Monoclonal antibody to type F Clostridium botulinum toxin

Hybridomas synthesizing monoclonal antibodies (MAbs) against type F Clostridium botulinum toxin were developed. MAb from one stable hybridoma, hybridoma 223, consisted of kappa light chains and an immunoglobulin G subclass 2a heavy chain. This MAb was used in a double-sandwich enzyme-linked immunosorbent assay to detect type F toxin in foods, culture fluids, and purified toxin preparations. The sensitivity of the double-sandwich enzyme-linked immunosorbent assay was approximately 10 mouse lethal doses of toxin per ml of toxic fluid.     

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.     

Comparative dose-survival curves of representative Clostridium botulinum type F spores with type A and B spores.

Radiation survival data of proteolytic (Walls 8G-F) and non-proteolytic (Eklund 83F) type F spores of Clostridium botulinum were compared with dose-response data of radiation-resistant type A (33A) and B (40B) spores. Strain Eklund 83F was as resistant as strain 33A, whereas strain Walls 8G-F was the most sensitive of the four strains tested. The methods suggested for computing both an initial shoulder and a D value for the dose-survival curves yielded results comparable to the graphic techniques used to obtain these two parameters.     

Comparative dose-survival curves of representative Clostridium botulinum type F spores with type A and B spores.

Radiation survival data of proteolytic (Walls 8G-F) and non-proteolytic (Eklund 83F) type F spores of Clostridium botulinum were compared with dose-response data of radiation-resistant type A (33A) and B (40B) spores. Strain Eklund 83F was as resistant as strain 33A, whereas strain Walls 8G-F was the most sensitive of the four strains tested. The methods suggested for computing both an initial shoulder and a D value for the dose-survival curves yielded results comparable to the graphic techniques used to obtain these two parameters.     

Sequence of the gene encoding type F neurotoxin of Clostridium botulinum

Primers designed to conserved regions of botulinum and tetanus clostridial toxins were used to amplify DNA fragments from non-proteolytic Clostridium botulinum type F (202F) DNA using polymerase chain reaction technology. The fragments were cloned and the complete nucleotide sequence of the gene encoding type F toxin determined. Analysis of the nucleotide sequence demonstrated the presence of an open frame encoding a protein of 1274 amino acids, similar to other botulinum neurotoxins. Upstream of the toxin gene is the end of an open reading frame which encodes the C-terminus of a protein with homology to non-toxic-non-hemagglutinin component of type C progenitor toxin.     

F型肉毒梭菌培养液可滤性实验的初步研究

目的通过探索F型肉毒梭菌培养液可滤性实验,为现有生产工艺提供科学的优化方案。方法分别采用不同型号的过滤器,通过不同的过滤组合对F型肉毒梭菌培养液进行澄清过滤。结果采用K700P过滤器(过滤精度6.0～15.0μm)对F型肉毒梭菌培养液进行一级澄清过滤,采用50P过滤器(过滤精度0.5～0.85μm)对料液进行二级澄清过滤,对F型肉毒梭菌培养液具有良好的澄清效果。结论可滤性实验的探索对于F型肉毒梭菌培养液批生产量的扩大具有切实可行的指导意义。     

Isolation and immunochemical study of the toxic complex of Cl. botulinum type F]

The toxic comples of Cl. botulinum, type F, was separated into the toxic and nontoxic protein fractions by the methods of ion exchange chromatography and gel filtration in accordance with a specially devised purification scheme. Highly purified, electrophoretically and serologically homogeneous toxin with a molecular weight of 150,000 and potency equal to 10 X 10(6) DLM per 1 mg of protein was isolated from the toxic fraction. The nontoxic protein component had faintly pronounced hemagglutinating properties and was essentially different from type A and B hemagglutinins. The toxic complex of Cl. botulinum, type F, was shown to contain a proteolytically active fraction.     

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.