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13,14-Dihydro-15-Keto-Prostaglandin F2-alpha (PGFM) ELISA Kit

Reactivity: Various Species Colorimetric Sandwich ELISA Fecal, Plasma, Serum, Tissue Culture Medium, Urine
Catalog No. ABIN577676
  • Target
    13,14-Dihydro-15-Keto-Prostaglandin F2-alpha (PGFM)
    Reactivity
    • 2
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    Various Species
    Detection Method
    Colorimetric
    Method Type
    Sandwich ELISA
    Minimum Detection Limit
    46.2 pg/mL
    Application
    ELISA
    Purpose
    The DetectX® 13,14-dihydro-15-keto-PGF2a (PGFM) Immunoassay kit is a patent pending assayfrom Dr. Martin Dehnhard at Leibniz Institute for Zoo & Wildlife Research, Berlin, Germany licensedexclusively and designed to quantitatively measure PGFM present in fecal extracts, urine, serumand plasma samples.
    Brand
    DetectX®
    Sample Type
    Fecal, Urine, Serum, Plasma, Tissue Culture Medium
    Analytical Method
    Quantitative
    Specificity
    Species Independent. Samples Types validated: Fecal Extracts, Urine, Serum, Plasma and Tissue Culture Media
    Cross-Reactivity (Details)
    No Cross Reactivity to Other Related PGF Metabolites
    Sensitivity
    20.8 pg/mL
    Characteristics
    The 13,14,Dihydro-15-keto-Prostaglandin F2alpha (PGFM) Enzyme Immunoassay kit measures PGFM present in non-invasively collected fecal and urine samples. The assay will also measure PGFM in plasma, serum and TCM samples. The kit has been developed for urine and extracted fecal samples and has been validated with Iberian lynx and other felid samples. After an hour incubation the plate is washed and substrate is added. The substrate reacts with the bound PGFM-peroxidase conjugate and the reaction is read at 450nm wavelength. PGFM has been suggested as a useful non-invasive pregnancy marker both in urine and fecal samples. It has been shown to be a precise, practical method for this application in these matrices for a number of species. Parallel courses were obtained when comparing urinary and fecal PGFM in a variety of felids and other species, and only a simple dilution of fecal extracts is necessary prior to analyses. Fecal PGFM analyses may allow pregnancy diagnosis in captive and free-ranging felids. Recent evidence has suggested that PGFM may also be a useful pregnancy marker in some other non-felid species.
    Components
    Coated Clear 96 Well Plates A clear plastic microtiter plate(s) with break-apart strips, coated with goat anti-rabbit IgG. 1 Or 5 Each
    PGFM Standard 13,14-dihydro-15-keto-PGF2a at 32,000 pg/mL in a special stabilizing solution. 125 Or 625 μL
    DetectX® PGFM Antibody A rabbit polyclonal antibody specific for PGFM. 3 mL Or 13 mL
    DetectX® PGFM Conjugate A PGFM-peroxidase conjugate in a special stabilizing solution. 3 mL Or 13 mL
    Assay Buffer Concentrate A 5X concentrate that must be diluted with deionized or distilled water. 28 mL Or 55 mL
    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 Notes
    This assay has been validated for dried fecal extracts, urine, serum, and plasma samples.
    Samples containing visible particulate should be centrifuged prior to using.
    Severely hemolyzed samples should not be used in this kit.
    All samples containing lipids may interfere with the mea- surement of PGFM.
    Samples containing high lipid content may be extracted as described below.
    A useful online resource for the extraction of bioactive lipids can be found at: http://lipidlibrary.aocs.org/topics/spe_alm/index.htm#ext .
    PGFM is identical across all species and we expect this kit may measure PGFM from sources other than those tested.
    The end user should evaluate recoveries of PGFM in other samples being tested.
    Assay Time
    1.5 h
    Plate
    Pre-coated
    Protocol
    A PGFM 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 IgG.
    A PGFM-peroxidase conjugate is added to the standards and samples in the wells.
    The binding reaction is initiated by the addition of a rabbit polyclonal antibody highly specific to PGFM to each well.
    After an hour incubation, the plate is washed and substrate is added.
    The substrate reacts with the bound PGFM-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 450 nm wavelength.
    The concentration of the PGFM in the sample is calculated, after making suitable 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 30-60 minutes.
    We recommend that all standards and samples be run in duplicate to allow the end user to accurately determine PGFM concentrations.
    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 seven test tubes as #1 through #7.
    Pipet 450 μL of Assay Buffer into tube #1 and 200 μL into tubes #2 to #7.
    The PGFM stock solution contains an organic solvent.
    Prerinse the pipet tip several times to ensure accurate delivery.
    Carefully add 50 μL of the PGFM stock solution to tube #1 and vortex completely.
    Take 200 μL of the PGFM solution in tube #1 and add it to tube #2 and vortex completely.
    Repeat the serial dilutions for tubes #3 through #7.
    The concentration of PGFM in tubes 1 through 7 will be 3,200, 1,600, 800, 400, 200, 100, and 50 pg/mL.
    Use all Standards within 2 hours of preparation.

