Bisphenol A ELISA

A 96-wells microtiter plate immunoassay for the fast, sensitive and easy quantification of Bisphenol A (BPA)

in water and milk

Bisphenol A (BPA)IntroductionBisphenol A (BPA) is produced in large quantities for use primarily in the production of polycarbonate plastics and epoxy resins. Polycarbonate plastics have many applications including use in food and drink packaging, e.g. water and baby bottles. Epoxy resins are used to coat metal products such as food cans, drink containers and water supply pipes. BPA can leach into food from the protective internal ep-oxy resin of canned food and from products such as food storage containers, water- and baby bottles. BPA in food and beverages accounts for the majori-ty of daily human exposure.

Figure 1. Molecule structure of BPA

Health risksBPA is a known endocrine disruptor, mimicking es-trogens and thyroid hormones. Many studies have found that laboratory animals exposed to low levels of BPA show elevated rates of diabetes, mammary and prostate cancers, decreased sperm count, re-productive problems, early puberty, obesity, and neurological problems.

Legislation and opinionsIn a draft scientific opinion report [1], the Europe-an Food Safety Authority (EFSA) recommends the decrease of the tolerable daily intake (TDI) from 50 ppb bw/ day to 5 ppb bw/day. At present there are no restrictions on the amount of BPA that can be present in a final plastic product, but the tendency of BPA to migrate from food contact materials has been acknowledged in the EU food law. EU legis-lation (Commission Directive 2002/72/EC) sets a Specific Migration Limit (SML) of 600 ppb BPA in food. The SML in Japan is 2500 ppb. The manufac-ture of BPA-containing baby bottles is prohibited since 2011 (EU Directive (2011/8/EU)). The French National Assembly and Senate voted in the end of 2012 to ban BPA from all food contact products by January 2015 (Proposition N°49).

BPA exposureAs described by the EFSA [1], our diet is the major source of exposure to BPA, with a highest estimat-ed average of 0,375 ppb of body weight per day for infants (till 3 years of age). Compared with non-canned food, systematic higher BPA concentrations

were found in canned food and 7 out of 17 canned food categories presented an average BPA concen-tration above 30 ppb with highest levels of around 200 ppb found in meat and fish products [1]. The National Workgroup for Safe Markets in the USA reported in 2010 that BPA was detected in 46 of 50 canned food samples with an average of 77 ppb and a highest level of 1140 ppb in canned beans [2]. In 2009, Health Canada analyzed 17 bottled water products for BPA and in 13 samples from dif-ferent polycarbonate bottled water products, BPA was detected from 0.50 (LOD) to 8.82 ppb, with an average of 1.5 ppb [3]).

BPA in the environment The median reported BPA concentrations in streams and rivers from 21 European countries and 13 US States were 0.016 and 0.5 ppb, respectively [4]. In 2010, the news media reported about a Japanese survey on ocean waters taken at 200 sites in 20 countries around the world [5]. They found BPA in water and sand at concentrations ranging from 10 to 500 ppb. The breakdown of soft and hard plas-tics and BPA-based paints on boats and ships were held responsible for these high levels.

References1. EFSA Draft opinion on BPA exposure, 20142. National Workgroup for Safe Market, 2010, No Silver Lin-ing, An investigation into Bisphenol A in canned foods. 3. Health Canada, 2009, Survey of Bisphenol A in bottled wa-ter products, ISBN: 978-1-100-12127-74. Cousins et al, 2002, Human and Ecological Risk Assessment, vol. 8, pp 1107-1135.5. Sato et al, 2011, Study on new ocean contamination de-rived from marine debris plastics: Investigation of costal area and the West Pacific Ocean. Proceedings of the Twenty-first International Offshore and Polar Engineering Conference (June 2011, Maui, Hawaii).

Assay PerformancePrinciple of the ELISA:This 96-well microtiter plate (12 strips, 8 wells each) inhibition ELISA uses secondary antibody-coated wells. The anti-BPA rabbit antiserum, BPA labelled with horseradish peroxidase (HRP), and standard solutions of BPA or samples are added to the wells. The anti-BPA binds to the anti-rabbit-coated plate and the binding of BPA-HRP is inhibited by free BPA from standards/ samples. After an incubation, the non-bound reagents are removed in a washing step. The amount of bound BPA-HRP is visualized by adding a substrate/chromo-gen solution (H2O2/TMB) which transforms the colourless chromogen into a coloured product.

• Add 100 µl stop solution, shake for a few sec.• Read the absorbance values at 450 nm.

Assay ResultsLimit of detection (LOD)The LOD of 0.005 ng/ml was calculated as the mean value of 20 analyzed blank water samples plus 3SD. The LOD of 0.42 ng/ml was calculated as the mean value of 20 analyzed blank milk samples plus 3SD.

Detection capability (CCβ)The CCβ values of 0.01 ng/ml for water and 0.25 ng/ml for milk were determined by analysis of 20 blank water or milk samples and 20 samples of wa-ter or milk spiked at 0.01 and 0.25 ng/ml, respec-tively.

RecoveryThe mean recoveries of 6 water samples spiked at 0.025, 0.05 and 0.075 ng/ml were found from 84 up to 90%. The mean recoveries of 6 milk samples spiked at 0.625, 1.25 and 2.5 ng/ml were found from 102 up to 108%.

Ordering information:For ordering the Bisphenol A ELISA kit, please use catalogue code 5221BPA

The reaction is stopped by adding sulphuric acid and the colour intensity is measured photometri-cally at 450 nm. The optical density is inversely pro-portional to the BPA concentration in the sample.

Figure 2. ELISA for BPA

The ELISA kit contains all reagents to perform the assay.

Good sensitivity for the calibration curve:

Figure 3. Calibration curve

Cross-reactivity with:2,2-Bis-(4-hydroxyphenyl)-propane (BPA) =100%4,4-Bis-(4-hydroxyphenyl)-valeric acid (BVA) = 85%Bis-(4-hydroxyphenyl)-sulfone (BPS) = 6%4,4’-Cyclohexylidenebisphenol (BPZ) = 4%Coumestrol = 1.5%2,2-Bis-(4-hydroxyphenyl)-methane = 0.1%Bisphenol A diglycidyl ether (BADGE) = 0.1%

Fast assay protocol:• Pipette 50 µl of each standard/sample (in duplicate) into wells and add 25 µl of BPA-HRP and 25 µl anti-BPA and shake for a few seconds.• Incubate for 1 hour in the dark at 4°C.• Discard the solution and wash 3 times.• Add 100 µl substrate solution.• Incubate for 30 min in the dark at RT.

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