CSB-E13093h人转铁蛋白（TF）ELISA试剂盒说明书

 Human transferrin（TF） ELISA Kit

（96T）

This immunoassay kit allows for the in vitro quantitative determination of human TF concentrations in serum, plasma.

Expiration date six months from the date of manufacture

 

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The microtiter plate provided in this kit has been pre-coated with an antibody specific to TF. Standards or samples are then added to the appropriate microtiter plate wells with a HRP-conjugated antibody preparation specific for TF to each microplate well and incubated. Then a TMB （3,3',5,5' tetramethyl-benzidine） substrate solution is added to each well. Only those wells that contain TF, HRP-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 ± 2 nm. The concentration of TF in the samples is then determined by comparing the

O.D. of the samples to the standard curve.

0.023nmol/L-1.5nmol/L. The standard curve concentrations used for the

ELISA’s were 1.5nmol/L, 0.75nmol/L, 0.375nmol/L, 0.187nmol/L,

0.094nmol/L, 0.047nmol/L, 0.023nmol/L.

This assay recognizes human TF. No significant cross-reactivity or interference was observed.

The minimum detectable dose of human TF is typically less than 0.006nmol/L.The sensitivity of this assay, or Lower Limit of Detection （LLD） was defined as the lowest protein concentration that could be differentiated from zero.

Assay plate 1 Standard 2 Sample Diluent 2 x 20 ml HRP-conjugate Diluent 1 x 20 ml HRP-conjugate 1 x 120μl

1 x 20 ml

Wash Buffer

（25×concentrate）

TMB Substrate 1 x 10 ml

Stop Solution 1 x 10 ml

1          Unopened test kits should be stored at 2-8C upon receipt and the microtiter plate should be kept in a sealed bag with desiccants to minimize exposure to damp air. The test kit may be used throughout the expiration date of the kit. Refer to the package label for the expiration date.

2          A microtiter plate reader with a bandwidth of 10 nm or less and an optical density range of 0-3 OD or greater at 450nm wavelength is acceptable for use in absorbance measurement.

 

1          Wash Buffer If crystals have formed in the concentrate, warm up to room temperature and mix gently until the crystals have compley dissolved. Dilute 20 ml of Wash Buffer Concentrate into deionized or distilled water to prepare 500 ml of Wash Buffer.

2          Standard Centrifuge the standard vial at 6000-10000rpm for 30s. Reconstitute the Standard with 1.0 ml of Sample Diluent. This reconstitution produces a stock solution of 1.5nmol/L.Allow the standard to sit for a minimum of 15 minutes with gentle agitation prior to making serial dilutions. The undiluted standard serves as the high standard （1.5nmol/L）. The Sample Diluent serves as the zero standard （0nmol/L）. Prepare fresh for each assay. Use within 4 hours and discard after use.

3          HRP-conjugate Dilute to the working concentration using HRP-conjugate Diluent（1:100）, respectively. The suggested 100-fold dilution can be achieved by adding 10 uL HRP-conjugate to 990uL of HRP-conjugate Diluent for 1ml working solution.

 

1           Microplate reader capable of measuring absorbance at 450 nm, with the correction wavelength set at 540 nm or 570 nm.

2           Pipettes and pipette tips.

3           Deionized or distilled water.

4         • Squirt bottle, manifold dispenser, or automated microplate washer. SAMPLE COLLECTION AND STORAGE

5           Serum Use a serum separator tube （SST） and allow samples to clot for 30 minutes before centrifugation for 15 minutes at 1000 x g. Remove serum and assay immediay or aliquot and store samples at -20°C. Avoid repeated freeze-thaw cycles.

6           Plasma Collect plasma using citrate, EDTA, or heparin as an anticoagulant. Centrifuge for 15 minutes at 1000 x g within 30 minutes of collection. Assay immediay or aliquot and store samples at-20°C. Avoid repeated freeze-thaw cycles.

 

Recommend to dilute the serum or plasma samples with Sample Diluent（1:20000） before test. The suggested 20000-fold dilution can be

achieved by adding 1μl sample to 99μl of Sample Diluent, Complete the 20000-fold dilution by adding 1μl of this solution to 199μl of Sample Diluent. The recommended dilution factor is for reference only. The optimal dilution factor should be determined by users according to their particular experiments.

1          Add 100μl Sample Diluent serves as the zero standard, Add 100μl of Standard or Sample per well. Cover with the adhesive strip. Incubate for 60min at 37°C.

2          Aspirate each well and wash, repeating the process three times for a total of three washes. Wash by filling each well with Wash Buffer （200μl） using a squirt bottle, multi-channel pipette, manifold dispenser or autowasher. Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against clean paper towels.

3          Add 100μl of HRP-conjugate working solution to each well. Incubate for 60min at 37°C. HRP-conjugate working solution may appear cloudy. Warm up to room temperature and mix gently until solution appears uniform.

4          Repeat the aspiration and wash five times as before.

5          Add 90μl of TMB Substrate to each well. Incubate for 20 minutes at 37°C. Keeping the plate away from drafts and other temperature fluctuations in the dark.

