Estrone-3-Sulfate (E1S) ELISA Kit

Catalog No. ABIN2815102

Product Details

Target: Estrone-3-Sulfate (E1S)

Reactivity: Various Species

Detection Method: Colorimetric

Method Type: Sandwich ELISA

Application: ELISA

Purpose: The DetectX® Estrone-3-Sulfate (E1S) Immunoassay kit uses a specifically generated antibody to measure E1S in a variety of matrices, including serum, plasma, urine and fecal samples.

Brand: DetectX®

Sample Type: Fecal, Urine, Serum, Plasma, Tissue Culture Medium

Analytical Method: Quantitative

Cross-Reactivity (Details): The following cross reactants were tested in the assay and calculated at the 50 % binding point. Steroid Cross Reactivity: Estrone-3-sulfate 100 %, Estrone 267 %, Estrone-3-glucuronide 200 %, 17-Estradiol 11.7 %, Estradiol-3-Glucuronide 5.7 %, Estradiol-3-Sulfate 5.0 %, Estradiol-17-Sulfate 0.2 %, Progesterone < 0.2 %, Estriol < 0.2 %, Cortisol < 0.2 %, Testosterone < 0.2 %, Pregnanediol < 0.2 %

Components: Coated Clear 96 Well Plates Clear 1 by 8 break-apart strip well plastic microtiter plate(s) coated with goat anti-rabbit IgG. 1 Or 5 Each
Estrone-3-Sulfate (E1S) Standard Estrone-3-Sulfate (E1S) at 40,000 pg/mL in a special stabilizing solution. 125 μL Or 625 μL
DetectX® Estrone-3-Sulfate (E1S) Antibody A rabbit polyclonal antibody specific for Estrone-3-Sulfate. 3 mL Or 13 mL Or -13ML DetectX® Estrone-3-Sulfate (E1S) Conjugate An Estrone-3-Sulfate-peroxidase conjugate in a special stabilizing solution. 3 mL Or 13 mL Or -13ML Assay Buffer Concentrate A 5X concentrate that should be diluted with deionized or distilled water. 28 mL Or 55 mL
Dissociation Reagent 1 mL Or 5 mL
Dissociation Reagent is to be used only with Serum and Plasma samples.
Wash Buffer Concentrate A 20X concentrate that should be diluted with deionized or distilled water. 30 mL Or 125 mL
TMB Substrate 11 mL Or 55 mL
Stop Solution A 1M solution of hydrochloric acid. CAUSTIC. 5 mL Or 25 mL
Plate Sealer 1 Or 5 Each

Material not included: Distilled or deionized water.
Repeater pipet with disposable tips capable of dispensing 25 μL, 50 μL and 100 μL.
A microplate shaker.
Colorimetric 96 well microplate reader capable of reading optical density at 450 nm.
Software for converting raw relative optical density readings from the plate reader and carrying out four parameter logistic curve (4PLC) fitting.
Contact your plate reader manufacturer for de- tails.

Application Details

Application Notes: This assay has been validated for serum, plasma, fecal, urine and tissue culture samples.
Samples containing visible particulate should be centrifuged prior to using.
Estrone-3-sulfate can be as- sayed in solid sample types by using one of the extraction protocols lit.asp.
Estrone-3-sulfate (E1S) is identical across all species and we expect this kit to measure estrone-3- sulfate from all sources.
The end user should evaluate recoveries of E1S in other sample matrices being tested.

Plate: Pre-coated

Protocol: The kit will quantitatively measure E1S present in diluted buffer samples and tissue culture media samples.
Please read the complete kit insert before performing this assay.
An E1S standard is provided to generate a standard curve for the assay and all samples should be read off the standard curve.
Standards or diluted samples are pipetted into a clear microtiter plate coated with an antibody to capture rabbit antibodies.
An E1S-peroxidase conjugate is added to the standards and samples in the wells.
The binding reaction is initiated by the addition of a polyclonal antibody to E1S to each well.
After a 2 hour incubation the plate is washed and substrate is added.
The substrate reacts with the bound E1S-peroxidase conjugate.
After a short incubation, the reaction is stopped and the intensity of the generated color is detected in a microtiter plate reader capable of measuring 450nm wavelength.
The concentration of the E1S in the sample is calculated, after making suit- able correction for the dilution of the sample, using software available with most plate readers.

