An Immunoassay for Dibutyl Phthalate Based on Direct Hapten Linkage to the Polystyrene Surface of Microtiter Plates

Abstract

BackgroundDibutyl 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 FindingsA 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 (IC50 = 106 ng/mL), the direct hapten coated format (IC50 = 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/SignificanceThe 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.

Exploiting genetic diversity in two European maize landraces for improving Gibberella ear rot resistance using genomic tools

“…In Germany, a recent survey on the prevalence of Fusarium species showed that F. graminearum (Fg) and F. verticillioides (Fv) were dominant, their relative occurrence depending on temperature and humidity (Pfordt et al 2020 ). F. graminearum (sexual stage: Gibberella zeae ) causes Gibberella ear rot (GER) which reduces the quantity and quality of maize kernels and more importantly, contaminates the grains with mycotoxins such as deoxynivalenol (DON) and zearalenone (ZON) (Trail 2009 ; Ding et al 2011 ; Martin et al 2012a ; Mesterházy et al 2012 ). These mycotoxins are associated with serious health problems such as kidney diseases, poor growth, and disorders of reproduction in animals and humans (Pinton and Oswald 2014 ; Zhou et al 2018 ).…”

Development of immunoassays for the determination of phthalates

“…Based on the problems mentioned above, we have developed an indirect competitive enzyme-based immunoassay strategy for PAE detection. Thus, we have summed up several indirect competitive enzyme-based immunoassays for PAE detection by conjugating enzyme with the secondary antibody to combine the primary antibody (Kuang et al, 2010(Kuang et al, , 2013Wei et al, 2011). This strategy effectively economizes the amount of the primary antibody and decreases non-specific adsorption and strengthens the sensitivity of the immunoassay (Li et al, 2019;Tang et al, 2015).…”

Characterization of the binding between phthalate esters and mouse PPARα for the development of a fluorescence polarization-based competitive binding assay

“…[10][11][12][13][14] Nevertheless, polyclonal antibody is restricted by immunized animals and cannot be produced unlimitedly. 15 Furthermore, it is difficult to produce a broad spectrum antibody that can recognize the whole group of PAEs. Hence, a fast and multi-residue method using relatively inexpensive equipment is desired for the high throughput screening (HTS) of PAEs.…”