6          Add 50μl of Stop Solution to each well when the first four wells containing the highest concentration of standards develop obvious blue color. If color change does not appear uniform, gently tap the plate to ensure thorough mixing.

7          Determine the optical density of each well within 30 minutes, using a microplate reader set to 450 nm.

 

Average the duplicate readings for each standard, control, and sample and subtract the average zero standard optical density. Create a standard curve by reducing the data using computer software capable of generating a four parameter logistic （4-PL） curve-fit. As an alternative, construct a standard curve by plotting the mean absorbance for each standard on the x-axis against the concentration on the y-axis and draw a best fit curve through the points on the graph. The data may be linearized by plotting the log of the TF concentrations versus the log of the O.D. and the best fit line can be determined by regression analysis. This procedure will produce an adequate but less precise fit of the data. If samples have been diluted, the concentration read from the standard curve must be multiplied by the dilution factor.

1           The kit should not be used beyond the expiration date on the kit label.

2           Do not mix or substitute reagents with those from other lots or sources.

3           Any variation in operator, pipetting technique, washing technique, incubation time or temperature, and kit age can cause variation in binding.

4         • This assay is designed to eliminate interference by soluble receptors, binding proteins, and other factors present in biological samples. Until all factors have been tested in the Immunoassay, the possibility of interference cannot be excluded. TECHNICAL HINTS

5           When mixing or reconstituting protein solutions, always avoid foaming.

6           To avoid cross-contamination, change pipette tips between additions of each standard level, between sample additions, and between reagent additions. Also, use separate reservoirs for each reagent.

7           When using an automated plate washer, adding a 30 second soak period following the addition of wash buffer, and/or rotating the plate 180 degrees between wash steps may improve assay precision.

8           To ensure accurate results, proper adhesion of plate sealers during incubation steps is necessary.

9           Substrate Solution should remain colorless until added to the plate. Keep Substrate Solution protected from light. Substrate Solution should change from colorless to gradations of blue.

10       Stop Solution should be added to the plate in the same order as the Substrate Solution. The color developed in the wells will turn from blue to yellow upon addition of the Stop Solution. Wells that are green in color indicate that the Stop Solution has not mixed thoroughly with the Substrate Solution.

 

人转铁蛋白（TF）酶联免疫分析试剂盒使用说明书

本试剂盒仅供研究使用

产品编号：CSB-E13093h

检测范围：0.023nmol/L –1.5nmol/L

zui低检测限：0.006nmol/L

特异性：本试剂盒可检测人 TF，且与其他相关蛋白无交叉反应。有效期：6个月预期应用：ELISA法定量测定人血清、血浆中 TF含量。

说明

1          2-8℃。

2          中、英文说明书可能会有不一致之处，请以英文说明书为准。

3          刚开启的酶联板孔中可能会含有少许水样物质，此为正常现象，不会对实验结果造成任何影响。

 

实验原理

用纯化的抗体包被微孔板，制成固相载体，往包被抗 TF抗体的微孔中加入标本或标准品、HRP标记的抗 TF抗体，经过*洗涤后用底物显色。底物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zui终的黄色。颜色的深浅和样品中的 TF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。

试剂盒组成及试剂配制

1.酶联板 （Assay plate ）：一块（96孔）。

2.标准品（Standard）： 2瓶（冻干品）。

3.酶结合物（ HRP-conjugate）: 1 x 120μl /瓶。

4.酶结合物稀释液 （HRP-conjugate Diluent） : 1×20ml/瓶。

5.样品稀释液 （Sample Diluent）： 2×20ml/瓶。

6.底物溶液（ TMB Substrate）： 1×10ml/瓶。

7.浓洗涤液（ Wash Buffer） 1×20ml/瓶，使用时每瓶用蒸馏水稀释 25倍。

8.终止液（ Stop Solution）： 1×10ml/瓶。

需要而未提供的试剂和器材

1.标准规格酶标仪

2.高速离心机

3.电热恒温培养箱

4.干净的试管和 Eppendof管

5.系列可调节移液器及吸头，一次检测样品较多时，用多通道移液器

6.蒸馏水，容量瓶等

标本的采集及保存

1.血清：全血标本请于室温放置 2小时或 4℃过夜后于 1000 x g离心 20分钟，取上清即可检测，或将标本放于 -20℃或 -80℃保存，但应避免反复冻融。

2.血浆：可用 EDTA或肝素作为抗凝剂，标本采集后 30分钟内于 2 -8°C 1000 x g离心 15分钟，或将标本放于 -20℃或 -80℃保存，但应避免反复冻融。

 

注：标本溶血会影响zui后检测结果，因此溶血标本不宜进行此项检测。

标本的稀释原则：

血清，血浆样本用样本稀释液进行 1:20000倍稀释后进行检测，具体操作如下：取 1μl 样本加入到 99μl 的样本稀释液（ 1:100稀释）中混匀，再从上述稀释液中取 1μl加入到 199μl 样本稀释液（ 1:200稀释）中混匀。得到的即为 1:20000倍稀释后的样本。此推荐稀释倍数仅供参考，用户应根据实验自行确定其*稀释倍数。

