Colony immunoblot assay of botulinal toxin

Goodnough, Michael C.; Hammer, B. W.; Sugiyama, Hiroshi; Johnson, Eric A.

doi:10.1128/aem.59.7.2339-2342.1993

Cited by 28 publications

(14 citation statements)

References 11 publications

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“…Due to the availability of monoclonal antibodies against HBL [13] and NHE (publication in preparation), the work described herein was focused on these toxin complexes. After some initial experiments using strains DSM 4384, producing HBL and NHE, and NVH 0075/95, which produces only NHE, it became clear that antibodies 2A3 and 1A8 were suitable for a colony blot procedure, similar to that described by Goodnough et al [22] for botulinal toxin. NC membranes turned out to be superior to PVDF membranes in view of handling and efficiency of antigen binding, allowing the components of HBL and NHE being transferred to the membrane (Fig.…”

Section: Discussionmentioning

confidence: 88%

Colony immunoblot assay for the detection of hemolysin BL enterotoxin producing

Moravek¹,

Wegscheider²,

Schulz³

et al. 2004

FEMS Microbiology Letters

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Bacillus cereus strains involved in food poisoning cases of the diarrheal type may produce two different enterotoxin complexes. To facilitate the identification of hemolysin BL-enterotoxin complex (HBL) and/or the nonhemolytic enterotoxin (NHE) producing colonies a colony immunoblot procedure was developed, which allows a fast and easy identification of the respective colonies from blood agar plates. The enterotoxins were transferred from the blood agar medium to a nitrocellulose membrane and the immobilized toxins were probed with monoclonal antibodies. The antibodies 2A3 and 1A8 allowed the specific detection of the B component of HBL and the nheA component of NHE. The assay enabled the reliable identification of HBL expressing colonies and differentiation from NHE producing but HBL negative colonies.

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Section: Discussionmentioning

confidence: 88%

Colony immunoblot assay for the detection of hemolysin BL enterotoxin producing

Moravek¹,

Wegscheider²,

Schulz³

et al. 2004

FEMS Microbiology Letters

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“…The use of immunosassays and ELISAs in the area of food microbiology is common. Immunoassays have been developed for the detection of toxins of sporeformers such as B. cereus toxins, and C. botulinum toxin (Day et al 1994;Goodnough et al 1993) as well as for the toxins of nonsporeformers, such as Staphylococcus aureus (Lapeyre et al 1988). Immunoassays have also been developed for the vegetative cells of both sporeformers and nonsporeformers (Hartman et al 1992).…”

Section: Introductionmentioning

confidence: 99%

MONOCLONAL ANTIBODY‐BASED ELISAS FOR THE DETECTION OF BACTERIAL SPORES ¹

Quinlan

Foegeding

1998

Rapid Methods Automation Mic

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Monoclonal antibodies against bacterial spores were used to develop ELISAs to detect a range of spores. ELISAs utilizing anti‐Bacillus spore antibodies detected spores of Bacillus megaterium ATCC 12872, Bacillus stearothermophilus ATCC 7953, Bacillus subtilis var. globigii and Bacillus cereus T. They did not detect Bacillus subtilis A, Bacillus coagulans ATCC 56177 or Bacillus licheniformis ATCC 9789. One ELISA utilizing anti‐Clostridium spore antibodies detected spores of Desulfotomaculum nigrificans ATCC 7946, Clostridium perfringens ATCC 3624 and B. stearothermophilus ATCC 7953, but not spores of Clostridum sporogenes PA3679 or Clostridium botulinum 62A. Whole milk, skim milk, gelatin and starch all interfered to some degree with the abilities of the assays to detect spores. Neither centrifugation nor immunomagnetic bead separation provided sufficient concentration of spores from the food matrix to be applicable to the ELISAs developed. These results demonstrate the ability to detect a range of spores using monoclonal antibody‐based ELISAs. Such assays may, however, be limited by sensitivity and interference from food components.

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“…In the case of colony blot, the protocol described by Goodnough et al [ 34 ] was modified as follows. After cultivation of P. pastoris /pPGKΔ3 α /L1 multicopy clones in YPD agar plates for 3 days at 30°C, the PVDF membranes were cut as discs and left standing with the surface colonies for 3 hours at 28°C.…”

Section: Methodsmentioning

confidence: 99%

Development of an IP-Free Biotechnology Platform for Constitutive Production of HPV16 L1 Capsid Protein Using thePichia pastoris PGK1Promoter

Mariz

Coimbra

Jesus

et al. 2015

BioMed Research International

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The human papillomavirus (HPV) L1 major capsid protein, which forms the basis of the currently available vaccines against cervical cancer, self-assembles into virus-like particles (VLPs) when expressed heterologously. We report the development of a biotechnology platform for HPV16 L1 protein expression based on the constitutive PGK1 promoter (PPGK1) from the methylotrophic yeast Pichia pastoris. The L1 gene was cloned under regulation of PPGK1 into pPGKΔ3 expression vector to achieve intracellular expression. In parallel, secretion of the L1 protein was obtained through the use of an alternative vector called pPGKΔ3α, in which a codon optimized α-factor signal sequence was inserted. We devised a work-flow based on the detection of the L1 protein by dot blot, colony blot, and western blot to classify the positive clones. Finally, intracellular HPV VLPs assembly was demonstrated for the first time in yeast cells. This study opens up perspectives for the establishment of an innovative platform for the production of HPV VLPs or other viral antigens for vaccination purposes, based on constitutive expression in P. pastoris.

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Colony immunoblot assay of botulinal toxin

Cited by 28 publications

References 11 publications

Colony immunoblot assay for the detection of hemolysin BL enterotoxin producing

Colony immunoblot assay for the detection of hemolysin BL enterotoxin producing

MONOCLONAL ANTIBODY‐BASED ELISAS FOR THE DETECTION OF BACTERIAL SPORES ¹

Development of an IP-Free Biotechnology Platform for Constitutive Production of HPV16 L1 Capsid Protein Using thePichia pastoris PGK1Promoter

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Colony immunoblot assay of botulinal toxin

Cited by 28 publications

References 11 publications

Colony immunoblot assay for the detection of hemolysin BL enterotoxin producing

Colony immunoblot assay for the detection of hemolysin BL enterotoxin producing

MONOCLONAL ANTIBODY‐BASED ELISAS FOR THE DETECTION OF BACTERIAL SPORES 1

Development of an IP-Free Biotechnology Platform for Constitutive Production of HPV16 L1 Capsid Protein Using thePichia pastoris PGK1Promoter

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MONOCLONAL ANTIBODY‐BASED ELISAS FOR THE DETECTION OF BACTERIAL SPORES ¹