Production and characterization of a monoclonal antibody against the sialidase of Gardnerella vaginalis using a synthetic peptide in a MAP8 format

Abstract

Bacterial vaginosis is one of the most frequent vaginal infections. Its main etiological agent is Gardnerella vaginalis, which produces several virulence factors involved in vaginal infection and colonization, in particular, sialidase (SLD), a potential clinical biomarker that participates in immune response modulation and mucus degradation. The main objective of this work was the production and evaluation of a monoclonal antibody against G. vaginalis sialidase and its validation in immunoassays. For immunization of mice, a synthetic multiantigenic peptide was used, and hybridomas were generated. After fusion, hybridomas were evaluated for antibody production and cloned by limited dilution. One clone producing IgG1 was selected and characterized by indirect ELISA, dot blot, and Western blot, and we also tested clinical isolates and HeLa cells infected with G. vaginalis. The results showed that the anti-SLD antibody recognized a single protein of~90 kDa that correlated with the estimated molecular weight of SLD. In addition, anti-SLD antibody recognized SLD from complete bacteria and from culture supernatants of infected Hela cells. In conclusion, our results showed that the anti-SLD antibody recognized SLD from different sources and could be considered a new tool for the diagnosis of bacterial vaginosis. Key Points • Anti-sialidase mAb was generated using a synthetic peptide • The mAb recognizes synthetic peptide and intact protein from multiple sources • The antibody was characterized by several immunological methods

Nanophotonic Sialidase Immunoassay for Bacterial Vaginosis Diagnosis

“…Existing quantitative approaches targeting SLD are based on the catalytic activity of this enzyme, , which can be affected by variation in temperature/pH or any process leading to denaturation upon sample collection . Conversely, nanoBV does not measure the catalytic activity of SLD but the enzyme directly, even in denatured conditionsprovided the corresponding binding sites can still be recognized by the developed mAb . We hypothesize that this is the origin of the SLD levels reported by nanoBV, which are not in agreement with those SLD levels resulting from the aforementioned method based on enzymatic hydrolysis of methoxyphenyl acetyl muramic acid via SLD, since this method generally reports SLD levels lower than 600 ng mL –1 . , Consequently, our research also opens a debate on the SLD levels found in vaginal swab samples depending on the employed analytical platform.…”

“…We also compared the optimized LOD of nanoBV with that of an indirect enzyme-linked immunosorbent assay (ELISA) previously reported by our team, whose limit of detection accounted for 0.031 ng mL –1 (see Figure S6, SI); hence, nanoBV was observed to be 2.5-fold more sensitive than the indirect ELISA in terms of limit of detection. The overall cost of both detection platforms targeting SLD was also compared.…”

“…A synthetic peptide of SLD was designed by our team and synthesized by Pepmic (Gusu District, Zushou, China). Full details related on this peptide were previously reported . Dimethyl sulfoxide (DMSO), biotin N -hydroxysuccinimide ester, Tween 20, phosphate-buffered saline (PBS), and sodium bicarbonate (NaHCO 3 ) were acquired from Sigma-Aldrich (St. Louis, MO); BSA (employed in the inmunobuffer) was purchased from ToCris Bioscience (Bristol, United Kingdom).…”

“…In this context, our research team recently developed a novel monoclonal antibody (mAb) against SLD, which has been validated via several immunological methods and demonstrated to recognize SLD with high specificity . Taking advantage of this mAb, we designed a single-step quantitative biosensing system for targeting SLD.…”

“…20−24 In this context, our research team recently developed a novel monoclonal antibody (mAb) against SLD, which has been validated via several immunological methods and demonstrated to recognize SLD with high specificity. 25 Taking advantage of this mAb, we designed a single-step quantitative biosensing system for targeting SLD. This biosensing concept has been recently reported to be technically sound as a virtually universal wash-free and single-step immunosensing platform.…”