development of a direct competitive enzyme-linked immunosorbent assay using a sensitive monoclonal...

6
Development of a Direct Competitive Enzyme-linked Immunosorbent Assay Using a Sensitive Monoclonal Antibody for Bisphenol A Chunmei Ju, 1, * Youhua Xiong, 2, * Aizhong Gao, 3 Tangbin Yang, 2 and Lei Wang 2 To set up an immunoassay-based method to detect Bisphenol A (BPA), we generated a monoclonal antibody (MAb) using a specially designed carboxyl derivative of BPA as the immunogen. BPA-HS was synthesized by reaction using BPA and succinic anhydride. The mice were immunized with the BPA-HS-BSA conjugate. The MAb was obtained from a hybridoma. In addition, we showed that the MAb was highly specific for BPA. The limit of detection was approximately 0.05 ng mL 1 (ppb) in assay buffer and 0.1 ng mL 1 (ppb) in water samples. The recoveries of BPA for water samples were from 90.8% to 114%, and coefficients of variation were from 15.6% to 39.4%. Thus, the ELISA method is a rapid and high throughput screening tool to detect BPA in water products. Introduction B isphenol A (BPA, 2,2-bis-(4-hydroxyphenyl)-propane; CAS Registry no. 80-05-7) is widely used as an interme- diate for binding, plasticizing, or hardening of plastics, paints/lacquers, binding materials, and filling-in materials. BPA is also a substrate for the production of polycarbonate resins (71%) and epoxy resins (27%). Furthermore, BPA is used as an additive for flame-retardants, brake fluids, and thermal papers. In 2005, the worldwide production of bi- sphenol A was 3,200,000 tons/year. (1) In Europe, four com- panies manufacture a total amount of 700,000 tons/year of BPA at six production sites, with one factory in Southern Spain producing more than 250,000 tons/year. (2,3) While frequent use and heating can break down the plastic and cause BPA to leach from plastic products into food, the higher the temperature the sooner the speed of releasing. (4) As a result, BPA gets into the human body and increases the plasma level of BPA, which has been identified as a potential estrogenic substance. It can stimulate cell proliferation of the male and female sexual organs, (5) causing reproductive ab- normalities in wildlife and disrupting endocrine function in animals and humans. (6–8) In addition, BPA is postulated to cause various kinds of cancers, such as prostate, testicular, and breast cancer, and has diverse pleiotropic effect on the brain and cardiovascular system. (9) Thus, a simple, selective, and sensitive analytical method for the detection of a trace amount of BPA in the environment has become a significant issue. Recently, the United States, Canada, and France have banned the use of BPA on food packaging and contact ma- terials; China also forbade the import of polycarbonate baby bottles containing BPA. To date, the methods most frequently used for the determination of BPA are high performance liquid chromatography (HPLC), (10) gas chromatography coupled with mass spectrometry (GC/MS), (11) liquid chro- matography with electrochemical detection (LC–ED), (12) and liquid chromatography coupled with mass spectrometry (LC- MS). (13,14) Although these methods are quantitative, they take a long time, need large sample volumes, generate large amounts of waste, and require bulky and expensive instru- ments. Kaddar and colleagues (15) developed a radioimmu- noassay using a polyclonal anti-BPA antibody (PAb), while the results show that the PAb is of low sensitivity and weak specificity. Marchesini and colleagues (16) established biosen- sor immunoassays for the detection of bisphenol A by em- ploying anti-BPA antibodies (polyclonal [PAbs] and monoclonal [MAbs]). However, this assay is too sensitive to be disrupted and the binding capacity of the immobilized MAbs decrease fast. Therefore, a rapid and high throughput screening method to detect BPA in liquid storage containers is highly desirable. In this study, we report the generation of a monoclo- nal antibody specifically recognizing BPA. Using the anti- body, we designed an enzyme-linked immunosorbent assay (ELISA) for detecting BPA in liquid storage con- tainers in a simplified and sensitive way. This method has 1 College of Veterinary Medicine, South China Agricultural University, Guangzhou, China. 2 College of Medicine, China Three Gorges University (CTGU), Yichang, China. 3 Guangzhou Ucando Biotechnology Co., Guangzhou, China. *These authors contributed equally to this work. HYBRIDOMA Volume 30, Number 1, 2011 ª Mary Ann Liebert, Inc. DOI: 10.1089/hyb.2010.0079 95

