ELISA                                  

Human RANKL

Catalogue Number 1R001

For the quantitative determination of Human receptor activator of nuclear factor kappa B ligand concentrations in serum- body fluid - celiac fluid - tissue homogenate - culture fluid.

This package insert must be read in its entirety before using this product.

FOR RESEARCH USE ONLY.

NOT FOR USE IN DIAGNOSTIC PROCEDURES.   

          http://www.rapidbio.org   

INTENDED USE AND TEST PRINCIPLE

This immunoassay kit allows for the quantitative determination of Human RANKL concentrations in serum, plasma, cell culture supernates and other biological fluids.

Add RANKL to pre-coated RANKL monoclonal antibody microelisa well, incubation; washing. Add HRP tagged RANKL antibodies. After another incubation and washing, remove the unbound enzyme, add Chromogen Solution A and B, the color of the liquid change into blue, and the color finally become yellow at the effect of acid. The depth of the color is positively correlated with concentration of the Porcine RANKL in samples.

MATERIALS REQUIRED BUT NOT SUPPLIED

1. 37 ℃ incubator

2. Standard microplate reader capable of measuring absorbance at 450 nm

3. Precision pipettes, disposable pipette tips and Absorbent paper

4. Distilled or deionized water

SAMPLE COLLECTION AND STORAGES

Serum - Use a serum separator tube and allow samples to clot for 30 minutes before centrifugation for 20 minutes at approximay 1000×g. Remove serum and assay immediay or aliquot and store samples at -20℃ or -80℃.Avoid repeated freeze-thaw cycles

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

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

Note:  The samples shoule be centrifugated dequay and no hemolysis or granule was allowed.

REAGENTS PROVIDED

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. 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.
11. Substrate Solution is easily contaminated. If bluish prior to use, do not use.
12. Substrate B contaFE 20% acetone, keep this reagent away from sources of heat or flame.
13. 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 50 μL of Standards or Samples to the appropriate well of the antibody pre-coated Microtiter Plate.
3. Add 10 μL of Biological to samples well.（DO not add Biological to standards well）.
4. Add 100 μL of Enzyme Conjugate to each well. Mix well. Complete mixing in this step is important. Cover and incubate for 1 hour at 37°C.
5. Prepare Substrate Solution no more than 15 minutes before end of incubation （see Preparation of Reagents）.
6. Wash the Microtiter Plate using one of the specified methods indicated below:
7. 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.
8. 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.
9. Add 50 μL Substrate A&B to each well. Cover and incubate for 15 minutes at 20-25°C.
10. Add 50 μL of Stop Solution to each well. Mix well.
11. Read the Optical Density （O.D.） at 450 nm using a microtiter plate reader within 30 minutes.

CALCULATION OF RESULTS

1. This standard curve is used to determine the amount in an unknown sample. The standard curve is generated by plotting the average O.D. （450 nm） obtained for each of the six standard concentrations on the vertical （Y） axis versus the corresponding concentration on the horizontal （X） axis.
2. First, calculate the mean O.D. value for each standard and sample. All O.D. values, are subtracted by the mean value of the zero standard before result interpretation. Construct the standard curve using graph paper or statistical software.
3. To determine the amount in each sample, first locate the O.D. value on the Y-axis and extend a horizontal line to the standard curve. At the point of intersection, draw a vertical line to the X-axis and read the corresponding concentration.
4. Any variation in operator, pipetting and washing technique, incubation time or temperature, and kit age can cause variation in result. Each user should obtain their own standard curve.
5. The sensitivity by this assay is 10 pmol/L.
6. Standard curve

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