人 T 细胞抑制因子 B7H4 ELISA试剂盒北京驰明瑞使用说明书

人T细胞抑制因子B7H4使用说明书产品编号：本试剂盒仅供科研使用，不得用于临床及诊断使用！操作步骤1.取出试剂盒，于室温（20-25℃）放置15-30分钟。

实验过程应在室温（20-25℃）内进行。

2.取出酶标板，按照标准品的次序分别加入100μl的标准品溶液于空白微孔中。

3.空白微孔中加入100μl的样品，空白对照加入100μl的蒸馏水；4.在各孔中加入50μl的酶标记溶液；（不含空白对照孔）5.将酶标板用封口胶密封后，37℃孵育反应1小时；（在孵育箱中保持稳定的温度与湿度）6.充分清洗酶标板3-5次，保持各孔有充足的水压；（浓缩洗涤液以1：100的比例与蒸馏水稀释）7.酶标板洗涤后用吸水纸彻底拍干；8.各孔加入显色剂A、B液各50μl；（不含空白对照孔）9.20-25℃下避光反应15分钟；10.各孔加入50μl终止液，终止反应；结果判断1.30分钟内在波长450nm的酶标仪上读取各孔的OD值；2.百分结合率计算：设S0管计数为B0，各标准管或样品管计数为B，非特异管计数为NSB，则百分结合率计算公式如下：B/ B0=(B－NSB)/( B0－NSB)×100%3.logit计算:各标准点或样品管的logit值计算公式如下:logit=ln(B/ B0)/(1－B/ B0)4.将标准品的OD均值与标准品0点的OD均相除，为标准点的百分结合率，在log-logit坐标纸上绘图。

5.Log-logit双对数标准曲线：坐标纸上横轴从左至右第一个1-9表示为第一个10进位，第二个1-9表示为第二个10进位。

第三个1-9表示为第三个10进位。

坐标纸纵轴为百分比（1-99），即各标准吸光值的百分结合率。

取一条通过各点的直线。

要求尽可能多的点在线上，同时剩余的点均匀分布在直线的两边。

样品也同样由吸光值计算百分结合率，再从纵轴上的相应结合率找到直线上的点，此点对应的横坐标浓度即为样品的浓度，无须换算。

6.人工处理：以标准浓度取log值为横坐标，对应的logit值为纵坐标在普通坐标纸上或以标准浓度为横坐标，对应的B/B0为纵坐标在logit-log坐标纸上画出标准曲线（理想化时是一条直线）。

根据待测样品的B/B0可以从坐标纸上查出样品的浓度值。

如果使用普通坐标纸，查出的数值应取反对数才是最后的浓度值。

7.自动处理：使用logit-log或四参数数据处理模式，由电脑自动计算得出结果。

8.敏感度：1.0ng/ml；9.图例Human B7H4 ELISA Kit96 TestsCatalogue Number:Store all reagents at 2-8°CFOR LABORATORY RESEARCH USE ONLY. NOT FOR THERAPEUTIC OR DIAGNOSTIC APPLICATIONS!PLEASE READ THROUGH ENTIRE PROCEDURE BEFORE BEGINNING!INTENDED USEThis BOGOO B7H4 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 B7H4 in the sample, this B7H4 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 B7H4 concentration. The concentration of B7H4 in the samples is then determined by comparing the O.D. of the samples to the standard curve.PRINCIPLE OF THE ASSAYThe coated well immunoenzymatic assay for the quantitative measurement of serum B7H4 utilizes a monoclonal anti-B7H4 and a B7H4-HRP conjugate. The assay asample and buffer are incubated together with anti-B7H4 antibody coated plate for sixty and washed. The diluted B7H4-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 B7H4 concentration since B7H4 from samples and B7H4-HRP conjugate compete for the anti-B7H4 antibody binding site. Since the number of sites is limited, as more sites are occupied by B7H4 from the sample, fewer sites are left to bind B7H4-HRP conjugate.Standards of known B7H4 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 B7H4. The unknown B7H4 concentration in each sample is interpolated from this curve.REAGENTS PROVIDEDAll reagents provided are stored at 2-8° C. Refer to the expiration date on the label.1.MICROTITER PLATE 96 wells2.ENZYME CONJUGATE 6.0 mL 1 vial3.STANDARD.1 0ng/ml 1 vial4.STANDARD.2 50ng/ml 1 vial5.STANDARD.3 100ng/ml 1 vial6.STANDARD.4 250ng/ml 1 vial7.STANDARD.5 500ng/ml 1 vial8.STANDARD.6 1000ng/ml 1 vial9.SUBSTRATE A 6.0 mL 1 vial10.SUBSTRATE B 6.0 mL 1 vial11.STOP SOLUTION 6.0 mL 1 vial12.WASH SOLUTION x100 10 mL 1 vial13.Instruction 1MATERIALS REQUIRED BUT NOT SUPPLIED1.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-channelpipette 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 asdesired.11.Tubes to prepare standard or sample dilutions.ASSAY PROCEDURE1. 1 .Prepare all Standards before starting assay procedure (see Preparation Reagents). It is recommended thatall Standards and Samples be added in duplicate to the Microtiter Plate.Standards or Samples to2.First, secure the desired number of coated wells in the holder, then add 100 μL ofthe 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 andincubate 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 wastecontainer. Using a squirt bottle, fill each well completely with distilled or de-ionized water, then aspiratecontents of the plate into a sink or proper waste container. Repeat this procedure four more times for a totalof FIVE washes. After final wash, invert plate, and blot dry by hitting plate onto absorbent paper or papertowels until no moisture appears. Note: Hold the sides of the plate frame firmly hen washing the plate toassure 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 paper towels until no moisture appears. It is recommended that the washer be set for a soaking time of 10seconds 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.BOGOO CALCULATION OF RESULTS1.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 standard0and 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 correspondingconcentrations 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 factorin 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 standardcurve.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 1.0ng/ml7.The standard curve presented here is an example of the data typically produced with this kit; however, yourresults will not be identical to these.8.Standard curve。