Startseite A one-step incubation ELISA kit for rapid determination of dibutyl phthalate in water, beverage and liquor
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A one-step incubation ELISA kit for rapid determination of dibutyl phthalate in water, beverage and liquor

  • Quing Sun , Yanli Chen , Fuxue Li , Minghong Jia EMAIL logo und Guoqing Shi EMAIL logo
Veröffentlicht/Copyright: 8. Juli 2019
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Open Chemistry
Aus der Zeitschrift Open Chemistry Band 17 Heft 1

Abstract

A one-step incubation ELISA kit based on monoclonal antibody against dibutyl phthalate (DBP) was developed. After optimizing concentrations of coating antigen, antibody and composition of the assay buffer, an inhibition curve was plotted. The IC50 is 29.6 ng·mL-1, and the detection limit for DBP is 3.6 ng·mL-1. Compared with other ELISA methods, this ELISA kit had a simpler sample preparation, costed less time for detection and could detect more types of sample. The recoveries of DBP in water, beverage and liquor samples were range from 78% to 110.4%, the range of coefficient of variations is 7.7-15.3%. The cross reactivity was very low (&1%) except that for butyl benzyl phthalate (3.9%) and the di-isobutyl phthalate (12.5%). The detection results in liquor showed good correlation with those from GC-MS. All data above indicated that this kit could be used as the fast and high-throughput screening of DBP in water, beverage and liquor.

Keywords: dibutyl phthalate; ELISA kit; one-step incubation; beverage; liquor

1 Introduction

Phthalate acid esters (PAEs) are ubiquitous industrial chemicals that are used as plasticizers, lubricants, cosmetics ingredients, and other applications. The commonly used PAEs are diethylhexyl phthalate (DEHP), dimethyl phthalate (DMP), butyl benzyl phthalate (BBP), dibutyl phthalate (DBP) and di-n-octyl phthalate (DOP) [1].

Several phthalates, including DBP, have demonstrated weak oestrogenic activity in vitro. A study of 168 men performed by Duty et al suggested that some phthalate monoesters are associated with lower sperm concentration, lower motility and increased percentage of sperm with abnormal morphology in humans [2]. In a study published in 2005, Swan reported that human phthalate exposure during pregnancy results in decreased anogenital distance in baby boys [3]. The phthalate-related syndrome of incomplete virilization in prenatally exposed rodents indicates the relationship between abnormal development of male genital and phthalate exposure. In recent studies, in utero exposure to phthalates in general has been shown to have anti-androgenic effects on the foetus [4,5]. Additionally, phthalates exposure could have side effects on the sexual development of women. In 2004-2007, a multiethnic longitudinal study of 1,151 girls (6-8 years of age) from five cities was performed to investigate the associations of phthalate and pubertal development. A positive trend was observed for phthalates with breast and pubic hair development [6]. In a zebrafish embryo toxicity test, DBP showed the highest toxicity, with a lethal concentration required to kill 50% of the population (LC50) of 0.63 mg·kg-1 in six PAEs. Additionally, BBP, DBP, DEHP and DINP demonstrated oestrogenic activity [7]. Due to their toxicity, DEHP, DBP and BBP have been identified as reprotoxic, category 2 compounds by EU in 2005 [8].

Due to exposure from dietary ingestion and many commonly used products, the PAEs have found their way into every corner of our lives. Based on the concentrations

of phthalate metabolites in 183 urine samples, the estimated daily intakes of DBP in China were 12.2 μg•kg-1 bw/day, exceeding the suggested tolerable daily intake of 10 μg•kg-1 bw/day by European Food Safety Authority [9]. Digestive intake was dominated the human exposure to phthalates [10]. Phthalate esters in food are mainly due to contamination from plastic food packaging and processing. Phthalates are fat-soluble compounds and can very easily enter the food via fats and oils, which then become phthalate-enriched.

Considered the health effects of phthalates, the Chinese Ministry of Health (MOH) released an administrative notice on the maximum residue limit (MRL) of phthalates in food and food additives on 13 June 2011 (MOH Notice 551): DEHP (1.5 mg·kg-1), DINP (9.0 mg·kg-1) and DBP (0.3 mg·kg-1). The US Food and Drug Administration (FDA) published a guidance document on the use of phthalates in products in 2012. The draft guidance recommends that the pharmaceutical industry should avoid the use of DBP and DEHP as excipients in drugs and biological products.

Chromatographic analyses including HPLC-MS, and GC-MS are the most used methods for PAEs determination [11, 12, 13, 14]. Although these methods are sensitive and accurate, they are also labourious, expensive, time-consuming and unsuitable for effectively monitoring the PAEs in environment and food. By contrast, the enzyme immunoassay method has excellent selectivity due to the high specificity of the antibodies, and therefore, the sample pre-treatment requirements are relatively simple and inexpensive. Multiple samples can be measured simultaneously, making this method suitable for the rapid detection and screening of a large number of samples.

