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)
Introduction
Bisphenol 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 epoxy resin of canned food and from products such as
food storage containers, water- and baby bottles.
BPA in food and beverages accounts for the majority of daily human exposure.
Figure 1. Molecule structure of BPA
Health risks
BPA is a known endocrine disruptor, mimicking estrogens 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, reproductive problems, early puberty, obesity, and
neurological problems.
Legislation and opinions
In a draft scientific opinion report [1], the European 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 legislation (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 manufacture 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 exposure
As described by the EFSA [1], our diet is the major
source of exposure to BPA, with a highest estimated average of 0,375 ppb of body weight per day for
infants (till 3 years of age). Compared with noncanned food, systematic higher BPA concentrations
were found in canned food and 7 out of 17 canned
food categories presented an average BPA concentration 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 different 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 plastics and BPA-based paints on boats and ships were
held responsible for these high levels.
References
1. EFSA Draft opinion on BPA exposure, 2014
2. National Workgroup for Safe Market, 2010, No Silver Lining, An investigation into Bisphenol A in canned foods.
3. Health Canada, 2009, Survey of Bisphenol A in bottled water products, ISBN: 978-1-100-12127-7
4. Cousins et al, 2002, Human and Ecological Risk Assessment,
vol. 8, pp 1107-1135.
5. Sato et al, 2011, Study on new ocean contamination derived 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 Performance
Principle 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.
The reaction is stopped by adding sulphuric acid
and the colour intensity is measured photometrically at 450 nm. The optical density is inversely proportional to the BPA concentration in the sample.
•
sec.
•
Add 100 µl stop solution, shake for a few
Read the absorbance values at 450 nm.
Assay Results
Limit 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 water or milk spiked at 0.01 and 0.25 ng/ml, respectively.
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)
4,4-Bis-(4-hydroxyphenyl)-valeric acid (BVA)
Bis-(4-hydroxyphenyl)-sulfone (BPS)
4,4’-Cyclohexylidenebisphenol (BPZ)
Coumestrol
2,2-Bis-(4-hydroxyphenyl)-methane
Bisphenol A diglycidyl ether (BADGE)
=100%
= 85%
= 6%
= 4%
= 1.5%
= 0.1%
= 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.
Recovery
The 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
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