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Estrone-3-Glucuronide ELISA Kit

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Estrone-3-Glucuronide ELISA Kit Item No. 501290 www.caymanchem.com Customer Service 800.364.9897 Technical Support 888.526.5351 1180 E. Ellsworth Rd · Ann Arbor, MI · USA TABLE OF CONTENTS GENERAL INFORMATION GENERAL INFORMATION 3 Materials Supplied Materials Supplied 4 Safety Data 4 Precautions 5 If You Have Problems Item Number Item 96 wells Quantity/Size 5 Storage and Stability 5 Materials Needed but Not Supplied 501291 Estrone-3-Glucuronide ELISA Antiserum 1 vial/100 dtn INTRODUCTION 6 Background 501292 Estrone-3-Glucuronide AP Tracer 1 vial/100 dtn 6 About This Assay 501293 Estrone-3-Glucuronide ELISA Standard 1 vial 7 Principle of Assay 8 Definition of Key Terms 400080 Tris Buffer Concentrate (10X) 1 vial/10 ml PRE-ASSAY PREPARATION 10 Buffer Preparation 411007 AP Wash Buffer Concentrate (150X) 1 vial/5 ml 11 Sample Preparation 400004/400006 Mouse Anti-Rabbit IgG Coated Plate 1 plate ASSAY PROTOCOL 16 Preparation of Assay-Specific Reagents 400089 pNPP Substrate Solution 2 vials/12 ml 18 Plate Set Up 19 Performing the Assay 400040 ELISA Tracer Dye 1 ea ANALYSIS 21 Calculations 400042 ELISA Antiserum Dye 1 ea 22 Performance Characteristics 400012 96-Well Cover Sheet 1 cover 30 Assay Summary 31 Plate Template RESOURCES 32 Troubleshooting If any of the items listed above are damaged or missing, please contact our Customer Service department at (800) 364-9897 or (734) 971-3335. We cannot 33 References accept any returns without prior authorization. 34 Notes GENERAL INFORMATION 3 If You Have Problems WARNING: THIS PRODUCT IS FOR RESEARCH ONLY - NOT FOR HUMAN OR VETERINARY DIAGNOSTIC OR THERAPEUTIC USE. Technical Service Contact Information ! Phone: 888-526-5351 (USA and Canada only) or 734-975-3888 Safety Data Fax: 734-971-3640 Email: [email protected] This material should be considered hazardous until further information becomes In order for our staff to assist you quickly and efficiently, please be ready to supply available. Do not ingest, inhale, get in eyes, on skin, or on clothing. Wash the lot number of the kit (found on the outside of the box). thoroughly after handling. Before use, the user must review the complete Safety Data Sheet, which has been sent via email to your institution. Storage and Stability Precautions This kit will perform as specified if stored as directed at -20°C and used before the expiration date indicated on the outside of the box. Please read these instructions carefully before beginning this assay. This kit may not perform as described if any reagent or procedure is replaced or Materials Needed But Not Supplied modified. When compared to quantification by LC/MS or GC/MS, it is not uncommon 1. A plate reader capable of measuring absorbance at 405 nm. for immunoassays to report higher analyte concentrations. While LC/MS or 2. Adjustable pipettes and a repeating pipettor. GC/MS analyses typically measure only a single compound, antibodies used 3. A source of pure water; glass distilled water or deionized water is acceptable. in immunoassays sometimes recognize not only the target molecule, but also NOTE: Ultra-Pure water is available for purchase from Cayman (Item No. structurally related molecules, including biologically relevant metabolites. In 400000). many cases, measurement of both the parent molecule and metabolites is more representative of the overall biological response than is the measurement of a 4. Materials used for Sample Preparation (see page 11). short-lived parent molecule. It is the responsibility of the researcher to understand the limits of both assay systems and to interpret their data accordingly. 4 GENERAL INFORMATION GENERAL INFORMATION 5 INTRODUCTION Principle of the Assay This assay is based on the competition between estrone-3-glucuronide and Background an estrone-3-glucuronide-alkaline phosphatase (AP) conjugate (Estrone-3- Glucuronide AP Tracer) for a limited amount of Estrone-3-Glucuronide ELISA Estrone-3-glucuronide is the predominant metabolite of estradiol in urine and Antiserum. Because the concentration of the Estrone-3-Glucuronide AP Tracer a urinary marker for the fertile window in women.1 Measurement of urinary is held constant while the concentration of estrone-3-glucuronide standards and glucuronides is a convenient, non-invasive method for detecting reproductive samples vary, the amount of Estrone-3-Glucuronide AP Tracer that is able to bind hormone levels compared to measuring plasma levels.2 Estrone-3-glucuronide, to the Estrone-3-Glucuronide ELISA Antiserum will be inversely proportional to along with pregnanediol-3-glucuronide, are indicators of female reproductive the concentration of estrone-3-glucuronide in the well. This antibody-estrone-3- health.2 glucuronide complex binds to a mouse anti-rabbit IgG that has been previously Literature has shown high plasma estrone levels, which directly correlate to attached to the well. The plate is washed to remove any unbound reagents and urinary