The first study of the antibodies against PAEs was reported in a patent by Yanaihar et al [15]. Zhuang et al. have developed several ELISA methods and prepared antibodies against PAEs [16, 17, 18, 19]. However, the antibodies used in these works were polyclonal antibodies. A monoclonal antibody specific to DBP was produced by Wei et al., who developed an indirect competitive enzyme-linked immunosorbent assay (icELISA) method employing direct coating of hapten on polystyrene microtiter plates [20]. The limit of detection (LOD) of their method was 0.426 ng·mL-1 for DBP. In their study, polystyrene was oxidized and directly linked to hapten. However, besides the additional time of the pre-coating process, the entire analysis required almost 3h and contained two incubation steps.

In this work, based on the DBP monoclonal antibody developed in our lab, a one-step incubation rapid ELISA kit for DBP was developed. After the optimization of icELISA, the kit could detect the DBP in water, beverages and liquor. The detection time was decreased to 30 min,which was faster than the methods mentioned above. The results of DBP detection by ELISA kit were consistent with the results of the detection by GC-MS.

2 Experimental

2.1 Reagents and chemicals

Dibutyl phthalate (DBP), diethylhexyl phthalate (DEHP), dimethyl phthalate (DMP), butylbenzyl phthalate (BBP), 4-nitrophthalic anhydride, and 1-butanol were purchased from J&K Scientific Ltd. (Beijing, China). Methyl 4-hydroxybenzoate, ethyl 4-hydroxybenzoate, isopropyl 4-hydroxybenzoate, butyl 4-hydroxybenzoate, isobutyl 4-hydroxybenzoate, pentyl 4-hydroxybenzoate, n-heptyl 4-hydroxybenzoate, and butyl formate were of analytical grade and were obtained from Sinopharm Chemical Reagent Beijing Co., Ltd. (Beijing, China). Bovine serum albumin (BSA), complete and incomplete Freund’s adjuvant, and goat anti-mouse IgG-HRP (IgG-HRP) were obtained from Sigma-Aldrich (St. Louis, MO, USA). HAT, HT, Cell-culture media (DMEM), foetal bovine serum and supplements were obtained from Gibco Life Technologies Corporation (Carlsbad, CA, USA). 3,3’,5’,5-Tetramethylbenzidine (TMB) substrates, blocking buffer and immobilized biomolecule stabilizer were purchased from Biopanda Diagnostics (UK). Clonotyping ELISA kit was obtained from SouthernBiotech, and 96-well microplates were purchased from Corning-Costar (Tewksbury, MA, USA). Chromatography-grade reagents such as hexane, methanol, ethanol, N,N-dimethylformamide (DMF) and dimethyl sulphoxide (DMSO) were purchased from Thermo Fisher Scientific (MA, USA). All of the other reagents were of analytical grade or higher.

2.2 Apparatus

The cells were incubated in a SANYO MCO-15AC incubator (Moriguchi-shi Osaka, Japan). For ELISAs, a plate washer (ELx50/8) purchased from BioTek (Bad Friedrichshall, Germany) was used. Immunoassay absorbance was recorded using a Thermo Scientific Multiskan MK3 Microplate spectrophotometer (Thermo Fisher Scientific, Inc. MA, USA). The UV-2802 UV/Vis spectrophotometer used in this work was purchased from UNICO Instruments Co., Ltd. (Shanghai, China).

An Agilent GC-MS System (7890A+5975C) with an Agilent DB-5MS column (30 m x 0.25 mm x 0.25 μm) (California, USA) was used.

2.3 Hapten synthesis

Concentrated sulphuric acid (1 mL) was added slowly to a solution of 4-nitrophthalic anhydride (2.5 g) in 50 mL 1-butanol. The mixture was stirred and heated at reflux (120°C) for 7 h. The residue was washed with 10 mL of 10% (w/v) aqueous Na2CO3 three times and was evaporated to remove water and 1-butanol at 80°C. The oily residue (0.15 g) was dissolved in benzene (28 mL), and zinc dust (0.28 g) was added to the solution. Then, hydrochloric acid (8.2 mL) was added in three portions. After 18 min of stirring at room temperature (RT), more zinc dust (0.28 g) was added, and the mixture was stirred at RT for 12 h. The benzene layer was washed with water (34 mL) and dried over anhydrous sodium sulphate (5 g) three times. The benzene layer was removed by evaporation, and a yellow crude solid of dibutyl 4-aminophthalate (DBAP) was obtained. The product was analysed by TLC and nuclear magnetic resonance spectroscopy (NMR).

DBAP (0.0466 g) was added to a stirred mixture of concentrated hydrochloric acid (0.1 mL), water (3 mL) and DMF (2 mL) at 5°C. NaNO2 (0.2 M) was added dropwise until the solution turned the potassium iodide starch test paper blue. The mixture was stirred at 5°C for 30 min. BSA (160 mg) in sodium borate (40 mL, pH 9.0) was added dropwise. The colour of the mixture changed from colourless to a reddish orange. After 2 h of stirring, the mixture was dialyzed against phosphate buffered saline (PBS, 10 mM, pH 7.4) that was changed twice a day for three days at 4°C. The mixture dialyzed was the DBAP-BSA conjugate (Figure 1).

Figure 1 The synthetic route of DBP-BSA.
Figure 1

The synthetic route of DBP-BSA.

