Human GSDMD ELISA Kit操作方法

Human GSDMD ELISA 操作方法

INTENDEDUSEAND TEST PRINCIPLE

ThisGSDMDELISA 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 ofGSDMDin the sample, thisGSDMDELISA 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 versusGSDMDconcentration. The concentration ofGSDMDin the samples is then determined by comparing the O.D. of the samples to the standard curve.

SAMPLE COLLECTION AND STORAGES

Serum- Use a serum separator tube and allow samples to clot for two hours at room temperature or overnight at4℃before centrifugation for 20 minutes at approximately 1000×g. Assay freshly prepared serumimmediately or store samples in aliquot at -20℃or -80℃for later use. Avoid repeated freeze/thaw cycles.

Plasma- Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples for 15 minutes at1000×g at 2-8℃within 30 minutes of collection. Remove plasma and assay immediately or store samplesin aliquot at -20℃or -80℃for later use. Avoid repeated freeze/thaw cycles.

Tissue homogenates- For general information, hemolysis blood may affect the result, so you shouldrinse the tissues with ice-cold PBS (0.01M, pH=7.4) to remove excess blood thoroughly. Tissuepieces should be weighed and then minced to small pieces which will be homogenized in PBS (thevolume depends on the weight of the tissue. 9mL PBS would be appropriate to 1 gram tissue pieces.Some protease inhibitor is recommended to add into the PBS.) with a glass homogenizer on ice. Tofurther break the cells, you can sonicate the suspension with an ultrasonic cell disrupter or subject it tofreeze-thaw cycles. The homogenates are then centrifugated for 5minutes at 5000×g to get thesupernate.

Cell culture supernatesand other biological fluids -Centrifuge samples for 20 minutes at 1000×g. Removeparticulates and assay immediately or store samples in aliquot at -20℃or -80℃for later use. Avoid repeatedfreeze/thaw cycles.

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

MATERIALSREQUIREDBUTNOT SUPPLIED

1.37 ℃ incubator

2.Standard microplate readercapableofmeasuringabsorbanceat450nm

3.Precision pipettes, disposable pipette tipsandAbsorbent paper

4.Distilled or deionized water

REAGENTS PROVIDED

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

Note:

1.Standard concentrationwas followed by:20, 10, 5, 2.5, 1.25, 0.625ng/mL.

2.If samples generate values higher than the highest standard,please dilute thesamples with Sample Diluent and repeat the assay.

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 fromRatblood 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 contain20% 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).

REAGENT PREPARATION ANDSTORAGE

WashSolution(1X) -Dilute 1 volume of Washsolution(20X) with19volumes of deionized or distilled water. WashSolutionis stable for 1 month at 2-8°C.

ASSAY PROCEDURE

1.Prepare all reagents before starting assay procedure. It is recommended that all Standards and Samples be added in duplicate to theMicrotiter plate.

2.Add50μlof Standard or Sample to the appropriate wells.Blank welldoesn’t add anyting.

3.Add100μlofEnzymeconjugate to standard wellsandsample wellsexcept the blank well,cover with anadhesive stripandincubatefor60 minutesat37°C.

4.Wash the Microtiter Plate 4 times.

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 completely with WashSolution(1X), then aspirate contents of the plate into a sink or proper waste container. Repeat this procedure for a total offourtimes. 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 firmlywhen washing the plate to assure that all strips remain securely in frame.

Automated Washing-Aspirate all wells, then wash platesfourtimes using WashBuffer(1X). Always adjust your washer to aspirate as much liquid as possible and set fill volume at 350μL/well/wash. After final wash, invert plate, and blot dry by hitting plate onto absorbent paper or paper towels until no moisture appears.

5.AddSubstrateA 50μl andSubstrateB 50μl toeach well.Gently mixandincubate for 15 minutes at37°C.Protect from light.

6.Add 50μlStop Solution toeach well. The color in the wells should change from blue toyellow. If the color in the wells is green or the color change does notappear uniform,gently tap the plate to ensure thorough mixing.

7.ReadtheOpticalDensity(O.D.)at450nmusingamicrotiterplatereaderwithin15minutes.

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 (X) axis versus the corresponding concentration on the horizontal (Y) axis.

2.First, calculate the mean O.D. value for each standard and sample. All O.D. Valuesare subtracted by the mean value of thebalnk wellbefore 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.Intra-assay CV(%)is less than 10% and Inter-assayCV(%)is less than 15%.

6.Assay range:0.625ng/mL–20ng/mL.

7. Sensitivity: The minimum detectable dose ofHumanGSDMDis typically less than0.1ng/mL.

8. Cross-reactivity: This assay recognizes recombinant and naturalHumanGSDMD. No significantcross-reactivity or interference was observed.

9.Storage: 2-8℃ (Use frequently); six months (-20℃)。

10.Standard curve

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