The quantitative analysis of estrogen levels in different brands of milk using ELISA
Kara Arbogast
Fall 2011
Abstract
Cows are given the growth hormones recombinant Bovine Growth Hormone and recombinant
Bovine Somatotropin to increase milk production where organic cows are not. To determine if these
hormones affect the levels of estrogen in the milk, analysis by Enzyme-Linked Immunosorbent Assay
(ELISA) is performed. Two different kits, Estrogen (E1/E2/E3) EIA kit and Cayman EIA Kit, both using an
Estradiol-enyme competition reaction were used for analysis. The estrogen (E1/E2/E3) kit had a
sensitivity of .0μg/L to 3.0μg/L and required a lot of sample preparation. Cayman EIA kit has a sensitivity
of 6.6 pg/mL to 4000 pg/mL. The Cayman EIA was used for different samples (table 3) testing for
interferences. Straight milk (no preparation) fell on the standard curve proving that sample preparation
is not necessary. In the future, estrogen levels for various types of milk can be efficiently analyzed and
compared.
Background:
Over the years, the dairy industry has been going through a scientific revolution to increase
production and efficiency. Recombinant Bovine Growth Hormone (rBGH) and recombinant Bovine
Somatotropin (sbST) are two hormones that dairy farmers have been giving their cows to increase milk
production(7). These hormones cause the cows to become more prone to infections and therefore need
an increased amount of antibiotics (6). Recent studies show that these extra hormones and antibiotics
may be taking a toll on our health by disrupting our endocrine system through the introduction of excess
levels of hormones (3, 6). Organic cows are not given sbST and rBGH and feed from open pastures.
Estrogen is a hormone that is critical for various areas of health such as regulating the female
reproductive system and maintaining the development of female gonads. High levels lead to many
health conditions such as breast, testicular, prostate, and ovarian cancer (4). To raise awareness and
decrease the ingestion of estrogen, estrogen levels are now being quantified in various food products (3,
4). A large source of our dietary estrogen comes from milk, either human, goat, bovine, etc, where the
estrogens cross the blood-milk barrier and into the milk that is later processes and consumed (4). A
study measured the levels of estrogen in raw bovine milk at different stages of pregnancies and in raw
bovine milk from cows that were not pregnant. The levels of estrogen increased in the cows during
pregnancy; causing an increase of estrogen in the milk they produced (4). Due to the increasing
economic need for milk over the years, the cows are being forced to reproduce more resulting with an
increasing amount of pregnant cows, causing a constant source of high estrogen milk (4).
This experiment uses an Enzyme-Linked Immunosorbent Assay kit, or ELISA, to quantitatively
measure and compare the estrogen levels in various types of milk such as organic (from cows that are
not treated with rBGH/sbST), non-organic, bottled, canned, as well as different brands. The ELISA kit
uses competition between the enzyme AChE, an estradiol-acetylcholinesterase conjugate, and free
estrogens that are present in the milk sample for an antibody that is fixed to the plate. The plate is then
treated with Ellman’s Reagent which contains acetylthiocholine and dithionitrobenzoic acid (DTNB). The
AChE present in the wells will hydrolyze the acetylthiocholine producing thiocholine. Thiocholine then
cleaves the disulfide bond present in the Ellman’s reagent producing 5-thio-2-nitrobenzoic acid (NTB2-).
NTB2- is a yellow compound that can quantified spectrophotometrically. The intensity of the yellow color
is with respect to the concentration of the AChE bound to the antibody so the exact concentration of
AChE can be quantitatively measured using absorbance. The concentration of AChE is inversely related
to the concentration of estrogen
There are many interferences that cause cross reactivity with the antibody, such as the presence
of trace organic solvents, affecting the results of ELISAs (5). Interferences will cause the maximum
binding of the tracer (AChE) and free estrogen to decrease and must be minimized as much as possible
(5). Interferences can be removed by purification methods such as solid-phase extraction (SPE) column,
high-performance liquid chromatography (HPLC), or by dilutions with ultra pure water. If dilutions
produce consistent results, then there is no interference affecting results. DSC-18 reversed phase SPE
columns were used to extract the estrogens from the supernatant of milk samples. DSC-18 columns are
polymerically bonded, 18% octadecyl, and endcapped and silica based (8). They work by trapping the
estrogens in the pores of the columns while the rest of the supernatant drains through. The estrogen
are then released from the column by adding 100% methanol due to a higher column affinity for
methanol. ELISAs were not originally designed to analyze milk samples; therefore a method to use the
ELISA for milk must be determined.
Two different ELISA kits were used to analyze the levels of estrogen; the ELISA kits both used an
estrodiol-enzyme conjugate. The first was the Estrogen (E1/E2/E3) EIA kit that was purchased from
Biosense Laboratories (1). The kit and its reagents were all stored at 4 degrees Celsius. The kit required
the milk to be purified using Solid-Phase Extraction column (SPE) to remove interferences before
analyzing. The process of adding the milk samples and the reagents to the ELISA kit was very time
sensitive. A chromagen solution was used as the coloring agent and an acidic stop solution was required
to stop the coloring reaction. The kit must incubate for 30 min before the absorbance can be measured.
