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。     

马免疫血浆的稳定性研究

目的了解不同温度条件对马免疫血浆外观和效价的稳定性影响,为马免疫血浆的采集、分离、贮存、运输及抗毒素生产提供数据支持。方法将破伤风类毒素及肉毒A、B、E、F型类毒素制备的马免疫血浆,分别放置于2~8℃、20℃、37℃3种温度下,并分别于0、1、3、6、12个月取样,依据《中国药典》三部(2010版)的检测方法和标准,对样品进行外观检查及效价检测。结果破伤风及肉毒A、B、E、F型马免疫血浆在2~8℃条件下,放置12个月稳定性良好,外观及效价均符合《中国药典》三部(2010版)规定要求。20℃与37℃下放置的马免疫血浆随着时间的延长,外观会发生不同程度的浑浊,效价也均有不同程度的降低,低效价组比中、高效价组的效价下降明显,且温度越高效价降低幅度越大。结论马免疫血浆在2~8℃条件下质量稳定。     

F型肉毒毒素的制备及类毒素免疫原性初探

将F型肉毒梭菌经适宜条件产毒培养后,以硫酸铵盐析和酸沉两种不同工艺制备的F型肉毒毒素,用分段脱毒法脱毒制备类毒素,分别免疫豚鼠、马匹后测定免疫血清抗体效价。结果显示,两种工艺制备的毒素其类毒素都具有较好的免疫原性。     

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。     

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。     

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...     

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     

E型肉毒类毒素的免疫原性(实验结果)

十几年来,有些国家(¨、(趴、。’已先后开始试制E型肉毒类毒素,以期用于人的自动免疫,做为E型肉毒中毒的预防措施之一。但是,不论在制造方法上,或在检定     

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...     

应用双抗原ELISA夹心法检测破伤风抗毒素效力的试验

作者建立了一套适用于破伤风抗毒素效力检测的双抗原夹心ELISA法,对破伤风类毒素免疫后的豚鼠和马匹分别进行了血清效价测定,并和传统的小鼠攻毒法的效力检测进行了比较,结果表明：该检测方法与小鼠攻毒法对不同水平的破伤风抗毒素效力检测,结果是一致的,有很好的相关性;此法省时、特异、灵敏,可成为另一种检测破伤风抗毒素的有效方法。     

Multiplex PCR assay for detection and identification of Clostridium botulinum types A, B, E, and F in food and fecal material.

Botulism is diagnosed by detecting botulinum neurotoxin and Clostridium botulinum cells in the patient and in suspected food samples. In this study, a multiplex PCR assay for the detection of Clostridium botulinum types A, B, E, and F in food and fecal material was developed. The method employs four new primer pairs with equal melting temperatures, each being specific to botulinum neurotoxin gene type A, B, E, or F, and enables a simultaneous detection of the four serotypes. A total of 43 C. botulinum strains and 18 strains of other bacterial species were tested. DNA amplification fragments of 782 bp for C. botulinum type A alone, 205 bp for type B alone, 389 bp for type E alone, and 543 bp for type F alone were obtained. Other bacterial species, including C. sporogenes and the nontoxigenic nonproteolytic C. botulinum-like organisms, did not yield a PCR product. Sensitivity of the PCR for types A, E, and F was 10(2) cells and for type B was 10 cells per reaction mixture. With a two-step enrichment, the detection limit in food and fecal samples varied from 10(-2) spore/g for types A, B, and F to 10(-1) spore/g of sample material for type E. Of 72 natural food samples investigated, two were shown to contain C. botulinum type A, two contained type B, and one contained type E. The assay is sensitive and specific and provides a marked improvement in the PCR diagnostics of C. botulinum.     

