C-Telopeptide of Type I Collagen ELISA Kit

Details zu Produkt Nr. ABIN2644529, Anbieter: Anmelden zum Anzeigen
Antigen
Reaktivität
Human
Alternativen
Kits mit alternativen Reaktivitäten:
8
8
7
2
2
1
1
Methodentyp
Sandwich ELISA
Detektionsbereich
25-800 ng/mL
Untere Nachweisgrenze
25 ng/mL
Applikation
ELISA
Optionen
Hersteller
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Hersteller Produkt- Nr.
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'Independent Validation' Siegel
Chargennummer 2014-2AX-2014-08AX
Validierte Anwendung ELISA
Positivkontrolle Human serum
Negativkontrolle Chicken plasma
Bewertung Signal was detected in positive control sample and not in negative control sample.
Primärantikörper
Kontrollen
  • Positive control: Human individual post-menopausal female serum (Biochemed, 750-NS-FI-POM) (positive)
  • Negative control: Chicken plasma (Sigma-Aldrich, p3266) – reconstituted at 1 mg/mL
  • Standard curve: 0, 25, 75, 175, 400, 800 ng/mL ICTP provided in the ELISA kit
  • Spike control: 800 ng/mL standard premixed with chicken plasma in a 1:1 ratio
Protokoll
  • 50 µL of standard and samples were added 48-well strip plates provided in the kit with 50 µL of HRP conjugate. All samples and standards were assayed in duplicate.
  • The microplate was covered and incubated at 37°C for 1 h.
  • Plate contents were discarded.
  • Content of the wells was discarded and wells were washed 3 times with 200 µL of distilled water with a 1 min soak for each wash.
  • 50 µL of Substrate A and Substrate B each was added to each well. The plate was covered and incubated at 37°C for 15 min.
  • 50 µL of the Stop Solution was added per well.
  • The entire sample was transferred into a 96-well plate (Nunc, Maxisorp)
  • The optical density (OD value) of each well was read immediately using a microplate reader set to 450 nm.
  • The duplicate readings for each sample were averaged and the average zero standard optical density subtracted. The corrected average-value was tabulated as Average Absorbance. A standard curve was generated by plotting the mean OD value for each standard on the x-axis against the concentration on the Y-axis using CurveExpert 1.4. A linear equation was used for the best fit through the points on the graph.
  • The CurveExpert Analyze feature was used to calculate human ICTP concentrations of the samples based on their Average Absorbance values.
Anmerkungen - Transfers of final samples for OD reading were necessitated by inability to read a 48-well plate in the standard plate carriage. This caused high variance between duplicates.
Validierungsbilder
ELISA validation image for C-Telopeptide of Type I Collagen ELISA Kit (ABIN367349) Figure 1: Graph of corrected-average absorbance (OD 450 nm) readings plotted for stan...
ELISA validation image for C-Telopeptide of Type I Collagen ELISA Kit (ABIN367349) Table 1: Table of absorbance readings (OD 450 nm) for standard curve, spike controls ...
Verwendungszweck For the quantitative determination of human C-telopeptide of type ? collagen (ICTP) concentrations in serum, plasma, tissue homogenates.
Proben Serum, Plasma, Tissue Homogenate
Analytische Methode Quantitative
Nachweismethode Colorimetric
Spezifität This assay has high sensitivity and excellent specificity for detection of human ICTP.
Kreuzreaktivität (Details) Limited by current skills and knowledge, it is impossible for us to complete the cross-reactivity detection between the target antigen and all analogues for other species. Therefore, cross reaction may still exist.
Sensitivität 12.5 ng/mL
Bestandteile
  • Assay plate (12 × 8 coated Microwells)
  • Standard (freeze dried)
  • Biotin-antibody (100 × concentrate)
  • HRP-avidin (100 × concentrate)
  • Biotin-antibody Diluent
  • HRP-avidin Diluent
  • Sample Diluent
  • Wash Buffer (25 × concentrate)
  • TMB Substrate
  • Stop Solution
  • Adhesive Strip (for 96 wells)
  • Instruction manual
Benötigtes Material
  • Microplate reader capable of measuring absorbance at 450nm, with the correction wavelength set at 540nm or 570nm.
  • An incubator which can provide stable incubation conditions up to 37°C ± 0.5°C.
  • Squirt bottle, manifold dispenser or automated microplate washer.
  • Absorbent paper for blotting the microtiter plate.
  • 100mL and 500mL graduated cylinders.
  • Deionized or distilled water.
  • Pipettes and pipette tips.
  • Test tubes for dilution.
Andere Bezeichnung C-telopeptide of type I collagen (CTX-I)
Applikationshinweise
  • The supplier is only responsible for the kit itself, but not for the samples consumed during the assay. The user should calculate the possible amount of the samples used in the whole test. Please reserve sufficient samples in advance.
  • Samples to be used within 5 days may be stored at 2-8°C, otherwise samples must be stored at -20°C (≤ 1 month) or -80°C (≤ 2 months) to avoid loss of bioactivity and contamination.
  • Grossly hemolyzed samples are not suitable for use in this assay.
  • If the samples are not indicated in the manual, a preliminary experiment to determine the validity of the kit is necessary.
  • Please predict the concentration before assaying. If values for these are not within the range of the standard curve, users must determine the optimal sample dilutions for their particular experiments.
  • Tissue or cell extraction samples prepared by chemical lysis buffer may cause unexpected ELISA results due to the impacts of certain chemicals.
  • Owing to the possibility of mismatching between antigens from another resource and antibodies used in this supplier's kits (e.g., antibody targets conformational epitope rather than linear epitope), some native or recombinant proteins from other manufacturers may not be recognized by this supplier's products.
  • Influenced by factors including cell viability, cell number and cell sampling time, samples from cell culture supernatant may not be recognized by the kit.
  • Fresh samples without long time storage are recommended for the test. Otherwise, protein degradation and denaturalization may occur in those samples and finally lead to wrong results.
Kommentare

