Hubei Key Laboratory of Genetic Regulation and Integrative Biology, College of Life Sciences, Huazhong Normal University, Wuhan, China.
PLoS One. 2011;6(12):e29196. doi: 10.1371/journal.pone.0029196. Epub 2011 Dec 27.
Dibutyl phthalate (DBP) is predominantly used as a plasticizer inplastics to make them flexible. Extensive use of phthalates in both industrial processes and other consumer products has resulted in the ubiquitous presence of phthalates in the environment. In order to better determine the level of pollution in the environment and evaluate the potential adverse effects of exposure to DBP, immunoassay for DBP was developed.
METHODOLOGY/PRINCIPAL FINDINGS: A monoclonal antibody specific to DBP was produced from a stable hybridoma cell line generated by lymphocyte hybridoma technique. An indirect competitive enzyme-linked immunosorbent assay (icELISA) employing direct coating of hapten on polystyrene microtiter plates was established for the detection of DBP. Polystyrene surface was first oxidized by permanganate in dilute sulfuric acid to generate carboxyl groups. Then dibutyl 4-aminophthalate, which is an analogue of DBP, was covalently linked to the carboxyl groups of polystyrene surface with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC). Compared with conjugate coated format (IC(50)=106 ng/mL), the direct hapten coated format (IC(50)=14.6 ng/mL) improved assay sensitivity after careful optimization of assay conditions. The average recovery of DBP from spiked water sample was 104.4% and the average coefficient of variation was 9.95%. Good agreement of the results obtained by the hapten coated icELISA and gas chromatography-mass spectrometry further confirmed the reliability and accuracy of the icELISA for the detection of DBP in certain plastic and cosmetic samples.
CONCLUSIONS/SIGNIFICANCE: The stable and efficient hybridoma cell line obtained is an unlimited source of sensitive and specific antibody to DBP. The hapten coated format is proposed as generally applicable because the carboxyl groups on modified microtiter plate surface enables stable immobilization of aminated or hydroxylated hapten with EDC. The developed hapten coated icELISA can be used as a convenient quantitative tool for the sensitive and accurate monitoring DBP in water, plastic and cosmetic samples.
邻苯二甲酸二丁酯（DBP）主要用作塑料的增塑剂，使其具有柔韧性。邻苯二甲酸酯在工业生产过程和其他消费产品中的广泛使用，导致其在环境中无处不在。为了更好地确定环境的污染程度，并评估接触邻苯二甲酸二丁酯的潜在不良影响，开发了邻苯二甲酸二丁酯的免疫测定方法。
方法/主要发现：采用淋巴细胞杂交瘤技术，从小鼠杂交瘤细胞系中制备出一种针对邻苯二甲酸二丁酯的单克隆抗体。建立了一种间接竞争酶联免疫吸附测定法（icELISA），采用直接包被邻苯二甲酸二丁酯在聚苯乙烯微量滴定板上的方法，用于检测邻苯二甲酸二丁酯。聚苯乙烯表面首先用稀硫酸中的高锰酸盐氧化，生成羧基。然后，邻苯二甲酸二丁酯 4-氨基酯，这是邻苯二甲酸二丁酯的类似物，通过 1-乙基-3-（3-二甲基氨基丙基）碳二亚胺盐酸盐（EDC）与聚苯乙烯表面的羧基共价连接。与缀合物包被格式（IC50=106ng/mL）相比，在仔细优化测定条件后，直接半抗原包被格式（IC50=14.6ng/mL）提高了测定的灵敏度。从加标水样中回收的邻苯二甲酸二丁酯的平均回收率为 104.4%，平均变异系数为 9.95%。用半抗原包被的 icELISA 和气相色谱-质谱法得到的结果具有良好的一致性，进一步证实了该 icELISA 用于检测某些塑料和化妆品样品中邻苯二甲酸二丁酯的可靠性和准确性。
结论/意义：获得的稳定、高效的杂交瘤细胞系是一种对邻苯二甲酸二丁酯具有高灵敏度和特异性的抗体的无限来源。由于修饰后的微量滴定板表面上的羧基基团可以通过 EDC 稳定地固定氨基化或羟化的半抗原，因此提出了半抗原包被格式，该格式具有普遍适用性。所建立的半抗原包被 icELISA 可作为一种方便的定量工具，用于水、塑料和化妆品样品中邻苯二甲酸二丁酯的灵敏、准确监测。