The quantitative analysis range for this kit was .0μg/L to 3.0μg/L(1). The second ELISA that is currently
being used is an Estradiol EIA kit that was purchased from Cayman Chemicals (2). This kit uses an AChE
conjugate as a competitor for E2. This kit must be stored at -20 degrees Celsius and is not time sensitive
due to the fact that it does not require a stop solution. No sample prep is required in this experiment for
the Cayman kit because the data for the straight milk samples fell on the standard curve. The Cayman
ELISA kit uses Ellman’s Reagent as a coloring agent and must incubate with mixing for 2 hours before the
absorbency can be measured. The quantitative analysis range for this kit is 6.6pg/mL to 4000pg/mL (2).
The Cayman kit will continue to be used because it is less expensive and no sample preparation is
necessary.
Methods:
Before the milk could analyzed by ELISA it needed to be prepared. The proteins were
precipitated out by using acetic acid and centrifuging. The centrifuge used was a Thermo Scientific Centi
Sorvall RC plus and was run at 6500RPM using a rotor code of 28 at 4 degrees Celsius. The estrogen was
then extracted out using a C-18 SPE column. The C-18 SPE column is activated by running 5 mL of 100%
methanol and then 5 mL of deionized water with a vacuum. The supernatant was then run through the
activated C-18 SPE column to purify sample and reduce the amount of interferences. The estrogen is
eluted from SPE column using 5mL 100% methanol. The ELISA kit requires a 10% concentration of
methanol, so the methanol is evaporated from the sample using a Buchi Rotovapor R-210 rotary
vaporizer and then reconstituted 1:1 using 10% methanol. The sample is then ready to be analyzed by
ELISA.
For the Estrogen (E1/E2/E3) Enzyme Immunosorbent assay kit, the E2 standards, antigen –
enzyme complex, wash solution, and chromogen solution for the ELISA were stored and prepared using
the instructions noted in the instruction manual (1). The standard Estradiol concentrations used to
create the standard curve were 0.1, 0.2, 0.4, 1.0, and 3.0g/L and are dilutes in 10% methanol (1). The
prepared milk samples and standards were added in double to a non-coated plate and transferred to
the ELISA kit plate in intervals of 15 seconds. Everything was run in duplicate. After an incubation period
of 60 minutes at room temperature, the plate was washed with the wash solution and all unbound
material removed. The chromogen solution, which is prepared within 15 minutes of using, is then added
to the wells in intervals of 15 seconds. The plate is then incubated for 30 minutes at room temperature
in order for the color to develop. After 30 minutes, 100μL of stop solution is added in intervals of 15
seconds to each well. Absorbance is measured at 450nm using a BIORAD Benchmark microplate reader
plate reader.
The Cayman kit does not require sample preparation because the sensitivity for estrogen is
higher and straight milk samples fall on the standard curve. The Estradiol standards, EIA buffer, samples,
Ellman’s reagent, and Tracer (AChE) were stored and prepared following the instructions provided by
the instruction manual (2). The concentrations used for the standards was 0.0, 6.6, 16.4, 41, 102.4, 256,
640, 1600, and 4000 pg/mL and are diluted in EIA buffer. The milk samples and standards are added in
double to a non-coated plate according to instructions in manual and then transferred the EIA kit plate
in intervals of 15 seconds. The plate is covered with film then placed on a shaker and incubated at room
temperature for 1 hour. The plate is then washed with wash buffer to remove all unbound material.
Within 15 minutes of being used the Ellman’s Reagent must be made and placed in the dark till ready to
use due to sensitivity to light. After the wells are washed and dry, the 200μL of Ellman’s Reagent is
added to each well and the kit is covered with film and incubated in the dark for two hours while
shaking. After two hours, the film is removed, and using a plate reader, the kit’s absorbance is measured
at a wavelength of 405nm using a BIORAD Benchmark microplate reader.
For the Cayman kit, each well has different amounts of each prepared solution. The amount of
solutions added to each well is listed in table 1. The ELISA plate is 12 wells by 8 wells, giving a 96 well
plate with removable wells. To be cost efficient, only partial plates are used at a time depending on
amount of samples. The first column of 8 wells is used for 2 wells of blanks, 3 wells of nonspecific
binding (NSB) and 3 wells of maximum binding (B0). Column 2 and 3 are used for the Estradiol standards
in duplicate. The rest of the wells are used for the milk samples in triplicates. The blanks are used to
eliminate background absorbance from the Ellman’s reagent. NSB gives the amount of tracer that might
bind to the well. B0 gives the maximum amount of tracer that can bind to the antibody without any free
Estradiol present. The total amount of solution in each well should add up to 150μL.
The data received from the microplate reader is sent to Microsoft excel where it can be
analyzed. The data is analyzed (Table 2, Table 3.) following the instructions in the analysis section of the
instruction manual (2). A standard curve is plotted (Figure 1.) using ratio of absorbance of the sample to
the absorbance of maximum binding (B0) and the Estradiol concentrations in each milk sample can be
determined.
