An enzyme labelled immunosorbent assay for measuring<i>Clostridium perfringens</i>epsilon toxin in gut contents

Weddell, W.; Worthington, R.W.

doi:10.1080/00480169.1985.35148

Cited by 12 publications

(10 citation statements)

References 4 publications

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“…perfringens can easily lose the plasmid encoding ε toxin during culture and isolation, the presence of the ε toxin gene cannot unambiguously prove the presence (and the quantity) of epsilon toxin and prototoxin and finally those techniques are time-consuming and time to results vary from several hours to several days. Thus, there have been many reports of alternative methods using immunoassays for direct ε toxin protein detection by counterimmunoelectrophoresis [16], latex agglutination test [17] and several sandwich enzyme immunoassays using either polyclonal antibodies [18–20] or monoclonal/polyclonal antibodies [16,21]. Three of these immunotechniques were compared with the mouse neutralization test [16], and results showed that there was a marked inconsistency among the four techniques to detect ε toxin in different spiked or naturally contaminated ovine intestinal contents (with presumptive or experimental enterotoxemia).…”

Section: Introductionmentioning

confidence: 99%

Highly sensitive sandwich immunoassay and immunochromatographic test for the detection of Clostridial epsilon toxin in complex matrices

et al. 2017

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Epsilon toxin is one of the four major toxins of Clostridium perfringens. It is the third most potent clostridial toxin after botulinum and tetanus toxins and is thus considered as a potential biological weapon classified as category B by the Centers for Disease Control and Prevention (CDC). In the case of a bioterrorist attack, there will be a need for a rapid, sensitive and specific detection method to monitor food and water contamination by this toxin, and for a simple human diagnostic test. We have produced and characterized five monoclonal antibodies against common epitopes of epsilon toxin and prototoxin. Three of them neutralize the cytotoxic effects of epsilon toxin in vitro. With these antibodies, we have developed highly sensitive tests, overnight and 4-h sandwich enzyme immunoassays and an immunochromatographic test performed in 20 min, reaching detection limits of at least 5 pg/mL (0.15 pM), 30 pg/mL (0.9 pM) and 100 pg/mL (3.5 pM) in buffer, respectively. These tests were also evaluated for detection of epsilon toxin in different matrices: milk and tap water for biological threat detection, serum, stool and intestinal content for human or veterinary diagnostic purposes. Detection limits in these complex matrices were at least 5-fold better than those described in the literature (around 1 to 5 ng/mL), reaching 10 to 300 pg/mL using the enzyme immunoassay and 100 to 2000 pg/mL using the immunochromatographic test.

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Section: Introductionmentioning

confidence: 99%

Highly sensitive sandwich immunoassay and immunochromatographic test for the detection of Clostridial epsilon toxin in complex matrices

et al. 2017

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“…5,6,12 Different techniques are routinely used to detect epsilon toxin in body fluids. 4,6,8,9,12,16 In the present study the 4 techniques tested showed marked differences in their ability to detect epsilon toxin in intestinal content and other body fluids. The PC-ELISA was the most sensitive technique regardless of fluid type.…”

Section: Discussionmentioning

confidence: 89%

“…Affinity-purified ovine antiepsilon toxin antibodies were prepared as previously described. 16 Briefly, highly purified epsilon prototoxin was converted to toxoid with formalin and used to hyperimmunize a sheep. Antibodies from the sheep serum were purified on an affinity chromatography column made by coupling purified epsilon toxin to AH Sepharose b with diethyl-3 (3-dimethyl amino propyl) carbodiimide, using the method described previously.…”

Section: Diagnostic Techniquesmentioning

confidence: 99%

“…Optical densities of 1.0 or higher indicated positive results. 16 The MC-ELISA was performed using a commercial ELISA kit d according to the manufacturer's instructions. Briefly, the samples were added to the wells of the ELISA plates to which a C. perfringens epsilon toxin monoclonal antibody was bound and the plates were incubated for 60 minutes at room temperature.…”

