人乙酰胆碱 ACH ELISA试剂盒 北京驰明瑞

人乙酰胆碱ACH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！2.微量加液器、吸头3.蒸馏水或去离子水以及滤纸操作步骤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50μl的标准品溶液于空白微孔中。

3.空白微孔中加入50μl的样品，空白对照加入50μl的蒸馏水；4.在样品孔中加入10μl的生物素；（不含空白对照孔,切忌：生物素只加样品孔！）5.在各孔中加入100μl的酶标记溶液；（不含空白对照孔）6.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）7.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）8.酶标板洗涤后用吸水纸彻底拍干；9.各孔加入显色剂A、B液各50μl；（不含空白对照孔）10.20-25℃下避光反应15分钟；11.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.以OD值为纵坐标，以标准品的浓度为横坐标，绘制曲线图；3.根据样品的OD值查找对应的浓度范围；4.敏感度：0.01 ng/ml5.图例Human Acetylcholine ELISA Kit48TestsCatalogue 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 ACH 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 ACH in the sample, this ACH 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 ACH concentration. The concentration of ACH in the samples is then determined by comparing the O.D. of the samples to the standard curve.PRINCIPLE OF THE ASSAYThis ACH enzyme linked immunosorbent assay applies a technique called a quantitative sandwich immunoassay. The microtiter plate provided in this kit has been pre-coated with a monoclonal antibody specific for ACH. Standards or samples are then added to the microtiter plate wells and ACH if present, will bind to the antibody pre-coated wells. In order to quantitatively determine the amount of ACH present in the sample, a standardized preparation of horseradish peroxidase (HRP)-conjugated polyclonal antibody, specific for ACH are added to each well to “sandwich” theACH immobilized on the plate. The microtiter plate undergoes incubation, and then the wells are thoroughly washed to remove all unbound components. Next, A and B substrate solution is added to each well. The enzyme (HRP) and substrate are allowed to react over a short incubation period.Only those wells that contain ACH and enzyme-conjugated antibody will exhibit a change in color. The enzyme-substrate reaction is terminated by the addition of a sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450 nm.REAGENTS PROVIDEDAll reagents provided are stored at 2-8° C. Refer to the expiration date on the label.1.MICROTITER PLATE 48 wells2.ENZYME CONJUGATE 10.0 mL 1 vial3.BIOTIN SOLUTION 1.0ml 1vial4.STANDARD.1 0ng/ml 1 vial5.STANDARD.2 0.5 ng/ml 1 vial6.STANDARD.3 1.0 ng/ml 1 vial7.STANDARD.4 2.5 ng/ml 1 vial8.STANDARD.5 5.0 ng/ml 1 vial9.STANDARD.6 10.0 ng/ml 1 vial10.SUBSTRATE A 6.0 mL 1 vial11.SUBSTRATE B 6.0 mL 1 vial12.STOP SOLUTION 6.0 mL 1 vial13.WASH SOLUTION x100 10 mL 1 vial14.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 de sired number of coated wells in the holder, then add 50 μL ofthe appropriate well of the antibody pre-coated Microtiter Plate.3.Add 10 μL of Biotin to Samples well.4.Add 100 μL of Conjugate to each well. Mix well. Complete mixing in this step is important. Cover andincubate for 1 hours 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 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.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 fil l 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.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.BOGOO CALCULATION OF RESULTS1.This standard curve is used to determine the amount in an unknown sample. The standard curve is generatedby 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 themean value of the zero standard before result interpretation. Construct the standard curve using graph paper orstatistical software.3.To determine the amount in each sample, first locate the O.D. value on the Y-axis and extend a horizontal lineto the standard curve. At the point of intersection, draw a vertical line to the X-axis and read thecorresponding concentration.4.Any variation in operator, pipetting and washing technique, incubation time or temperature, and kit age cancause variation in result. Each user should obtain their own standard curve.5.The sensitivity by this assay is 0.01 ng/ml6.Standard curve。