    Sample Preparation

    Extracted Samples We have a detailed Extraction Protocol available on our website at: http://www.ArborAssays.com/resources/lit.asp. The ethanol concentration in the final Assay Buf- fer dilution added to the well should be <5 % . Urine Samples Urine samples should be diluted ≥ 1:8 with the supplied Assay Buffer prior running in the assay. Serum and

    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 (B0 or 0 pg/mL).
      5. Add 25 μL of the DetectX® PGFM Conjugate to each well using a repeater pipet.
      6. Add 25 μL of the DetectX® PGFM 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 1 hour. If the plate is not shaken signals bound will be approximately 50 % 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 PGFM 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 MyAssays™ online tool from http://www.myassays.com/arbor-assays-pgfm-enzyme-immunoassay-kit.assay to calculate the data.
    QR code for Data Analysis: *MyAssays logo is a registered trademark of MyAssays Ltd. typical data Sample Mean OD Net OD % B/B0 PGFM Conc. (pg/mL) NSB 0.044 0 - - Standard 1 0.148 0.104 19.8 3,200 Standard 2 0.208 0.164 31.3 1,600 Standard 3 0.248 0.204 38.9 800 Standard 4 0.331 0.287 54.8 400 Standard 5 0.395 0.351 67.0 200 Standard 6 0.482 0.438 83.6 100 Standard 6 0.526 0.482 92.0 50 B0 0.568 0.524 100.0 0 Sample 1 0.202 0.158 30.2 1,493 Sample 2 0.315 0.271 51.6 450.1 Always run your own standard curve for calculation of results.
    Do not use this data.

    Restrictions
    For Research Use only
  • 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.
  • Roberts, Brown, Kersey, Snyder, Durrant, Kouba: "Use of urinary 13,14, dihydro-15-keto-prostaglandin F2α (PGFM) concentrations to diagnose pregnancy and predict parturition in the giant panda (Ailuropoda melanolecua)." in: PLoS ONE, Vol. 13, Issue 5, pp. e0195599, (2018) (PubMed).

    Fernández-Alvarez, Llorente-Izquierdo, Mayoral, Agra, Boscá, Casado, Martín-Sanz: "Evaluation of epigenetic modulation of cyclooxygenase-2 as a prognostic marker for hepatocellular carcinoma." in: Oncogenesis, Vol. 1, pp. e23, (2013) (PubMed).

  • Target
    13,14-Dihydro-15-Keto-Prostaglandin F2-alpha (PGFM)
    Alternative Name
    PGFM
    Background
    In many species, uterine and placental Prostaglandin F2alpha (PGF2a) is involved in the regulation of reproductive and pregnancy-related processes such as embryonic development, initiation of parturition, and resumption of ovarian activity1. In domestic ruminants, uterine tissue is a primary source of PGF2a, and secretion of uterine PGF2a is a key regulator for the cyclical regression of PGFM the corpus luteum2-4. Prostaglandin F2a is metabolized to PGFM (13,14- dihydro-15-keto-PGF ) during the first passage through the lungs52a . PGFM has a longer half-life in peripheral circulation than PGF and has been applied as a useful analytical marker of PGF 62a 2a . PGFM has been suggested as a useful non-invasive marker of pregnancy when measured in both urine and fecal samples7. It has been shown to be a precise, practical method for this application in these matrices. Parallel courses were obtained when comparing urinary and fecal PGFM in a variety of felids and other species, and only a simple dilution of fecal extracts is necessary prior to analyses. Fecal PGFM analyses may allow pregnancy diagnosis in captive and free-ranging felids. Recent evidence has suggested that PGFM may also be a useful pregnancy marker in some other non-felid species8
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