人（Human）Ⅶ型胶原（ColⅦ）ELISA检测试剂盒

使用说明书

检测原理

试剂盒采用双抗体一步夹心法酶联免疫吸附试验（ELISA）。往预先包被Ⅶ型胶原（ColⅦ）抗体的包被微孔中，依次加入标本、标准品、HRP标记的检测抗体，经过温育并洗涤。用底物TMB显色，TMB在过氧化物酶的催化下转化成蓝色，并在酸的作用下转化成终的黄色。颜色的深浅和样品中的Ⅶ型胶原（ColⅦ）呈正相关。用酶标仪在450nm 波长下测定吸光度（OD 值），计算样品浓度。

试剂盒性能

1. 准确性：标准品线性回归与预期浓度相关系数R值，大于等于0.9900。

2. 灵敏度：检测浓度小于0.1 ng/mL。

3. 特异性：不与其它可溶性结构类似物交叉反应。

4. 重复性：板内、板间变异系数均小于15%。

5. 贮藏：2-8℃，避光防潮保存。

6. 有效期：6个月

免责声明

1. 试剂盒仅供研究使用，不得用于临床实验或人体实验，否则所产生的一切后果，由实验者承担，本公司概不负责。

2. 严格按照说明书操作，实验者违反说明书操作，后果由实验者承担。

FOR RESEARCH USE ONLY.

NOT FOR USE IN DIAGNOSTIC PROCEDURES.

HumanCollagen TypeⅦ(ColⅦ) ELISA Kit instruction

Intended use

This ColⅦELISA kit is intended Laboratory for Research use only ａnd is not for use in diagnostic or therapeutic procedures.The S 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 ColⅦin the sample, this ColⅦELISA 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 ColⅦconcentration. The concentration of ColⅦin 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 for 10 minutes at approximately 3000×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 for 30 minutes at 3000×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 are matchedfor optimal performance. Useonly thereagentssuppliedby manufacturer.

2. Donot removemicroplatefrom the storage baguntilneeded. Unusedstripsshouldbe stored at2-8°Cin their pouchwith the desiccantprovided.

3. Mix all reagents before using.

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

Materials supplied

Note: Standard (S0→ S5) concentration was followed by:0,1.5,3,6,12,24 ng/ml.

Reagent preparation

20×wash solution:Dilute with Distilled or deionized water1:20.

Assay procedure

1. Prepare allreagentsbeforestartingassayprocedure. ItisrecommendedthatallStandardsand Samplesbe addedin duplicateto the MicroelisaStripplate.

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

3. Add Sample: Add Sample10μl then add Sample 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 process four times for a total of five washes.Wash by filling each well with Wash Solution (400μl) using a squirt bottle, manifolddispenseror autowasher. Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Solution 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 mix and incubate for 15 minutes at 37°C. Protect from light.

7. Add 50μl S 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.)at450nmusinga microtiterplatereaderwithin15minutes.

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 is0.1ng/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!