Screening and characterization of new monoclonal anti-benzo[a]pyrene antibodies using automated flow-through microarray technology

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental pollutants, which can cause cancer in humans. The maximum tolerable limit of benzo[a]pyrene (B[a]P) in drinking water was set to 10ng/L by the European Commission (Council Directive 98/83/EC), because of its highly carcinogenic and mutagenic effect on humans. In the present investigation, mice were immunized with B[a]P-bovine serum albumin conjugates and 110 generated hybridoma cell lines screened by different techniques to identify clones that produce anti-B[a]P antibodies. Subsequently, a new automated flow-through biochip noncompetitive direct chemiluminescence immunoassay (CLEIA) was compared with conventional indirect and direct enzyme-linked immunosorbent assays (ELISAs). It was demonstrated that the microchip-based screening method compared to ELISA was fast and very sensitive with use of only nanoliter volumes of supernatant. Forty clones could be evaluated in less than 5min. Six high affinity monoclonal antibodies with different cross-reactivities (CR) for individual PAHs were identified by the chip-based assay and indirect microtiter plate ELISA. In comparison, the direct ELISA in the microtiter plate failed to identify three of these clones. The four antibodies with the highest affinity had half maximum inhibitory concentrations (IC₅₀ values) between 0.31 and 0.92μg/L for B[a]P. Affinity constants of these four antibodies were determined by surface plasmon resonance using a water soluble B[a]P-peptide. The observed CR pattern of the four monoclonal antibodies for 16 tested PAHs was quite different. Only one specific antibody for B[a]P was observed, while others were more suitable for class-specific PAH determination.