C型肉毒毒素的制备及类毒素保护力初探

将C型肉毒梭菌经适宜条件的产毒培养后纯化,并进行相关鉴定。制备的C型肉毒毒素用分段脱毒法脱毒,并进行类毒素保护力的初步研究。以不同蛋白含量C型肉毒类毒素免疫小鼠后攻毒,结果显示,蛋白含量为0.625μg的类毒素免疫2针或蛋白含量为1.25μg的类毒素免疫1针均可保护50LD50的C型肉毒毒素攻击。蛋白含量为5μg的C型肉毒类毒素与福氏不完全佐剂配制的抗原免疫小鼠3次所得抗血清的保护力(Anti LD50/ml)为4.3×104。说明用该纯化工艺制备的C型肉毒类毒素具有很好的免疫原性,作为抗原成分用于C型肉毒疫苗和C型肉毒抗毒素的研究和生产具有较好的应用潜力。     

用皂土法制造型特异性肉毒诊断血清

用皂土为载体与类毒素结合方法及破伤风类毒素抗原抗体絮状反应方法去除A、B、C、D、E、F型肉毒抗血清原料中的异型和异种抗毒素(破伤风抗毒素)。制备的A、B、C、D、E、F型肉毒诊断血清每1m l均能中和相应型的肉毒毒素10000LD50以上,而中和异型肉毒毒素或破伤风毒素均低于5 LD50;A、B、C、D、E、F各型混合后的混合型血清每1m l能中和各型肉毒毒素亦大于10000 LD50,中和破伤风毒素低于5 LD50,即效价和特异性符合规程要求。     

F型肉毒类毒素的免疫原性(实验结果)

两批精制F型肉毒类毒素,按不同剂量用氢氧化铝予以吸附,分别用豚鼠进行免疫原性 试验。结果表明,每毫升含60结合单位的吸附样品,0.5毫升一次皮下注射豚鼠,30天后即足以耐过1,000个致死量的F型肉毒毒素攻击,42天后的血清抗毒效价增在0.2单位/毫升以上。 用豚鼠做吸附精制F型肉毒类毒素免疫原性试验中,毒素攻击法似较血清效价测定法为优越。     

A型肉毒聚合类毒素的制备及免疫原性研究

通过制备A型肉毒聚合类毒素,研究获得较好的免疫原性和反应原性的方法.通过碳二亚胺法,聚合A型肉毒类毒素,免疫小鼠,用ELISA检测抗体,成功地制备了A型肉毒聚合类毒素,免疫小鼠获得高效价免疫血清,且该免疫血清具有中和活性,研究A型肉毒聚合类毒素抗原的作用机制,获得具有中和活性的保护性抗体,对肉毒毒素中毒的防治具有重要意...     

肉毒毒素和类毒素抗原

用毒素或福尔马林处理的类毒素免疫家兔制备抗肉毒血清(A和E型)。用类毒素连续吸收抗类毒素,能完全吸收掉小鼠保护效价;但其在吸收抗毒素时,却保留较高的保护效价,而此只能为毒素所除去。由于肉毒类毒素和毒素的明显差别,所以能将两者区分为免疫抗元和(或)抗血清吸收抗元。     

高效单价特异抗A型肉毒兔血清的制备

目前关于纯的肉毒类毒素及其抗血清的制备研究较少。纯抗原及纯抗血清不仅对肉毒毒素结构和功能的研究起重要作用,而且对于提高类毒素的免疫效果与抗毒素的治疗效果;减     

肉毒神经毒素抑制剂的研究进展

肉毒神经毒素(botulinum neurotoxins,BoNTs)是由梭状芽孢杆菌属分泌的外毒素,是目前已知毒性最强的生物类毒素.BoNTs共分为7种血清型(A-G),其中A型导致的肉毒中毒最为常见.由于肉毒毒素的强毒性及易于制备,其已被列为A类生物恐怖制剂.目前,针对肉毒中毒的有效治疗手段为早期注射抗毒素血清.但...     

