Development of an enzyme linked immunosorbent assay for dehydroabietic acid in pulp mill effluents
Creators: name: Li, Kai
Publisher: name: University of British Columbia
Year: 2009

description: An enzyme-linked immunosorbent assay (ELISA) was developed for the quantification of resin acids in softwood pulp mill effluents. Various antigens were synthesized and polyclonal antibodies were successfully raised by immunizing rabbits with DHAM-SUC-KLH or DHA-KLH, which represent antigens with and without a spacer arm between the hapten and the carrier protein. Different ELISA formats were evaluated for their assay sensitivities and the direct ELISA was applied to both chemi-thermomechanical pulp (CTMP) mill effluents and bleached kraft mill effluents (BKME), as well as fish bile for the quantification of resin acids. A GC protocol involving extraction, derivatization and GC quantification was developed as a standard method and used to compare the results with ELISA. The ELISA formats played an important role in the assay sensitivities and the 50% inhibition concentration (I₅₀) for DHA ranged from 12.3 to 113.2 μg/L. Indirect ELISA with the biotin-streptavidin system showed the highest assay sensitivity. Direct ELISA had an I₅₀ of 49.7 μg/L, with a detection limit of 4.5 μg/L. It was considered the method of choice due to its simple assay protocol and the reasonably good sensitivity that was obtained. However, when we compared two polyclonal antibodies derived from the antigen with and without a spacer arm, no significant differences were observed in either assay sensitivity or cross-reactivity. Both polyclonal antibodies showed high cross-reactivities with the abietic type resin acids. Thus the polyclonal antibodies appeared to be group specific and could be used to quantify total abietic type resin acids. Various assay parameters, such as the concentrations of coating antigen and antibodies used, the incubation temperature and time, pH, ionic strength and metallic ions, were each evaluated for their influence on the assay sensitivity. The direct ELISA method was used to quantify DHA equivalents in both BKME and CTMP effluents. For CTMP effluents, which usually contain high levels of resin acids together with a high chemical oxygen demand (COD) and a high biochemical oxygen demand (BOD), a large dilution factor was required to bring the resin acid concentration into the quantification window. The DHA equivalents determined by ELISA compared favourably with total abietic type resin acids determined by GC, and also correlated well with total resin acids. While dilution could be used to reduce the background to a negligible level for CTMP samples containing high resin acid concentrations, this method was not suitable for samples containing low resin acid concentrations such as BKME. A significant background effect was encountered and the background interference could not be eliminated by dilution, as this brought the resin acid concentration to below the detection limit of the ELISA. Various techniques, such as filtration, extraction and fractionation were used to try to eliminate this background effect. However, all these methods were unsuccessful. By using a bio-treated BKME as the diluent and creating a standard curve using the same bio-treated BKME we were able to eliminate the background interference. Although the assay sensitivity decreased, the DHA equivalents determined in the BKME by the ELISA method compared favourably with the amount of total abietic type and the total resin acids as determined by GC. The direct ELISA was also used for the quantification of resin acids in fish bile. Good recoveries were obtained with a DHA spiked control bile sample. Direct ELISA of a limited number of bile samples from fish exposed to pulp mill effluents indicated that this immunochemical method did not require sample hydrolysis and much less sample was needed when compared to the GC method (1-5 μl for ELISA vs 100-500 μl for GC). This work represents the first attempt at developing an antibody-based immunochemical method for the detection and quantification of resin acids in softwood pulp mill effluents. Compared with conventional instrumental methods such as GC, HPLC and GC-MS, the ELISA method was able to overcome several drawbacks of these alternative methods such as, extensive sample pre-treatment, expensive instrumentation and low sample output. Although not intended to replace the conventional analysis, ELISA will be particularly useful in the screening of large number of samples which can produce results within a short period of time.