2.4 Preparation of antibodies

DBAP-BSA (100 μg) in 0.1 mL sterilized PBS was emulsified with an equal volume of Freund’s complete adjuvant. This solution was injected intraperitoneally into each of several 7-week-old female BALB/c mice. After 3 weeks, the mice were boosted with another 100 μg DBAP-BSA that was emulsified with Freund’s incomplete adjuvant (1:1). Ten days after injection, serum was collected from the caudal vein of each mouse, and titres of antisera were determined by indirect ELISA. A positive mouse was sacrificed for cell fusion after four immunizations. The spleen was removed aseptically, and splenocytes were fused with the cells of the Sp2/0 murine myeloma cell line in the presence of polyethylene glycol, molecular weight 4000. After fusion, the cells were suspended in DMEM HAT medium containing 16% foetal bovine serum (FBS) and added to 96-well microplates. After 7 days, culture supernatants from each well were assayed using indirect competitive ELISA. Hybridoma cells in the ELISA-positive wells were subcloned by the limiting dilution method. Female BALB/c mice were treated with intraperitoneal injection of pristine (0.2 mL), and then intraperitoneally injected with cloned hybridoma cells (1.0×107 in PBS). Antibodies were purified from the ascitic fluids by protein-A-agarose affinity chromatography [21].

2.5 Optimization of icELISA

Checkerboard titration was used to select the appropriate concentrations of coated antigen, antibody and IgG-HRP. The checkerboard assays were performed using indirect competitive ELISA (ic-ELISA).

Microplates were coated overnight at 4°C with 100 μL per well of DBAP-BSA in 0.05 M sodium carbonate buffer (pH 9.6). After the incubation at 4°C, the microtiter plates were washed three times with PBST (0.01 M, pH=7.4, 0.05% Tween 20) after each incubation step. The wells were blocked with 200 μL blocking buffer. After another washing step, the plates were vacuum dried, vacuum sealed and stored in 4°C.

2.6 Development of ELISA methods

After the temperature of the microplates reached the room temperature, 50 μL/well standard or sample solution, 50 μL/well antibody and 50 μL/well HRP-conjugated IgG were added and incubated at 37°C for 20 min. Following a washing step, 100 μL/well TMB substrates were added. The colour development was stopped after 10 min by adding 50 μL/well stop solution (2 M H2SO4). The absorbance was detected at 492 nm with 630 nm as reference wavelength.

2.7 Optimization of the assay buffer

Methanol, ethanol, DMF and DMSO were mixed with PBST in several ratios, and then, DBP was dissolved in the buffer for the preparation of the standard solution.

2.8 Development of the standard curve of ELISA kits

DBP was diluted with assay buffer to the concentrations of 0, 5, 15, 45, 135, and 405 ng·mL-1. After the ELISA procedure, the results were expressed as the binding percentage:

Binding (%)=(B/B0)×100,

where B is the mean absorbance in the presence of the DBP standard sample or analysed sample and B0 is the mean absorbance in the absence of DBP. A standard curve was plotted based on the binding (%) and DBP concentrations.

2.9 Sample preparation for ELISA

For the bottled water and carbonated beverages, the sample (5 g) was transferred to a clean, dried glass tube, and chromatography-grade hexane (2 mL) was added. For liquor and some beverages such as orange juice or tea drinks, the sample (1 g) was transferred to the tube, and water (4 mL) and chromatography-grade hexane (2 mL) was added. All tubes were capped and vibrated. After vibrating for 3 min, 1 mL of the supernatant was transferred to another clean, dried glass vessel and evaporated naturally. The residues were dissolved in assay buffer (1 mL) for a further ELISA test. The samples for which the DBP content exceeded the highest concentration of the standard solution were diluted with assay buffer and re-analysed.

2.10 Accuracy, specificity and precision of the ELISA kit

The LOD was determined as the DBP concentration that gives a 10% inhibition (IC10). Three concentration levels of DBP in fortified samples were investigated: 0, 0.3 and 0.6 μg·mL-1. Three sets of five samples each purchased from a supermarket were spiked with the DBP standard. The IC50 value is obtained by calculating DBP concentration corresponding to 50% of B0. Cross-reactivity (CR) for each DBP analogue was calculated according to [20,22]:

CR%=(IC50value of DBP)/(IC50value of DBP analogue)×100

2.11 Storage stability study

Excess ELISA kits were stored at 4°C and were used to perform the standard curve experiments every month. Another set of kits was stored at 37°C and was used to carry out the ELISA method measurements every day. The B0 and IC50 values of each kit were investigated.

2.12 Validation with GC-MS

Overall, 24 liquor samples collected from the markets were evaluated with an ELISA kit and the national standard method (GB/T 21911-2008, Determination of phthalate esters in foods). The sample (5 mL) was added to hexane (2 mL) and vortexed vigorously for 1 min. After standing to allow the formation of distinct layers, 1 mL of the upper layer was transferred for GC-MS analysis.

DBP was detected by gas chromatographic with a DB-5MS column and with helium as the carrier gas .1 microliter of sample was injected in splitless mode at 250°C with the oven program as follows: 60°C, for 1 min; increase at a rate of 20° C·min-1 up to 220 °C, hold for 1 min; and increase at a rate of 5°C·min-1 up to 280°C, hold for 4 min. The MS analysis was performed in the selected ion monitoring (SIM) mode, and the detector was operated in the electron impact (EI) mode at 70 eV. The transfer line and source temperatures were both maintained at 280°C. DBP SIM ions (m/z): 149, 223, 205, and 104.