B型肉毒毒素重链抗原表位的预测及表位合成肽抗原性的检测

目的:预测B型肉毒毒素蛋白抗原表位,研究表位合成肽的生物学活性。方法:利用生物信息学,结合Anthwine分析软件及网络平台,预测B型肉毒毒素蛋白重链的B细胞抗原表位。人工合成4条(P1,P2,P3,P4)表位多肽,与抗B型肉毒毒素类毒素的马血清反应;采用腹腔注射的方式用合成肽免疫BALB/c小鼠,检测免疫血清与合成肽的结合;对免疫小鼠腹腔注射B型肉毒毒素。结果:合成的多肽能与抗B型肉毒毒素的类毒素的马血清结合;多肽免疫小鼠后能产生针对多肽的抗体,其中P1、P3、P4产生的抗体对受毒素攻击的小鼠具有保护作用。结论:成功预测了抗原表位,合成的表位多肽具有较好的抗原性。     

肉毒A、B、E、F型抗毒素血浆的试生产与检定

按照《中国生物制品规程》(2000版)的规定,在肉毒A、B、E、F型抗毒素血浆试生产前,制定了肉毒A、B、E、F型类毒素马匹免疫计划及相应抗毒素血浆效价测定方法,免疫结果显示：肉毒A、B、E、F型类毒素免疫马匹成功率分别为75%、75%、60%、100%。     

IEF纯化重组A型肉毒毒素保护性抗原

在大肠杆菌中高效表达的重组A型肉毒毒素保护性抗原（rBoNTaH468),是以包涵体形式存在,将表达菌株发酵后,裂解菌体,制备包涵体,溶解后的包涵体溶液经样品处理,通过等地电聚焦制备型电泳纯化,纯化的重组A型肉毒毒素保护性抗原（rBoNTaH468)纯度高于90%,产量及回收率高,纯化的重组表达产物酶联检测具有结合活性,这为下一步A型肉毒毒素抗毒素的研制打下基础。     

Toxoid of Clostridium botulinum type F: purification and immunogenicity studies.

Toxin from Clostridium botulinum type F was recovered from dialysis cultures and partially purifed by: (i) ammonium sulfate and ethanol precipitation; (ii) O-(diethylaminoethyl)-cellulose chromatography; or (iii) diethylaminoethyl-cellulose chromatography followed by O-(carboxymethyl)-cellulose chromatography. Toxin purities as reflected by specific activity were 1.83 X 10(6), 9.8 X 10(6), and 2.0 X 10(7) mouse 50% lethal doses (LD50)/mg of N, respectively, for toxins purified by the three methods. The toxins were converted to toxoids by incubation at 35 C in the presence of 0.3 to 0.45% formalin for 21 to 35 days. Toxoids were immunogenic in guinea pigs, as demonstrated by serum antitoxin response and the immunized animals' resistance to challenge by type F botulinal toxin. The immune response to type F toxoids was lower when toxoids of serotypes A, B, C, D, and E were combined with the type F toxoid than when the type F toxoid only was administered. The toxoid prepared from the most highly purified toxin (method [iii]) conferred the highest immunity in guinea pigs at a given dose level. A relation between serum antitoxin level and resistance to challenge was observed. At least 50% of the groups of guinea pigs with 0.015 antitoxin units or more per ml survived challenge by 10(5) mouse LD50 of type F botulinal toxin. A dose of 3.75 mug of N of the most highly purified type F toxoid in combination with the other five serotypes of botulinal toxoid invoked an immune response in guinea pigs comparable to that considered adequate for the other toxoids.     

