Monoclonal Antibody-Based ELISA for Detection of Clostridium perfringens Alpha-Toxin

A monoclonal antibody-based ELISA was developed to detect alpha-toxin present in Clostridium perfringens bacterial cell lysates and cell-free culture supernatants, Monoclonal antibodies against C. perfringens alpha-toxin were produced in hybridoma tissue culture supernatants and in BALB/c mice ascites fluid, The monoclonal antibodies obtained from hybridoma culture supernatant and ascites fluid showed identical antigen specificity, but the latter showed a higher titer, with a 50% endpoint at 1/4,000. The monoclonal antibodies were specific for phospholipase C produced by C. perfringens, but not by Bacillus cereus. The lower limit of phospholipase C detection was 16 ng/ml. The dose-dependent relationship between absorption at 490 nm and concentration of phospholipase C diluted in HEPES (N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid) or Trypticase glucose yeast broth fit a four-parameter and a quadratic model, respectively. The monoclonal antibody-based ELISA developed is a rapid, sensitive and specific detection method and can be used for quantitative characterization of C. perfringens alpha-toxin.

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Separation of Monoclonal Antibodies from Cell-Culture Supernatants and Ascites Fluid Using Thiophilic Agarose

Monoclonal Antibody Protocols ◽

10.1385/0-89603-308-2:177 ◽

2003 ◽

pp. 177-182

Author(s):

Thorkild C. Bøg-Hansen

Keyword(s):

Cell Culture ◽

Monoclonal Antibodies ◽

Ascites Fluid ◽

Culture Supernatants

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Contraction of the rat isolated aorta caused by Clostridium perfringens alpha toxin (phospholipase C): evidence for the involvement of arachidonic acid metabolism

British Journal of Pharmacology ◽

10.1111/j.1476-5381.1989.tb11931.x ◽

1989 ◽

Vol 97 (1) ◽

pp. 119-124 ◽

Cited By ~ 34

Author(s):

Yoshio Fujii ◽

Jun Sakurai

Keyword(s):

Arachidonic Acid ◽

Phospholipase C ◽

Clostridium Perfringens ◽

Acid Metabolism ◽

Arachidonic Acid Metabolism ◽

Alpha Toxin ◽

Isolated Aorta

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Histamine release from mast cells by highly purified phospholipase C (alpha-toxin) and theta-toxin from Clostridium perfringens

Toxicon ◽

10.1016/0041-0101(74)90261-x ◽

1974 ◽

Vol 12 (2) ◽

pp. 219

Keyword(s):

Mast Cells ◽

Histamine Release ◽

Phospholipase C ◽

Clostridium Perfringens ◽

Alpha Toxin

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Characterization of a Broadly Reactive Monoclonal Antibody against Norovirus Genogroups I and II: Recognition of a Novel Conformational Epitope

Journal of Virology ◽

10.1128/jvi.00891-07 ◽

2007 ◽

Vol 81 (22) ◽

pp. 12298-12306 ◽

Cited By ~ 44

Author(s):

Tomoyuki Shiota ◽

Michio Okame ◽

Sayaka Takanashi ◽

Pattara Khamrin ◽

Makiko Takagi ◽

...

Keyword(s):

Monoclonal Antibody ◽

Monoclonal Antibodies ◽

Conformational Epitope ◽

The Novel ◽

Antigen Specificity ◽

Virus Like Particle ◽

The Family ◽

Immunological Diagnosis ◽

Further Development

ABSTRACT Norovirus, which belongs to the family Caliciviridae, is one of the major causes of nonbacterial acute gastroenteritis in the world. The main human noroviruses are of genogroup I (GI) and genogroup II (GII), which were subdivided further into at least 15 and 18 genotypes (GI/1 to GI/15 and GII/1 to GII/18), respectively. The development of immunological diagnosis for norovirus had been hindered by the antigen specificity of the polyclonal antibody. Therefore, several laboratories have produced broadly reactive monoclonal antibodies, which recognize the linear GI and GII cross-reactive epitopes or the conformational GI-specific epitope. In this study, we characterized the novel monoclonal antibody 14-1 (MAb14-1) for further development of the rapid immunochromatography test. Our results demonstrated that MAb14-1 could recognize 15 recombinant virus-like particles (GI/1, 4, 8, and 11 and GII/1 to 7 and 12 to 15) and showed weak affinity to the virus-like particle of GI/3. This recognition range is the broadest of the existing monoclonal antibodies. The epitope for MAb14-1 was identified by fragment, sequence, structural, and mutational analyses. Both terminal antigenic regions (amino acid positions 418 to 426 and 526 to 534) on the C-terminal P1 domain formed the conformational epitope and were in the proximity of the insertion region (positions 427 to 525). These regions contained six amino acids responsible for antigenicity that were conserved among genogroup(s), genus, and Caliciviridae. This epitope mapping explained the broad reactivity and different titers among GI and GII. To our knowledge, we are the first group to identify the GI and GII cross-reactive monoclonal antibody, which recognizes the novel conformational epitope. From these data, MAb14-1 could be used further to develop immunochromatography.

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Naturally Occurring Clostridium perfringens Nontoxic Alpha-Toxin Variant as a Potential Vaccine Candidate against Alpha-Toxin-Associated Diseases

Infection and Immunity ◽

10.1128/iai.69.11.7194-7196.2001 ◽

2001 ◽

Vol 69 (11) ◽

pp. 7194-7196 ◽

Cited By ~ 15

Author(s):

Heike Schoepe ◽

Christian Pache ◽

Axel Neubauer ◽

Heidrun Potschka ◽

Tobias Schlapp ◽

...

Keyword(s):

Phospholipase C ◽

Mutant Strain ◽

Clostridium Perfringens ◽

Hemolytic Activity ◽

Vaccine Candidate ◽

Alpha Toxin ◽

Wild Type ◽

Potential Vaccine Candidate ◽

Naturally Occurring ◽

Potential Vaccine

ABSTRACT Clostridium perfringens mutant strain 121A/91 shows neither enzymatic (phospholipase C) nor hemolytic activity. Nevertheless, the cpa gene and the corresponding alpha-toxin variant are detectable. Vaccination with this genetically constructed alpha-toxin variant, rAT121/91, induces antibodies capable of significantly reducing activities induced by wild-type toxin. Thus, rAT121/91 could be a useful vaccine candidate.

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