DEFa3 ELISA Kit (Defensin, alpha 3, Neutrophil-Specific)

Details for Product DEFa3 ELISA Kit No. ABIN419782, Anbieter: Anmelden zum Anzeigen
Antigen
  • DEFA1
  • DEFA3
  • DEFT1P
  • DEF3
  • HNP-3
  • HNP3
  • HP-3
  • HP3
  • Defa27
  • Defcr-3
  • Defcr3
  • defensin, alpha 3, neutrophil-specific
  • alpha-defensin 1A
  • defensin, alpha 1 and alpha 3, variable copy number locus
  • defensin, alpha, 3
  • DEFA3
  • MNP1A
  • DEFA1A3
  • Defa3
Alternativen
Human DEFa3 ELISA Kit
Reaktivität
Human
Alternativen
Kits mit alternativen Reaktivitäten:
5
1
Methodentyp
Sandwich ELISA
Detektionsbereich
0.47-30 ng/mL
Untere Nachweisgrenze
0.47 ng/mL
Applikation
ELISA
Optionen
Hersteller
Anmelden zum Anzeigen
Hersteller Produkt- Nr.
Anmelden zum Anzeigen

Bilder

'Independent Validation' Siegel
Chargennummer L131225503
Validierte Anwendung ELISA
Positivkontrolle Human serum
Negativkontrolle Mouse serum
Bewertung Minimal levels of signal detected in negative control. Percent recovery of the spiked samples shows that there is matrix interference. Dilution of >50 fold is required for accurate measurement of the analyte in human serum samples.
Primärantikörper
Kontrollen
  • Positive control: normal human serum
  • Negative control: mouse serum
  • Standard curve: serial two-fold dilutions from 30 ng/ml [30, 15, 7.5, 3.75, 1.875, 0.9375, 0] were generated from the standard provided in the kit using standard diluent buffer.
  • Spike control: standard diluted in human or mouse serum [6 ng/mL].
  • Control lyophilized powder was dissolved in 150uL standard diluent buffer (7.39 ng/mL)
Protokoll
  • All reagents in the ELISA kit were brought up to room temperature (RT) before use.
  • 100 µl of each sample was added per well to the micro ELISA plate well. All samples and standards were assayed in triplicate.
  • The plate was covered with sealer (provided in kit) and incubated for 120 mins at 37°C.
  • Liquid was removed from each well by pipette.
  • Detection Reagent A was diluted 100 fold in Assay Diluent A. 100 µl of diluted detection reagent A was added to each well and the plate was sealed. The plate was tapped to ensure mixing and incubated for 60 mins at 37°C.
  • Wells were washed with 300 µl wash buffer three times. Each wash involved fully aspirating the liquid from each well by pipette. After the last wash the plate was inverted against clean absorbent paper to remove any remaining liquid.
  • Detection Reagent B was diluted 100 fold in Assay Diluent B. 100 µl of diluted detection reagent B was added to each well and the plate was sealed. The plate was tapped to ensure mixing and incubated for 30 mins at 37°C.
  • Wells were washed with 300 µl wash buffer five times. Each wash involved fully aspirating the liquid from each well by pipette. After the last wash the plate was inverted against clean absorbent paper to remove any remaining liquid.
  • 90 µl of Substrate Solution was added to each well and the plate was covered with a new plate sealer. The plate was tapped to ensure mixing and incubated at room temperature in the dark.
  • After about 10 mins, when an apparent gradient appeared in the standard wells, the reaction was terminated by adding 50 µl of Stop Solution to each well.
  • The optical density (OD value) of each well was read using a micro-plate reader set to 450 nm.
  • The triplicate readings for each sample were averaged and the average zero standard optical density subtracted. A standard curve was generated by plotting the mean OD value for each standard on the y-axis against the concentration on the x-axis using Softmax Pro softare.
  • The equation y = (A-D)/(1 + (x/C)^B) + D was used to calculate IL-6 concentrations of the samples based on their average OD values.
Anmerkungen - Percent recovery of the spiked samples shows that there is matrix interference. Dilution of >50 fold is required for accurate measurement of the analyte in human serum samples.
Validierungsbilder
ELISA validation image for Defensin, alpha 3, Neutrophil-Specific (DEFa3) ELISA Kit (ABIN419782) Figure 1: Graph of corrected-average absorbance (OD 450 nm) readings plotted for stan...
ELISA validation image for Defensin, alpha 3, Neutrophil-Specific (DEFa3) ELISA Kit (ABIN419782) Table 1: ELISA. DEFa3 could be detected in human serum (positive control) after 10 fo...
ELISA validation image for Defensin, alpha 3, Neutrophil-Specific (DEFa3) ELISA Kit (ABIN419782) Table 2: Value for Average Reading is derived from the average of three readings (OD ...
Verwendungszweck The kit is a sandwich enzyme immunoassay for in vitro quantitative measurement of DEFa3 in Serum,Plasma,Biological Fluids
Proben Serum, Plasma, Tissue Homogenate, Cell Lysate, Cell Culture Supernatant, Biological Fluids
Analytische Methode Quantitative
Nachweismethode Colorimetric
Spezifität