Development of enrichment semi-nested PCR for Clostridium botulinum types A, B, E, and F and its application to Korean environmental samples

An enrichment semi-nested PCR procedure was developed for detection of Clostridium botulinum types A, B, E, and F. It was applied to sediment samples to examine the prevalence of C. botulinum in the Korean environment. The first pair of primers for the semi-nested PCR was designed using a region shared by the types A, B, E, and F neurotoxin gene sequences, and the second round employed four nested primers complementary to the BoNT/A, /B, /E, and /F encoding genes for simultaneous detection of the four serotypes. Positive results were obtained from the PCR analysis of five of 44 sediments (11%) collected from Yeong-am Lake in Korea; all were identified as deriving from type B neurotoxin (bontb) genes. Two of the C. botulinum type B organisms were isolated, and their bontb genes sequenced. The deduced amino acid sequences of BoNT/B showed 99.5 and 99.8% identity with the amino acid sequence of accession no. AB084152. Our data suggest that semi-nested PCR is a useful tool for detecting C. botulinum in sediments, and renders it practicable to conduct environmental surveys.     

A Pyrolysis Gas-liquid Chromatography Study of Clostridium botulinum and Related Organisms

Low resolution pyrolysis gas-liquid chromatography could differentiate the following groups of Clostridium botulinum and related organisms: (1) Cl. botulinum type A. proteolytic types B and F and Cl. sporogenes ; (2) Cl. botulinum types C and D. and (3) Cl. botulinum type E and non-proteolytic types B and F. Toxin types A and B could be distinguished from type E and from type F.     

Genetic interrelationships of saccharolytic Clostridium botulinum types B, E and F and related clostridia as revealed by small-subunit rRNA gene sequences

Abstract The phylogenetic interrelationships of saccharolytic C. botulinum types B, E and F together with eleven other saccharolytic clostridia were examined by 16S rRNA gene sequencing. Comparative analysis of the sequence data revealed that the saccharolytic C. botulinum types B, E and F were highly related and represents a single genetic group. Strains of C. barati and C. butyricum that produce botulinal neurotoxin revealed almost 100% 16S rRNA sequence identity with their respective non-toxigenic counterparts and were phylogenetically distinct from saccharolytic C. botulinum (types B, E and F). Proteolytic C. botulinum type F was shown to be phylogenetically remote from the saccharolytic C. botulinum group. The implications of the sequence data for the taxonomy of the C. botulinum complex are discussed.     

Detection of Clostridium botulinum types A, B, E and F in foods by PCR and DNA probe

A PCR procedure was developed for the detection of Clostridium botulinum in foods. PCR products were detected in agarose gels and by Southern hybridization. The sensitivity of PCR was tested in broth cultures and in canned asparagus, dry cured ham and honey. The sensitivity of the method in broth was high (2·1–8·1 cfu ml⁻¹) for types A and B, but rather low (10⁴ cfu ml⁻¹) for types E and F. However, after enrichment at 37°C for 18 h, it was possible to detect Cl. botulinum types A, B, E and F in food samples at initial levels of about 1 cfu 10 g⁻¹ of food. This PCR detection protocol provides a sensitive and relatively rapid technique for the routine detection of Cl. botulinum in foods.     

Fluorescent Studies in the Genus Clostridium. II. A rapid method for differentiating Clostridium botulinum types A, B and F, types C and D, and type E

S ummary : Fluorescent labelled specific antisera have been used to distinguish between types A, B and F, types C and D, and type E of Clostridium botulinum .     

Development of a combined selection and enrichment PCR procedure for Clostridium botulinum Types B, E, and F and its use to determine prevalence in fecal samples from slaughtered pigs

A specific and sensitive combined selection and enrichment PCR procedure was developed for the detection of Clostridium botulinum types B, E, and F in fecal samples from slaughtered pigs. Two enrichment PCR assays, using the DNA polymerase rTth, were constructed. One assay was specific for the type B neurotoxin gene, and the other assay was specific for the type E and F neurotoxin genes. Based on examination of 29 strains of C. botulinum, 16 strains of other Clostridium spp., and 48 non-Clostridium strains, it was concluded that the two PCR assays detect C. botulinum types B, E, and F specifically. Sample preparation prior to the PCR was based on heat treatment of feces homogenate at 70 degrees C for 10 min, enrichment in tryptone-peptone-glucose-yeast extract broth at 30 degrees C for 18 h, and DNA extraction. The detection limits after sample preparation were established as being 10 spores per g of fecal sample for nonproteolytic type B, and 3.0 x 10(3) spores per g of fecal sample for type E and nonproteolytic type F with a detection probability of 95%. Seventy-eight pig fecal samples collected from slaughter houses were analyzed according to the combined selection and enrichment PCR procedure, and 62% were found to be PCR positive with respect to the type B neurotoxin gene. No samples were positive regarding the type E and F neurotoxin genes, indicating a prevalence of less than 1.3%. Thirty-four (71%) of the positive fecal samples had a spore load of less than 4 spores per g. Statistical analysis showed that both rearing conditions (outdoors and indoors) and seasonal variation (summer and winter) had significant effects on the prevalence of C. botulinum type B, whereas the effects of geographical location (southern and central Sweden) were less significant.     