Upload: lei

Post on 23-Dec-2016

213 views

Category:

Documents


1 download

TRANSCRIPT

Development of a Direct Competitive Enzyme-linkedImmunosorbent Assay Using a Sensitive Monoclonal

Antibody for Bisphenol A

Chunmei Ju,1,* Youhua Xiong,2,* Aizhong Gao,3 Tangbin Yang,2 and Lei Wang2

To set up an immunoassay-based method to detect Bisphenol A (BPA), we generated a monoclonal antibody(MAb) using a specially designed carboxyl derivative of BPA as the immunogen. BPA-HS was synthesized byreaction using BPA and succinic anhydride. The mice were immunized with the BPA-HS-BSA conjugate. TheMAb was obtained from a hybridoma. In addition, we showed that the MAb was highly specific for BPA. Thelimit of detection was approximately 0.05 ng mL�1 (ppb) in assay buffer and 0.1 ng mL�1 (ppb) in water samples.The recoveries of BPA for water samples were from 90.8% to 114%, and coefficients of variation were from 15.6%to 39.4%. Thus, the ELISA method is a rapid and high throughput screening tool to detect BPA in waterproducts.

Introduction

B isphenol A (BPA, 2,2-bis-(4-hydroxyphenyl)-propane;CAS Registry no. 80-05-7) is widely used as an interme-

diate for binding, plasticizing, or hardening of plastics,paints/lacquers, binding materials, and filling-in materials.BPA is also a substrate for the production of polycarbonateresins (71%) and epoxy resins (27%). Furthermore, BPA isused as an additive for flame-retardants, brake fluids, andthermal papers. In 2005, the worldwide production of bi-sphenol A was 3,200,000 tons/year.(1) In Europe, four com-panies manufacture a total amount of 700,000 tons/year ofBPA at six production sites, with one factory in SouthernSpain producing more than 250,000 tons/year.(2,3)

While frequent use and heating can break down the plasticand cause BPA to leach from plastic products into food, thehigher the temperature the sooner the speed of releasing.(4) Asa result, BPA gets into the human body and increases theplasma level of BPA, which has been identified as a potentialestrogenic substance. It can stimulate cell proliferation of themale and female sexual organs,(5) causing reproductive ab-normalities in wildlife and disrupting endocrine function inanimals and humans.(6–8) In addition, BPA is postulated tocause various kinds of cancers, such as prostate, testicular, andbreast cancer, and has diverse pleiotropic effect on the brainand cardiovascular system.(9) Thus, a simple, selective, andsensitive analytical method for the detection of a trace amountof BPA in the environment has become a significant issue.

Recently, the United States, Canada, and France havebanned the use of BPA on food packaging and contact ma-terials; China also forbade the import of polycarbonate babybottles containing BPA. To date, the methods most frequentlyused for the determination of BPA are high performanceliquid chromatography (HPLC),(10) gas chromatographycoupled with mass spectrometry (GC/MS),(11) liquid chro-matography with electrochemical detection (LC–ED),(12) andliquid chromatography coupled with mass spectrometry (LC-MS).(13,14) Although these methods are quantitative, they takea long time, need large sample volumes, generate largeamounts of waste, and require bulky and expensive instru-ments. Kaddar and colleagues(15) developed a radioimmu-noassay using a polyclonal anti-BPA antibody (PAb), whilethe results show that the PAb is of low sensitivity and weakspecificity. Marchesini and colleagues(16) established biosen-sor immunoassays for the detection of bisphenol A by em-ploying anti-BPA antibodies (polyclonal [PAbs] andmonoclonal [MAbs]). However, this assay is too sensitive tobe disrupted and the binding capacity of the immobilizedMAbs decrease fast. Therefore, a rapid and high throughputscreening method to detect BPA in liquid storage containers ishighly desirable.