This assay has high sensitivity and excellent specificity for detection of Defensin Alpha 3, Neutrophil Specific (DEFa3).
No significant cross-reactivity or interference between Defensin Alpha 3, Neutrophil Specific (DEFa3) and analogues was observed.

Kreuzreaktivität (Details) No significant cross-reactivity or interference between Defensin Alpha 3, Neutrophil Specific (DEFa3) and analogues was observed.
Sensitivität 0.21 ng/mL
Bestandteile
  • Pre-coated, ready to use 96-well strip plate
  • Plate sealer for 96 wells
  • Standard Diluent
  • Assay Diluent A
  • Assay Diluent B
  • Stop Solution
  • Standard
  • Detection Reagent A
  • Detection Reagent B
  • TMB Substrate
  • Wash Buffer (30 × concentrate)
  • Instruction manual
Benötigtes Material
  • Microplate reader with 450 nm filter.
  • Precision single or multi-channel pipettes and disposable tips.
  • Eppendorf Tubes for diluting samples.
  • Deionized or distilled water.
  • Absorbent paper for blotting the microtiter plate.
  • Container for Wash Solution
Andere Bezeichnung DEFa3 (DEFa3 ELISA Kit Abstract)
UniProt P59666
Pathways Cellular Response to Molecule of Bacterial Origin
Applikationshinweise
  • Limited by the current condition and scientific technology, we cannot completely conduct the comprehensive identification and analysis on the raw material provided by suppliers. So there might be some qualitative and technical risks to use the kit.
  • The final experimental results will be closely related to validity of the products, operation skills of the end users and the experimental environments. Please make sure that sufficient samples are available.
  • Kits from different batches may be a little different in detection range, sensitivity and color developing time.
  • Do not mix or substitute reagents from one kit lot to another. Use only the reagents supplied by manufacturer.
  • Protect all reagents from strong light during storage and incubation. All the bottle caps of reagents should be covered tightly to prevent the evaporation and contamination of microorganism.
  • There may be some foggy substance in the wells when the plate is opened at the first time. It will not have any effect on the final assay results. Do not remove microtiter plate from the storage bag until needed.
  • Wrong operations during the reagents preparation and loading, as well as incorrect parameter setting for the plate reader may lead to incorrect results. A microplate plate reader with a bandwidth of 10nm or less and an optical density range of 0-3 O.D. or greater at 450 ± 10nm wavelength is acceptable for use in absorbance measurement. Please read the instruction carefully and adjust the instrument prior to the experiment.
  • Even the same operator might get different results in two separate experiments. In order to get better reproducible results, the operation of every step in the assay should be controlled. Furthermore, a preliminary experiment before assay for each batch is recommended.
  • Each kit has been strictly passed Q.C test. However, results from end users might be inconsistent with our in-house data due to some unexpected transportation conditions or different lab equipments. Intra-assay variance among kits from different batches might arise from above factors, too.
  • Kits from different manufacturers for the same item might produce different results, since we have not compared our products with other manufacturers.
Kommentare

Information on standard material:
The standard might be recombinant protein or natural protein, that will depend on the specific kit. Moreover, the expression system is E.coli or yeast or mammal cell. There is 0.05% proclin 300 in the standard as preservative.

