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 reagentproducing 5-thio-2-nitrobenzoic acid (NTB2-).NTB2- is a yellow compound that can quantified spectrophotometrically. The intensity of the yellowcolor 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 decreaseand 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. Thecentrifuge used was a Thermo Scientific CentiSorvall 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 BuchiRotovapor R-210rotary vaporizer and then reconstituted 1:1 using 10% methanol. Thesample 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 3.0g/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 platein 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, whichwere 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 / EIA Buffer / Standard/ sample / Tracer (AChE) / AntibodyBlank / - / - / - / -
NSB / 100μL / - / 50μL / -
B0 / 50μL / - / 50μL / 50μL
Standard/sample / - / 50μL / 50μL / 50μL
Table 2. Data Analysis for Estradiol standards
lineated dataStandards / concentration / standard - NSB / B/Bo / log (concentration) / logit (b/Bo
1 / 4000 / 0.25 / 0.0235 / 0.052948 / 3.60206 / -1.25253
2 / 1600 / 0.2835 / 0.057 / 0.128427 / 3.20412 / -0.83165
3 / 640 / 0.3355 / 0.109 / 0.245588 / 2.80618 / -0.4874
4 / 256 / 0.405 / 0.1785 / 0.402178 / 2.40824 / -0.17215
5 / 102.4 / 0.5075 / 0.281 / 0.633121 / 2.0103 / 0.236963
6 / 41 / 0.595 / 0.3685 / 0.830267 / 1.612784 / 0.68945
7 / 16.4 / 0.6615 / 0.435 / 0.980098 / 1.214844 / 1.692365
8 / 6.6 / 0.7425 / 0.516 / 1.162599 / 0.819544 / #NUM!
Table 3. Data Analysis for milk samples
samples / Sample Identification / Raw Absorbance Reading / Absorbance – NSB / B/B0 / logit (B/Bo) / Interpolation from the Standard Curve / 10^interpolation / Final Concentration in pg/mL1 / straight milk / 0.653 / 0.4265 / 0.96094 / 1.3910 / 1.1608 / 14.480 / 14.480
2 / diluted 50% milk / 0.7095 / 0.03917 / 0.088246 / -1.0141 / 3.2835 / 1920.7 / 3841.3
3 / supernatant milk / 0.6435 / 0.6435 / 1.4498 / #NUM! / #NUM! / #NUM! / too high
4 / diluted 50% supernatantmilk / 0.6995 / 0.2556 / 0.57604 / 0.13313 / 2.2709 / 186.60 / 373.20
Figure 1. Standard curve
Table 4. Different Sample Preparations to test for interferences.(5)
solutionStraight milk / 50 uL
50% diluted milk / 25 uL milk and 25 uL EIA buffer
Precipitaed milk (treated with acetic acid) / 50uL of supernatant
50% diluted precipitated milk / 25 uL supernatant and 25 uL EIA buffer
Spiked milk / uL straight milk and 25 uL of 500 pg/mL estradiol
Spiked Supernatant / 25 uL milk supernatant and 25 uL of 500 pg/mL estradiol
Spike EIA buffer / 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.
7.Tunick, Micheal. Dairy Innovations over the Past 100 Years.Journal of Agricultural and Food Chemistry.2009, 57.18. 8093-8097.
8.Discover Reversed-Phase SPE products. Thermo Scientific; Bellefonte, PA. sigma-aldrich.com/supelco.