Ethical approval: The conducted research is not related to either human or animal use.

3 Results and discussion

3.1 Preparation and characterization of monoclonal antibody

Five monoclonal antibodies showed good affinity to DBP (data not shown). The antibodies titers were assessed by indirect competitive ELISA (icELISA). The antibody of 1D2 had good titers and the lowest IC50 value for DBP. The isotype of antibody 1D2 was IgG1 and the light chain was kappa, as determined by the SouthernBiotech Clonotyping ELISA kit. The affinity constant of antibody reached 6.3×107 L·mol-1, which was evaluated by indirect non-competitive ELISA as described by Beatty et al23.

3.2 Optimization of ELISA

To optimize the conditions of the coating process, the type, pH and concentration of the coating buffer were investigated carefully. Citrate buffer, phosphate buffered saline and carbonic acid buffer were compared in this work. Among these solutions, the carbonic acid buffer was found to be the most efficient buffer (data not shown). The B0 values under different pH and different concentrations of carbonic acid buffer are shown in Figure 2. The highest B0 value was obtained when the buffer concentration was 50 mM at pH 9.6, indicating that maximum immobilization of DBP-BSA conjugates in the plate well occurred. Therefore, the carbonic acid buffer (50 mM, pH=9.6) was chosen as the coating buffer in the subsequent experiments.

Figure 2 Effect of pH and concentrations of carbonic acid buffer on B0 value (n=3).
Figure 2

Effect of pH and concentrations of carbonic acid buffer on B0 value (n=3).

In this experiment, organic solvents were utilized to increase the solubility of DBP20 and modify the interaction between the DBP and its monoclonal antibody in the assay buffer. Four water-miscible organic solvents such as methanol, ethanol, DMF and DMSO were used to prepare the standard solution and the sample solutions. As shown in Figure 3, compared to the DMF and DMSO solutions, the B0 values were higher in the methanol or ethanol solutions, indicating greater DBP antibody binding onto the immobilized DBP-BSA conjugates. The experimental group with a higher B0 and lower IC50 value means that the solution was more suitable for the specific binding between the DBP antibody and the antigenic determinant of DBP. When 20% methanol was used as the standard solution, the highest B0 value and lowest IC50 value were obtained (Figure 3a); therefore, this solution was selected for further optimization assay.

Figure 3 The influence of organic solvent on the B0 and IC50.
Figure 3

The influence of organic solvent on the B0 and IC50.

The results of the checkerboard determination (Table 1) indicated that the concentrations of DBP-BSA, antibody and HPR-IgG all affected the IC50 value. The IC50 value changed slightly with the changes in the HRP-IgG concentration, while the changes in the concentrations of DBP-BSA and antibody had a marked effect on the IC50 value. While lower IC50 values were obtained at lower antigen and antibody concentrations, the B0 value was lower and the relative standard deviation (RSD) was higher at the same time. For a commercial ELISA kit, the B0 value was critical for long-term storage, and the RSD was critical for the accuracy of the final results. Taking these results together, we chose the concentration of the DBP-BSA used for coating to be 0.4 μg·mL-1; the concentration of antibody was 100 ng·mL-1, and that of IgG-HRP was 1:4000, for which the B0 value was higher than 1.5, the RSD was less than 10% and the IC50 value was low.

Table 1

Optimization of concentrations of coating antigen, antibody and IgG-HRP.

Concentration of antigen/(μg·mL-1)Concentration of antibody/(μg·mL-1)Dilution ratio of HRP-IgGB0RSD%IC/(ng·mL-1)50
0.81001/40002.5397.8%74
501/20002.1676.9%63
501/40001.8137.3%48
251/20001.29111.6%29
0.41001/20002.0177.6%44
1001/40001.7268.5%35
501/20001.44213.2%22
501/40001.10815.1%16
0.22001/20001.39016.7%45
2001/40001.23816.3%37
1001/20001.08517.9%16
1001/40000.940----

3.3 Standard curves of icELISA for DBP

The standard curve was obtained under the optimized conditions described above (Figure 4). The concentrations of DBP were 0, 5, 15, 45, 135, and 405 ng·mL-1. The IC50 value was calculated as 29.6 ng·mL-1, and LOD (IC10) was calculated as 3.6 ng·mL-1. Thus, this kit is less sensitive than Wei’s immunoassay for DBP (IC50=14.6 ng·mL-1) [20] but is similar to Kuang’s assay (IC50=33.6 ng·mL-1) [24]. However, both of their methods require two-step incubations and more than 1 h, while the incubation time of this kit was less than theirs and the IC50 was sufficiently low for the MRL in China (0.3 μg·mL-1 in food).

Figure 4 The Standard curve for DBP detection by ELISA.
Figure 4

The Standard curve for DBP detection by ELISA.

3.4 Cross-reactivity

Eleven phthalate esters were tested for cross reactivity (CR) using the ELISA kit. As shown in Table 2, for all of the tested compounds, the obtained CR values were very low except those for BBP (3.9%) and DIBP (12.5%).