In this study, we report the generation of a monoclo-nal antibody specifically recognizing BPA. Using the anti-body, we designed an enzyme-linked immunosorbentassay (ELISA) for detecting BPA in liquid storage con-tainers in a simplified and sensitive way. This method has

1College of Veterinary Medicine, South China Agricultural University, Guangzhou, China.2College of Medicine, China Three Gorges University (CTGU), Yichang, China.3Guangzhou Ucando Biotechnology Co., Guangzhou, China.*These authors contributed equally to this work.

HYBRIDOMAVolume 30, Number 1, 2011ª Mary Ann Liebert, Inc.DOI: 10.1089/hyb.2010.0079

95

the potential to be developed into an alternative tool forBPA detection.

Materials and Methods

Chemicals and reagents

All chemicals, unless otherwise stated, were analytical re-agent grade. Bisphenol A, urea hydrogen peroxide, dicyclo-hexylcarbodiimide (DCC), Freund’s adjuvant (Freund’scomplete adjuvant and Freund’s incomplete adjuvant), bo-vine serum albumin (BSA), ovalbumin (OVA), horseradishperoxidase (HRP), polyethylene glycol 1450 (PEG 1450, 50%,w/v) and N-hydroxysuccinimide (NHS) were from Sigma-Aldrich (St. Louis, MO). 3,30,5,50-tetramethylbenzidine (TMB),dimethyl sulphoxide (DMSO), dimethylformamide (DMF),Tween-20, succinic anhydride, and pyridine were from Sino-pharm Chemical Reagent Co. (Shanghai, China). Hypox-anthine/aminopterin/thymidine (HAT), hypoxanthine/thymidine (HT), and DMEM with l-glutamine culture me-dium were bought from Gibco (Invitrogen, Carlsbad, CA).Fetal calf serum was from Guangzhou Zhanchen Bio-tech Co.(Guangzhou, China). Peroxidase-labeled goat anti-mouse IgG(HþL) (HRP-IgG) was from Jackson Immunoresearch La-boratories (West Grove, PA).

Instruments

Microtiter plates were from Nunc (Roskilde, Denmark).The microplate reader was Synergy-HT from Biotek(Winooski, VT). Cell culture plates (24 and 96 wells) andculture flasks were from Costar (Cambridge, MA). Water waspurified by a MilliQ purification system from Millipore (Bill-erica, MA). The LC-MS system model API 3000 was fromApplied Biosystems (Bedford, MA).

Buffers and solutions

The following buffers were used in the experiments: (1)phosphate-buffered saline (PBS; 10 mmol L�1 sodiumphosphate, 137 mmol L�1 NaCl, 2.7 mmol L�1 KCl [pH 7.5])for the dilution of antibodies and the preparation of stan-dard solutions; (2) stock solution of BPA at a concentrationof 1 mg mL�1 was prepared in methanol and diluted tostandard solutions with PBS; (3) the coating buffer was0.1 M carbonate buffer (pH 9.6); (4) the blocking buffer was1% OVA (m/v) in PBS; (5) phosphate buffer saline (PBS)with 0.8% (w/v) NaCl (pH 7.2); (6) PBST, a PBS buffercontaining 0.05% Tween-20 (v/v), was used for washing; (7)the substrate buffer was 0.1 M citrate (pH 5.5); (8) the per-oxidase substrate solution was prepared by mixing 200 mL1% (w/v) TMB in DMSO with 64 mL 0.75% (w/v) to 20 mLsubstrate buffer; (9) the enzymatic reaction was stoppedwith 2 M H2SO4.

