Preparation Of Monoclonal Antibodies And Immunoassay Study For Pyrene And Naphthalene

Polycyclic aromatic hydrocarbon (PAHs), as a group of individual organic compoundscomposed of two or more blended aromatic rings, are important environmental pollutants and arewidely present in water, soil and air. Some studies showed that PAHs could cause gastric cancer,lung cancer, bladder cancer and esophagus cancer while the immune system, nervous system andendocrine system were damaged in different extent. The animal experiments results also provedthat PAHs had powerful carcinogens or potent carcinogens. PAHs are been getting getting moreand more attention due to the injury to human. Up to now, the conventional techniques for PAHdetection are cell detection, thin-layer chromatographic scanning (TLCS), spectrophotometry, gaschromatograph (GC), high performance liquid chromatography (HPLC), capillary electrophoresis(CE) and diode matrix method. However, the dependence on expensive equipment and skilledanalysts limits their widespread use. Immunologic detection method having the advantages of lowcost and high sensitivity, could be used for rapid detection of PAH in real sample. In this study, themonoclonal antibodies against pyrene and naphthalene were prepared. Four detecting methods,including indirect competition ELISA, immunochromatographic strip, real-time immuno-PCR(RT-IPCR) and improved RT-IPCR were developed for detection of pyrene and some homologuesin water samples.The conjugates of OVA-pyrene, OVA-naphthalene, BSA-pyrene and BSA-naphthalene wereprepared using NHS method and confirmed using ultraviolet (UV)-visible spectroscopy andnondenaturing agarose gel electrophoresis. After the procedures of immunization, cell fusion,hybridoma selection and cloning, two monoclonal antibody (McAb) against pyrene andnaphthalene respectively were produced. The isotypes of the McAb were IgG1and IgG3, theaverage affinities were3.23×109L/mol and4.17×108L/mol, the titers were1:2.56×106and1:3.2×105.Two competitive enzyme-linked immunosorbent assays (ELISAs) were developed fordetection of pyrene and naphthalene in water sample. The detection limit of the pyrene assay was65.08pg/mL. The linear range for pyrene was from117.16to3986.97pg/mL and the averagerecoveries of sample was98.7±1.3%. The detection limit of the naphthalene assay was2.0ng/mL. The linear range for naphthalene was from3.78to172.33ng/mL and the average recoveryof sample was96.1±1.8%.The McAbs of pyrene and naphthalene were conjugate with gold nanoparticles (AuNPs,20nm diameter) respectively, then conformed by using ultraviolet (UV)-visible spectroscopy and transmission electron microscope. Immunochromatographic strips were prepared and theexperiment condition was optimized. The detection range of pyrene strip was from10ng/mL to50ng/mL and naphthalene strip was from25ng/mL to125ng/mL. The stability of the assay showedthat the loss of color intensity in8weeks was not significant. The application study demonstratedthat the pyrene and naphthalene strips could be applied to real water samples.Two RT-IPCR assays were developed based on pyrene McAb and naphthalene McAb. Thelinear range of RT-IPCR assay for pyrene was from100fg/mL to1ng/mL, the average recoveryof sample was98.6±4.1%. The linear range of RT-IPCR assay for pyrene was from10pg/mL to10ng/ml, the average recovery of sample was96.7±6.4%.Two improved RT-IPCR assays were developed based on pyrene McAb and naphthaleneMcAb. The linear range of RT-IPCR assay for pyrene was from1pg/mL to1ng/ml, the averagerecovery of sample was96.9±4.8%. The linear range of RT-IPCR assay for pyrene was from100pg/mL to100ng/ml, the average recovery of sample was97.4±4.9%.Fluorene, phenanthrene, pyrene, chrysene, benzo[a]pyrene, naphthalene, benzo[a]anthracene,fluoranthene and indeno[1,2,3-cd]pyrene were tested using Gas Chromatograph-MassSpectrometer (GC-MS). The results showed that, compared with GC-MS, the sensitivity ofcompetitive ELISA, RT-IPCR and improved RT-IPCR increased dozens of times to thousands oftimes.Sum up, in this study, competitive ELISA, immunochromatographic strips, RT-IPCR andimproved RT-IPCR were developed using pyrene McAb and naphthalene McAb. Compare withtraditional method such as GC-MS, the advantages of these methods were low cost and highsensitivity. These methods could be used for rapid detection of PAH in real water samplesselectively.