Human transferrin（TF） ELISA Kit
Catalog No. CSB-E13093h
（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
FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES.
廈門慧嘉生物長期經營ELISA試劑盒及抗體、細胞因子、生化試劑、耗材等生物試劑產品。誠信經營，價格實惠，服務周到，質量有保證。歡迎廣告老師來詢！: : 1048735792 或登陸/download（向客服人員索取原版說明書）
PRINCIPLE OF THE ASSAY
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.
DETECTION RANGE
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.
SPECIFICITY
This assay recognizes human TF. No significant cross-reactivity or interference was observed.
SENSITIVITY
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.
MATERIALS PROVIDED
Reagent Quantity
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
STORAGE
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.
REAGENT PREPARATION
Bring all reagents to room temperature before use for 30min.
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.
OTHER SUPPLIES REQUIRED
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.
Note: Grossly hemolyzed samples are not suitable for use in this assay.
If samples generate values higher than the highest standard, dilute the samples with the Sample Diluent and repeat the assay.
SAMPLE PREPARTION
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.
ASSAY PROCEDURE
Bring all reagents and samples to room temperature before use. It is recommended that all samples, standards, and controls be assayed in duplicate. All the reagents should be added directly to the liquid level in the well. The pipette should avoid contacting the inner wall of the well.
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.
CALCULATION OF RESULTS
Using the professional soft "Curve Exert 1.3" to make a standard curve is recommended, which can be downloaded from our web.
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.
LIMITATIONS OF THE PROCEDURE
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.
人轉鐵蛋白（TF）酶聯免疫分析試劑盒使用說明書
本試劑盒僅供研究使用
產品編號：CSB-E13093h
檢測范圍：0.023nmol/L –1.5nmol/L
zui低檢測限：0.006nmol/L
特異性：本試劑盒可檢測人 TF，且與其他相關蛋白無交叉反應。有效期：6個月預期應用：ELISA法定量測定人血清、血漿中 TF含量。
說明
1 2-8℃。
2 中、英文說明書可能會有不一致之處，請以英文說明書為準。
3 剛開啟的酶聯板孔中可能會含有少許水樣物質，此為正?，F象，不會對實驗結果造成任何影響。
實驗原理
用純化的抗體包被微孔板，制成固相載體，往包被抗 TF抗體的微孔中加入標本或標準品、HRP標記的抗 TF抗體，經過*洗滌后用底物顯色。底物在過氧化物酶的催化下轉化成藍色，并在酸的作用下轉化成zui終的黃色。顏色的深淺和樣品中的 TF呈正相關。用酶標儀在 450nm波長下測定吸光度（OD值），計算樣品濃度。
試劑盒組成及試劑配制
1.酶聯板 （Assay plate ）：一塊（96孔）。
2.標準品（Standard）： 2瓶（凍干品）。
3.酶結合物（ HRP-conjugate）: 1 x 120μl /瓶。
4.酶結合物稀釋液 （HRP-conjugate Diluent） : 1×20ml/瓶。