Table 2

Cross reactivity for ELISA kit analysis of DBP analogue.

CompoundIC50 /(ng·mL-1)Cross-reactivity %
dibutyl phthalate(DBP)29.6±1.2100
butyl benzyl phthalate(BBP)762.8±23.23.9
di(2-ethylhexyl)phthalate(DEHP)>9100&1
di-isobutyl phthalate(DIBP)236.1±16.012.5
methyl 4-hydroxybenzoate>9100&1
ethyl 4-hydroxybenzoate>9100&1
isopropyl 4-hydroxybenzoate>9100&1
butyl 4-Hydroxybenzoate>9100&1
isobutyl 4-Hydroxybenzoate>9100&1
pentyl 4-Hydroxybenzoate>9100&1
heptyl 4-Hydroxybenzoate>9100&1
butyl formate>9100&1

3.5 Recoveries in some consumer products

The detection of DBP in bottled water and liquor has been reported by Zhang et al. and Xu et al., respectively [16,25]. These two types of samples were reasonably clean compared to the beverages, and therefore, these two samples did not require dilution prior to extraction. For the detection of DBP in carbonated beverages and tea-flavoured beverages, Sun et al. developed a method using

Table 3

Recoveries of DBP spiked in samples (n=5).

SamplesSpiked level/ (μg·mL-1)Average Recovery (%)RSD (%)
Bottled water0.3110.47.7
0.6102.58.0
Carbonated beverages0.387.910.0
0.697.915.3
Juices and tea drinks0.380.39.5
0.698.013.0
Liquor0.381.710.2
0.678.07.8

ultrasonic processing for 30 min three times [26]. This sample processing requires much time and is not suitable for a rapid ELISA kit, and hence, we developed a new preparation for the beverages in this work.

Five samples from a retail store were spiked with 0.3 and 0.6 μg·mL-1 of DBP. The samples were mixed, stored at room temperature overnight, and then analysed using the ELISA kit. The recoveries of DBP in various samples ranged from 78% to 110.4%, and the range of the coefficient of variations was 7.7-15.3% (Table 3). The high recoveries indicated that the sample preparation could extract the DBP with satisfactory results.

Since the ELISA kit exhibited approximate and acceptable recoveries and the MRL requirements of DBP in

food is 0.3 μg·mL-1, the ELISA kit could be applied to screen DBP residues in water, beverage, and liquor.

3.6 Storage stability study

Table 4 shows the summarized results of the accelerated stability study of the ELISA kit. According to the reference, 7 days at 37°C is equivalent to 1 year at 4°C [27]. The kits stored at 37°C showed performance that was similar to that of the kits stored at 4°C. The results collectively indicate that the DBP ELISA kit has a shelf life of at least 1 year.

3.7 Validation of the assay with GC-MS

Overall, 24 liquor samples were evaluated with the ELISA kit and the GC-MS. The typical GC-MS chromatogram for DBP is shown in Figure 5a DBP was not detected in 6 samples using both methods, and the DBP values were in the range of 0.05-1.76 mg·kg-1(GC-MS) in the other 18 samples. Good correlation was obtained between the results of the ELISA and GC-MS methods, as characterized by the linear equation y = 0.983x, R2 = 0.9625. This result further confirmed the reliability of the ELISA kit for the detection of DBP (Figure 5b)

Figure 5 Validation of the assay with GC-MS. a, A typical GC-MS chromatogram for DBP in liquor samples. b, correlation of GC-MS and ELISA analysis of certain liquor.
Figure 5

Validation of the assay with GC-MS. a, A typical GC-MS chromatogram for DBP in liquor samples. b, correlation of GC-MS and ELISA analysis of certain liquor.

Table 4

Results for accelerated stability of ELISA kits. (n=3).

Stored at 4 °CStored at 37 °C
storage period (months)B0IC50 /(ng·mL-1)storage period (days)B0IC50 /(ng·mL-1)
02.06629.602.06429.6
11.98531.721.96532.7
21.95430.641.82728.6
41.88526.561.82130.5
61.89332.291.77334.2
81.93530.1121.65935.1
101.78828.0
121.76925.8
141.72324.7

  1. *N/A: not available

3.8 Comparison with other ELISA methods

Three published ELISA methods for DBP detection were compared with the present method as shown in Table 5. Although the ELISA method reported in reference 26 is the most sensitive, it is also the most time-consuming. However, taking all factors together, the present method has the best combination of high sensitivity and analysis speed. Furthermore, the method has the more extensive applications for food than the other methods do.

Table 5

Comparison of Several ELISA methods.

This methodELISA 1ELISA 2Biotin-Streptavidin ELISA
Antibodymonoclonal antibodymonoclonal antibodypolyclonal antibodypolyclonal antibody
Sample typewater, beverage and liquorLiquorWhite wineBeverages and drinking Water
Estimated assay time60 minN/A*65 minMore than 200 min
IC50 (ng·mL-1)29.633.6292.10.36
LOD (ng·mL-1)3.621.564.50.005
SpecificityHighHighModerateHigh
AccuracyGoodGoodGoodGood
ReferenceThis article242526

4 Conclusion

A simple, fast and accurate ELISA kit for DBP was developed in this work. The IC50 was 29.6 ng·mL-1, and the LOD of DBP was 3.6 ng·mL-1. A simple sample preparation could alleviate or eliminate the matrix effect for water, beverages and liquor. The recoveries of DBP in the samples ranged from 78% to 110.4%, and the range of the coefficient of variations was 7.7-15.3%. The detection results in liquor showed good correlation to those obtained by GC-MS. Compared to other ELISA methods, this kit requires less time and can detect more types of samples than can the other ELISA methods for DBP; additionally, this kit has a shelf life of at least 1 year. The kit can be applied to the fast and high-throughput screening for DBP in water, beverages and liquor.

