| Polycyclic aromatic hydrocarbons(PAHs) were a large group of organic compounds with two or more benzene rings, known as carcinogens and mutagens. PAHs were widely distributed in the environment including atmosphere, soil, river and food. PAHs contributed to the high potential for environment polluted and health hazard. In recent years, immunological detection assay has been developed to monitor PAHs in the environment because of its high-specificity,high-sensitivity and simple-operation.In this study, acenaphthene-3-butyricacid(3-ACE) with 4-carbon spacer arm and acenaphthene-5-methanoicacid(5-ACE) with one spacer arm were linked to bovine serum albumin(BSA) and ovalbumin(OVA) by active ester method to produce immunogen(3-ACEBSA and 5-ACE-BSA) and detection antigen 3-ACE-OVA. Compared serum titer and specificity, 4-carbon spacer arm immunogen(3-ACE-BSA) induced higher specific antibody against acenaphthene(ACE). 3-ACE-BSA was used to product monoclonal antibody against ACE(ACE-Mc Ab), the splenocytes from immnnized mice were fused with sp2/0, and the C4 was selected by limited dilution method. The ascite was purified using affinity chromatography. The subclass, affinity and specificity of the monoclonal antibody were also studied. Indirect competitive enzyme-linked immunosorbent assay(ic-ELISA) and immunomagnetic indirect competitive enzyme-linked immunosorbent assay(IMB-ic-ELISA) based on monoclonal antibody against ACE were developed and optimized.Immunizing antigen acenaphthene-3-butyricacid(3-ACE) with 4-carbon spacer arm induced high sensitivity antibody. The subclass of the positive hybridoma cells was Ig G2 b. The titers of ascite and monoclonal antibody were 1.28×106 and 0.32×106. The antibody affinity constant was1.48×108. The cross-reactivity reactions of ACE-Mc Ab with Naphthalene, Fluorene, Chrysene and Benzo[a]pyrene were 13.18%, 7.94%, 4.17%, 20.89% respectively.The equation of linear regression with ACE in ic-ELISA was y=22.598 x – 57.313, the correlation coefficient was 0.9923. The detection limit of ic-ELISA was 0.953 ng/m L, and the linear detection range was from 2.636 ng/m L to 1.191 μg/m L. Three water samples(gathered fromYitong River, South Lake Park and tap water of Changchun city, Jilin Provence) were spiked with ACE, for the ic-ELISA, the average recoveries were 93.69 – 108.58%, 89.54 – 109.2% and 102.43– 108.36% respectively, and the average coefficient of variation was from 5.57% to 6.32%. The results of detection of five water samples(obtained from Jingyuetan, South Lake Park, Yitong River, Nouth Lake Park and tap water) for the ic-ELISA were 0 ng/m L, 1.14 ng/m L, 0 ng/m L, 0ng/m L, 1.61 ng/m L respectively.The equation of linear regression with ACE in IMB-ic-ELISA was y=16.934 x – 30.240, the correlation coefficient was 0.9961. The detection limit of IMB-ic-ELISA was 0.238 ng/m L, and the linear detection range was from 0.927 ng/m L to 3.236 μg/m L. Three water samples(gathered from Yitong River, South Lake Park and tap water of Changchun city, Jilin Provence) were spiked with ACE, for the IMB-ic-ELISA, the average recoveries were 90.91 – 100.62%、92.74 – 98.16%' 93.97 – 103.35% respectively, and the average coefficient of variation was from 3.14% to3.15%. The results of detection of five water samples(obtained from Jingyuetan, South Lake Park,Yitong River, Nouth Lake Park and tap water) for the IMB-ic-ELISA were 0.95 ng/m L, 1.17ng/m L, 0.83 ng/m L, 0.69 ng/m L, 1.91 ng/m L respectively. |
PDF Full Text Request