Clostridium botulinum type C toxin

Summary The purification and crystallization of type C botulinum toxin along with its physical characteristics are described. The shape of Clostridium botulinum type C toxin molecule is globular like a pressed ball with a 7.4 nm diameter and a 4.3 urn thickness. The molecular volume is approximately 185 nl and the molecular weight is 141 000. The toxin molecule is composed of two parts, which are separable under appropriate conditions. These parts have some differences in the electrophoretic properties, amino acid distribution, immunological, and functional characteristics. The toxin molecule can be reconstituted by association of S-S bond between the two chains. The expression of the toxicity requires that the fragments of the polypeptide chain carrying the necessary information be functionally organized for the proper development of the specific tertiary structure for active conformation.     

Stabilization of botulinum toxin type A during lyophilization.

Botulinum toxin for medical use is diluted to very low concentrations (nanograms per milliliter); when it is preserved by lyophilization, considerable loss of activity can occur. In the present study, conditions that gave > 90% recovery of the toxicity after lyophilization of solutions containing 20 to 1,000 mouse 50% lethal doses per ml were found. Toxicity was recovered upon drying 0.1 ml of toxin solution when the pH was maintained below 7 and bovine or human serum albumins were used as stabilizers. Various other substances tested with albumin, including glucose, sucrose, trehalose, mannitol, glycine, and cellibiose, did not increase recovery on drying.     

Stabilization of botulinum toxin type A during lyophilization.

Botulinum toxin for medical use is diluted to very low concentrations (nanograms per milliliter); when it is preserved by lyophilization, considerable loss of activity can occur. In the present study, conditions that gave > 90% recovery of the toxicity after lyophilization of solutions containing 20 to 1,000 mouse 50% lethal doses per ml were found. Toxicity was recovered upon drying 0.1 ml of toxin solution when the pH was maintained below 7 and bovine or human serum albumins were used as stabilizers. Various other substances tested with albumin, including glucose, sucrose, trehalose, mannitol, glycine, and cellibiose, did not increase recovery on drying.     

治疗用A型肉毒毒素的制备及其质量控制

我国治疗用Ａ型肉毒毒素采用酸等电点沉淀,磷酸盐提取,核糖核酸酶处理,ＤＥＡＥ Ａ５０离子交换层析,硫酸铵浓缩及自然结晶等程序从Ａ型肉毒梭菌培养物中提取,并经稀释、冻干而成。它毒力强、纯度高、性能稳定,宜于长期保存。经生化、免疫学测定,毒素系神经毒素和血凝素的复合体,纯度为２５～３．０×１０７ＬＤ５０（小鼠,下同）／ｍｇｐｒ,ＯＤ２６０／ＯＤ２８０≤０．５５不仅达到了美国ＦＤＡ对注射用Ａ型肉毒毒素的质量要求,而且在冻干损失,稳定性方面还优于美国制品。     

In vitro acetylcholinesterase inhibition by type A botulinum toxin

Type A botulinum toxin was studied for its ability to inhibit the action of acetyl-cholinesterase. The chromogenic substrate, indophenyl acetate, was used for assay of enzyme activity. Inhibition of enzyme function was detected through use of both 6.6 x 10(-6) mg (20 ld(50)) and 6.6 x 10(-10) mg (2 x 10(-3)ld(50)) of type A botulinal toxin. Control assays were performed by use of both homologous antitoxin and heterologous antitoxins (types B and E). Enzyme inhibition was effectively prevented by use of homologous antitoxin only. The inhibition noted was specific and reproducible for given substrate, enzyme, and toxin concentrations.     

A型肉毒毒素ELISA鉴别试验方法的建立

目的建立鉴定A型肉毒毒素的ELISA鉴别试验方法以替代传统的动物试验法。方法采用现代免疫学技术,制备马源性和兔源性抗A型肉毒毒素多克隆抗体,建立了双抗体夹心ELISA,并就此初步进行方法学验证。结果所建立的ELISA具有良好的特异性、灵敏度、精密度和耐用性,具有替代动物试验方法的良好前景。结论在进一步验证和确认之后,该方法有望正式成为可用于鉴定A型肉毒毒素的试验方法以替代动物试验法。     

Attomolar detection of botulinum toxin type A in complex biological matrices

Background

A highly sensitive, rapid and cost efficient method that can detect active botulinum neurotoxin (BoNT) in complex biological samples such as foods or serum is desired in order to 1) counter the potential bioterrorist threat 2) enhance food safety 3) enable future pharmacokinetic studies in medical applications that utilize BoNTs.

Methodology/Principal Findings

Here we describe a botulinum neurotoxin serotype A assay with a large immuno-sorbent surface area (BoNT/A ALISSA) that captures a low number of toxin molecules and measures their intrinsic metalloprotease activity with a fluorogenic substrate. In direct comparison with the “gold standard” mouse bioassay, the ALISSA is four to five orders of magnitudes more sensitive and considerably faster. Our method reaches attomolar sensitivities in serum, milk, carrot juice, and in the diluent fluid used in the mouse assay. ALISSA has high specificity for the targeted type A toxin when tested against alternative proteases including other BoNT serotypes and trypsin, and it detects the holotoxin as well as the multi-protein complex form of BoNT/A. The assay was optimized for temperature, substrate concentration, size and volume proportions of the immuno-sorbent matrix, enrichment and reaction times. Finally, a kinetic model is presented that is consistent with the observed improvement in sensitivity.

Conclusions/Significance

The sensitivity, specificity, speed and simplicity of the BoNT ALISSA should make this method attractive for diagnostic, biodefense and pharmacological applications.     

Thermal inactivation of type E botulinum toxin

The theoretical required cooking times for inactivation of type E Clostridium botulinum toxin (5,000 ld(50) mouse units per 0.5 ml) in haddock fillets of various sizes were calculated by graphical integration of the toxin inactivation rate and heat penetration data. The results indicated that normal cooking procedures should suffice to inactivate this amount of toxin. This conclusion was substantiated by the following additional experimental observations which revealed that the original experiments had been conducted under conservative conditions. First, maximal heat stability of the toxin was found to occur at about pH 5.5, with decreasing resistance upon increasing pH. The theoretical cooking times were based on destruction of the toxin at pH 6.7. The pH of radio-pasteurized inoculated haddock, when toxin production had occurred, was on the alkaline side, at which condition the toxin is heat-labile. Second, when spoilage was discernible in radio-pasteurized inoculated haddock, the toxin titer was low, about 50 ld(50) mouse units per 0.5 ml. Third, the toxin was adequately inactivated in toxic fillets after deep-fat frying for 3 min at 375 F (190.6 C) or after pan frying for 5 min per side at 400 F (204.4 C). Fourth, in this study, residual toxin activity was assayed by intraperitoneal injection of mice. It was shown that the oral toxic dose was 50 to 100 times greater than the intraperitoneal toxic dose.