标准品的稀释原则： 2瓶，使用前于 6000-10000rpm离心 30秒。每瓶临用前以样品稀释液稀释至 1ml，盖好后静置 10分钟以上，然后反复颠倒 /搓动以助溶解，其浓度为 1.5nmol/L，做系列倍比稀释后，分别稀释 1.5nmol/L, 0.75nmol/L, 0.375nmol/L, 0.187nmol/L, 0.094nmol/L, 0.047nmol/L,

0.023nmol/L，样品稀释液直接作为标准浓度 0nmol/L，临用前 15分钟内配制，用完丢弃，下次检测使用新鲜配置的标准品。

如配制 0.75nmol/L标准品：取 0.5ml（不要少于 0.5ml）1.5nmol/L的上述标准品加入含 0.5ml样品稀释液的 Eppendorf管中，混匀即可，其余浓度以此类推。

酶结合物的稀释原则：

打开瓶盖前请离心，收集瓶壁上的溶液。临用前用酶结合物稀释液稀释，稀释前根据预先计算好的每次实验所需的总量配制（每孔 100μl），实际配制时应多配制 0.1-0.2ml。如 10μl酶结合物加 990μl酶结合物稀释液的比例配制，轻轻混匀，在使用前一小时内配制。洗液的

洗液的配制

将浓洗涤液移至室温平衡半小时，取浓洗涤液，根据当批检测数量，用蒸馏水 1:25稀释，混匀后备用。

操作步骤

实验开始前，请提前配置好所有试剂，试剂或样品稀释时，均需混匀，混匀时尽量避免起泡。每次检测都应该做标准曲线。如样品浓度过高时，用样品稀释液进行稀释，以使样品符合试剂盒的检测范围。

1.加样：分别设标准孔、待测样品孔。加 100μl样品稀释液作为标准品 S0孔。余孔分别加标准品或待测样品 100μl，注意不要有气泡，加样将样品加于酶标板孔底部，尽量不触及孔壁，轻轻晃动混匀，酶标板加上盖或覆膜，37℃反应 60分钟。为保证实验结果有效性，每次实验请使用新的标准品溶液。

2.温育后，弃去孔内液体，甩干，洗板 3次，每次浸泡 1-2分钟，200μl/每孔，甩干，zui后用力在洗水纸上拍干。

3.每孔加酶结合物 100μl，空白孔不加。混匀， 37℃， 60分钟。

4.温育后，弃去孔内液体，甩干，洗板 5次，每次浸泡 1-2分钟，200μl/每孔，甩干，zui后用力在洗水纸上拍干。

5.依序每孔加底物溶液 90μl，37℃避光显色 20分钟。

6.依序每孔加终止溶液 50μl，终止反应（此时蓝色立转黄色）。终止液的加入顺序应尽量与底物液的加入顺序相同。为了保证实验结果的准确性，底物反应时间到后应尽快加入终止液。

7.用酶联仪在 450nm波长依序测量各孔的光密度（ OD值）。在加终止液后 15分钟以内进行检测。

实验备注

1.用户在初次使用试剂盒时，应将各种试剂管离心数分钟，以便试剂集中到管底。

2.每次实验留一孔作为空白调零孔，该孔不加任何试剂，只是zui后加底物溶液及终止液。测量时先用此孔调 OD值至零。

3.为防止样品蒸发，试验时将反应板放于铺有湿布的密闭盒内，酶标板加上盖或覆膜。

4.未使用完的酶标板或者试剂，请于 2-8℃保存。

5.建议检测样品时均设双孔测定，以保证检测结果的准确性。

 

洗板方法

手工洗板方法：吸去（不可触及板壁）或甩掉酶标板内的液体；在实验台上铺垫几层吸水纸，酶标板朝下用力拍几次；将推荐的洗涤缓冲液至少 0.2ml注入孔内，浸泡 1-2分钟。根据需要，重复此过程数次。自动洗板：如果有自动洗板机，应在熟练使用后再用到正式实验过程中。

计算

以标准物的浓度为纵坐标（对数坐标），OD值为横坐标（普通坐标），在半对数坐标纸上绘出标准曲线，根据样品的 OD值由标准曲线查出相应的浓度；再乘以稀释倍数；或用标准物的浓度与 OD值计算出标准曲线的直线回归方程式，将样品的 OD值代入方程式，计算出样品浓度，再乘以稀释倍数，即为样品的实际浓度。

注意事项

1          本操作说明适用于 48T试剂盒， 48T试剂盒中酶联板、标准品及酶结合物的量减半。

2          当混合蛋白溶液时应尽量轻缓，避免起泡。

3          洗涤过程非常重要，不充分的洗涤易造成假阳性。

4          一次加样时间控制在 5分钟内，如标本数量多，推荐使用排枪加样。

5          请每次测定的同时做标准曲线，做复孔。

6          如标本中待测物质含量过高，请先稀释后再测定，计算时请zui后乘以稀释倍数。

7          底物请避光保存。

8          不要用其它生产厂家的试剂替换试剂盒中的试剂。