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Clinical and Diagnostic Laboratory Immunology, July 2004, p. 752-757, Vol. 11, No. 4
1071-412X/04/$08.00+0     DOI: 10.1128/CDLI.11.4.752-757.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Use of Bispecific Antibodies in Molecular Velcro Assays Whose Specificity Approaches the Theoretical Limit of Immunodetection for Bordetella pertussis

X. L. Tang,1 M. S. Peppler,2 R. T. Irvin,2 and M. R. Suresh1*

Faculty of Pharmacy and Pharmaceutical Sciences,1 Department of Medical Microbiology & Immunology, Faculty of Medicine, University of Alberta, Edmonton, Alberta, Canada T6G 2N82

Received 25 August 2003/ Returned for modification 24 November 2003/ Accepted 14 March 2004

A bispecific monoclonal antibody (bsMAb) that detects Bordetella pertussis, the causative agent of whooping cough, and horseradish peroxidase (HRPO) has been developed by use of the quadroma technology. A quadroma, P123, was produced by fusing two well-characterized hybridomas against the bacterium and the enzyme and was subcloned to obtain a stable bsMAb-secreting cell line. The quadroma was theoretically expected to produce up to 10 different molecular species of immunoglobulins, so secreted bispecific antibody was complexed with excess HRPO and the HRPO-bsMAb complex was purified in one step by benzhydroxamic acid-agarose affinity cochromatography. An ultrasensitive homosandwich molecular "velcro" enzyme-linked immunosorbent assay for the detection of B. pertussis whole bacteria with HRPO-bsMAb was established in both microplate and nasopharyngeal swab formats. This assay demonstrates a high sensitivity that approaches the theoretical limit of detection of one bacterium. This new nanoprobe can be used to develop a new generation of assays that are simple, inexpensive alternatives to quantitative PCR and that can be used by clinical laboratories. This strategy of homosandwich assays with solid-phase monospecific antibodies and solution-phase bsMAb with specificity for the same repeating surface determinants can be applied to generate ultrasensitive immunodiagnostic assays for viruses and bacteria.

* Corresponding author. Mailing address: Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Alberta, Canada T6G 2N8. Phone: (780) 492-9233. Fax: (780) 492-1217. E-mail: msuresh{at}pharmacy.ualberta.ca.

Clinical and Diagnostic Laboratory Immunology, July 2004, p. 752-757, Vol. 11, No. 4
1071-412X/04/$08.00+0     DOI: 10.1128/CDLI.11.4.752-757.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.





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