Information on reagents:
The stop solution used in the kit is sulfuric acid with concentration of 1 mol/L. And the wash solution is TBS. The standard diluent contains 0.02 % sodium azide, assay diluent A and assay diluent B contain 0.01% sodium azide. Some kits can contain is BSA in them.

Information on antibodies:
The provided antibodies and their host vary in different kits.

Probenmenge 100 μL
Testdauer 3 h
Plattentyp Pre-coated
Protokoll The test principle applied in this kit is Sandwich enzyme immunoassay. The microtiter plate provided in this kit has been pre-coated with an antibody specific to Defensin Alpha 3, Neutrophil Specific (DEFa3). Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated antibody specific to Defensin Alpha 3, Neutrophil Specific (DEFa3). Next, Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. After TMB substrate solution is added, only those wells that contain Defensin Alpha 3, Neutrophil Specific (DEFa3), biotin-conjugated antibody and enzyme-conjugated Avidin will exhibit a change in color. The enzyme-substrate reaction is terminated by the addition of sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450nm ± 10nm. The concentration of Defensin Alpha 3, Neutrophil Specific (DEFa3) in the samples is then determined by comparing the O.D. of the samples to the standard curve.
Aufbereitung der Reagenzien
  • Bring all kit components and samples to room temperature (18-25°C) before use.
  • Standard - Reconstitute the Standard with 1.0mL of Standard Diluent, kept for 10 minutes at room temperature, shake gently(not to foam). The concentration of the standard in the stock solution is 3,000pg/mL. Please prepare 7 tubes containing 0.5mL Standard Diluent and produce a double dilution series. Mix each tube thoroughly before the next transfer. Set up 7 points of diluted standard such as 3,000pg/mL, 1,500pg/mL, 750pg/mL, 375pg/mL, 187.5pg/mL, 93.75pg/mL, 46.88pg/mL, and the last EP tubes with Standard Diluent is the blank as 0pg/mL.
  • Assay Diluent A and Assay Diluent B - Dilute 6mL of Assay Diluent A or B Concentrate(2×) with 6mL of deionized or distilled water to prepare 12 mL of Assay Diluent A or B. (In fact, more than 6mL Assay Diluent A and Assay Diluent B are contained in the bottles. Therefore, in every test, please precisely pipette required amount of Diluent and make double dilution in a new container. The prepared working dilution cannot be frozen.)
  • Detection Reagent A and Detection Reagent B - Briefly spin or centrifuge the stock Detection A and Detection B before use. Dilute to the working concentration with working Assay Diluent A or B, respectively (1:100).
  • Wash Solution - Dilute 20mL of Wash Solution concentrate (30×) with 580mL of deionized or distilled water to prepare 600 mL of Wash Solution (1×).
  • TMB substrate - Aspirate the needed dosage of the solution with sterilized tips and do not dump the residual solution into the vial again.
Note:
  • Making serial dilution in the wells directly is not permitted.
  • Prepare standard within 15 minutes before assay. Please do not dissolve the reagents at 37°C directly.
  • Please carefully reconstitute Standards or working Detection Reagent A and B according to the instruction, and avoid foaming and mix gently until the crystals are completely dissolved. To minimize imprecision caused by pipetting, use small volumes and ensure that pipettors are calibrated. It is recommended to suck more than 10µL for once pipetting.
  • The reconstituted Standards, Detection Reagent A and Detection Reagent B can be used only once.
  • If crystals have formed in the Wash Solution concentrate (30×), warm to room temperature and mix gently until the crystals are completely dissolved.
  • Contaminated water or container for reagent preparation will influence the detection result.
Probennahme Serum: Allow samples to clot for two hours at room temperature or overnight at 4°C before centrifugation for 20 minutes at approximately 1000 × g. Assay immediately or store samples in aliquot at -20°C or -80°C. Avoid repeated freeze/thaw cycles.

Plasma: Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples for 15 minutes at 1000 × g within 30 minutes of collection. Remove plasma and assay immediately or store samples in aliquot at -20°C or -80°C. Avoid repeated freeze/thaw cycles.