Section: Diagnostic Techniquesmentioning

confidence: 99%

“…The techniques most commonly used to detect epsilon toxin in intestinal contents and other body fluids are the mouse neutralization test (MNT), 12 several enzyme-linked immunosorbent assays (ELISAs), 4,8,9,16 and counterimmunoelectrophoresis (CIEP). 6 There is little information available in the literature about the comparative ability of these methods to detect epsilon toxin in intestinal contents and other body fluids.…”

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confidence: 99%

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Comparison of Four Techniques for the Detection of Clostridium Perfringens Type D Epsilon Toxin in Intestinal Contents and other Body Fluids of Sheep and Goats

et al. 2003

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Abstract. Polyclonal capture enzyme-linked immunosorbent assay (PC-ELISA), monoclonal capture ELISA (MC-ELISA), mouse neutralization test (MNT), and counterimmunoelectrophoresis (CIEP), were compared for their ability to detect epsilon toxin in intestinal contents and body fluids of sheep and goats. When used to evaluate intestinal contents of sheep artificially spiked with epsilon prototoxin, PC-ELISA detected 0.075 mouse lethal dose (MLD) 50 /ml, whereas the MNT, MC-ELISA, and CIEP detected 6, 25, and 50 MLD 50 /ml, respectively. Amounts of epsilon toxin detected by PC-ELISA, MC-ELISA, MNT, and CIEP in sheep pericardial fluid artificially spiked with epsilon prototoxin were 0.075, 0.75, 6, and 200 MLD 50 /ml, respectively. For assaying epsilon toxin in aqueous humor, PC-ELISA and MC-ELISA detected 0.075 MLD 50 / ml, whereas CIEP detected 200 MLD 50 /ml (MNT was not evaluated). When 51 samples of intestinal contents of sheep and goats (32 positive and 19 negative to MNT) were analyzed by the other 3 techniques, the relative sensitivity of PC-ELISA, MC-ELISA, and CIEP was 93.75, 84.37, and 37.50%, respectively. The specificity of PC-ELISA, MC-ELISA, and CIEP was 31.57, 57.89, and 84.21%, respectively. The absolute sensitivity of PC-ELISA, MC-ELISA, CIEP, and MNT was 90.90, 69.69, 15.15, and 54.54%. The absolute specificity of the 4 techniques was 100%. These results show that there is a marked inconsistency among techniques routinely used to detect Clostridium perfringens epsilon toxin. Until more consistent results are achieved, the diagnosis of enterotoxemia should not only be based solely on epsilon toxin detection, but also on clinical and pathological data.

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Selection of a Clostridium perfringens type D epsilon toxin producer via dot-blot test

et al. 2009

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Clostridium perfringens type D produces enterotoxemia, an enteric disease in ruminants, also known as pulpy kidney disease. Caused by epsilon toxin, enterotoxemia is a major exotoxin produced by this microorganism. Epsilon toxin is also the main component of vaccines against this enteric disorder. In this study, a standardized dot-blot was used to choose strains of C. perfringens type D that are producers of epsilon toxin. Clones producing epsilon toxin were chosen by limiting dilution; after three passages, lethal minimum dose titers were determined by soroneutralization test in mice. These clones produced epsilon toxin 240 times more concentrated than the original strain. The presence of the epsilon toxin gene (etx) was verified by polymerase chain reaction. All clones were positive, including those determined to be negative by dot-blot tests, suggesting that mechanisms in addition to the presence of the etx gene can influence toxin production. The dot-blot test was efficient for the selection of toxigenic colonies of C. perfringens type D and demonstrated that homogeneous populations selected from toxigenic cultures produce higher titers of epsilon toxin.

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An enzyme labelled immunosorbent assay for measuringClostridium perfringensepsilon toxin in gut contents

Cited by 12 publications

References 4 publications

Highly sensitive sandwich immunoassay and immunochromatographic test for the detection of Clostridial epsilon toxin in complex matrices

Highly sensitive sandwich immunoassay and immunochromatographic test for the detection of Clostridial epsilon toxin in complex matrices

Comparison of Four Techniques for the Detection of Clostridium Perfringens Type D Epsilon Toxin in Intestinal Contents and other Body Fluids of Sheep and Goats

Selection of a Clostridium perfringens type D epsilon toxin producer via dot-blot test

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