BPA hapten derivatives (BPA-HS) for conjugationwith proteins

BPA (4.5 g) dissolved in 10 mL pyridine in a conical flaskwas mixed by slowly adding 2 g succinic anhydride. Themixture solution was then heated to 708C and stirred for 3 h.Thin-layer chromatography of the reaction mixture demon-strated formation of a product (elution in 10% methanol inchloroform). After removal of pyridine by evaporation, the

resulting mixture was crude BPA-HS, which was furtherpurified by recrystallization in methanol.

Synthesis of BPA-HS-protein conjugates

BPA-HS was conjugated to bovine serum albumin (BSA) byan active ester method.(17–19) The carboxylic acid on the hap-ten was activated with DCC and NHS to produce an activeester, which then reacted with the amine groups on BSA orOVA to form amide bonds. BPA-HS (32.8 mg), NHS (11.5 mg),and DCC (20.6 mg) were each dissolved in 0.5 mL 1,4-dioxane.NHS, followed by DCC, was added slowly to the BPA-HSsolution. This activation reaction and carried out overnight atroom temperature with continuous stirring. The reactionmixture was centrifuged (2000 g, 10 min) and the supernatantwas added very slowly to a BSA solution (340 mg BSA dis-solved in 5 mL PBS). The mixture was stirred overnight at 48Cto complete the conjugation reaction. The mixture was thendialyzed against phosphate buffer (0.1 mol L-1 [pH 7.4]) for 3days (two changes of buffer per day). BPA-HS-BSA wasfreeze-dried and stored at �208C. Similarly, BPA-HS-OVAwas synthesized as a coating conjugate. The complete antigenBPA-HS-BSA was identified by the ultraviolet and infraredspectrophotometers.

BPA-HS-HRP tracer

BPA-HS was conjugated to HRP by an active estermethod.(20) BPA-HS (1.64 mg), NHS (0.58 mg), and DCC(1.03 mg) were dissolved in DMF (0.5 mL). The mixture wasstirred at room temperature overnight. The activated haptenwas centrifuged (2500 rpm/10 min) and 113.3mL of superna-tant was added dropwise under stirring to 3.4 mg of HRP in0.13 mol L�1 NaHCO3 (600 mL) in order to obtain molar ratio5:1 (hapten-HRP). The conjugation mixture was stirred atroom temperature for 5 h, and the mixture was then dialyzedagainst phosphate buffer (0.1 mol L-1 [pH 7.4]). The tracerobtained was diluted with the same volume of glycerol andstored at �208C until use.

Immunization of mice and productionof monoclonal antibodies

Three Balb/c female mice, 8–10 weeks old, were immu-nized by intraperitoneal injection. BPA-HS-BSA (50 mg) im-munogen was diluted in 100mL sterile PBS and emulsified inFreund’s complete adjuvant for the primary immunization.Booster doses of BPA-HS-BSA (50 mg) in Freund’s incompleteadjuvant were then injected into the above mice once every2 weeks for three times. Blood was obtained via retro-orbitalvenous plexus at day 12 after the third booster immunizationand stored overnight at 48C. Then antisera were collectedfrom the blood by centrifugation at 4000 rpm for 10 min. Themouse that exhibited the highest titer by indirect ELISA wasimmunized with a final dose of BPA-HS-BSA (50mg) for fu-sion experiment.

Anti-BPA monoclonal antibodies were produced using thehybridoma technique. The splenocytes from the immunizedmice were fused with the SP2/0 myeloma cells. Hybridomaswere selected in a HAT medium (DMEM medium containing15% FBS). The cultures in 96-well plates were maintained in a5% CO2 incubator at 378C. When hybridoma colonies ap-peared, they were expanded in the HT medium. Hybridoma

96 JU ET AL.

culture supernatants were screened by an indirect competi-tive ELISA (icELISA) with BPA as the competitor. BPA-HS-OVA conjugate was used as the coating conjugate. Thehybridoma that secreted antibodies specific to BPA was sub-cloned by the serial dilution method. Colonies of interest werepropagated, frozen (overnight at �808C) in culture mediumcontaining 10% dimethyl sulphoxide, and stored in liquidnitrogen.

