unlabelled type B toxin did. The inhibition by the small-molecular-weight fragment (fragment II) was less than that by fragment I. These findings suggest that type B toxin binds to synaptosomes mainly with some part of fragment I. The binding of labelled type A and E derivative toxins was inhibited by either of the unlabelled type A or E derivative toxins, but not by type B derivative toxin. It is concluded that synaptosomes of rat brain possess relatively specific binding sites for botulinum toxin types.     

Formulation and characterization of immunoreactive tetanus toxoid biodegradable polymer particles

Poly lactide-co-glycolide and polylactide polymer particles entrapping immunoreactive tetanus toxoid (TT) were prepared with a view to developing a single shot controlled release vaccine formulation. Denaturation of TT by dichloromethane (DCM) during primary emulsification stage of particle formulation was minimized by incorporation of an optimal amount of rat serum albumin (RSA) in the internal aqueous phase. Incorporation of RSA as a stabilizer during the primary emulsification stage of polymer particle formulation protected the immunoreactivity of TT, enhanced its encapsulation efficiency and also led to uniform polymer particle formation. Use of sonication, both during primary and secondary emulsification processes, resulted in formation of nanoparticles whereas microparticles were formed when the secondary emulsion was carried out by homogenization. Immunoreactive TT particles made from different polymers incorporating stabilizers released antigen continuously for more than four months in vitro. Single injection of both type of particles encapsulating stabilized TT elicited anti-TT antibody titers in rats for more than five months, which was higher than that obtained with TT injected in saline. Anti-TT antibody titers in vivo were in accordance with the in vitro release characteristics of immunoreactive TT from the particles. Immune responses with hydrophobic polymer particles were better than those made using hydrophilic polymers. These results indicate the importance of protecting the immunoreactivity of TT during formation of polymer particles for sustained and improved antibody response.     

The effects of induced hypoxia on the cardiovascular system in dogs poisoned with tetanus toxin

Summary Tetanus toxicity was induced in dogs by injecting the toxin subcutaneously in the groin. On developing generalised toxic symptoms, these dogs were characterised by signs of increased sympathetic discharge to the cardiovascular system as evidenced by high basal values of blood pressure, heart rate and LV dP/dt max. Mild to moderate hypoxia induced by ventilation with 10% O₂ in N₂ had no appreciable effect on the cardiovascular variables. However, moderate to severc hypoxia induced by ventilation with 7% O₂ in N₂ further increased the sympathetic discharge to the heart and blood vessels resulting in increases in heart rate, LV dP/dt max and blood pressure. These responses were abolished by adrenergic blockers. The responses in the tetanus dogs were identical to those seen in dogs without tetanus toxicity. Atropine or moderate lactic acidaemia did not alter the responses to hypoxia. Beta-adrenergic blockers appear to be useful drugs in the control of tetanus patients who show evidence of increased sympathetic activity or who develop hypoxaemia.     

Pore formation by tetanus toxin,its chain and fragments in neuronal membranes and evaluation of the underlying motifs in the structure of the toxin molecule

The pore-forming activity of tetanus toxin, its chains and fragments was studied on membrane patches from spinal cord neurons of fetal mice using the outside-out patch-clamp configuration.1. The dichain tetanus toxin forms pores at pH 5, but not at pH 7.4. The elementary pore conductance is 38.4±1.1 pS and nonselective for small cations. The open probability of the pores is voltage-dependent and increases with membrane depolarisation. The pores activate at +80 mV with a time constant of about 20 ms and deactivate at –80 mV with two time constants of about 2 ms and 10 ms. Besides the elementary pore conductance, larger pore conductances which are multiples of the elementary conductance were observed. With increasing conductances, their frequency of occurrence decreases exponentially.2. The light chain of tetanus toxin alone does not form pores in neuronal membranes at pH 5 or at pH 7.4.3. The heavy chain of tetanus toxin forms pores at pH 5 as well as at pH 7.4. The single pore conductance increases from 35.0±1.2 pS at pH 5 to 43.2±1.8 pS at pH 7.4. The pores allow mono- and divalent cations and chloride ions to pass. Only at pH 5 do they have a voltage dependence with time constants identical to those obtained with tetanus toxin.4. Secondary structure predictions show a high density of presumably helically organized elements in fragment ₂ (45 kDa) of the heavy chain between residues 700–850. By investigating all predicted helices having a size of 18 residues or more, we characterized three surface-seeking regions with sufficiently high amphiphilic character to be considered as candidates for pore formation. Fragment C (50 kDa) lacking this region did not show pore-forming activity, whereas all fragments of the heavy chain, which contain these amphiphilic helices (fragment 2 and 86/91 kDa fragments), were effective in forming pores.5. The fact that tetanus toxin does not form pores at pH 7.4 indicates that the extracellular tetanus toxin or its light chain do not pass through the plasma membrane directly via pores. Therefore we hypothesize that tetanus toxin reaches an acidic intracellular compartment via receptor-mediated endocytosis. Correspondence to: F. Dreyer at the above address     

