本试剂盒只能用于科学研究，不得用于医学诊断
人（Human）白细胞介素 6（IL-6）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
先包被白细胞介素6（IL-6）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，经过温育并彻底洗涤。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白细胞介素6（IL-6）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。
样品收集、处理及保存方法
1. 血清：使用不含热原和内毒素的试管，操作过程中避免任何细胞
刺激，收集血液后，3000 转离心 10 分钟将血清和红细胞迅速小心地
分离。
2. 血浆：EDTA、柠檬酸盐或肝素抗凝。3000 转离心 30 分钟取上清。
3. 细胞上清液：3000 转离心 10 分钟去除颗粒和聚合物。
4. 组织匀浆：将组织加入适量生理盐水捣碎。3000 转离心 10 分钟
取上清。
5. 保存：如果样本收集后不及时检测，请按一次用量分装，冻存于
-20℃，避免反复冻融，在室温下解冻并确保样品均匀地充分解冻。
自备物品
1. 酶标仪（450nm）
2. 高精度加样器及枪头：0.5-10uL、2-20uL、20-200uL、200-1000uL
3. 37℃恒温箱
操作注意事项
1. 试剂盒保存在 2-8℃，使用室温平衡 20 分钟。从冰箱取出的
浓缩洗涤液会有结晶，这属于正常现象，水浴加热使结晶完全溶解
后再使用。
2. 实验中不用的板条应立即放回自封袋中，密封（低温干燥）保存。
3. 浓度为 0 的 S0 号标准品即可视为阴性对照或者空白；按照说明
书操作时样本已经稀释 5 倍，终结果乘以 5 才是样本实际浓度。
4. 严格按照说明书中标明的时间、加液量及顺序进行温育操作。
5. 所有液体组分使用充分摇匀。
试剂盒组成
名称 96 孔配置 48 孔配置 备注
微孔酶标板 12 孔×8 条 12 孔×4 条 无
标准品 0.3mL*6 管 0.3mL*6 管 无
样本稀释液 6mL 3mL 无
检测抗体-HRP 10mL 5mL 无
20×洗涤缓冲液 25mL 15mL 按说明书进行稀释
底物 A 6mL 3mL 无
底物 B 6mL 3mL 无
终止液 6mL 3mL 无
封板膜 2 张 2 张 无
说明书 1 份 1 份 无
自封袋 1 个 1 个 无
注：标准品（S0-S5）浓度依次为：0、3、6、12、24、48 pg/mL
试剂的准备
20×洗涤缓冲液的稀释：蒸馏水按 1：20 稀释，即 1 份的 20×洗涤缓
冲液加 19 份的蒸馏水。
洗板方法
1. 手工洗板：甩尽孔内液体，每孔加满洗涤液，静置 1min 后甩尽
孔内液体，在吸水纸上拍干，如此洗板 5 次。
2. 自动洗板机：每孔注入洗液 350μL，浸泡 1min，洗板 5 次。
操作步骤
1. 从室温平衡 20min 后的铝箔袋中取出所需板条，剩余板条用自封
袋密封放回 4℃。
2. 设置标准品孔和样本孔，标准品孔各加不同浓度的标准品 50μL；
3. 样本孔先加待测样本 10μL，再加样本稀释液 40μL；空白孔不
加。
4. 除空白孔外，标准品孔和样本孔中每孔加入辣根过氧化物酶
（HRP）标记的检测抗体 100μL，用封板膜封住反应孔，37℃水浴
锅或恒温箱温育 60min。
5. 弃去液体，吸水纸上拍干，每孔加满洗涤液，静置 1min，甩去
洗涤液，吸水纸上拍干，如此重复洗板 5 次（也可用洗板机洗板）。
6. 每孔加入底物 A、B 各 50μL，37℃避光孵育 15min。
7. 每孔加入终止液 50μL，15min 内，在 450nm 波长处测定各孔的
OD 值。
结果判断
绘制标准曲线：在 Excel 工作表中，以标准品浓度作横坐标，对应
OD 值作纵坐标，绘制出标准品线性回归曲线，按曲线方程计算各样
本浓度值。
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：低检测浓度小于 1.0 pg/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. Human Interleukin 6 (IL-6) ELISA Kit instruction
Intended use
This IL-6 ELISA 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 IL-6 in the sample, this
IL-6 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 IL-6 concentration. The concentration of
IL-6 in the samples is then determined by comparing the O.D. of the samples to the
standard curve.
Sample collection 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. Do not substitute reagents from one kit to another. Standard, conjugate and
microplates are matched for optimal performance. Use only the reagents supplied by
manufacturer. 2. Do not remove microplate from the storage bag until needed. Unused strips
should be stored at 2-8°C in their pouch with the desiccant provided.
3. Mix all reagents before using.
Remove all kit reagents from refrigerator ａnd allow them to reach room temperature
( 20-25°C)
Materials supplied
Name 96 determinations 48 determinations
Microelisa stripplate 12*8strips 12*4strips
Standard 0.3ml*6tubes 0.3ml*6tubes
Sample Diluent 6.0ml 3.0ml
HRP-Conjugate reagent 10.0ml 5.0ml
20X Wash solution 25ml 15ml
Chromogen Solution A 6.0ml 3.0ml
Chromogen Solution B 6.0ml 3.0ml
S Solution 6.0ml 3.0ml
Closure plate membrane 2 2
User manual 1 1
Sealed bags 1 1
Note: Standard (S0 → S5) concentration was followed by:0,3,6,12,24,48 pg/ml
Reagent preparation
20×wash solution:Dilute with Distilled or deionized water 1:20.
Assay procedure
1. Prepare all r e a g e n ts before starting assay procedure. It is recommended that all Standards ａnd Samples be added in duplicate to the Microelisa Stripplate.
2. Add standard: Set Standard wells, testing sample wells. Add standard 50μl to
standard well. 3. Add Sample: Add testing sample 10μl then add Sample Diluent 40μl to testing
sample well; Blank well doesn’t add anyting.
4. Add 100μl of HRP-conjugate reagent to each well, cover with an adhesive strip
and incubate for 60 minutes at 37°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,
manifold dispenser or autowasher. Complete removal of liquid at each step is
essential to good performance. After the last wash, remove any remaining Wash
Solution by aspirating or decanting. 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 to yellow. 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. Read the Optical Density (O.D.) at 450 nm using a microtiter plate reader
within 15 minutes. 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 is 1.0 pg/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!

原创作者：上海诚竺生物科技有限公司