5.樣品稀釋液 （Sample Diluent）： 2×20ml/瓶。
6.底物溶液（ TMB Substrate）： 1×10ml/瓶。
7.濃洗滌液（ Wash Buffer） 1×20ml/瓶，使用時每瓶用蒸餾水稀釋 25倍。
8.終止液（ Stop Solution）： 1×10ml/瓶。
需要而未提供的試劑和器材
1.標準規格酶標儀
2.高速離心機
3.電熱恒溫培養箱
4.干凈的試管和 Eppendof管
5.系列可調節移液器及吸頭，一次檢測樣品較多時，用多通道移液器
6.蒸餾水，容量瓶等
標本的采集及保存
1.血清：全血標本請于室溫放置 2小時或 4℃過夜后于 1000 x g離心 20分鐘，取上清即可檢測，或將標本放于 -20℃或 -80℃保存，但應避免反復凍融。
2.血漿：可用 EDTA或肝素作為抗凝劑，標本采集后 30分鐘內于 2 -8°C 1000 x g離心 15分鐘，或將標本放于 -20℃或 -80℃保存，但應避免反復凍融。
注：標本溶血會影響zui后檢測結果，因此溶血標本不宜進行此項檢測。
標本的稀釋原則：
血清，血漿樣本用*進行 1:20000倍稀釋后進行檢測，具體操作如下：取 1μl 樣本加入到 99μl 的*（ 1:100稀釋）中混勻，再從上述稀釋液中取 1μl加入到 199μl *（ 1:200稀釋）中混勻。得到的即為 1:20000倍稀釋后的樣本。此推薦稀釋倍數僅供參考，用戶應根據實驗自行確定其*稀釋倍數。
標準品的稀釋原則： 2瓶，使用前于 6000-10000rpm離心 30秒。每瓶臨用前以樣品稀釋液稀釋至 1ml，蓋好后靜置 10分鐘以上，然后反復顛倒 /搓動以助溶解，其濃度為 1.5nmol/L，做系列倍比稀釋后，分別稀釋 1.5nmol/L, 0.75nmol/L, 0.375nmol/L, 0.187nmol/L, 0.094nmol/L, 0.047nmol/L,
0.023nmol/L，樣品稀釋液直接作為標準濃度 0nmol/L，臨用前 15分鐘內配制，用完丟棄，下次檢測使用新鮮配置的標準品。
如配制 0.75nmol/L標準品：取 0.5ml（不要少于 0.5ml）1.5nmol/L的上述標準品加入含 0.5ml樣品稀釋液的 Eppendorf管中，混勻即可，其余濃度以此類推。
酶結合物的稀釋原則：
打開瓶蓋前請離心，收集瓶壁上的溶液。臨用前用酶結合物稀釋液稀釋，稀釋前根據預先計算好的每次實驗所需的總量配制（每孔 100μl），實際配制時應多配制 0.1-0.2ml。如 10μl酶結合物加 990μl酶結合物稀釋液的比例配制，輕輕混勻，在使用前一小時內配制。洗液的
洗液的配制
將濃洗滌液移至室溫平衡半小時，取濃洗滌液，根據當批檢測數量，用蒸餾水 1:25稀釋，混勻后備用。
操作步驟
實驗開始前，請提前配置好所有試劑，試劑或樣品稀釋時，均需混勻，混勻時盡量避免起泡。每次檢測都應該做標準曲線。如樣品濃度過高時，用樣品稀釋液進行稀釋，以使樣品符合試劑盒的檢測范圍。
1.加樣：分別設標準孔、待測樣品孔。加 100μl樣品稀釋液作為標準品 S0孔。余孔分別加標準品或待測樣品 100μl，注意不要有氣泡，加樣將樣品加于酶標板孔底部，盡量不觸及孔壁，輕輕晃動混勻，酶標板加上蓋或覆膜，37℃反應 60分鐘。為保證實驗結果有效性，每次實驗請使用新的標準品溶液。
2.溫育后，棄去孔內液體，甩干，洗板 3次，每次浸泡 1-2分鐘，200μl/每孔，甩干，zui后用力在洗水紙上拍干。
3.每孔加酶結合物 100μl，空白孔不加?；靹颍?37℃， 60分鐘。
4.溫育后，棄去孔內液體，甩干，洗板 5次，每次浸泡 1-2分鐘，200μl/每孔，甩干，zui后用力在洗水紙上拍干。
5.依序每孔加底物溶液 90μl，37℃避光顯色 20分鐘。
6.依序每孔加終止溶液 50μl，終止反應（此時藍色立轉黃色）。終止液的加入順序應盡量與底物液的加入順序相同。為了保證實驗結果的準確性，底物反應時間到后應盡快加入終止液。
7.用酶聯儀在 450nm波長依序測量各孔的光密度（ OD值）。在加終止液后 15分鐘以內進行檢測。
實驗備注
1.用戶在初次使用試劑盒時，應將各種試劑管離心數分鐘，以便試劑集中到管底。
2.每次實驗留一孔作為空白調零孔，該孔不加任何試劑，只是zui后加底物溶液及終止液。測量時先用此孔調 OD值至零。
3.為防止樣品蒸發，試驗時將反應板放于鋪有濕布的密閉盒內，酶標板加上蓋或覆膜。
4.未使用完的酶標板或者試劑，請于 2-8℃保存。
5.建議檢測樣品時均設雙孔測定，以保證檢測結果的準確性。
洗板方法
手工洗板方法：吸去（不可觸及板壁）或甩掉酶標板內的液體；在實驗臺上鋪墊幾層吸水紙，酶標板朝下用力拍幾次；將推薦的洗滌緩沖液至少 0.2ml注入孔內，浸泡 1-2分鐘。根據需要，重復此過程數次。自動洗板：如果有自動洗板機，應在熟練使用后再用到正式實驗過程中。
計算
以標準物的濃度為縱坐標（對數坐標），OD值為橫坐標（普通坐標），在半對數坐標紙上繪出標準曲線，根據樣品的 OD值由標準曲線查出相應的濃度；再乘以稀釋倍數；或用標準物的濃度與 OD值計算出標準曲線的直線回歸方程式，將樣品的 OD值代入方程式，計算出樣品濃度，再乘以稀釋倍數，即為樣品的實際濃度。
注意事項
1 本操作說明適用于 48T試劑盒， 48T試劑盒中酶聯板、標準品及酶結合物的量減半。
2 當混合蛋白溶液時應盡量輕緩，避免起泡。
3 洗滌過程非常重要，不充分的洗滌易造成假陽性。
4 一次加樣時間控制在 5分鐘內，如標本數量多，推薦使用排槍加樣。
5 請每次測定的同時做標準曲線，做復孔。
6 如標本中待測物質含量過高，請先稀釋后再測定，計算時請zui后乘以稀釋倍數。
7 底物請避光保存。
8 不要用其它生產廠家的試劑替換試劑盒中的試劑。