PCR and gene probe identification of botulinum neurotoxin A-, B-, E-, F-, and G-producing Clostridium spp. and evaluation in food samples.

A degenerate primer pair was selected to amplify specifically a 260-bp DNA fragment from Clostridium botulinum types A, B, E, F, and G, and five individual probes allowed identification of each toxinotype by hybridization of the PCR products. The 72 strains of different Clostridium species tested and 11 other bacterial species commonly found in food samples gave an amplification product. This assay was able to detect 1 C. botulinum type A or B and 10 C. botulinum type E strains per reaction. With 184 artificially contaminated food samples, after an 18-h enrichment step, the sensitivity was 10 bacteria per g of sample and the correlation with the mouse bioassay reached 95.6%.     

Multiplex PCR Assay for Detection and Identification of Clostridium botulinum Types A, B, E, and F in Food and Fecal Material

Botulism is diagnosed by detecting botulinum neurotoxin and Clostridium botulinum cells in the patient and in suspected food samples. In this study, a multiplex PCR assay for the detection of Clostridium botulinum types A, B, E, and F in food and fecal material was developed. The method employs four new primer pairs with equal melting temperatures, each being specific to botulinum neurotoxin gene type A, B, E, or F, and enables a simultaneous detection of the four serotypes. A total of 43 C. botulinum strains and 18 strains of other bacterial species were tested. DNA amplification fragments of 782 bp for C. botulinum type A alone, 205 bp for type B alone, 389 bp for type E alone, and 543 bp for type F alone were obtained. Other bacterial species, including C. sporogenes and the nontoxigenic nonproteolytic C. botulinum-like organisms, did not yield a PCR product. Sensitivity of the PCR for types A, E, and F was 10² cells and for type B was 10 cells per reaction mixture. With a two-step enrichment, the detection limit in food and fecal samples varied from 10⁻² spore/g for types A, B, and F to 10⁻¹ spore/g of sample material for type E. Of 72 natural food samples investigated, two were shown to contain C. botulinum type A, two contained type B, and one contained type E. The assay is sensitive and specific and provides a marked improvement in the PCR diagnostics of C. botulinum.     

Detection of type A, B, E, and F Clostridium botulinum neurotoxins in foods by using an amplified enzyme-linked immunosorbent assay with digoxigenin-labeled antibodies

An amplified enzyme-linked immunosorbent assay (ELISA) for the detection of Clostridium botulinum complex neurotoxins was evaluated for its ability to detect these toxins in food. The assay was found to be suitable for detecting type A, B, E, and F botulinum neurotoxins in a variety of food matrices representing liquids, solid, and semisolid food. Specific foods included broccoli, orange juice, bottled water, cola soft drinks, vanilla extract, oregano, potato salad, apple juice, meat products, and dairy foods. The detection sensitivity of the test for these botulinum complex serotypes was found to be 60 pg/ml (1.9 50% lethal dose [LD50]) for botulinum neurotoxin type A (BoNT/A), 176 pg/ml (1.58 LD50) for BoNT/B, 163 pg/ml for BoNT/E (4.5 LD50), and 117 pg/ml for BoNT/F (less than 1 LD50) in casein buffer. The test could also readily detect 2 ng/ml of neurotoxins type A, B, E, and F in a variety of food samples. For specificity studies, the assay was also used to test a large panel of type A C. botulinum, a smaller panel of proteolytic and nonproteolytic type B, E, and F neurotoxin-producing Clostridia, and nontoxigenic organisms using an overnight incubation of toxin production medium. The assay appears to be an effective tool for large-scale screening of the food supply in the event of a botulinum neurotoxin contamination event.