壳聚糖固定化胰蛋白酶亲和层析抑肽酶的条件及影响因素

利用壳聚糖固定化胰蛋白酶作为亲和吸附剂从牛肺中直接分离纯化抑防酶。该方法在适当的温度（35℃）、pH值（pH1．5）、离子强度（1．0mol／LKCI）以及吸附条件（176FIP／g）下，纯化的抑肽酶活性回收率达83％，比活可达5000kIU／mg以上。     

Limited proteolysis of single-chain tetanus toxin by tissue enzymes,in cultured brain tissue and during retrograde axonal to the spinal cord

Summary Single-chain toxin was investigated in vitro and in vivo for limited proteolysis into the fully active two-chain toxin. Plasmin from serum, elastase and gelatinase from leucocytes, as well as clostripain from C. histolyticum cleaved single-chain toxin and increased by that way its ability to inhibit [³H]noradrenaline release in vitro. Cultured mouse brain generated fragments from ¹²⁵I-single-chain toxin which were cell-associated. Some of them comigrated in electrophoresis with light and heavy chain after mercaptolysis. When injected i. v. into rats, ¹²⁵I-single-chain-toxin disappeared from the blood with a half-life of about 11 h without signs of nicking. However, after its injection into the triceps surae muscle both single- and two-chain toxin were found in the ipsilateral ventral horn of the spinal cord. Thus single-chain toxin is subjected to limited proteolysis by enzymes involved in tissue damage, by cultured brain tissue, and during or after its retrograde axonal transport to the spinal cord. Limited proteolysis is necessary for the release of the light chain known to mediate the action of toxin on several systems.     

刺桐胰蛋白酶抑制剂的高效表达、纯化、亲和介质的制备及其在r-PA纯化中的应用

刺桐胰蛋白酶抑制剂(ETI)是一种丝氨酸蛋白酶抑制剂,能够特异性结合胰蛋白酶及组织型纤溶酶原激活剂,抑制其活性。因此ETI可以作为配体与固定相偶联制备亲和层析介质,用于tPA(组织型纤溶酶原激活剂)及其突变体的纯化。利用基因工程方法构建了ETI的高表达工程菌株,诱导表达后目的蛋白占总蛋白的39.6%,经包涵体变性、复性、纯化,制备了纯度高于96%的ETI样品,收率达到了63.5%。利用纯化的ETI,偶联制备了亲和层析介质,偶联率达到99%。用制备的ETI亲和层析介质纯化瑞替普酶(r-PA),获得的r-PA样品纯度高于95%,生物学活性高于5×105U/mg。本研究为ETI的进一步生产及应用奠定了基础。     

Metabolic fate of 125I-tetanus toxin in the spinal cord of rats and cats with early local tetanus

