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1、 1 人免疫球蛋白 IgG 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！ 试 剂 组 成 名称 规格 数量 保存 包被平底微孔板 96 孔 1 可拆卸板 2-8密封冷藏 酶结合物 6 毫升 1 瓶 2-8冷藏 标准品 1 毫升 6 瓶 2-8冷藏 显色剂 A 6 毫升 1 瓶 2-8冷藏 显色剂 B 6 毫升 1 瓶 2-8避光冷藏 终止液 6 毫升 1 瓶 2-8冷藏 浓缩洗涤液（100 倍 稀释） 10 毫升 1 瓶 2-8冷藏 使用说明书 1 份 自备物品 1.酶标仪（尽量提前预热） 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
2、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 100l 的标准品溶液于空白微孔中。 3. 空白微孔中加入 100l 的样品，空白对照加入 100l 的蒸馏水； 4. 在各孔中加入 50l 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 50l ；
3、（不含空白对照孔） 9. 20-25下避光反应 15 分钟； 10. 各孔加入 50l 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=(BNSB)/( B0NSB)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)/(1B/ 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-l
4、ogit 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2
5、 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 3 Human immunoglobulin G ELISA Kit 96 Tests Catalogue Number: HEI018 Store all reagents at 2-8 C FOR LABORATORY RESEAR
6、CH USE ONLY. NOT FOR THERAPEUTIC OR DIAGNOSTIC APPLICATIONS!PLEASE READ THROUGH ENTIRE PROCEDURE BEFORE BEGINNING! INTENDED USE This BOGOO IGG 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 bl
7、ue to yellow and the intensity of the color is measured at 450 nm using a spectrophotometer. In order to measure the concentration of IGG in the sample, this IGG ELISA Kit includes a set of calibration standards. The calibration standards are assayed at the same time as the samples and allow the ope
8、rator to produce a standard curve of Optical Density versus IGG concentration. The concentration of IGG in the samples is then determined by comparing the O.D. of the samples to the standard curve. PRINCIPLE OF THE ASSAY The coated well immunoenzymatic assay for the quantitative measurement of serum
9、 IGG utilizes a monoclonal anti-IGG and a IGG-HRP conjugate. The assay asample and buffer are incubated together with anti-IGG antibody coated plate for sixty and washed. The diluted IGG-HRP conjugate is then added to each well and incubated. After the incubation period, the wells are decanted and w
10、ashed 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 spectrop
11、hotometrically at 450nm in a microplate reader. The intensity of the color is inversely proportional to the IGG concentration since IGG from samples and IGG-HRP conjugate compete for the anti-IGG antibody binding site. Since the number of sites is limited, as more sites are occupied by IGG from the
12、sample, fewer sites are left to bind IGG-HRP conjugate.Standards of known IGG 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 IGG. The unknown IGG concentration in each sa
13、mple is interpolated from this curve. REAGENTS PROVIDED All reagents provided are stored at 2-8 C. Refer to the expiration date on the label. 1. MICROTITER PLATE 96 wells 4 2. ENZYME CONJUGATE 6.0 mL 1 vial 3. STANDARD.1 0ng/ml 1 vial 4. STANDARD.2 50ng/ml 1 vial 5. STANDARD.3 100ng/ml 1 vial 6. ST
14、ANDARD.4 250ng/ml 1 vial 7. STANDARD.5 500ng/ml 1 vial 8. STANDARD.6 1000ng/ml 1 vial 9. SUBSTRATE A 6.0 mL 1 vial 10. SUBSTRATE B 6.0 mL 1 vial 11. STOP SOLUTION 6.0 mL 1 vial 12. WASH SOLUTION x100 10 mL 1 vial 13. Instruction 1 MATERIALS REQUIRED BUT NOT SUPPLIED 1. Microplate reader capable of m
15、easuring 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, prefer
16、ably with disposable plastic tips. (A manifold multi-channel pipette 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 as desired. 11. Tubes to prepare standard or sample dilu