Characteristics of Clostridium botulinum Type F Isolated from the Pacific Coast of the United States

Some of the physiological and biochemical characteristics of a type F strain recently isolated from the United States were studied and compared with those of the prototype Langeland type F strain. The recent isolates were nonproteolytic, fermented sucrose and ribose, produced spores of low thermal resistance, produced a protoxin activated by trypsin, and grew and produced toxin at 38 F (3.3 C) from a spore inoculum. The prototype Langeland strain was proteolytic, did not ferment sucrose or ribose, and produced spores of relatively high thermal resistance, and the toxin of 3-day-old cultures was not activated by trypsin. Approximately two to three times the minimal lethal dose (MLD) of type F toxin from either Langeland or nonproteolytic strains was cross-neutralized by 1,000 anti-MLD of type E antitoxin. Antitoxin serums prepared by immunizing rabbits with the toxoid of the nonproteolytic type F isolate neutralized the toxin of the Langeland strain, but did not show cross-neutralization with the toxins of other types of Clostridium botulinum.     

Efficacy of a type C botulism vaccine in green-winged teal

We tested the efficacy of a single dose of Botumink toxoid for protecting wild green-winged teal (Anas crecca) during botulism epizootics caused by Clostridium botulinum type C. We challenged control and immunized ducks with four different doses of type C botulinum toxin to determine the LD50 for this species and to evaluate vaccine protection. Fewer immunized ducks were affected with botulism than control ducks, indicating that a single dose of Botumink toxoid could increase the survival of ducks during epizootics. However, the frequency of immunized ducks with signs of botulism increased with the challenge dose of botulinum toxin. Even at doses of botulinum toxin approximately 2 to 4 green-winged teal LD50, about 50% of the immunized ducks were affected. We believe an improved vaccine or a better delivery system is required to justify immunization of wild birds for experimental survival studies.     

Evaluation of a monoclonal antibody-based immunoassay for detecting type B Clostridium botulinum toxin produced in pure culture and an inoculated model cured meat system

A monoclonal antibody-based amplified ELISA method for detecting Clostridium botulinum type B toxin was evaluated for its ability to detect the toxin in the supernatant fluid of pure cultures and after growth from Cl. botulinum spores inoculated into pork slurries. Slurries containing NaCl (1.5–4.5%w/v) and polyphosphate (0.3%w/v) were either unheated or heated 80°C/5 min followed by 70°C/2 h before incubation at 15°, 20° or 27°C. Presence of specific toxin was confirmed by mouse bioassay and results were compared with those of the amplified ELISA method. A total of 48 strains, consisting of 38 Cl. botulinum and 10 Cl. sporogenes (putrefactive anaerobes), and 140 slurry samples were tested. Cultures of eight out of nine strains of type B Cl. botulinum and 73 of 101 slurry samples containing type B toxin were positive by ELISA; the remaining 28 slurry samples contained type B toxin at levels below or close to the detection limit (20 LD₅₀/ml) of the type B ELISA. No falsepositive reactions occurred with Cl. botulinum types A, C, D, E or F, or with the 10 strains of Cl. sporogenes. Toxin produced by one strain of Cl. botulinum type B (NCTC 3807) was not detected by this single monoclonal antibody-based amplified ELISA. With a mixture of two monoclonal antibodies, however, the toxin from NCTC 3807 could be detected without reducing the sensitivity of the ELISA.     

Evaluation of a monoclonal antibody-based immunoassay for detecting type B Clostridium botulinum toxin produced in pure culture and an inoculated model cured meat system

