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Duck IL-10 ELISA Kit

Prod.No.  15R025

FROM:RB

For the quantitative in vitro determination of  IL-10 concentrations in Duck supernates, serum, plasma and tissue.

FOR RESEARCH USE ONLY.

NOT FORUSE IN DIAGNOSTIC PROCEDURES.

TABLE OF CONTENTS

Contents                        Page

TABLE OF CONTENTS.. 2

INTENDED USE.. 3

PRINCIPLE.. 3

WARNINGS AND PRECAUTIONS.. 4

MATERIALS PROVIDED WITH THE KIT. 7

MATERIALS REQUIRED BUT NOT PROVIDED.. 7

STORAGR CONDITIONS.. 8

REAGENT PREPARATION.. 9

SPECIMEN COLLECTION AND PREPARATION.. 9

ASSAY PROCEDURE.. 10

CALCULATION OF RESULTS.. 13

REFERENCES.. 14

INTENDED USE

An enzyme immunoassay for the quantitative in vitro diagnostic measurement of Duck IL-10 in cell culture supernates, serum, plasma and tissue.

PRINCIPLE

The kit assay Duck IL-10 level in the sample，use Purified Duck IL-10 antibody to coat microtiter plate wells, make solid-phase antibody, then add IL-10 to wells, Combined IL-10 antibody which With HRP labeled , become antibody - antigen - enzyme-antibody complex, after washing Compley, Add TMB substrate solution,TMB substrate becomes blue color At HRP enzyme-catalyzed, reaction is terminated by the addition of a sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450 nm. The concentration of IL-10 in the samples is then determined by comparing the O.D. of the samples to the standard curve.

WARNINGS AND PRECAUTIONS

• This kit is for in vitro diagnostic use only. For professional use only.
• All reagents of this test kit which contain human serum or plasma have been tested and confirmed negative for HIV I/II, HBsAg and HCV by FDA approved procedures. All reagents, however, should be treated as potential biohazards in use and for disposal.
• Before starting the assay, read the instructions compley and carefully. Use the valid version of the package insert provided with the kit. Be sure that everything is understood.
• The microplate contains snap-off strips. Unused wells must be stored at 2 °C to 8 °C in the sealed foil pouch and used in the frame provided.
• Pipetting of samples and reagents must be done as quickly as possible and in the same sequence for each step.
• Use reservoirs only for single reagents. This especially applies to the substrate reservoirs. Using a reservoir for dispensing a substrate solution that had previously been used for the conjugate solution may turn solution colored. Do not pour reagents back into vials as reagent contamination may occur.
•  Mix the contents of the microplate wells thoroughly to ensure good test results. Do not reuse microwells.
• Do not let wells dry during assay; add reagents immediay after completing the rinsing steps.
• Allow the reagents to reach room temperature （21-26°C） before starting the test. Temperature will affect the absorbance readings of the assay. However, values for the patient samples will not be affected.
• Never pipet by mouth and avoid contact of reagents and specimens with skin and mucous membranes.
• Do not smoke, eat, drink or apply cosmetics in areas where specimens or kit reagents are handled.
• Wear disposable latex gloves when handling specimens and reagents. Microbial contamination of reagents or specimens may give false results.
• Handling should be done in accordance with the procedures defined by an appropriate national biohazard safety guideline or regulation.
• Do not use reagents beyond expiry date as shown on the kit labels.
• All indicated volumes have to be performed according to the protocol. Optimal test results are only obtained when using calibrated pipettes and microtiterplate readers.
• Do not mix or use components from kits with different lot numbers. It is advised not to exchange wells of different plates even of the same lot. The kits may have been shipped or stored under different conditions and the binding characteristics of the plates may result slightly different.
• Avoid contact with Stop Solution containing 0.5 M H2SO4. It may cause skin irritation and burns.
• Some reagents contain Proclin, BND and/or MIT as preservatives. In case of contact with eyes or skin, flush immediay with water.
• TMB substrate has an irritant effect on skin and mucosa. In case of possible contact, wash eyes with an abundant volume of water and skin with soap and abundant water. Wash contaminated objects before reusing them. If inhaled, take the person to open air.
• Chemicals and prepared or used reagents have to be treated as hazardous waste according to the national biohazard safety guideline or regulation.
• For information on hazardous substances included in the kit please refer to Material Safety Data Sheets

MATERIALS PROVIDED WITH THE KIT

MATERIALS REQUIRED BUT NOT PROVIDED

• Microplate reader capable of measuring absorbance at 450 nm.
• Precision pipettes to deliver 2 ml to 1 ml volumes.
• Adjustable 10ml -100ml pipettes for reagent preparation.
• Adjustable 10ml -100ml pipettes for reagent preparation.
• 100 ml and 1 liter graduated cylinders.
• Calibrated adjustable precision pipettes, preferably with disposable plastic tips. （A manifold multi-channel pipette is desirable for large assays.）
• Absorbent paper.
• 37°C incubator.
• Distilled or deionized water.
• Data analysis and graphing software. Graph paper: linear （Cartesian）,log-log or semi-log, or log-logit as desired.
•  Tubes to prepare standard or sample dilutions.

STORAGR CONDITIONS

1. When stored at 2 °C to 8 °C unopened reagents will retain reactivity until expiration date.
2. Do not use reagents beyond this date. Opened reagents must be stored at 2 °C to 8 °C.
3. Microtiter wells must be stored at 2 °C to 8 °C. Once the foil bag has been opened, care should be taken to close it tightly again.
4. Opened kits retain activity for 8 weeks if stored as described above.

