Monoclonal antibody-based immunoassay for type A Clostridium botulinum toxin is comparable to the mouse bioassay

A monoclonal antibody (BA11) has been produced against Clostridium botulinum type A neurotoxin by the fusion of myeloma cells (P3 NS1/1-Ag4-1) with spleen cells from BALB/c mice immunized with botulinum type A neurotoxoid. The antibody bound specifically to botulinum type A neurotoxin, showing no cross-reactivity with types B and E botulinum toxins or with any of several other bacterial toxins tested. The monoclonal antibody did not bind to botulinum type A neurotoxin which had been denatured with sodium dodecyl sulfate and bound only weakly to each of the separated heavy and light subunits of the neurotoxin, suggesting a conformational requirement for the antigenic determinant of the antibody. A sensitive immunoassay for C. botulinum type A toxin with monoclonal antibody BA11 in conjunction with an enzyme amplication system has been developed which allows detection of 5 to 10 mouse 50% lethal doses ml-1 of purified neurotoxin. The assay was equally sensitive when applied to the detection of crude toxin in food stuffs; the average value for the minimum level of detectable toxin in extracts of tinned salmon or corned beef was 9 +/- 3.1 mouse 50% lethal doses ml-1.     

Monoclonal antibody-based immunoassay for type A Clostridium botulinum toxin is comparable to the mouse bioassay.

A monoclonal antibody (BA11) has been produced against Clostridium botulinum type A neurotoxin by the fusion of myeloma cells (P3 NS1/1-Ag4-1) with spleen cells from BALB/c mice immunized with botulinum type A neurotoxoid. The antibody bound specifically to botulinum type A neurotoxin, showing no cross-reactivity with types B and E botulinum toxins or with any of several other bacterial toxins tested. The monoclonal antibody did not bind to botulinum type A neurotoxin which had been denatured with sodium dodecyl sulfate and bound only weakly to each of the separated heavy and light subunits of the neurotoxin, suggesting a conformational requirement for the antigenic determinant of the antibody. A sensitive immunoassay for C. botulinum type A toxin with monoclonal antibody BA11 in conjunction with an enzyme amplication system has been developed which allows detection of 5 to 10 mouse 50% lethal doses ml-1 of purified neurotoxin. The assay was equally sensitive when applied to the detection of crude toxin in food stuffs; the average value for the minimum level of detectable toxin in extracts of tinned salmon or corned beef was 9 +/- 3.1 mouse 50% lethal doses ml-1.     

肉毒毒素体外检测方法的研究进展

肉毒毒素是肉毒梭状芽胞杆菌在生长繁殖过程中产生的一种神经毒素,对人和动物具有强毒性和高致死性,在世界范围内,肉毒中毒的案例时有发生,病情严重者一般2~3 d即可死亡。肉毒毒素产品所用的体内检测方法(小鼠生物法)的结果变异系数大,不利于肉毒毒素产品含量的标准化控制。因而,建立准确且灵敏度高的肉毒毒素体外检测方法,在临床治疗及制品的质量控制方面都具有重要意义,现就常用的肉毒毒素体外检测方法作一阐述。     

Development of an in vitro activity assay as an alternative to the mouse bioassay for Clostridium botulinum neurotoxin type A

Currently, the only accepted assay with which to detect active Clostridium botulinum neurotoxin is an in vivo mouse bioassay. The mouse bioassay is sensitive and robust and does not require specialized equipment. However, the mouse bioassay is slow and not practical in many settings, and it results in the death of animals. Here, we describe an in vitro cleavage assay for SNAP-25 (synaptosome-associated proteins of 25 kDa) for measuring the toxin activity with the same sensitivity as that of the mouse bioassay. Moreover, this assay is far more rapid, can be automated and adapted to many laboratory settings, and has the potential to be used for toxin typing. The assay has two main steps. The first step consists of immunoseparation and concentration of the toxin, using immunomagnetic beads with monoclonal antibodies directed against the 100-kDa heavy chain subunit, and the second step consists of a cleavage assay targeting the SNAP-25 peptide of the toxin, labeled with fluorescent dyes and detected as a fluorescence resonance energy transfer assay. Our results suggest that the sensitivity of this assay is 10 pg/ml, which is similar to the sensitivity of the mouse bioassay, and this test can detect the activity of the toxin in carrot juice and beef. These results suggest that the assay has a potential use as an alternative to the mouse bioassay for analysis of C. botulinum type A neurotoxin.     

