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

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 solutionphase bsMAb with specificity for the same repeating surface determinants can be applied to generate ultrasensitive immunodiagnostic assays for viruses and bacteria.Whooping cough (pertussis) is a vaccine-preventable disease caused by the gram-negative bacterium Bordetella pertussis. Despite required vaccination in most countries, the incidence of whooping cough is still unacceptably high and has recently emerged in the adolescent age group (2, 10). Accurate diagnosis of B. pertussis is essential to proper surveillance of whooping cough, and improved diagnostic methods are desirable.One of the present methods for the diagnosis of B. pertussis infection is a direct fluorescent-antibody assay that uses a fluorescence-labeled monoclonal antibody (MAb) directed against the lipopolysaccharide (LPS) in the outer membrane of the bacterium (14). Unlike many other bacteria, B. pertussis produces one predominant antigenic type of LPS molecule (17), making it a good specific target for immunodiagnosis. Like other endotoxins, B. pertussis LPS is a durable molecule that can easily withstand the conditions encountered during handling and transport. The detection of antibody to LPS is a good strategy for the diagnosis of B. pertussis infection, but the method could be improved to make it easier to use and a more sensitive reagent is needed (20).Here we report on the development and characterization of a bispecific MAb (bsMAb) against horseradish peroxidase (HRPO) and B. pertussis LPS that could be used for enzymebased detection of soluble LPS antigen and whole B. pertussis bacteria in clinical samples, immunochemical structural studies, and serological characterization of B. pertussis LPS, with some potential advantage over present MAb-or polyclonal antibody-labeled immunoassays. In particular, ...

Bispecific Antibodies for Diagnostic Applications

“…The sensitivity of detection was high since as few as ten bacteria could be detected compared to the controls (Tang et al 2004). The BsMAb-based assay has unique advantages in being highly sensitive and easy to perform.…”

“…Interestingly, binding of B. pertussis to the anti-B. pertussis LPS monoclonal antibody-coated solid phase was found to be irreversible despite extensive washing suggesting a unique molecular velcro effect (Tang et al 2004). The use of BsMAb in the immunoassay allowed ultrasensitive detection of B. pertussis since it avidly captured multiple exposed LPS molecules on the bacterial surface.…”

“…Our laboratory therefore developed a highly sensitive and simple BsMAb-based immunodiagnostic assay for the detection of whole B. pertussis and soluble B. pertussis LPS (Tang et al 2004). The assay is useful not only for analyzing clinical samples but also for immunochemical structural studies and serological characterization of B. pertussis LPS.…”

“…BsMAb was subsequently purified by affinity chromatography using benzhydroxamicacid agarose column (Husereau and Suresh 2001) and used in a homosandwich ELISA immunoassay. Heat-killed B. pertussis BP347 by itself or spiked into nasopharyngeal aspirates was first bound by the anti-LPS specific monoclonal antibody, and then the BsMAb was added as the detection antibody (Tang et al 2004). Refer to Fig.…”

“…The BsMAb is subsequently purified by affinity chromatography using m-aminophenylboronic acid agarose column . The general format of the immunodiagnostic assay involves coating Dengue virus antigen-specific monoclonal antibody as the capture antibody on calcium alginate tipped swabs with aluminum/or plastic shafts as per previously published protocol (Tang et al 2004). The swab is then blocked and later incubated with the specific antigen spiked in serum, after which, the purified BsMAb is used as the detection antibody.…”