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Human β-defensins 2 ELISA Kit
（96T）
? This immunoassay kit allows for the in vitro quantitative determination of human
β-defensins 2 concentrations in serum, plasma.
? Expiration date six months from the date of manufacture
? FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES.
2
PRINCIPLE OF THE ASSAY
The microtiter plate provided in this kit has been pre-coated with an
antibody specific to β-defensins 2. Standards or samples are then added to
the appropriate microtiter plate wells with a biotin-conjugated antibody
preparation specific for β-defensins 2 and Avidin conjugated to Horseradish
Peroxidase （HRP） is added 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 β-defensins 2, biotin-conjugated antibody
and enzyme-conjugated Avidin 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 β-defensins 2 in the
samples is then determined by comparing the O.D. of the samples to the
standard curve.
DETECTION RANGE
62.5 pg/ml-4000 pg/ml. The standard curve concentrations used for the
ELISA’s were 4000 pg/ml, 2000 pg/ml, 1000 pg/ml, 500 pg/ml, 250 pg/ml,
125 pg/ml, 62.5 pg/ml.
SPECIFICITY
This assay recognizes human β-defensins 2. No significant cross-reactivity
or interference was observed.
SENSITIVITY
The minimum detectable dose of human β-defensins 2 is typically less than
15.6 pg/ml.
The sensitivity of this assay, or Lower Limit of Detection （LLD） was defined
as the lowest protein concentration that could be differentiated from zero.
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MATERIALS PROVIDED
Reagent Quantity
Assay plate 1
Standard 2
Sample Diluent 1 x 20 ml
Biotin-antibody Diluent 1 x 10 ml
HRP-avidin Diluent 1 x 10 ml
Biotin-antibody 1 x 120μl
HRP-avidin 1 x 120μl
Wash Buffer
1 x 20 ml
（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. The test kit may be used
throughout the expiration date of the kit, provided it is stored as
prescribed above. Refer to the package label for the expiration date.
2. Opened test plate should be stored at 2-8?C in the aluminum foil bag
with desiccants to minimize exposure to damp air. The kits will remain
stable until the expiring date shown, provided it is stored as prescribed
above.
3. 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.
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REAGENT PREPARATION
Bring all reagents to room temperature before use.
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 4000 pg/ml. 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 （4000 pg/ml）. The Sample Diluent serves as the zero
standard （0 pg/ml）. Prepare fresh for each assay. Use within 4 hours
and discard after use.
3. Biotin-antibody Centrifuge the vial before opening. Dilute to the
working concentration using Biotin-antibody Diluent（1:100）,
respectively.
4. HRP-avidin Centrifuge the vial before opening. Dilute to the working
concentration using HRP-avidin Diluent（1:100）, respectively.
Precaution: The Stop Solution provided with this kit is an acid solution. Wear
eye, hand, face, and clothing protection when using this material.
OTHER SUPPLIES REQUIRED
? Microplate reader capable of measuring absorbance at 450 nm, with
the correction wavelength set at 540 nm or 570 nm.
? Pipettes and pipette tips.
? Deionized or distilled water.
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? Squirt bottle, manifold dispenser, or automated microplate washer.
? An incubator which can provide stable incubation conditions up to 37°C
±0.5°C.
SAMPLE COLLECTION AND STORAGE
? Serum Use a serum separator tube （SST） and allow samples to clot
for 30 minutes before centrifugation for 15 minutes at 1000 g. Remove
serum and assay immediay or aliquot and store samples at -20°C.
Centrifuge the sample again after thawing before the assay. Avoid
repeated freeze-thaw cycles.
? Plasma Collect plasma using citrate, EDTA, or heparin as an
anticoagulant. Centrifuge for 15 minutes at 1000 g within 30 minutes of
collection. Assay immediay or aliquot and store samples at -20°C.
Centrifuge the sample again after thawing before the assay. Avoid
repeated freeze-thaw cycles.
Note: Grossly hemolyzed samples are not suitable for use in this assay.
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 of Standard, Blank, or Sample per well. Cover with the
adhesive strip. Incubate for 2 hours at 37°C.
2. Remove the liquid of each well, don’t wash.
3. Add 100μl of Biotin-antibody working solution to each well. Incubate
for 1 hour at 37°C. Biotin-antibody working solution may appear
cloudy. Warm up to room temperature and mix gently until solution
appears uniform.
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4. Aspirate each well and wash, repeating the process three times for a
total of three washes. Wash: Fill each well with Wash Buffer （200μl） and
let it stand for 2 minutes, then remove the liquid by flicking the plate
over a sink. The remaining drops are removed by patting the plate on a
paper towel. Complete removal of liquid at each step is essential to
good performance.
5. Add 100μl of HRP-avidin working solution to each well. Cover the
microtiter plate with a new adhesive strip. Incubate for 1 hour at 37°C.
6. Repeat the aspiration and wash five times as step 4.
7. Add 90μl of TMB Substrate to each well. Incubate for 15-30 minutes at
37°C. Keeping the plate away from drafts and other temperature
fluctuations in the dark.
8. 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.
9. 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
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points on the graph. The data may be linearized by plotting the log of the
β-defensins 2 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
? The kit should not be used beyond the expiration date on the kit label.
? Do not mix or substitute reagents with those from other lots or sources.
? It is important that the Standard Diluent selected for the standard curve
be consistent with the samples being assayed.
? If samples generate values higher than the highest standard, dilute the
samples with the appropriate Standard Diluent and repeat the assay.
? Any variation in Standard Diluent, operator, pipetting technique,
washing technique, incubation time or temperature, and kit age can
cause variation in binding.