The mouse bioassay for the detection of estrogenic activity in rodent diets: I. A standardized method for conducting the mouse bioassay

A standardized procedure was developed for conducting the mouse bioassay for detecting estrogenic activity in rodent diets. Studies were conducted with CD-1 mice to determine the appropriate weaning age and length of bioassay period. Uterine growth curves were generated from mice weaned at 15 days of age and fed a negative control diet until 28 days of age. These mice showed slow regular increases in uterine weights from 15 22 days of age followed by rapid uterine growth in some mice from 24 to 28 days of age. Estrogenic bioassays using female mice weaned at 15 days of age and fed the positive control diets containing 4 or 6 ppb diethylstilbestrol (DES) demonstrated significant (P less than 0.05) increases in uterine weight and in uterus to body weight (U:BW) ratios over those of mice fed the negative control diet without DES for 5, 7 or 9 days after weaning. In contrast, mice weaned at 17 days of age showed significant (P less than 0.05) increases in uterine weight and in U:BW ratios only at 5 days after weaning. Six ppb DES was required in the positive control diet to produce a 1.5 fold increase in the U:BW ratio over those of mice fed the negative control diet. It was concluded that mice should be weaned at 15 days of age and that the bioassay period should be terminated at 7 days, when the mice are 22 days old, for best reproducible results. The criteria for a valid bioassay should include the demonstration of a significant statistical increase in the U:BW ratios of mice fed the DES positive diet over those of mice fed the negative control diet.     

Biochemical resistance detection: an alternative to bioassay

Insecticide resistance is an increasing problem in vector control programmes. Until recently, the usual means of detecting it has been by bioassay, requiring the use of relatively large numbers of insects and insecticide-impregnated test papers which may be difficult to prepare and store reproducibly. William Brogdon argues for the use of biochemical microplate assays which are cheaper and easier to use, permit up to 30 assays to be made on a single insect, and give more reproducible results.     

Simplified purification method for Clostridium botulinum type E toxin

Clostridium botulinum type E toxin was purified in three chromatography steps. Toxin extracted from cells was concentrated by precipitation and dissolving in a small volume of citrate buffer. When the extract was chromatographed on DEAE-Sephadex without RNase or protamine treatment, the first protein peak had most of the toxin but little nucleic acid. When the toxic pool was applied to a carboxymethyl Sepharose column, toxin was recovered in the first protein peak in its bimolecular complex form. The final chromatography step at 4 degrees C on a DEAE-Sephacel column at a slightly alkaline pH purified the toxin (Mr, 145,000) by separating the nontoxic protein from the complex. At least 1.5 mg of pure toxin was obtained from each liter of culture, and the toxicity was 6 X 10(7) 50% lethal doses per mg of protein. These values are significantly higher than those previously reported.     

Differentiation of Clostridium difficile toxin from Clostridium botulinum toxin by the mouse lethality test

The mouse lethality test is the most sensitive method for confirming the diagnosis of infant botulism. Both Clostridium difficile and Clostridium botulinum produce heat-labile toxins which are lethal for mice and can be found in the feces of infants. These two toxins can be distinguished from one another in this assay when both are present in the same fecal specimen because they appear to be immunologically distinct toxins.     

Bovine serum eliminates rapid nonspecific toxic reactions during bioassay of stored fish for Clostridium botulinum toxin

When stored fish or some fish products were tested for the presence of Clostridium botulinum toxin, nonspecific toxic reactions in mice often occurred, rendering the bioassay inconclusive. The nonspecific toxic reactions were mediated by the gram-negative microbiota, inherent to the fish, which were the source of lethal, heat-stable endotoxins. The treatment of assay samples with bovine serum eliminated nonspecific reactions through the interaction of constituent serum immunoglobulin M (IgM) with endotoxic material. Removal of IgM from bovine serum through treatment with protein A or concanavalin A resulted in a loss of protective activity.     