Minghong Jia: Beijing Key Laboratory of Detection and Control of Spoilage Organisms and Pesticide Residues in Agricultural Products- Beijing University of Agriculture, Beijing, 102206, China

Acknowledgement

We greatly appreciate the support of the Fundamental Research Funds for the Central Universities (FRF-AS-17-010), International cooperation project of the Ministry of Agriculture (NYB201501), Beijing Municipal Commission of Rural Affairs (20140131), the Key Technologies R&D program of Tianjin (12ZCZDSY13900), and the Open Project Program of Beijing Key Laboratory of Detection and Control of Spoilage Organisms and Pesticide Residues in Agricultural Products- Beijing University of Agriculture.

  1. Conflict of interest: Authors declare no conflict of interest.

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Received: 2018-05-04
Accepted: 2018-09-08
Published Online: 2019-07-08

© 2019 Qing Sun et al., published by De Gruyter

This work is licensed under the Creative Commons Attribution 4.0 Public License.

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https://doi.org/10.1515/chem-2019-0052
Schlagwörter für diesen Artikel
dibutyl phthalate; ELISA kit; one-step incubation; beverage; liquor
Creative Commons
BY 4.0

Artikel in diesem Heft

  1. Regular Articles
  2. Research on correlation of compositions with oestrogenic activity of Cistanche based on LC/Q-TOF-MS/MS technology
  3. Efficacy of Pyrus elaeagnifolia subsp. elaeagnifolia in acetic acid–induced colitis model
  4. Anti-inflammatory and antinociceptive features of Bryonia alba L.: As a possible alternative in treating rheumatism
  5. High efficiency liposome fusion induced by reducing undesired membrane peptides interaction
  6. Prediction of the Blood-Brain Barrier Permeability Using RP-18 Thin Layer Chromatography
  7. Phytic Acid Extracted from Rice Bran as a Growth Promoter for Euglena gracilis
  8. Development of a validated spectrofluorimetric method for assay of sotalol hydrochloride in tablets and human plasma: application for stability-indicating studies
  9. Topological Indices of Hyaluronic Acid-Paclitaxel Conjugates’ Molecular Structure in Cancer Treatment
  10. Thermodynamic properties of the bubble growth process in a pool boiling of water-ethanol mixture two-component system
  11. Critical Roles of the PI3K-Akt-mTOR Signaling Pathway in Apoptosis and Autophagy of Astrocytes Induced by Methamphetamine
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  13. Specific, highly sensitive and simple spectrofluorimetric method for quantification of daclatasvir in HCV human plasma patients and in tablets dosage form
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  25. Aqueous Micro-hydration of Na+(H2O)n=1-7 Clusters: DFT Study
  26. A proposed image-based detection of methamidophos pesticide using peroxyoxalate chemiluminescence system
  27. Phytochemical screening and estrogenic activity of total glycosides of Cistanche deserticola
  28. Biological evaluation of a series of benzothiazole derivatives as mosquitocidal agents
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  36. Free Radical Scavenging Activity of Essential Oil of Eugenia caryophylata from Amboina Island and Derivatives of Eugenol
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  42. Complex formation in a liquid-liquid extraction-chromogenic system for vanadium(IV)
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  44. Analysis and Biological Evaluation of Arisaema Amuremse Maxim Essential Oil
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  69. The anti-corrosion behaviour of Satureja montana L. extract on iron in NaCl solution
  70. Interleukin-4, hemopexin, and lipoprotein-associated phospholipase A2 are significantly increased in patients with unstable carotid plaque
  71. A comparative study of the crystal structures of 2-(4-(2-(4-(3-chlorophenyl)pipera -zinyl)ethyl) benzyl)isoindoline-1,3-dione by synchrotron radiation X-ray powder diffraction and single-crystal X-ray diffraction
  72. Conceptual DFT as a Novel Chemoinformatics Tool for Studying the Chemical Reactivity Properties of the Amatoxin Family of Fungal Peptides
  73. Occurrence of Aflatoxin M1 in Milk-based Mithae samples from Pakistan
  74. Kinetics of Iron Removal From Ti-Extraction Blast Furnace Slag by Chlorination Calcination
  75. Increasing the activity of DNAzyme based on the telomeric sequence: 2’-OMe-RNA and LNA modifications
  76. Exploring the optoelectronic properties of a chromene-appended pyrimidone derivative for photovoltaic applications
  77. Effect of He Qi San on DNA Methylation in Type 2 Diabetes Mellitus Patients with Phlegm-blood Stasis Syndrome
  78. Cyclodextrin potentiometric sensors based on selective recognition sites for procainamide: Comparative and theoretical study
  79. Greener synthesis of dimethyl carbonate from carbon dioxide and methanol using a tunable ionic liquid catalyst