Reagent Preparation:

Allow the kit reagents to come to room temperature for 30 minutes.
We recommend that all standards and samples be run in duplicate to allow the end user to accurately determine E1S con- centrations.
Ensure that all samples have reached room temperature and have been diluted as appropriate prior to running them in the kit.
Assay Buffer Dilute Assay Buffer Concentrate 1:5 by adding one part of the concentrate to four parts of deion- ized water.
Once diluted this is stable at 4 °C for 3 months.
Wash Buffer Dilute Wash Buffer Concentrate 1:20 by adding one part of the concentrate to nineteen parts of deionized water.
Once diluted this is stable at room temperature for 3 months.
Standard Preparation Label six test tubes as #1 through #6.
Pipet 450 μL of Assay Buffer into tube #1 and 150 μL into tubes #2 to #6.
The Estrone-3-Sulfate stock solution contains an organic solvent.
Prerinse the pipet tip several times to ensure accurate delivery.
Carefully add 50 μL of the estrone-3-sulfate stock solution to tube #1 and vortex completely.
Take 100 μL of the estrone-3-sulfate solution in tube #1 and add it to tube #2 and vortex completely.
Repeat the serial dilutions for tubes #3 through #6.
The concentration of estrone-3-sulfate in tubes 1 through 6 will be 4,000, 1,600, 640, 256, 102.4, and 40.96 pg/mL.
Use all Standards within 2 hours of preparation.

Sample Preparation:

Serum and Plasma Samples Serum and plasma samples should be diluted with an equal volume of the supplied Dissociation Reagent. The diluted samples should then be further diluted ≥ 1:50 with the supplied Assay Buffer prior running in the assay. NOTE: Dissociation Reagent is to be used only with Serum and Plasma samples. Dried Fecal Samples: The ethanol concentration in the final Assay Buf- fer dilution added to the well should be <1 % . Urine Samples Urine samples should be diluted at least 1:8 times with the provided Assay Buffer. For comparison to creatinine as a urine volume marker please see our NIST-calibrated 2 plate and 10 plate Urinary Creatinine Detection kits, K002-H1 and K002-H5.

Assay Procedure:

1. Use the plate layout sheet on the back page to aid in proper sample and standard identification. Determine the number of wells to be used and return unused wells to the foil pouch with desiccant. Seal the ziploc plate bag and store at 4ºC.
   2. Pipet 50 μL of samples or standards into wells in the plate.
   3. Pipet 75 μL of Assay Buffer into the non-specific binding (NSB) wells.
   4. Pipet 50 μL of Assay Buffer into wells to act as maximum binding wells (Bo or 0 pg/mL).
   5. Add 25 μL of the DetectX® Estrone-3-Sulfate Conjugate to each well using a repeater pipet.
   6. Add 25 μL of the DetectX® Estrone-3-Sulfate Antibody to each well, except the NSB wells, using a repeater pipet.
   7. Gently tap the sides of the plate to ensure adequate mixing of the reagents. Cover the plate with the plate sealer and shake at room temperature for 2 hours. If the plate is not shaken signals bound will be approximately 35 % lower.
   8. Aspirate the plate and wash each well 4 times with 300 μL wash buffer. Tap the plate dry on clean absorbent towels.
   9. Add 100 μL of the TMB Substrate to each well, using a repeater pipet. 10. Incubate the plate at room temperature for 30 minutes without shaking. 11. Add 50 μL of the Stop Solution to each well, using a repeater pipet. 12. Read the optical density generated from each well in a plate reader capable of reading at 450 nm. 13. Use the plate reader's built-in 4PLC software capabilities to calculate estrone-3- sulfate (E1S) concentration for each sample.

Calculation of Results:

Average the duplicate OD readings for each standard and sample.
Create a standard curve by reducing the data using the 4PLC fitting routine on the plate reader, after subtracting the mean OD's for the NSB.
The sample concentrations obtained, calculated from the %B/B0 curve, should be multiplied by the dilution factor to obtain neat sample values.
Or use the online tool from http://www.myassays.com/arbor-assays-estrone-3-sulfate-(e1s)-eia- kit.assay to calculate the data. *The MyAssays logo is a registered trademark of MyAssays Ltd. typical data Sample Mean OD Net OD % B/B0 E1S Conc. (pg/mL) NSB 0.047 0.000 - - Standard 1 0.165 0.118 13.1 4,000 Standard 2 0.249 0.202 22.4 1,600 Standard 3 0.394 0.347 38.5 640 Standard 4 0.577 0.530 58.8 256 Standard 5 0.775 0.728 80.8 102.4 Standard 6 0.888 0.841 93.3 40.96 B0 0.948 0.901 100.0 0 Sample 1 0.475 0.428 47.5 423.9 Sample 2 0.678 0.631 70.0 165.5 Always run your own standard curve for calculation of results.
Do not use this data.
Conversion Factor: 100 pg/mL of E1S is equivalent to 268.5 pM.