? 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
? Centrifuge vials before opening to collect contents.
? When mixing or reconstituting protein solutions, always avoid foaming.
? 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.
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? 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.
? To ensure accurate results, proper adhesion of plate sealers during
incubation steps is necessary.
? Substrate Solution should remain colorless or light blue until added to
the plate. Keep Substrate Solution protected from light. Substrate
Solution should change from colorless or light blue to gradations of
blue.
? 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.
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人β -防御素2 酶聯免疫分析
試劑盒使用說明書
檢測范圍：62.5 pg/ml-4000 pg/ml
zui低檢測限：15.6 pg/ml
特異性：本試劑盒可檢測人β-defensins 2，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β-defensins 2 含量。
說明
1 試劑盒保存：未開封的試劑盒應儲存于2-8℃；開封后的酶標板應與干燥
劑一起儲存于鋁箔袋中置于2-8℃保存。僅在此儲存條件下，產品在有效
期內可正常使用。
2 濃洗滌液低溫保存會有鹽析出，稀釋時可在水浴中加溫助溶。
3 中、英文說明書可能會有不一致之處，請以英文說明書為準。
4 剛開啟的酶聯板孔中可能會含有少許水樣物質，此為正常現象，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β-defensins 2 抗體
的微孔中依次加入標本或標準品、*化的抗β-defensins 2 抗體、HRP
標記的親和素，經過*洗滌后用底物TMB 顯色。TMB 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β-defensins 2 呈正相關。用酶標儀在450nm 波長下測定吸光度（OD 值），
1 0
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. 樣品稀釋液（Sample Diluent）： 1×20ml/瓶。
4. *標記抗體稀釋液（Biotin-antibody Diluent）： 1×10ml/瓶。
5. 辣根過氧化物酶標記親和素稀釋液（HRP-avidin Diluent） 1×10ml/瓶。
6. *標記抗體（Biotin-antibody）： 1×120μl/瓶（1：100）。
7. 辣根過氧化物酶標記親和素（HRP-avidin）： 1×120μl/瓶（1：100）。
8. 底物溶液（TMB Substrate）： 1×10ml/瓶。
9. 濃洗滌液（Wash Buffer） 1×20ml/瓶，使用時每瓶用蒸餾水稀釋25 倍。
10. 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1000g 離心20 分鐘，
取上清即可立即檢測；或進行分裝，并將標本放于-20℃或-80℃保存，但
應避免反復凍融。解凍后的樣品應再次離心，然后檢測。
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 g 離心15 分鐘，取上清即可立即檢測；或進行分裝，并將標本放于
-20℃或-80℃保存，但應避免反復凍融。解凍后的樣品應再次離心，然后
檢測。
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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4000 pg/ml，做系列倍比稀釋后，分別稀釋4000
pg/ml，2000 pg/ml，1000 pg/ml， 500 pg/ml，250 pg/ml，125 pg/ml，62.5
pg/ml，樣品稀釋液直接作為標準濃度0 pg/ml，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2000 pg/ml 標準品：取0.5ml（不要少于0.5ml）4000 pg/ml 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。
*標記抗體的稀釋原則：
打開瓶蓋前請離心，收集瓶壁上的溶液。臨用前以*標記抗體稀釋液稀
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 *標記抗體加990μl *標記抗體
稀釋液的比例配制，輕輕混勻，在使用前一小時內配制。
辣根過氧化物酶標記親和素的稀釋原則：
打開瓶蓋前請離心，收集瓶壁上的溶液。臨用前以辣根過氧化物酶標記親和
素稀釋液稀釋，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 辣根過氧化物酶標記親和素
加990μl 辣根過氧化物酶標記親和素稀釋液 的比例配制，輕輕混勻，在使用
前一小時內配制。
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。加樣時，槍頭應直接對
準液面，切勿沿孔壁加樣。
1 加樣：分別設空白孔、標準孔、待測樣品孔。空白孔加樣品稀釋液100μl，
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，
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37℃反應120 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 棄去液體，甩干，不用洗滌。每孔加*標記抗體工作液 100μl（取1μl
*標記抗體加99μl *標記抗體稀釋液的比例配制，輕輕混勻，
在使用前一小時內配制），37℃,60 分鐘。
3 溫育60 分鐘后，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。
4 每孔加辣根過氧化物酶標記親和素工作液（同*標記抗體工作液）
100μl，37℃，60 分鐘。
5 溫育60 分鐘后，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。
6 依序每孔加底物溶液90μl，37℃避光顯色（15-30 分鐘內，此時肉眼可
見標準品的前3-4 孔有明顯的梯度藍色，后3-4 孔顯色不明顯，即可終止）。
7 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。
8 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. 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。
3. 未使用完的酶標板或者試劑，請于2-8℃保存。標準品、*標記抗體
工作液、辣根過氧化物酶標記親和素工作液請依據所需的量配置使用。請
勿重復使用已稀釋過的標準品、*標記抗體工作液、或辣根過氧化物
酶標記親和素工作液。
4. 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。
1 3
計算
請從我們的下載專業軟件"Curve Exert 1.3"，并根據提示制作標準曲線。
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. 本操作說明也適用于48T 試劑盒， 48T 試劑盒中酶聯板、標準品、生物
素標記抗體及辣根過氧化物酶標記親和素減半。
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. 在配制標準品、檢測溶液工作液時，請以相應的稀釋液配制，不能混淆。
8. 底物請避光保存。
9. 不要用其它生產廠家的試劑替換試劑盒中的試劑。