Summary Local tetanus was elicited in rats and cats by intramuscular injection of ¹²⁵I-tetanus toxin. After different times spinal radioactivity was extracted with either non-ionic (Lubrol PX) or ionic (sodium dodecyl sulfate, SDS) detergents and compared with native or ¹²⁵I-toxin by gel filtration, SDS-gel electrophoresis, immunological procedures, and toxicity tests. In double-isotope experiments, ¹³¹I-toxin was added to the extracts as standard.In rats, the bulk of extracted material was indistinguishable from native toxin. However, there was a slight shift of the extracted material towards smaller molecular weights in gel filtration with Lubrol. In gel filtration with SDS, the toxin peak was followed by some tailing of ¹²⁵I radioactivity. Accordingly a small part of extracted radioactivity moves faster than the standard in SDS disc gel electrophoresis. These findings taken together indicate some degradation in vivo.Adsorption to solid-phase antibodies indicated that more than 80% of the radioactivity extracted from rats was still immunoreactive. It yielded a zone confluent with extrinsic toxin in immunodiffusion.The spinal cord Lubrol extract from rats was still toxic in the expected range. Due to the very small amounts of toxin present, no precise toxicity data could be given.In cats, there was also some evidence for radioactive split products in both SDS gel filtration and disc gel electrophoresis. The patterns closely resembled those obtained with extracts from rat spinal cord.SDS extracts from rat and cat spinal cords, poisoned with ¹²⁵I tetanus toxin in vivo, were also subjected to SDS disc gel electrophoresis followign reduction with dithioerythritol (DTE). They yielded large and small chains of the same size as did native toxin.In vitro, extensive degradation with brain homogenate from rats took place at pH 3.65, but not at pH 7.5. This indicates that lysosomal degradation is not a major metabolic pathway of tetanus toxin in vivo, although it is possible in principle.It is concluded that a) unlike other toxins, tetanus toxin is not necessarily degraded during its cellular uptake, b) the bulk of radioactive material is indistinguishable, following its neuronal ascent, from native or labeled toxin, c) a part of the radioactivity is recovered as split products.     

Acidification of the cytosol inhibits the uptake of tetanus toxin in NG108-15 and NBr-10A neurohybridoma cells

The influence of cytosol acidification on the uptake of two-chain tetanus toxin (TeTX)¹ by neurohybridoma cells NG 108-15 and NBr-l0A was investigated with two established techniques, the NH4C1 pulse method and the pH-clamp method. With the former, the extracellular pH is maintained at its physiological value, but is set to different values with the latter. - Acidification of the cytoplasm with an NH4Cl pulse retarded the uptake of TeTX by both NG 108-15 and NBr-l0A cells. This result provides further evidence for a vesicular endocytotic uptake of TeTX. In contrast, acidification of both the external medium and the cytoplasm (pH-clamp method) resulted in a net increase of toxin uptake. This result is explained as follows: Acidification of the extracellular environment has been shown to facilitate the uptake of tetanus toxin, and under pH clamp conditions, this effect is stronger than the simultaneous retardation of the toxin uptake by acidification also of the cytosol.Abbreviations used BCECF-AM 2,7-bis-(-2-carboxyethyl)-5(and -6)-carboxyfluorescein acetoxy methyl ester - BSA bovine serum albumin - HEPES N-(-2-hydroxyethyl) piperazine-N' (-ethanesulfonic acid) - TeTX nicked tetanus toxin - TRF diferric transferrin     

微柱亲和层析分离肝癌特异性AFP组分及其临床价值

目的 分析肝癌特异性AFP组分的层析特性及其对肝癌诊断的临床价值.方法 选用微层析柱,以小扁豆凝集素(LCA)琼脂糖4B装柱,将血清稀释后上样,根据AFP对LCA结合能力差异,使用不同的洗脱液将不同亲和力的AFP洗脱,分别测定各部分的AFP浓度,计算肝癌特异性AFP比例,分析AFP-L3与总AFP、肝癌大小和分化程度间的关系.结果 糖蛋白AFP在亲和层析柱上,肝癌与良性肝病表达AFP呈不同的层析洗脱曲线;AFP-L3与血清总AFP水平无相关(P＞0.05),但与肝癌的大小和分化程度显著相关(P＜0.01).单结节型与弥漫型肝癌两组间的血AFP水平差异亦有统计学意义(P＜0.01).结论 使用微柱亲和层析法分析肝癌AFP其糖蛋白部分与良性肝病明显不同,AFP-L3是诊断肝癌特异标志物.     

高效液相亲和层析介质的制备和对基因重组α-干扰素的纯化

以硅胶为基质，经表面改性和单克隆抗体固定化，制备了高效液相亲和层析介质。在改性反应中，以γ-缩甘油氧基丙基三甲氧基硅烷为改性剂，比较了水相法键合与有机相法键合的硅烷化率。用合成的α干扰素单克隆抗体高效液相亲和介质纯化由大肠杆菌表达的基因重组人α干扰素，层析过程的活性回收率为94％，纯化倍数为79倍，比活为7.94×106IU／mg。