For ascites production, two Balb/c female mice were used.The mice were treated by intraperitoneal injection with 0.5 mLof Freund’s incomplete adjuvant. After 10 days, each mousewas intraperitoneally injected with 1�107 hybridoma cells.After 15 days, a total of 7.4 mL of ascitic fluid was collectedfrom the two mice, incubated overnight at 48C, and centri-fuged at 48C for 10 min. The supernatant was finally obtainedand stored at �208C.

ELISA procedure

Indirect ELISA was carried out as follows: (1) 100 mL ofcoating conjugate BPA-HS-OVA diluted with the coatingbuffer at 0.1 mg mL�1 were added into a microtiter plate andincubated at 48C overnight. (2) Plates were washed three

times using 300 mL/well of the washing buffer and then100mL of antibody diluted with PBS were added to each well.(3) After incubation for 30 min at 378C, the unbound com-pounds were washed away. (4) 100mL of HRP-IgG (workingconcentration recommend 1:5000) were then added to eachwell and incubated for 30 min at 378C. (5) After washing fourtimes, 100 mL of substrate solution was added into each welland incubated for 15 min at 378C. (6) The enzymatic reactionwas stopped by the stopping solution (50mL/well) and then

FIG. 1. The structures of BPA, BPA-HS, BHPVA, BPA-HS active ester and BPA-HS conjugated with protein.

Table 1. Molecular Ion and Fragment Ions

of BPA-HS Under Mass Spectrometry Detection

StructureMolecular

weightMeasured

m/z values

HOC6H4C(CH3)2C6H4OOC(CH2)2COOH

328.3 328.2

þHOC6H4C(CH3)2C6H4OOC(CH2)2COOH

329.3 329.1

HOC6H4C(CH3)2C6H4OOC(CH2)2- 274.3 274.2-OC6H4C(CH3)2C6H4O- 226.3 226.4

DIRECT COMPETITIVE ELISA FOR BISPHENOL A 97

absorbance at 450 nm was measured. The procedure for icE-LISA was similar to that of indirect ELISA, except for adding50 mL of BPA standard solution (competitor) and 50mL ofantibody solutions to every well after washing in step 2.

Direct competitive ELISA (dcELISA) tests were carried outusing the following procedure: 100mL of antibody dilutedwith coating buffer was pipetted into a microtiter plate andincubated at 48C overnight. Plates were washed three timesusing 300mL/well of washing buffer solution and 50 mL ofstandard solution or sample, and 50 mL of tracer solution wereadded to each well. Unbound compounds were removed bywashing solution after incubation for 30 min at 378C. 100 mL ofthe substrate solution were then added to each well and theenzymatic reaction was stopped after 15 min incubation at378C by the addition of 50 mL/well of stopping solution. Ab-sorbance values were measured at 450 nm and the concen-tration of the analyte in a sample was calculated from thecalibration curve.

Monoclonal antibody titer and sensitivity

The monoclonal antibody titer was measured using theindirect ELISA method. The ascites titer with an absorbance of2.0 was defined as the maximum dilution. The sensitivity tofree BPA was determined using the dcELISA. To prepare a

standard curve, 50mL of BPA standard solution (prepared inPBS at 0, 0.1, 0.3, 0.9, 2.7, and 8.1 ng mL�1 [ppb]) was added inthe dcELISA. The signals obtained in the presence of variousBPA concentrations and without competitor (maximal signal)were referred to as B and B0, respectively. The inhibition ratiowas obtained by dividing B with B0 (B/B0). A linear standardcurve was prepared by plotting log (BPA concentration)versus the inhibition ratio. The limit of detection (LOD) wascalculated as the standard deviation from the mean signalmeasured from the blank wells three times.

Assessment of monoclonal antibody specificity

The cross-reactivity of monoclonal antibody to variousdyes and their related derivatives was tested using thedcELISA described, which was determined by measuringtheir IC50 values in the dcELISA. The IC50 values (50% inhi-bition levels) were used for calculation of cross-reactivity(CR50%) using the formula: CR50%¼ 100�IC50 value of BPA/IC50 value of competitor.