Detection wavelength: 450 nm

Information on standard material:
Some standards are from natural resource, some are recombinant protein, but the recombinant protein will not expressed from Baculovirus. Generally it's CHO cells, but that depends.The formulation of auxiliary material in the standard is classified, but it dosen't contain any poisonous substance. Proclin 300 (1:3000) is used as preservative.

Information on reagents:
Most of the stop solution are 1 N H2SO4, a few is not. The formulation of wash solution is classified. None of the components contains (sodium) azide, thimerosal, 2-mercaptoethanol (2-ME) or any other poisonous materials. Some could contain BSA. For the sandwich method kits, the sample diluent, antibody diluent, enzyme diluent and standard all contain BSA.

Information on antibodies:
The provided antibodies and their host vary in different kits. Some are affinity purified, some are Protein A or G purified

Probenmenge 100 μL
Testdauer 1 - 4.5 h
Plattentyp Pre-coated
Protokoll This assay employs the quantitative sandwich enzyme immunoassay technique. Antibody specific for ICTP has been pre-coated onto a microplate. Standards and samples are pipetted into the wells with a Horseradish Peroxidase (HRP) conjugated antibody specific for ICTP. Following a wash to remove any unbound reagent, a substrate solution is added to the wells and color develops in proportion to the amount of ICTP bound in the initial step. The color development is stopped and the intensity of the color is measured.
Probennahme
  • Serum: Use a serum separator tube (SST) and allow samples to clot for two hours at room temperature or overnight at 4 °C before centrifugation for 15 minutes at 1000 × g. Remove serum and assay immediately or aliquot and store samples at -20 °C or -80 °C. Avoid repeated freeze-thaw cycles.
  • Plasma: Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge for 15 minutes at 1000 × g at 2-8 °C within 30 minutes of collection. Assay immediately or aliquot and store samples at -20 °C or -80 °C. Avoid repeated freeze-thaw cycles.
  • Tissue Homogenates: Rinse 100 mg tissue with 1× PBS, homogenize in 1mL of 1× PBS and store overnight at -20 °C. After two freeze-thaw cycles to break the cell membranes, centrifuge the homogenates for 5 minutes at 5000 × g, 2-8 °C. Remove and assay the supernate immediately. Alternatively, aliquot and store samples at -20 °C or -80 °C. Centrifuge the sample again after thawing before the assay. Avoid repeated freeze-thaw cycles.
Ergebnisberechnung

Average the duplicate readings for each standard and sample and subtract the average zero standard optical density.
Create a standard curve by reducing the data using computer software capable of generating a four parameter logistic (4-PL) curve fit. As an alternative, construct a standard curve by plotting the mean absorbance for each standard on the x-axis against the concentration on the y-axis and draw a best fit curve through the points on the graph. The data may be linearized by plotting the log of the target antigen concentration versus the log of the O.D. and the best fit line can be determined by regression analysis. This procedure will produce an adequate but less precise fit of the data.
If samples have been diluted, the concentration read from the standard curve must be multiplied by the dilution factor.

Testpräzision Intra-assay precision (precision within an assay): Three samples of known concentration were tested twenty times on one plate to assess precision.
Inter-assay precision (precision between assays): Three samples of known concentration were tested in twenty assays to assess precision.
  • Intra-assay: CV% less than 8%
  • Inter-assay: CV% less than 10%
Beschränkungen Nur für Forschungszwecke einsetzbar
Vorsichtsmaßnahmen The Stop Solution provided with this kit is an acid solution. Wear eye, hand, face and clothing protection when using this material.
Handhabung
  • The kit should not be used beyond the expiration date on the kit label.
  • Do not mix or substitute reagents with those from other lots or sources.
  • If samples generate values higher than the highest standard, dilute the samples with Sample Diluent and repeat the assay.
  • Any variation in Sample Diluent, operator, pipetting technique, washing technique, incubation time/temperature and kit age can cause variation in binding.
  • This assay is designed to eliminate interference by soluble receptors, binding proteins and other factors present in biological samples. Until all factors have been tested in the Immunoassay, the possibility of interference cannot be excluded.
Lagerung 4 °C/-20 °C
Informationen zur Lagerung May be stored at 2-8°C for up to 1 month. For long term storage, please store at -20°C. Try to keep assay plate in a sealed aluminium foil bag and avoid dampness.
Haltbarkeit 6 months
Bilder des Herstellers
ELISA image for C-Telopeptide of Type I Collagen ELISA Kit (ABIN2644529) Typical standard curve
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