FOR RESEARCH USE ONLY.

NOT FOR USE IN DIAGNOSTIC PROCEDURES.

MouseLegumain (LGMN) ELISA Kit instruction

Intended use

ThisLGMN 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 ofLGMN in the sample, thisLGMN 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 versusLGMN concentration. The concentration ofLGMN in the samples is then determined by comparing the O.D. of the samples to the standard curve.

Samplecollection and storages

Serum - Use a serum separator tube and allow samples to clot for 30 minutes before centrifugation for10 minutes at approximately3000×g. Remove serum and assay immediately 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 for30 minutes at3000×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 immediately or aliquot and store samples at -20℃or -80℃. Avoid repeated freeze-thaw cycles.

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

Materials required but not supplied

1. Standard microplate reader(450nm)

2. Precision pipettes and Disposable pipette tips.

3. 37 ℃ incubator

Precautions

1.Donot substitutereagentsfromone kit to another.Standard, conjugateandmicroplates arematchedfor optimal performance. Useonlythereagentssuppliedbymanufacturer.

2. Donot removemicroplatefrom thestorage baguntilneeded. Unusedstripsshouldbestored at2-8°Cintheir pouchwith the desiccantprovided.

3.Mix all reagents before using.

Remove allkit reagentsfrom refrigerator and allow them toreachroom temperature( 20-25°C)

Materials supplied

Note:Standard (S0→S5) concentration was followedby:0,7.5,15,30,60,120 ng/ml.

Reagent preparation

20×wash solution:Dilute withDistilled or deionized water1:20.

Assay procedure

1.Prepare allreagentsbeforestartingassayprocedure. ItisrecommendedthatallStandardsand Samplesbe addedin duplicatetotheMicroelisaStripplate.

2.Addstandard:Set Standard wells,testing sample wells.Add standard 50μl to standard well.

3. AddSample: Add testing sample10μlthen addSample Diluent 40μl to testing sample well; Blank welldoesn’t add anyting.

4. Add100μlofHRP-conjugate reagentto each well,cover with an adhesive stripandincubatefor60 minutes at37°C.

5.Aspirate each well and wash, repeating the processfour times for a total of fivewashes.Wash by filling each well with WashSolution (400μl) using a squirt bottle, manifolddispenseror autowasher. Complete removal of liquid at each step is essential to goodperformance. After the last wash, remove any remaining WashSolution by aspirating ordecanting. Invert the plate and blot it against clean paper towels.

6.Add chromogen solution A 50μl and chromogen solution B 50μl to each well. Gently mixandincubate for 15 minutes at37°C.Protect from light.

7.Add 50μlStop Solution to each well. The color in the wells should change from blue toyellow. If the color in the wells is green or the color change does not

appear uniform,gently tap the plate to ensure thorough mixing.

8.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 (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 is1.0 ng/ml

6. Standard curve

Storage： 2-8℃.

validity：six months.

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