FIG. 2. Representative dose response curves for BPA in dcELISA for 3C7 ascites antibody. B/B0 is the normalized responserelative to the zero standard. Coating monoclonal antibody dilution¼ 1:4�103, BPA-HS-HRP dilution¼ 1:2000. The regres-sion curve equation is as follows: y¼�36.678�þ44.322, with a correlation coefficient of 0.9943.

Table 2. Cross-reactivity (CR50%) of Monoclonal

Antibody with Various Compounds

Competitor in dcELISA CR50 (%)

BPA 100BPA-HS 129.3BHPVA 18.9Diethylstilbestrol 5.1p-Methylphenol <0.01Phenol <0.01

Table 3. Recoveries and Coefficients of Variation

of Sample Fortified with BPA

BPAFortified

(ng mL�1)Test

(ng mL�1) Recovery (%) CV (%)

Sample 1

0.1 0.114� 0.040 114 35.11 1.075� 0.267 107.5 24.8

10 10.218� 1.952 102.1 19.1

Sample 2

0.1 0.106� 0.042 106 39.41 1.024� 0.273 102.4 26.7

10 10.096� 1.736 100.9 17.2

Sample 3

0.1 0.097� 0.035 97 36.91 1.035� 0.303 103.5 29.3

10 9.083� 1.417 90.8 15.6

N¼ 5.

98 JU ET AL.

Sample preparation

The following commercial samples were purchased in hy-permarket: mineral (or purified) water in liquid storage con-tainers (all samples deriving from different batches). Eachkind of sample where BPA was demonstrated to be absentwas considered as the blank and used for validation purposes.

Samples were fortified at this stage by addition of BPAstandard solution in PBS buffer to give fortified calibrationstandards equivalent to 0, 0.1, 0.3, 0.9, 2.7, and 8.1 ng BPA permL sample (the water in liquid storage containers). The resultwas analysed by the direct competitive ELISA. The recoveryand coefficient of variation of BPA in sample were performed.

ELISA analysis and validation

The blank water samples were used for validation studyusing the dcELISA protocol to assess the range of matrix in-terferences in the assay and to provide data for determinationof false positive rates. Coefficients of variation (CV) weredetermined by the analysis of the above samples fortified withBPA at 0.1, 1, and 10 ng mL�1 (ppb). The recovery (%) of thefortified BPA was calculated using the equation: conc. mea-sured/conc. fortified�100. Repeatability of the method wasestablished using fortified duplicate blanks at the levels 0.1, 1,and 10 ng mL�1 (ppb).

Results and Discussion

Hapten design and synthesis of BPA conjugates

BPA are not able to initiate an immune response, so BPA-HS-BSA was used to produce antibody in this study. Since theabsence of a known coupling group such as NH2, COOH, andSH on BPA, a BPA derivative (BPA-HS) (Fig. 1) was synthe-sized following derivatization with succinic anhydride (HS)and identified by mass spectrum (MS) after purification. Asolution of the BPA-HS (*100 mg mL�1 in methanol) wasscanned in both positive and negative modes of LC-MS elec-trospray to confirm its identity. In positive mode, the pro-tonated molecular ion [MþH]þ m/z 329.1 and the moleculepeak m/z 328.2 were seen. In negative mode, the moleculepeak m/z 327.1 and the deprotonated molecular ion m/z 226.3,274.2 were seen. In negative mode, the relation between theprimary deprotonated molecular ion of BPA-HS and structureof BPA-HS molecule is shown in Table 1.

By an active ester method, the carboxylic acid group onBPA-HS was conjugated with the amino groups on BSA un-der the effect of NHS and DCC, forming amide bonds. It isdifferent from selected BHPVA (Fig. 1) as hapten, producingBHPVA-HS with amide bond on a different site.(21)

Monoclonal antibody titer and sensitivity

Two fusion experiments were carried out to obtain the spe-cific monoclonal antibody. The titer of the monoclonal antibodywas determined by checkerboard titration. The optimal condi-tions of ELISA were chosen when the OD value was about 2.0.Likewise, the optimal concentration of the coating conjugatewas found to be 0.2mg mL�1. Under the optimal conditions, wefound that the titer of the ascites antibody was 1�105.

