Use of bispecific antibodies in molecular velcro assays whose specificity approaches the theoretical limit of immunodetection for Bordetella pertussis.
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.
Tang
X L
XL
Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Alberta, Canada T6G 2N8.
Peppler
M S
MS
Irvin
R T
RT
Suresh
M R
MR
D000818:Animals; D000907:Antibodies, Bacterial / Q000276:immunology; D000911:Antibodies, Monoclonal / Q000276:immunology; D001886:Bordetella pertussis / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005434:Flow Cytometry; D006735:Horseradish Peroxidase / Q000276:immunology*; D006825:Hybridomas / Q000276:immunology; D012680:Sensitivity and Specificity
D000907:Antibodies, Bacterial; D000911:Antibodies, Monoclonal; D006735:Horseradish Peroxidase