Problems in immunoassay development of small and volatile molecules - Benzene, Toluene, and Xylenes (BTX)

Indirect and direct enzyme immunoassay formats for the detection of the volatile organic compounds benzene, toluene, and xylenes have been developed based on polyclonal antibodies. In the indirect ELISA, the detection limit for the five analytes (equimolar amounts) in water was 210 μg/L and the center point value of the calibration curve was 1.7 mg/L. Because of the volatility of the analytes special precautions were necessary. Best conditions for assay procedure were found with an incubation at 4°C and without covering the reaction vessel (microtiter plate or test tube) with a sealer (Parafilm®) during the competitive step. An incubation time of 10 min was sufficient and resulted in good sensitivity. With the addition of 10% dipolar aprotic organic solvent the analytes could be kept in water and the base value of the calibration curve could be decreased. With the use of a non-ionic surfactant the stability of the assay could not be improved. In contrast, the addition of Genapol C 080 or Triton X-100 led to interference with the ELISA. However, a slight increase in sensitivity was obtained (IC₅₀: 0.9 mg/L) by a transfer of the microtiter plate assay to test tubes, but with some loss of reproducibility. The tube ELISA can be used as a semiquantitative screening test.