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

Authors
Publisher
American Society for Microbiology
Publication Date
Source
PMC
Keywords
  • Microbial Immunology
Disciplines
  • Biology
  • Medicine

Abstract

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.

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