estrone-3-glucuronide levels, are associated with estrogen receptor- then pNPP Substrate Solution is added to the well. The product of this enzymatic 3 positive breast cancers. Hormone receptor status is the main factor in planning reaction has a distinct yellow color and absorbs strongly at 405 nm. The intensity treatment and can be treated with hormone therapies, including tamoxifen and of this color, determined spectrophotometrically, is proportional to the amount aromatase inhibitors. In addition to its role in breast cancer, circulating estrogens, of Estrone-3-Glucuronide AP Tracer bound to the well, which is inversely including estrone metabolized from oral hormone replacement therapy, have proportional to the amount of free estrone-3-glucuronide present in the well been shown to increase thrombin generation leading to higher risk for blood during the incubation; or clots, myocardial infarctions, deep vein thrombosis and strokes.4-6 Absorbance ∝ [Bound Estrone-3-Glucuronide AP Tracer] ∝ 1/[Estrone-3-Glucuronide] About This Assay A schematic of this process is shown in Figure 1, on page 8 Cayman’s Estrone-3-Glucuronide ELISA Kit is a competitive assay that can be used for quantification of estrone metabolites in urine, plasma and serum. This assay has a range from 2.7-6,000 pg/ml with a midpoint of approximately 125.6 pg/ml (50% B/B0) and a sensitivity of approximately 23.2 pg/ml (80% B/B0). 6 INTRODUCTION INTRODUCTION 7 Standard Curve: a plot of the %B/B0 values versus concentration of a series of wells containing various known amounts of analyte. Dtn: determination, where one dtn is the amount of reagent used per well. Cross Reactivity: numerical representation of the relative reactivity of this assay Wells are pre-coated with 1. Incubate with tracer, = Mouse Anǎ-Rabbit IgG mouse anǎ-rabbit IgG and anǎbody, and either towards structurally related molecules as compared to the primary analyte of interest. blocked with a proprietary standard or sample. = Blocking proteins formulaǎon of proteins. Biomolecules that possess similar epitopes to the analyte can compete with the = Alkaline Phosphatase linked to Estrone-3-Glucuronide (tracer) assay tracer for binding to the primary antibody. Substances that are superior to the = Specific anǎbody to Estrone-3-Glucuronide analyte in displacing the tracer result in a cross reactivity that is greater than 100%. = Free Estrone-3-Glucuronide Substances that are inferior to the primary analyte in displacing the tracer result in a cross reactivity that is less than 100%. Cross reactivity is calculated by comparing the mid-point (50% B/B ) value of the tested molecule to the mid-point (50% B/B ) 2. Wash to remove all 3. Develop the well with 0 0 unbound reagents. pNPP soluǎon. value of the primary analyte when each is measured in assay buffer using the following formula: 50% B/B value for the primary analyte Figure 1. Schematic of the ELISA % Cross Reactivity = 0 x 100% [ 50% B/B0 value for the potential cross reactant ] Definition of Key Terms Lower Limit of Detection (LLOD): the smallest measure that can be detected with Blank: background absorbance caused by pNPP Substrate Solution. The blank reasonable certainty for a given analytical procedure. The LLOD is defined as a point, absorbance should be subtracted from the absorbance readings of all the other wells. two standard deviations away from the mean zero value. Total Activity (TA): total enzymatic activity of the alkaline phosphatase-linked tracer. NSB (Non-Specific Binding): non-immunological binding of the tracer to the well. Even in the absence of specific antibody a very small amount of tracer still binds to the well; the NSB is a measure of this low binding. B0 (Maximum Binding): maximum amount of the tracer that the antibody can bind in the absence of free analyte. %B/B0 (%Bound/Maximum Bound): ratio of the absorbance of a particular sample or standard well to that of the maximum binding (B0) well. 8 INTRODUCTION INTRODUCTION 9 PRE-ASSAY PREPARATION Sample Preparation This assay has been demonstrated to work with urine, human plasma and human Buffer Preparation serum without extraction. Proper sample storage and preparation are essential for consistent and accurate results. Please read this section thoroughly before Store all diluted buffers at 4°C; they will be stable for about two months. beginning the assay. 1. Tris Buffer Preparation General Precautions Dilute the contents of one vial of Tris Buffer Concentrate (10X) (Item No. • All samples must be free of organic solvents prior to assay. 400080) with 90 ml of Pure Water. Be certain to rinse the vial to remove • Samples should be assayed immediately after collection; samples that any salts that may have precipitated. NOTE: It is normal for the concentrated cannot be assayed immediately should be stored at -80°C. buffer to contain crystalline salts after thawing. These will completely dissolve • Samples of rabbit origin may contain antibodies which interfere with the upon dilution with water.
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