Assay Precision: Three serum samples treated with Dissociation Reagent and diluted with Assay Buffer were run in replicates of 20 in an assay.
Inter Assay Precision:
Three serum samples treated with Dissociation Reagent and diluted with Assay Buffer were run in duplicates in fourteen assays run over multiple days by three operators.

Restrictions: For Research Use only

Handling

Preservative: Sodium azide

Precaution of Use: As with all such products, this kit should only be used by qualified personnel who have had labo- ratory safety instruction.
The complete insert should be read and understood before attempting to use the product.
The antibody coated plate needs to be stored desiccated.
The silica gel pack included in the foil ziploc bag will keep the plate dry.
The silica gel pack will turn from blue to pink if the ziploc has not been closed properly.
This kit utilizes a peroxidase-based readout system.
Buffers, including other manufacturers' Wash Buffers, containing sodium azide will inhibit color production from the enzyme.
Make sure all buffers used for samples are azide free.
Ensure that any plate washing system is rinsed well with deionized water prior to using the supplied Wash Buffer as prepared on Page 8.
The Stop Solution is acid.
The solution should not come in contact with skin or eyes.
Take appro- priate precautions when handling this reagent.

Storage: 4 °C,RT

Storage Comment: All components of this kit should be stored at 4°C until the expiration date of the kit.

References

Goudet, Nadal-Desbarats, Douet, Savoie, Staub, Venturi, Ferchaud, Boulot, Prunier: "Salivary and urinary metabolome analysis for pre-puberty-related biomarkers identification in porcine." in: Animal : an international journal of animal bioscience, pp. 1-11, (2018) (PubMed).

Higuchi, Endo, Hanamura, Gohno, Niwa, Yamaguchi, Horiguchi, Hayashi: "Contribution of Estrone Sulfate to Cell Proliferation in Aromatase Inhibitor (AI) -Resistant, Hormone Receptor-Positive Breast Cancer." in: PLoS ONE, Vol. 11, Issue 5, pp. e0155844, (2016) (PubMed).

Target Details

Target: Estrone-3-Sulfate (E1S)

Background: Estrone-3-sulfate, C18H22O5S, (1, 3, 5(10)-Estratrien-3-ol-17-one sulfate, E1S) is synthesized in the fetal or cotyledonary portion of the placentome1. Production rates of E1S are high in both males and females, with males producing 77 μg/day, and in women in early follicular phase, 95 μg/day and in early luteal phase, 182 μg/day2. Estrone sulfate, present in plasma in a higher concentration than either unconjugated estrone or estradiol in nonpregnant women and normal men, appears to originate almost entirely from a conjugation of estrone and converted estradiolin nonglandular tissues3. Estrone sulfate is only slowly cleared from plasma, thus its concentration does not fluctuate markedly during the day4,5. Estrone sulfate is quantitatively the most important circulating estrogen. In postmenopausal women with breast cancer, estrone sulfate concentrations in plasma have the same order of mag- nitude. Breast tumors contain sulfatase activity6 and can convert estrone sulfate into estradiol7. Consequently, estrone sulfate provides a continuous supply of estrogens to hormone-responsive tumors. Estrone-3-Sulfate, E1S O HO S O OH Cryptorchidism is a condition in which one or both testicles fail to descend into the scrotum, and it is considered to be a prevalent defect in horses8,9. Bilaterally cryptorchid stallions do not pro- duce viable spermatozoa but often exhibit normal secondary sexual characteristics such as libido, because of testosterone production by the interstitial cells of the retained testes. Bilateral cryp- torchids, must be differentiated from geldings who exhibit stallion like behavior. Thus, the correct laboratory diagnosis of this condition is very important, especially when exploratory abdominal surgery is considered for the removal of retained testes. Several investigators have suggested measuring testosterone and estrone sulfate serum levels as reliable diagnostic aids for the condi- tion8,9