REAGENT PREPARATION

Bring all reagents to room temperature before use

SPECIMEN COLLECTION AND PREPARATION

Serum-Use a serum separator tube（SST） and allow samples to clot for 30minutes before centrifugation for 15minutes at approximay 1000 xg.Remove serum and assay immediay or aliquot and store samples at -20°C or -80°C.

Plasma-Collect plasma using EDTA or heparin as an anticoagulant.Centrifuge samples for 15 minutes at 1000 x g at 2-8°C within 30minutes of collection. Store samples at -20°C or -80°C.Avoid repeated freeze-thaw cycles.

Cell culture fluid and other biological fluids-Remove particulates by centrifugation and assay immediay or aliquot and store samples at -20°C or -80°C.Avoid repeated freeze-thaw cycles

ASSAY PROCEDURE

1. General Remarks
   • All reagents and specimens must be allowed to come to room temperature before use. All reagents must be mixed without foaming.
   • Once the test has been started, all steps should be completed without interruption.
   • Use new disposal plastic pipette tips for each standard, control or sample in order to avoid cross contamination.
   • Absorbance is a function of the incubation time and temperature. Before starting the assay, it is recommended that all reagents are ready, caps removed, all needed wells secured in holder, etc. This will ensure equal elapsed time for each pipetting step without interruption.
   • As a general rule the enzymatic reaction is linearly proportional to time and temperature.
   • Determine absorption with an ELISA reader at 450 nm against 620 nm as reference. If no reference wavelength is available, read only at 450nm. If the extinction of the highest standard exceeds the measurement range of the photometer, absorption must be measured immediay at 405 nm against 620 nm as reference.
     1. Assay Procedure

1. Dilute and add sample to Standard: set 10 Standard wells on the ELISA plates coated, add Standard 100μl to the first and the second well, then add Standard dilution 50μl to the first and the second well, mix; take out 100μl form the first and the second well then add it to the third and the forth well separay. then add Standard dilution 50μl to the third and the forth well ,mix ; then take out 50μl from the third and the forth well discard, add 50μl to the fifth and the sixth well ,then add Standard dilution 50μl to the fifth and the sixth well, mix ; take out 50μl from the fifth and the sixth well and add to the seventh and the eighth well, then add Standard dilution 50μl to the seventh and the eighth well ,mix ; take out 50μl from the seventh and the eighth well and add to the ninth and the tenth well, add Standard dilution 50μl to the ninth and the tenth well, mix , take out 50μl from the ninth and the tenth well  discard（add Sample 50μl to each well after Diluting ,（density: 150 pg/ml，100 pg/ml，50 pg/ml，25pg/ml，12.5 pg/ml）.

1. add sample：Set blank wells separay （blank comparison wells don’t add sample and HRP-Conjugate reagent, other each step operation is same）. testing sample well. add Sample dilution 40μl to testing sample well, then add testing sample 10μl （sample final dilution is 5-fold）, add sample to wells , don’t touch the well wall as far as possible, and Gently mix.
2. Incubate: After closing plate with Closure plate membrane ,incubate for 30 min at 37℃.
3. Configurate liquid: 30-fold （or 20-fold）wash solution diluted 30-fold （or 20-fold） with distilled water and reserve.
4. washing：Uncover Closure plate membrane, discard Liquid, dry by swing, add washing buffer to every well, still for 30s then drain, repeat 5 times, dry by pat.
5. add enzyme：Add HRP-Conjugate reagent 50μl to each well, except  blank well.
6. incubate：Operation with 3.
7. washing：Operation with 5.
8. color：Add Chromogen Solution A 50ul and Chromogen Solution B to each well, evade the light preservation for 15 min at 37℃
9. Stop the reaction：Add Stop Solution50μl to each well, Stop the reaction（the blue color change to yellow color）.
10. assay：take blank well as zero , Read absorbance at 450nm after Adding Stop Solution and within 15min.

CALCULATION OF RESULTS

• Calculate the average absorbance values for each set of standards, controls and patient samples.
• Construct a standard curve by plotting the mean absorbance obtained from each standard against its.
• concentration with absorbance value on the vertical（Y） axis and concentration on the horizontal （X） axis.
• Using the mean absorbance value for each sample determine the corresponding concentration from the standard curve.
• Automated method: The results in the IFU have been calculated automatically using a 4 PL.
• （4 Parameter Logistics） curve fit. 4 Parameter Logistics is the preferred calculation method. Other data.
• reduction functions may give slightly different results.
• The concentration of the samples can be read directly from this standard curve. Samples with.
• concentrations higher than that of the highest standard have to be further diluted. For the calculation of.
• the concentrations this dilution factor has to be taken into account.

REFERENCES

REF  : Cat.-No.: / Kat.-Nr.: / No.- Cat.: / Cat.-No.: / N.º Cat.: / N.–Cat

LOT  : Lot-No.: / Chargen-Bez.: / No. Lot: / Lot-No.: / Lote N.º: / Lotto n.:

    :No. of Tests: / Kitgröße: / Nb. de Tests: / No. de Determ.: / N.º de Testes: / Quantità dei tests:

  : Keep away from heat or direct sun light. / Vor Hitze und direkter Sonneneinstrahlung schützen. /

Garder à l’abri de la chaleur et de toute exposition lumineuse. / Manténgase alejado del calor o la

luz solar directa. / Manter longe do calor ou luz solar directa. / Non esporre ai raggi solari.

  : Store at: / Lagern bei: / Stocker à: / Almacene a :/ Armazenar a :/ Conservare a: