Rabbit Interleukin 6 ELISA Kit

96 Tests

Catalogue Number: E04I0006

Store all reagents at 2-8°C

Collect sample: serum or blood plasma

FOR LABORATORY RESEARCH USE ONLY. NOT FOR THERAPEUTIC OR DIAGNOSTIC APPLICATIONS!PLEASE READ THROUGH ENTIRE PROCEDURE BEFORE BEGINNING!

INTENDED USE

This B.G IL-6 ELISA kit is intended Laboratory for Research use only and is not for use in diagnostic or therapeutic procedures.The Stop Solution changes the color from blue to yellow and the intensity of the color is measured at 450 nm using a spectrophotometer. In order to measure the concentration of IL-6 in the sample, this IL-6 ELISA Kit includes a set of calibration standards. The calibration standards are assayed at the same time as the samples and allow the operator to produce a standard curve of Optical Density versus IL-6 concentration. The concentration of IL-6 in the samples is then determined by comparing the O.D. of the samples to the standard curve.

PRINCIPLE OF THE ASSAY

The coated well immunoenzymatic assay for the quantitative measurement of serum IL-6 utilizes a monoclonal anti-IL-6 and a IL-6-HRP conjugate. The assay asample and buffer are incubated together with anti-IL-6 antibody coated plate for sixty and washed. The diluted IL-6-HRP conjugate is then added to each well and incubated. After the incubation period, the wells are decanted and washed three times. The wells are then incubated with a substrate for the enzyme. The product of the enzyme-substrate reaction forms a blue colored complex. Finally, a stopping solution is added to stop the reaction, which will then turn the solution yellow.The intensity of color is measured spectrophotometrically at 450nm in a microplate reader. The intensity of the color is inversely proportional to the IL-6 concentration since IL-6 from samples and IL-6-HRP conjugate compete for the anti-IL-6 antibody binding site. Since the number of sites is limited, as more sites are occupied by IL-6 from the sample, fewer sites are left to bind IL-6-HRP conjugate.Standards of known IL-6 concentrations are run concurrently with the samples being assayed and a standard curve is plotted relating the intensity of the color （Optical Density） to the concentration of IL-6. The  unknown IL-6 concentration in each sample is interpolated from this curve.

REAGENTS PROVIDED

All reagents provided are stored at 2-8° C. Refer to the expiration date on the label.

1.          MICROTITER PLATE  96 wells

2.          ENZYME CONJUGATE  6.0 mL  1 vial

3.          STANDARD.1  0pg/ml   1 vial

4.          STANDARD.2  50pg/ml   1 vial

5.          STANDARD.3  100pg/ml   1 vial

6.          STANDARD.4  250pg/ml   1 vial

7.          STANDARD.5  500pg/ml   1 vial

8.          STANDARD.6  1000pg/ml   1 vial

9.          SUBSTRATE A  6.0 mL  1 vial

10.       SUBSTRATE B  6.0 mL  1 vial

11.       STOP SOLUTION  6.0 mL  1 vial

12.       WASH SOLUTION x100  10 mL  1 vial

13.       Instruction   1

SAMPLE COLLECTION AND STORAGE

Serum- Use a serum separator tube（SST） and allow samples to clot for 30minutes before centrifugation for 15minutes at approximay 1000 x g.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.

NOTE: Serum, plasma, and cell culture fluid samples to be used within 7

days may be stored at 2-8°C, otherwise samples must stored at -20°C（≤2months） or -80°C（≤6months） to avoid loss of bioactivity and contamination. Avoid freeze-thaw cycles .When performing the assay slowly bring samples to room temperature.

DO NOT USE HEAT-TREATED SPECIMENS.

MATERIALS REQUIRED BUT NOT SUPPLIED

1.          Microplate reader capable of measuring absorbance at 450 nm.

2.          Precision pipettes to deliver 2 ml to 1 ml volumes.

3.          Adjustable 10ml -100ml pipettes for reagent preparation.

4.          Adjustable 10ml -100ml pipettes for reagent preparation.

5.          100 ml and 1 liter graduated cylinders.

6.          Calibrated adjustable precision pipettes, preferably with disposable plastic tips. （A manifold multi-channel pipette is desirable for large assays.）

7.          Absorbent paper.

8.          37°C incubator.

9.          Distilled or deionized water.

10.       Data analysis and graphing software. Graph paper: linear （Cartesian）, log-log or semi-log, or log-logit as desired.

11.       Tubes to prepare standard or sample dilutions.

PRECAUTIONS

1.          Do not substitute reagents from one kit lot to another. Standard, conjugate and microtiter plates are matched for optimal performance. Use only the reagents supplied by manufacturer.

2.          Allow kit reagents and materials to reach room temperature （20-25°C） before use. Do not use water baths to thaw samples or reagents.

3.          Do not use kit components beyond their expiration date.

4.          Use only deionized or distilled water to dilute reagents.

5.          Do not remove microtiter plate from the storage bag until needed. Unused strips should be stored at 2-8°C in their pouch with the desiccant provided.

6.          Use fresh disposable pipette tips for each transfer to avoid contamination.

7.          Do not mix acid and sodium hypochlorite solutions.

8.          Serum and plasma should be handled as potentially hazardous and capable of transmitting disease. Disposable gloves must be worn during the assay procedure, since no known test method can offer complete assurance that products derived from human blood will not transmit infectious agents. Therefore, all blood derivatives should be considered potentially infectious and good laboratory practices should be followed.

9.          All samples should be disposed of in a manner that will inactivate viruses.

10.       Solid Waste: Autoclave 60 min. at 121°C.

11.       Liquid Waste: Add sodium hypochlorite to a final concentration of 1.0%. The waste should be allowed to stand for a minimum of 30 minutes to inactivate the viruses before disposal.

12.       Substrate Solution is easily contaminated. If bluish prior to use, do not use.

13.       Substrate B contains 20% acetone, keep this reagent away from sources of heat or flame.

14.       Remove all kit reagents from refrigerator and allow them to reach room temperature （ 20-25°C）.

ASSAY PROCEDURE

1.          Prepare all Standards before starting assay procedure （see Preparation Reagents）. It is recommended that all Standards and Samples be added in duplicate to the Microtiter Plate.

2.          First, secure the desired number of coated wells in the holder, then add 100 μL of Standards or Samples to the appropriate well of the antibody pre-coated Microtiter Plate.

3.          Add 50 μL of Conjugate to each well. Mix well. Complete mixing in this step is important. Cover and incubate for 1 hours at 37°C.

4.          Prepare Substrate Solution no more than 15 minutes before end of incubation （see Preparation of Reagents）.

5.          Wash the Microtiter Plate using one of the specified methods indicated below:

6.          Manual Washing: Remove incubation mixture by aspirating contents of the plate into a sink or proper waste container. Using a squirt bottle, fill each well compley with distilled or de-ionized water, then aspirate contents of the plate into a sink or proper waste container. Repeat this procedure four more times for a total of FIVE washes. After final wash, invert plate, and blot dry by hitting plate onto absorbent paper or paper towels until no moisture appears. Note: Hold the sides of the plate frame firmly hen washing the plate to assure that all strips remain securely in frame.

7.          Automated Washing: Aspirate all wells, then wash plate FIVE times using distilled or de-ionized water. Always adjust your washer to aspirate as much liquid as possible and set fill volume at 350 μL/well/wash （range: 350-400 μL）. After final wash, invert plate, and blot dry by hitting plate onto absorbent paper or paper towels until no moisture appears. It is recommended that the washer be set for a soaking time of 10 seconds or shaking time of 5 seconds between washes.

8.          Add 50 μL Substrate A&B to each well. Cover and incubate for 15 minutes at 20-25°C.

9.          Add 50 μL of Stop Solution to each well. Mix well.

10.       Read the Optical Density （O.D.） at 450 nm using a microtiter plate reader within 30 minutes.

B.G CALCULATION OF RESULTS

1.        Calculate the mean absorbance value A₄₅₀ for each set of reference standards and samples. 

2.        Divide the average A450 value for each standard, control and test sample by the average A450 of standard0 and multiply by 100 to obtain %B/B0 for each sample.

3.        Prepare a standard curve by plotting the average absorbance of each standard versus the corresponding concentrations of the standards on linear-log graph paper or the %B/B0 value for each standard versus the corresponding concentration of the standard on linear-log or logit-log graph paper. logit=ln（B/ B0）/（1－B/ B0）

4.        Any values obtained for diluted samples must be further converted by applying the appropriate dilution factor in the calculations.

5.        The standard density is a X, the B/ B0 is a Y, sitting to mark the paper in the logit- log up draw a standard curve.According to the B/ B0 that need to be measured the sample can from sit to mark the density value that the paper looks up the sample up.

6.        The sensitivity by this assay is 1.0pg/ml

7.        The standard curve presented here is an example of the data typically produced with this kit; however, your results will not be identical to these.

8.        Standard curve

Disposal Note Safety

1.          This kit contains materials with small quantities of sodium azide. Sodium azide reacts with lead and copper plumbing to form explosive metal azides. Upon disposal, flush drains with a large volume of water to prevent azide accumulation. Avoid ingestion and contact with eyes, skin and mucous membranes. In case of contact, rinse affected area with plenty of water. Observe all federal, state and local regulations for disposal.

2.          All blood components and biological materials should be handled as potentially hazardous. Follow universal precautions as established by the Centers for Disease Control and Prevention and by the Occupational Safety and Health Administration when handling and disposing of infectious agents.

Additional Materials Required Quality Control

1.          When not in use, kit components should be refrigerated. All reagents should be warmed to room temperature before use.

2.          Microtiter plates should be allowed to come to room temperature before opening the foil bags. Once the desired number of strips has been removed, immediay reseal the bag and store at 2 - 8°C to maintain plate integrity.

3.          Samples should be collected in pyrogen/endotoxin-free tubes.

4.          Samples should be frozen if not analyzed shortly after collection. Avoid multiple freeze-thaw cycles of frozen samples. Thaw compley and mix well prior to analysis.

5.          When possible, avoid use of badly hemolyzed or lipemic sera. If large amounts of particulate matter are present, centrifuge or filter prior to analysis.

6.          It is recommended that all standards, controls and samples be run in duplicate.

7.          When pipetting reagents, maintain a consistent order of addition from well-to-well. This ensures equal incubation times for all wells.

8.          Cover or cap all reagents when not in use.

9.          Do not mix or interchange different reagent lots from various kit lots.

10.       Do not use reagents after the kit expiration date.

11.       Read absorbances within 2 hours of assay completion.

12.       The provided controls should be run with every assay. If control values fall outside pre-established ranges,the accuracy of the assay is suspect.

13.       All residual wash liquid must be drained from the wells by efficient

aspiration or by decantation followed by tapping the plate forcefully on absorbent paper. Never insert absorbent paper directly into the wells.

14.       Because Stabilized Chromogen is light sensitive, avoid prolonged exposure to light. Also avoid contact between Stabilized Chromogen and metal, or color may develop.

15.       Incomplete washing will adversely affect the test outcome. All washing must be performed with Wash Buffer provided.

16.       Washing can be performed manually as follows: compley aspirate the liquid from all wells by gently lowering anaspiration tip （aspiration device） into the bottom of each well. Take care not to scratch the inside of the well.

17.       After aspiration, fill the wells with at least 0.4 mL of diluted wash solution. Let soak for 15 to 30 seconds, then aspirate the liquid. Repeat as directed under ASSAY METHOD. After the washing procedure, the plate is inverted and tapped dry on absorbent tissue.

18.       Alternatively, the wash solution may be put into a squirt bottle. If a squirt bottle is used, flood the plate with wash buffer, compley filling all wells. After the washing procedure, the plate is inverted and tapped dry on absorbenttissue.

19.       If using an automated washer, the operating instructions for washing equipment should be carefully followed.

20.       Assay Procedure Preliminary notes: Do not mix reagents from different lots.It is recommended that assays be performed in duplicate.Standards and samples must be assayed at the same time.Avoid exposing the substrate to direct sunlight.