Development of an in vitro bioassay for Clostridium botulinum type B neurotoxin in foods that is more sensitive than the mouse bioassay.

A novel, in vitro bioassay for detection of the botulinum type B neurotoxin in a range of media was developed. The assay is amplified by the enzymic activity of the neurotoxin's light chain and includes the following three stages: first, a small, monoclonal antibody-based immunoaffinity column captures the toxin; second, a peptide substrate is cleaved by using the endopeptidase activity of the type B neurotoxin; and finally, a modified enzyme-linked immunoassay system detects the peptide cleavage products. The assay is highly specific for type B neurotoxin and is capable of detecting type B toxin at a concentration of 5 pg ml(-1) (0.5 mouse 50% lethal dose ml(-1)) in approximately 5 h. The format of the test was found to be suitable for detecting botulinum type B toxin in a range of foodstuffs with a sensitivity that exceeds the sensitivity of the mouse assay. Using highly specific monoclonal antibodies as the capture phase, we found that the endopeptidase assay was capable of differentiating between the type B neurotoxins produced by proteolytic and nonproteolytic strains of Clostridium botulinum type B.     

Simplified purification method for Clostridium botulinum type E toxin.

Clostridium botulinum type E toxin was purified in three chromatography steps. Toxin extracted from cells was concentrated by precipitation and dissolving in a small volume of citrate buffer. When the extract was chromatographed on DEAE-Sephadex without RNase or protamine treatment, the first protein peak had most of the toxin but little nucleic acid. When the toxic pool was applied to a carboxymethyl Sepharose column, toxin was recovered in the first protein peak in its bimolecular complex form. The final chromatography step at 4 degrees C on a DEAE-Sephacel column at a slightly alkaline pH purified the toxin (Mr, 145,000) by separating the nontoxic protein from the complex. At least 1.5 mg of pure toxin was obtained from each liter of culture, and the toxicity was 6 X 10(7) 50% lethal doses per mg of protein. These values are significantly higher than those previously reported.     

Bovine serum eliminates rapid nonspecific toxic reactions during bioassay of stored fish for Clostridium botulinum toxin.

When stored fish or some fish products were tested for the presence of Clostridium botulinum toxin, nonspecific toxic reactions in mice often occurred, rendering the bioassay inconclusive. The nonspecific toxic reactions were mediated by the gram-negative microbiota, inherent to the fish, which were the source of lethal, heat-stable endotoxins. The treatment of assay samples with bovine serum eliminated nonspecific reactions through the interaction of constituent serum immunoglobulin M (IgM) with endotoxic material. Removal of IgM from bovine serum through treatment with protein A or concanavalin A resulted in a loss of protective activity.     

Enzyme-linked immunosorbent assay for detection of Clostridium botulinum toxin type A.

The enzyme linked immunosorbent assay using the so-called "double-sandwich technique" has been applied to determine botulinum toxin type A. By this assay, 50-100 mouse ip LD50 of toxin type A can be detected. No cross-reaction occurs with botulinum toxins of other types tested. In all probability this is due to the high specificity of the antiserum prepared against the toxic component of type A toxin.     

Enzyme-linked immunosorbent assay for detection of Clostridium botulinum type E toxin.

The enzyme-linked immunosorbent assay using the "double-sandwich" technique was utilized to determine Clostridium botulinum type E toxin. With this technique, about 80 mouse intraperitoneal 50% lethal doses of toxin could be detected. Cross-reaction was hardly observed with C. botulinum type A and B toxins. No cross-reaction was observed with culture supernatants of C. botulinum type C or other Clostridium strains. In all probability this was due to the high specificity of the antiserum prepared aginst the toxic component of type E toxin.     

Enzyme-linked immunosorbent assay for detection of Clostridium botulinum type E toxin.

The enzyme-linked immunosorbent assay using the "double-sandwich" technique was utilized to determine Clostridium botulinum type E toxin. With this technique, about 80 mouse intraperitoneal 50% lethal doses of toxin could be detected. Cross-reaction was hardly observed with C. botulinum type A and B toxins. No cross-reaction was observed with culture supernatants of C. botulinum type C or other Clostridium strains. In all probability this was due to the high specificity of the antiserum prepared aginst the toxic component of type E toxin.     

Detection of estrogenicity by bioassay on the mouse mammary gland in vivo

The wide chemical diversity of estrogenic compounds precludes an accurate prediction of estrogenic activity on the basis of chemical structure or radioimmunological assay and thus requires that the potency of these compounds is defined by bioassay. The mammary duct growth response in intact prepubertal and adult gonadectomized female and male mice of the C3H/Di strain was used to assess the estrogenicity of synthetic compounds or their derivatives. The vehicle for tested compounds should be free of estrogenic and other hormonal effects. Olive oil or sunflower oil exerted estrogenic activities and were thus unsuitable as vehicles for the tested compounds. The absence of estrogenic activity, high solubility of different steroid hormones, and the low incidence of the inflammatory reactions at the injection site were achieved by using a vehicle containing benzyl alcohol, benzyl benzoate, butylhydroxyanisole, butylhydroxytoluene, ethyl oleate and ethanol. The bioassay was primarily designed to examine the effect of tested compounds on mammogenesis. The duration of hormone treatment was chosen to be long enough for induction of duct growth but too short to induce lobuloalveolar differentiation. Females were treated for 10 days, males for 15 days. The proportional volume occupied by mammary epithelial structures was estimated by the modified Chalkley's technique. The mean coefficient of variation of quantitative evaluation of 10 different mammary glands obtained by two operators varied between 3.2 and 17.4%. The mean coefficient of variation of quintuplicate determinations of each mammary gland by one operator was 10.1%, and 11.1% by the other. The correlation coefficient between results of two operators was 0.994. Estrogens are primarily defined by their ability to increase the mitotic activity of female secondary sex organs. However, our results have shown that progesterone alone, if administered in a high dose, stimulates mammary growth in both intact prepubertal and OV-X female mice similarly as the synthetic progestatial steroid norethindrone with inherent estrogenic properties. In contrast, progesterone alone had no effect, in young intact or adult castrated males, but norethindrone did stimulate mammary growth. These results demonstrated that the mammary gland of males is a suitable model for estrogen screening.     

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.     

一种改进的粉虱成虫生物测定方法

生物测定是检测害虫抗药性的一项重要技术。利用100mL插口圆底聚丙烯离心管对现在应用较多的粉虱成虫生物测定方法——琼脂保湿浸叶法进行了改进。改进后的方法不影响粉虱成虫的持续取食,具有操作简单、结果重复性好及无需对成虫麻醉等优点。同时发现茄子叶片对于B型烟粉虱Bemisia tabaci(Gennadius)B-biotype和温室白粉虱Trialeurodes vaporariorum(Westwood)成虫均是一种非常适合的生测材料。利用该方法分3次独立测定了烯啶虫胺对B型烟粉虱和温室白粉虱混合日龄成虫的毒力,结果具有很好的重复性。     

Immunodiffusion method for detection of type A Clostridium botulinum

A simple gel immunodiffusion agar procedure was developed for detecting toxigenic strains of Clostridium botulinum type A. The method consisted of overlaying colonies grown on thin-layer tryptone-peptone-glucose-yeast extract agar with gel diffusion agar containing desired levels of C. botulinum type A antitoxin. Concentric precipitin zones formed around colonies of C. botulinum type A. Strains of C. botulinum type A were detected by this procedure. However, C. botulinum type B reacted to a lesser degree with this system. No reaction was noted with types E, F, Langeland, F8G, Clostridium perfringens, or with strains of nontoxigenic Clostridium sporogenes. Thickness of the plating medium, incubation time and temperature, environmental growth conditions, and levels of both agar an antitoxin were important factors affecting the efficiency of the procedure, whereas the age of the culture (used as inoculum) was not critical. Thin agar medium (5 ml per plate [15 by 100 mm]) containing 1.5% agar gave consistent results, but more agar limited diffusion, and lower levels encouraged spreaders. The optimal concentration of antitoxin incorporated in to the gel diffusion agar overlay was 1.2 IU/ml gel diffusion agar. Rabbit type A antitoxin prepared with purer immunizing agent gave similar reactions. The addition of type A antitoxin in tryptone-peptone-glucose-yeast extract agar medium before inoculation with type A C. botulinum showed promising results.