Representative ELISA curves for BPA were presented inFigure 2. The ratios of B/B0 refer to the standard dose re-sponses. B0 ranged from 1.5 to 2 for both ELISA methods. As a

result, the assays showed a high sensitivity. The LOD for thedcELISA was approximately 0.05 ng mL�1(ppb), with IC50 of0.7 ng mL�1 (ppb) for BPA.

Monoclonal antibody specificity and isotypes

Specificity of the MAb in optimized assays was tested bymeasuring the cross-reactivity using BPA-related compounds.The cross-reactivity was determined by dcELISA describedabove. The chemical compounds and their corresponding cross-reactivity are shown in Table 2. The data showed that the im-munoassays for BPA are highly selective. We found a highcross-reactivity with BPA-HS (Table 2), but only low cross-reactivity with BHPVA (18.9%). No cross-reactivity was de-tected with other related compounds shown in Table 2.

Through identifying the heavy and light chain isotypes ofantibodies by SBA Clonotyping System/HRP kit, the MAbbelongs to IgG1 type.

ELISA validation

Twenty different samples (identical matrix) from retail out-lets were used as blank samples for the determination of LODand detection capability of the ELISA procedure at differentfalse positive and negative rates. The determination of LOD wasbased on 20 blank samples accepting no false positive rates(averageþ 3 SD). LOD in different samples are listed in Table 3.The LOD was approximately 0.1 ng mL�1 (ppb) in the twomatrices. The accuracy and precision were evaluated (5 repli-cates) by spiking BPA at 0.1, 1, and 10 ng mL�1 (ppb). Therecoveries of BPA in water samples were from 90.8 to 114% andcoefficients of variation were from 15.6 to 39.4% (Table 3).

Conclusion

We have generated a monoclonal antibody specific forBPA. This antibody is useful for determining the concentra-tions of BPA within the curve range shown in Figure 2. ThedcELISA using this antibody is sensitive enough to detectthose substances at a very low concentration level reaching toppb in water samples. Currently, HPLC and/or LC-MS/MSmethods are used to detect the presence of BPA. However, ourdata showed that the dcELISA method is as sensitive as theLC-MS/MS method and is more sensitive than the HPLCmethod. Thus, using the monoclonal antibody developed inthis study, the dcELISA method is a sensitive and simplifiedalternative for the detection of BPA in water samples.

Author Disclosure Statement

The authors have no financial conflicts to declare.

References

1. Environmental Protection Agency: Cross-species mode ofaction information assessment: a case study of bisphenol A.Government Reports Announcements & Index, Issue 26,National Center for Environmental Assessment, 2005.

2. European Commission: Risk assessment report of 4,4-iso-propylideden diphenol (Bisphenol-A). Italy, Ispra, 2003.

3. New Cartagena LEXAN resin plant represents GE’s largestinvestment in Spain. Polym News 2005;30:158–160.

4. Lyons G: Bisphenol A: a known endocrine disruptor. A WWFEuropean Toxics Programme Report. WWF-UK, 2000:1–15.

DIRECT COMPETITIVE ELISA FOR BISPHENOL A 99

5. Wetherill YB, Petre CE, Monk KR, Puga A, and Knudsen KE:The xenoestrogen bisphenol A induces inappropriate an-drogen receptor activation and mitogenesis in prostatic ad-enocarcinoma cells. Mol Cancer Ther 2002;1:515–524.

6. Segner H, Caroll K, Fenske M, Janssen CR, Maack G, PascoeD, Schafers C, Vandenbergh GF, Watts M, and Wenzel A:Identification of endocrine-disrupting effects in aquaticvertebrates and invertebrates. Environ Saf 2003;54:302–314.

7. Choi KC, and Jeung EB: The biomarker and endocrine dis-ruptors in mammals. J Reprod Dev 2003;49:337–345.

8. Soto AM, Justicia H, Wray JW, and Sonnenschein C:p-Nonyl-phenol: an estrogenic xenobiotic released frommodified polystyrene. Environ Health Perspect 1991;92:167–173.

9. Steinmetz R, Mitchner NA, Grant A, Allen DL, Bigsby RM,and BenJonathan N: The xenoestrogen bisphenol A inducesgrowth, differentiation, and c-fos gene expression in the fe-male reproductive tract. Endocrinology 1998;139:2741–2747.

10. Estevez MC, Galve R, Sanchez-Baeza F, and Marco MP:Direct competitive enzyme-linked immunosorbent assay forthe determination of the highly polar short-chain sulfophe-nyl carboxylates. Anal Chem 2005;77:5283–5293.

11. Meesters RJW, and Schroder HF: Simultaneous determina-tion of 4-nonylphenol and bisphenol A in sewage sludge.Anal Chem 2002;74:3566–3574.

12. D’Antuono A, Dall’Orto VC, Balbo AL, Sobral S, and Re-zzano I: Determination Of Bisphenol A in food-simulatingliquids using LCED with a chemically modified alectrode.J Agric Food Chem 2001;49:1098–1101.

13. Inoue K, Kato K, Yoshimura Y, Makino T, and Nakazawa H:Determination of bisphenol A in human serum by high-performance liquid chromatography with multi-electrodeelectrochemical detection. J Chromatogr B Biomed Sci Appl2000;749:17–23.

14. Usami M, Mitsunaga K, and Ohno Y: Estrogen receptorbinding assay of chemicals with a surface plasmon reso-nance biosensor. J Steroid Biochem Mol Biol. 2002;81:47–55.

15. Kaddar N, Bendridi N, Harthe C, Rolland de Ravel M, Bi-envenu A-L, Cuilleron C-Y, Mappus E, Pugeat M, and De-

chaud H: Development of a radioimmunoassay for themeasurement of Bisphenol A in biological samples. AnalChim Acta 2009;645:1–4.

16. Marchesini GR, Meulenberg E, Haasnoot W, and Irthc H:Biosensor immunoassays for the detection of bisphenol A.Anal Chim Acta 2005;528:37–45.

17. Vass M, Diblikova I, Cernoch I, and Franek M: ELISA forsemicarbazide and its application for screening in foodcontamination. Anal Chim Acta 2008;608:86–94.

18. Aizhong G, Qiaolin C, Yu C, Jing L, and Lingwen Z: Pre-paration of monoclonal antibodies against a derivative ofsemicarbazide as a metabolic target of nitrofurazone. AnalChim Acta 2007;592:58–63.

19. Chunmei J, Yong T, Huiying F, and Jinding C: Enzyme-linked immunosorbent assay (ELISA) using a specificmonoclonal antibody as a new tool to detect Sudan dyes andPara red. Anal Chim Acta 2008;621:200–206.

20. Iva D, Kevin MC, Glenn K, and Milan F: Monoclonalantibody-based ELISA for the quantification of nitrofuranmetabolite 3-amino-2-oxazolidinone in tissues using a sim-plified sample preparation. Anal Chim Acta 2005;540:285–292.

21. Andre K, Chun-Ri L, Chun-Feng J, Keun Woo L, Sang-HoonL, Kwang-Jae S, Nam Gyu P, Dong-Kyoo K, Shin-Won K,Yoon-Bo S, and Jang-Su P: A sensitive and reliable quanti-fication method for Bisphenol A based on modified com-petitive ELISA method. Chemosphere 2007;68:1204–1209.

Address correspondence to:Dr. Lei Wang

College of MedicineChina Three Gorges University (CTGU)

Yichang 443002China

E-mail: [email protected]

Received: July 24, 2010Accepted: September 28, 2010

100 JU ET AL.