Development of Polyclonal Antibodies Against Domoic Acid for Their Use in Electrochemical Biosensors

Abstract

The current work describes the raising of polyclonal antibodies (pAbs) against domoic acid (DA), an algal toxin produced by the diatom Pseudonitzschia pungens. They were screened for sensitivity and selectivity using a competitive enzyme-linked immunosorbent assay (ELISA). The antiserum produced against a keyhole limpet haemocyanin (DA-KLH) conjugate displayed a high affinity for free DA. The optimized horseradish peroxidase (HRP) ELISA had a detection limit of 0.6 ng mL−1 (ppb) and a working range of 0.8–300 ppb DA applying a streptavidin-biotin amplification system (ABC system). Furthermore this antiserum did not cross-react with similar chemical structures and algal toxins such as kainic acid, aspartic acid, glutamic acid, geranic acid and 2-methyl-3-butenoic acid. When the ELISA was compared using an alkaline phosphatase (AP) label we found the system to behave in a similar manner to the optimized HRP system but the linear range was smaller in the high DA concentration range. These pAbs were then used in the optimization of a screen-printed electrode (SPE) system for measurement of DA. A disposable screen-printed carbon electrode coupled with amperometric detection of p-aminophenol at +300 mV vs. Ag/AgCl, produced by the enzyme AP, was used for signal measurement. The sensor incorporates a relevant range for toxin detection, by which humans become ill (Iverson, F.; Truelove, J. Toxicology and seafood toxins: domoic acid. Natural Toxins 1994, 2, 334–339.) with detection limits achieved at SPE to the order of ppb. The SPE system is simple and cost-effective due to its disposable nature, and analysis time is complete in 30 min. In addition, recovery experiments on DA for both ELISA and SPE highlighted the functionality of these systems yielding a ±12% deviation for the true value for the ELISA using AP and ±25% for the sensor.