A monoclonal antibody-based amplified ELISA method for detecting Clostridium botulinum type B toxin was evaluated for its ability to detect the toxin in the supernatant fluid of pure cultures and after growth from Cl. botulinum spores inoculated into pork slurries. Slurries containing NaCl (1.5-4.5% w/v) and polyphosphate (0.3% w/v) were either unheated or heated 80 degrees C/5 min followed by 70 degrees C/2 h before incubation at 15 degrees, 20 degrees or 27 degrees C. Presence of specific toxin was confirmed by mouse bioassay and results were compared with those of the amplified ELISA method. A total of 48 strains, consisting of 38 Cl. botulinum and 10 Cl. sporogenes (putrefactive anaerobes), and 140 slurry samples were tested. Cultures of eight out of nine strains of type B Cl botulinum and 73 of 101 slurry samples containing type B toxin were positive by ELISA; the remaining 28 slurry samples contained type B toxin at levels below or close to the detection limit (20 LD50/ml) of the type B ELISA. No false-positive reactions occurred with Cl. botulinum types A, C, D, E or F, or with the 10 strains of Cl. sporogenes. Toxin produced by one strain of Cl. botulinum type B (NCTC 3807) was not detected by this single monoclonal antibody-based amplified ELISA. With a mixture of two monoclonal antibodies, however, the toxin from NCTC 3807 could be detected without reducing the sensitivity of the ELISA.     

Response of type B and E Botulinum toxins to purified sulfhydryl-dependent protease produced by Clostridium botulinum type F.

A sulfhydryl-dependent protease (SHP) was purified from a culture of Clostridium botulinum type F. The enzyme can activate type E progenitor toxin completely but type B progenitor toxin only partially. This may suggest that SHP by itself could completely activate the toxin of proteolytic C. botulinum types A and F in culture. The toxicity of type E progenitor toxin potentiated by the treatment with SHP persisted, whereas that of derivative toxin decreased rapidly by further incubation with SHP. This may indicate that only the progenitor toxin, the complex of the toxic and nontoxic components, activated by SHP withstands the subsequent exposure to the enzyme in cultures of proteolytic C. botulinum.     

Correlation of Clostridium botulinum type C antitoxin titers in mink and guinea pigs to protection against type C intoxication in mink

In USA, the potency of commercial vaccines containing Clostridium botulinum type C toxoid is determined by a mink vaccination-challenge assay outlined in the Code of Federal Regulations, Title 9, Part 113.110. A more humane potency test is desired, and this study provides preliminary data in support of a serological assay that correlates post-vaccination antitoxin titers of guinea pigs to vaccine efficacy in mink. Mink and guinea pigs were injected with varying dilutions of a vaccine containing C. botulinum type C toxoid. Blood samples were collected from each animal prior to challenging the mink with type C toxin. Serum antitoxin titers of mink and guinea pigs were measured by a mouse protection test, and the results were compared to the outcome of the toxin challenge in mink. A dose-dependent antitoxin response was observed in guinea pigs vaccinated with the critical dilutions of vaccine bracketing the minimum protective dose in mink. These preliminary data suggest that it may be possible to correlate post-vaccination antitoxin titers in guinea pigs to vaccine efficacy in mink. This correlation could be used as the basis for a more humane potency test for C. botulinum type C toxoids.     

Evaluation of a monoclonal antibody-based immunoassay for detecting type A Clostridium botulinum toxin produced in pure culture and an inoculated model cured meat system

A monoclonal antibody-based amplified enzyme-linked immunosorbent assay (ELISA) method for detecting Clostridium botulinum type A toxin was evaluated for its ability to detect the toxin in the supernatant fluid of pure cultures and after growth from Cl. botulinum spores inoculated into pork slurries. Slurries containing NaCl (1.5-4.5% w/v) and polyphosphate (0.3% w/v) were either unheated or heated, 80 degrees C/5 min + 70 degrees C/2 h, before storage at 15 degrees, 20 degrees or 27 degrees C. The presence of specific toxin was confirmed by mouse bioassay and results compared with those of the amplified ELISA method. A total of 49 strains, 39 Cl. botulinum and 10 Cl. sporogenes (putrefactive anaerobes), and 95 slurry samples were tested. Fourteen of 15 strains of type A Cl. botulinum and 34 of 36 slurry samples containing type A toxin were positive by ELISA. No false positive reactions occurred with Cl. botulinum types B, C, D, E and F, or with the 10 strains of Cl. sporogenes. However, toxin produced by one strain of Cl. botulinum type A (NCTC 2012) was not detected by the amplified ELISA.