  80. Nonisothermal Cold Crystallization Kinetics of Poly(lactic acid)/Bacterial Poly(hydroxyoctanoate) (PHO)/Talc
  81. Enhanced adsorption of sulfonamide antibiotics in water by modified biochar derived from bagasse
  82. Study on the Mechanism of Shugan Xiaozhi Fang on Cells with Non-alcoholic Fatty Liver Disease
  83. Comparative Effects of Salt and Alkali Stress on Antioxidant System in Cotton (Gossypium Hirsutum L.) Leaves
  84. Optimization of chromatographic systems for analysis of selected psychotropic drugs and their metabolites in serum and saliva by HPLC in order to monitor therapeutic drugs
  85. Electrocatalytic Properties of Ni-Doped BaFe12O19 for Oxygen Evolution in Alkaline Solution
  86. Study on the removal of high contents of ammonium from piggery wastewater by clinoptilolite and the corresponding mechanisms
  87. Phytochemistry and toxicological assessment of Bryonia dioica roots used in north-African alternative medicine
  88. The essential oil composition of selected Hemerocallis cultivars and their biological activity
  89. Mechanical Properties of Carbon Fiber Reinforced Nanocrystalline Nickel Composite Electroforming Deposit
  90. Anti-c-myc efficacy block EGFL7 induced prolactinoma tumorigenesis
  91. Topical Issue on Applications of Mathematics in Chemistry
  92. Zagreb Connection Number Index of Nanotubes and Regular Hexagonal Lattice
  93. The Sanskruti index of trees and unicyclic graphs
  94. Valency-based molecular descriptors of Bakelite network BNmn
  95. Computing Topological Indices for Para-Line Graphs of Anthracene
  96. Zagreb Polynomials and redefined Zagreb indices of Dendrimers and Polyomino Chains
  97. Topological Descriptor of 2-Dimensional Silicon Carbons and Their Applications
  98. Topological invariants for the line graphs of some classes of graphs
  99. Words for maximal Subgroups of Fi24‘
  100. Generators of Maximal Subgroups of Harada-Norton and some Linear Groups
  101. Special Issue on POKOCHA 2018
  102. Influence of Production Parameters on the Content of Polyphenolic Compounds in Extruded Porridge Enriched with Chokeberry Fruit (Aronia melanocarpa (Michx.) Elliott)
  103. Effects of Supercritical Carbon Dioxide Extraction (SC-CO2) on the content of tiliroside in the extracts from Tilia L. flowers
  104. Impact of xanthan gum addition on phenolic acids composition and selected properties of new gluten-free maize-field bean pasta
  105. Impact of storage temperature and time on Moldavian dragonhead oil – spectroscopic and chemometric analysis
  106. The effect of selected substances on the stability of standard solutions in voltammetric analysis of ascorbic acid in fruit juices
  107. Determination of the content of Pb, Cd, Cu, Zn in dairy products from various regions of Poland
  108. Special Issue on IC3PE 2018 Conference
  109. The Photocatalytic Activity of Zns-TiO2 on a Carbon Fiber Prepared by Chemical Bath Deposition
  110. N-octyl chitosan derivatives as amphiphilic carrier agents for herbicide formulations
  111. Kinetics and Mechanistic Study of Hydrolysis of Adenosine Monophosphate Disodium Salt (AMPNa2) in Acidic and Alkaline Media
  112. Antimalarial Activity of Andrographis Paniculata Ness‘s N-hexane Extract and Its Major Compounds
  113. Special Issue on ABB2018 Conference
  114. Special Issue on ICCESEN 2017
  115. Theoretical Diagnostics of Second and Third-order Hyperpolarizabilities of Several Acid Derivatives
  116. Determination of Gamma Rays Efficiency Against Rhizoctonia solani in Potatoes
  117. Studies On Compatibilization Of Recycled Polyethylene/Thermoplastic Starch Blends By Using Different Compatibilizer
  118. Liquid−Liquid Extraction of Linalool from Methyl Eugenol with 1-Ethyl-3-methylimidazolium Hydrogen Sulfate [EMIM][HSO4] Ionic Liquid
  119. Synthesis of Graphene Oxide Through Ultrasonic Assisted Electrochemical Exfoliation
  120. Special Issue on ISCMP 2018
  121. Synthesis and antiproliferative evaluation of some 1,4-naphthoquinone derivatives against human cervical cancer cells
  122. The influence of the grafted aryl groups on the solvation properties of the graphyne and graphdiyne - a MD study
  123. Electrochemical modification of platinum and glassy carbon surfaces with pyridine layers and their use as complexing agents for copper (II) ions
  124. Effect of Electrospinning Process on Total Antioxidant Activity of Electrospun Nanofibers Containing Grape Seed Extract
  125. Effect Of Thermal Treatment Of Trepel At Temperature Range 800-1200˚C
  126. Topical Issue on Agriculture
  127. The effect of Cladophora glomerata exudates on the amino acid composition of Cladophora fracta and Rhizoclonium sp.
  128. Influence of the Static Magnetic Field and Algal Extract on the Germination of Soybean Seeds
  129. The use of UV-induced fluorescence for the assessment of homogeneity of granular mixtures
  130. The use of microorganisms as bio-fertilizers in the cultivation of white lupine
