Yasuhiro Goda

Fully automated immunoassay system of endocrine disrupting chemicals using monoclonal antibodies chemically conjugated to bacterial magnetic particles

The development of a rapid and high-throughput detection system for endocrine disrupting chemicals (EDCs) has been required in the recent years. A fully automated immunoassay system was described for the detection of EDCs, such as alkylphenol ethoxylates (APEs), bisphenol A (BPA) and linear alkylbenzene sulfonates (LASs), using monoclonal antibodies chemically conjugated to bacterial magnetic particles (BMPs) and alkaline phosphatase (ALP)-conjugated EDCs. EDC concentrations were evaluated by the decrease in luminescence based on the competitive reaction of EDCs and ALP-conjugated EDCs. Full automation of the BMP-based immunoassay was achieved by using an automated eight-way pipet moving at X-, Y- and Z-direction and a B/F separation unit. B/F separation was performed on the tip surface of eight-way pipet with a retractable magnet mounted close to the pipet tip. Immunoreactions were saturated after 10 min, and the assay was completed within 15 min. The detection ranges for APE, BPA and LAS were 6.6 ppb–66 ppm, 2.3 ppt–2.3 ppm, and 35 ppt–35 ppm, respectively. This BMP-based immunoassay system has advantages due to its high sensitivity and rapid measurement of samples.

Piezoelectric immunosensor for bisphenol A based on signal enhancing step with 2-methacrolyloxyethyl phosphorylcholine polymeric nanoparticle

An immunoassay in which BPA competed with a BPA-horseradish peroxidase conjugate for binding to anti-BPA antibodies, coupled to a piezoelectric (PZ) immunosensor, was able to detect 0.1 ng mL(-1) BPA. To enhance the sensitivity of the assay, we tested nanoparticles approximately 200 nm in diameter, coupled to anti-BPA antibodies, to increase the mass change on the surface of the immunosensor and thereby increase the frequency shift detected. This second step, using nanoparticles coated with anti-BPA antibodies, improved the sensitivity of the assay by approximately eight times at BPA concentrations below 10 ng mL(-1). Field emission-scanning electron microscopy (FE-SEM) showed that polymeric 2-methacrolyloxyethyl phosphorylcholine (MPC) nanoparticles coupled to antibodies remained monodisperse on the surface of the immunosensor and therefore produced stable signals in the immunosensors. Since the frequency shift detected in the assay mainly originated from the mass change on the surface of the PZ crystal, the colloidal stability of the antibody-conjugated particles used in the enhancement step played an extremely important role in achieving a stable and highly sensitive signal.

Molecular analysis of specificity of anti-nonylphenol polyethoxylate single-chain antibody fragments by grafting and designed point mutations.

Alkylphenol polyethoxylates and alkylphenols are widely distributed contaminants in the environment. Two anti-alkylphenol polyethoxylate monoclonal antibodies MOF3-139 and AP-14 were established to measure these chemicals by enzyme immunoassays in previous studies. Interestingly, these two monoclonal antibodies showed different specificity; AP-14 cross-reacts with nonylphenoxyacetic acid and nonylphenol, whereas MOF3-139 does not. To understand the molecular basis of the difference in specificity, single-chain Fv (scFv) antibodies derived from the monoclonal antibodies were each produced in Escherichia coli cells and characterized in competitive enzyme-linked immunosorbent assay. The scFv antibodies exhibited comparable reactivity profiles to the derived parent monoclonal antibodies. It was found that the VH domain of AP-14 play an important role in the cross-reaction when specificity tests were performed using variable domain-swapped scFv antibodies. An experiment using complementarity-determining region (CDR)-grafted scFv antibodies revealed that CDR1 and CDR2 of AP-14 are involved in the cross-reaction to nonylphenoxyacetic acid and nonylphenol, respectively. Site-directed mutagenesis was introduced in both regions and the assay revealed that 33rd Thr and 35th His in VH domain of AP-14 were highly involved in the cross-reaction with nonylphenoxyacetic acid and that 33rd Thr, 57th Asp, and 59th Glu were involved in the cross-reaction with nonylphenol. The findings herein would contribute to the antibody engineering for specificity modification and to the generation of an alkylphenol-specific recombinant antibody by antibody engineering.