Biological Fluids: Centrifuge samples for 20 minutes at 1000 × g. Remove particulates and assay immediately or store samples in aliquot at -20 °C or -80 °C for later use. Avoid repeated freeze/thaw cycles.
Aufbereitung der Proben
  • We are 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.
  • 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. Sample should be diluted by 0.01mol/L PBS(PH=7.0-7.2).
  • If the samples are not indicated in the manual, a preliminary experiment to determine the validity of the kit is necessary.
  • Tissue or cell extraction samples prepared by chemical lysis buffer may cause unexpected ELISA results due to the impacts from certain chemicals.
  • Due to the possibility of mismatching between antigen from other origin and antibody used in our kits (e.g.antibody targets conformational epitope rather than linear epitope), some native or recombinant proteins from other manufacturers may not be recognized by our products.
  • Influenced by the factors including cell viability, cell number or sampling time, samples from cell culture supernatant may not be detected by the kit.
  • Fresh samples without long time storage is recommended for the test. Otherwise, protein degradation and denaturalization may occur in those samples and finally lead to wrong results.
Testdurchführung
  1. Prepare all reagents, samples and standards,
  2. Add 100μL standard or sample to each well. Incubate 2 hours at 37 °C,
  3. Aspirate and add 100μL prepared Detection Reagent A. Incubate 1 hour at 37 °C,
  4. Aspirate and wash 3 times,
  5. Add 100μL prepared Detection Reagent B. Incubate 30 minutes at 37 °C,
  6. Aspirate and wash 5 times,
  7. Add 90μL Substrate Solution. Incubate 10-20 minutes at 37 °C,
  8. Add 50μL Stop Solution. Read at 450nm immediately.
Ergebnisberechnung

Average the duplicate readings for each standard, control, and samples and subtract the average zero standard optical density. Create a standard curve on log-log graph paper, with DEFa3 concentration on the y-axis and absorbance on the x-axis. Draw the best fit straight line through the standard points and it can be determined by regression analysis. Using some plot software, for instance, curve expert 1.30, is also recommended. If samples have been diluted, the concentration read from the standard curve must be multiplied by the dilution factor.
In order to make the calculation easier, we plot the O.D. value of the standard (X-axis) against the known concentration of the standard (Y-axis), although concentration is the independent variable and O.D. value is the dependent variable. However, the O.D. values of the standard curve may vary according to the conditions of assay performance (e.g. operator, pipetting technique, washing technique or temperature effects), plotting log of the data to establish standard curve for each test is recommended. Typical standard curve below is provided for reference only.

Testpräzision

Intra-assay Precision (Precision within an assay): 3 samples with low, middle and high level Defensin Alpha 3, Neutrophil Specific (DEFa3) were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, middle and high level Defensin Alpha 3, Neutrophil Specific (DEFa3) were tested on 3 different plates, 8 replicates in each plate.
CV(%) = SD/meanX100
Intra-Assay: CV<10%
Inter-Assay: CV<12%

Beschränkungen Nur für Forschungszwecke einsetzbar
Vorsichtsmaßnahmen The Stop Solution suggested for use with this kit is an acid solution. Wear eye, hand, face, and clothing protection when using this material.
Handhabung

The stability of kit is determined by the loss rate of activity. The loss rate of this kit is less than 5 % within the expiration date under appropriate storage condition.
To minimize extra influence on the performance, operation procedures and lab conditions, especially room temperature, air humidity, incubator temperature should be strictly controlled. It is also strongly suggested that the whole assay is performed by the same operator from the beginning to the end.

Lagerung 4 °C
Informationen zur Lagerung
  • For unopened kit: All the reagents should be kept according to the labels on vials. The Standard, Detection Reagent A, Detection Reagent B and the 96-well strip plate should be stored at -20°C upon receipt while the others should be at 4°C.
  • For opened kit: When the kit is opened, the remaining reagents still need to be stored according to the above storage condition. Besides, please return the unused wells to the foil pouch containing the desiccant pack, and reseal along entire edge of zip-seal.
    Note: It is highly recommended to use the remaining reagents within 1 month provided this is within the expiration date of the kit.
  • For ELISA kit, 1 day storage at 37°C can be considered as 2 months at 4°C, which means 3 days at 37°C equaling 6 months at 4°C.
Haltbarkeit 6 months
Bilder des Herstellers
ELISA image for DEFa3 ELISA Kit (Defensin, alpha 3, Neutrophil-Specific) (ABIN419782) Defensin, alpha 3, Neutrophil-Specific (DEFa3) ELISA Kit