Results and Discussion:
The Cayman EIA is sensitive to interference from molecules such as organic solvents, which were
previously used in the other ELISA. To test for interferences, we performed different sample
preparations (listed in table 4) used in other studies (5) and analyzed them by ELISA. However the
results of the ELISA did not show until days after the test was run. We think the data was not run
accurately due to the fact that the test was not stored at the -20 degrees Celsius that it was supposed to
be at but instead at 4 degrees Celsius. Even though the samples cannot be read, many of the samples
turned yellow which is promising that the kit may work for samples without preparation. The second
time we ran the Cayman EIA kit, we used milk, diluted milk, centrifuged supernatant of milk, and
centrifuged supernatant of diluted milk as the samples. All of the samples fell on the standard curve
(Table 3. And Figure 1.). More milk samples can now be analyzed in the future without using sample
preparation.
Table 1. Amount of each solution for each well
Well
Blank
NSB
B0
Standard/sample
EIA Buffer
100μL
50μL
-
Standard/ sample
50μL
Tracer (AChE)
50μL
50μL
50μL
Antibody
50μL
50μL
Table 2. Data Analysis for Estradiol standards
Standards concentration
1
4000
2
1600
3
640
4
256
5
102.4
6
41
7
16.4
8
6.6
0.25
0.2835
0.3355
0.405
0.5075
0.595
0.6615
0.7425
standard NSB
0.0235
0.057
0.109
0.1785
0.281
0.3685
0.435
0.516
B/Bo
0.052948
0.128427
0.245588
0.402178
0.633121
0.830267
0.980098
1.162599
lineated data
log
(concentration)
3.60206
3.20412
2.80618
2.40824
2.0103
1.612784
1.214844
0.819544
logit
(B/Bo)
Interpolation
from the
Standard Curve
logit
(b/Bo
-1.25253
-0.83165
-0.4874
-0.17215
0.236963
0.68945
1.692365
#NUM!
Table 3. Data Analysis for milk samples
Raw
Sample
Absorbance Absorbance
samples Identification Reading
– NSB
1 straight milk
0.653
0.4265
diluted 50%
2 milk
0.7095
supernatant
3 milk
0.6435
0.6435
diluted 50%
supernatant
4 milk
0.6995
0.2556
B/B0
Final
10^
Concentration
interpolation in pg/mL
0.96094
1.3910
1.1608
14.480
14.480
0.03917 0.088246
-1.0141
3.2835
1920.7
3841.3
1.4498
#NUM!
0.57604 0.13313
#NUM!
2.2709
#NUM!
186.60
too high
373.20
Figure 1. Standard curve
2
1.5
Logit (B/Bo)
1
0.5
0
-0.5
1
1.25
-1
-1.5
-2
1.5
1.75
2
2.25
2.5
2.75
3
3.25
3.5
y = -1.1311x + 2.7063
R² = 0.9578
Log (Concentration)
Table 4. Different Sample Preparations to test for interferences.(5)
Straight milk
50% diluted milk
Precipitaed milk
(treated with acetic
acid)
50% diluted
precipitated milk
Spiked milk
Spiked Supernatant
Spike EIA buffer
solution
50 uL
25 uL milk and 25 uL EIA buffer
50uL of supernatant
25 uL supernatant and 25 uL EIA buffer
uL straight milk and 25 uL of 500 pg/mL estradiol
25 uL milk supernatant and 25 uL of 500 pg/mL estradiol
25 uL EIA buffer and 25 uL 500 pg/mL estradiol)
References:
1. Enzyme (E1/E2/E3) EIA Prod. No. L22000403. Product Description: Biosense Laboratories AS:
Bergen, Norway
2.
Estradiol EIA kit, Item NO. 582251. Cayman Chemical Company, Ann Arbor, MI. 2011
3.
Guenther, K., V. Heinke, B. Thiele, E. Kleist, H. Prast, and T. Raecker. Endocrine
Disrupting Nonylphenols Are Ubiquitous in Food. Environmental Science & Technology
2002. 36. 1676-1680.
4.
Malekinejad, H. P. Scherpenisse and A. Bergwerff. Naturally Occurring Estrogens in Processed
Milk and in Raw Milk (from Gestated Cows). Journal of Agriculture. Food Chemistry. 2006,
54. 9785–9791
5. Maxey, K.M., Maddipati, K.R. andBirkmeier, J. Interference in enzyme immunoassays. Journal of
Clinical Immunoassay. 1992.15. 116-120.
6. Le Breton, Marie-He’le’ne, Rohereau-Roulet, Sandrine, Sylvain Che’reau, Gaud Pinel, Thierry
Delatour, and Bruno Le Bizec. Identifiaction of Cows Treated with Recombinant Bovine
Somatotropin. Journal of Agricultural and Food Chemistry. 2010. 58.2. 729-733.
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Chemistry. 2009, 57.18. 8093-8097.
8. Discover Reversed-Phase SPE products. Thermo Scientific; Bellefonte, PA. sigmaaldrich.com/supelco.