  131. Lyophilized apples on flax oil and ethyl esters of flax oil - stability and antioxidant evaluation
  132. Production of phosphorus biofertilizer based on the renewable materials in large laboratory scale
  133. Human health risk assessment of potential toxic elements in paddy soil and rice (Oryza sativa) from Ugbawka fields, Enugu, Nigeria
  134. Recovery of phosphates(V) from wastewaters of different chemical composition
  135. Special Issue on the 4th Green Chemistry 2018
  136. Dead zone for hydrogenation of propylene reaction carried out on commercial catalyst pellets
  137. Improved thermally stable oligoetherols from 6-aminouracil, ethylene carbonate and boric acid
  138. The role of a chemical loop in removal of hazardous contaminants from coke oven wastewater during its treatment
  139. Combating paraben pollution in surface waters with a variety of photocatalyzed systems: Looking for the most efficient technology
  140. Special Issue on Chemistry Today for Tomorrow 2019
  141. Applying Discriminant and Cluster Analyses to Separate Allergenic from Non-allergenic Proteins
  142. Chemometric Expertise Of Clinical Monitoring Data Of Prolactinoma Patients
  143. Chemomertic Risk Assessment of Soil Pollution
  144. New composite sorbent for speciation analysis of soluble chromium in textiles
  145. Photocatalytic activity of NiFe2O4 and Zn0.5Ni0.5Fe2O4 modified by Eu(III) and Tb(III) for decomposition of Malachite Green
  146. Photophysical and antibacterial activity of light-activated quaternary eosin Y
  147. Spectral properties and biological activity of La(III) and Nd(III) Monensinates
  148. Special Issue on Monitoring, Risk Assessment and Sustainable Management for the Exposure to Environmental Toxins
  149. Soil organic carbon mineralization in relation to microbial dynamics in subtropical red soils dominated by differently sized aggregates
  150. A potential reusable fluorescent aptasensor based on magnetic nanoparticles for ochratoxin A analysis
  151. Special Issue on 13th JCC 2018
  152. Fluorescence study of 5-nitroisatin Schiff base immobilized on SBA-15 for sensing Fe3+
  153. Thermal and Morphology Properties of Cellulose Nanofiber from TEMPO-oxidized Lower part of Empty Fruit Bunches (LEFB)
  154. Encapsulation of Vitamin C in Sesame Liposomes: Computational and Experimental Studies
  155. A comparative study of the utilization of synthetic foaming agent and aluminum powder as pore-forming agents in lightweight geopolymer synthesis
  156. Synthesis of high surface area mesoporous silica SBA-15 by adjusting hydrothermal treatment time and the amount of polyvinyl alcohol
  157. Review of large-pore mesostructured cellular foam (MCF) silica and its applications
  158. Ion Exchange of Benzoate in Ni-Al-Benzoate Layered Double Hydroxide by Amoxicillin
  159. Synthesis And Characterization Of CoMo/Mordenite Catalyst For Hydrotreatment Of Lignin Compound Models
  160. Production of Biodiesel from Nyamplung (Calophyllum inophyllum L.) using Microwave with CaO Catalyst from Eggshell Waste: Optimization of Transesterification Process Parameters
  161. The Study of the Optical Properties of C60 Fullerene in Different Organic Solvents
  162. Composite Material Consisting of HKUST-1 and Indonesian Activated Natural Zeolite and its Application in CO2 Capture
  163. Topical Issue on Environmental Chemistry
  164. Ionic liquids modified cobalt/ZSM-5 as a highly efficient catalyst for enhancing the selectivity towards KA oil in the aerobic oxidation of cyclohexane
  165. Application of Thermal Resistant Gemini Surfactants in Highly Thixotropic Water-in-oil Drilling Fluid System
  166. Screening Study on Rheological Behavior and Phase Transition Point of Polymer-containing Fluids produced under the Oil Freezing Point Temperature
  167. The Chemical Softening Effect and Mechanism of Low Rank Coal Soaked in Alkaline Solution
  168. The Influence Of NO/O2 On The NOx Storage Properties Over A Pt-Ba-Ce/γ-Al2O3 Catalyst
  169. Special Issue on the International conference CosCI 2018
  170. Design of SiO2/TiO2 that Synergistically Increases The Hydrophobicity of Methyltrimethoxysilane Coated Glass
  171. Antidiabetes and Antioxidant agents from Clausena excavata root as medicinal plant of Myanmar
  172. Development of a Gold Immunochromatographic Assay Method Using Candida Biofilm Antigen as a Bioreceptor for Candidiasis in Rats
  173. Special Issue on Applied Biochemistry and Biotechnology 2019
  174. Adsorption of copper ions on Magnolia officinalis residues after solid-phase fermentation with Phanerochaete chrysosporium
  175. Erratum
  176. Erratum to: Sand Dune Characterization For Preparing Metallurgical Grade Silicon
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Heruntergeladen am 22.9.2025 von https://www.degruyterbrill.com/document/doi/10.1515/chem-2019-0052/html
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