Pig macrophage migration inhibitory factor (glycosylation-inhibiting factor) (MIF) ELISA Kit

(96T)

This immunoassay kit allows for the in vitro quantitative determination of pig MIF 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 MIF. Sta-n-dards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated antibody preparation specific for MIF a-n-d Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well a-n-d incubated. Then a TMB (3,3',5,5' tetramethyl-benzidine) substrate solution is added to each well. Only those wells that contain MIF, biotin-conjugated antibody a-n-d enzyme-conjugated Avidin will exhibit a change in color. The enzyme-substrate reaction is terminated by the addition of a sulphuric acid solution a-n-d the color change is measured spectrophotometrically at a wavelength of 450 nm ±2 nm. The concentration of MIF in the samples is then determined by comparing the O.D. of the samples to the sta-n-dard curve.

1.25 ng/ml-80 ng/ml. The sta-n-dard curve concentrations used for the

ELISA’s were 80 ng/ml, 40 ng/ml, 20 ng/ml, 10 ng/ml, 5 ng/ml, 2.5

ng/ml,1.25 ng/ml.

This assay recognizes pig MIF. No significant cross-reactivity or interference was observed.

The minimum detectable dose of pig MIF is typically less than 0.312 ng/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.

Assay plate 1 Sta-n-dard 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

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 a-n-d 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 ba-n-dwidth of 10 nm or less a-n-d 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 a-n-d 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          Sta-n-dard Centrifuge the sta-n-dard vial at 6000-10000rpm for 30s. Reconstitute the Sta-n-dard with 1.0 ml of Sample Diluent. This reconstitution produces a stock solution of 80 ng/ml. Allow the sta-n-dard to sit for a minimum of 15 minutes with gentle agitation prior to making serial dilutions. The undiluted sta-n-dard serves as the high sta-n-dard (80 ng/ml). The Sample Diluent serves as the zero sta-n-dard (0 ng/ml). Prepare fresh for each assay. Use within 4 hours a-n-d 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.

 

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

2           Pipettes a-n-d pipette tips.

3           Deionized or distilled water.

4           Squirt bottle, manifold dispenser, or automated microplate washer.

5         ? An incubator which can provide stable incubation conditions up to 37°C ±0.5°C. SAMPLE COLLECTION A-N-D STORAGE

Serum Use a serum separator tube (SST) a-n-d allow samples to clot for 30 minutes before centrifugation for 15 minutes at 1000 g. Remove serum a-n-d assay immediay or aliquot a-n-d 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 a-n-d store samples at -20°C. Centrifuge the sample again after thawing before the assay. Avoid repeated freeze-thaw cycles.

 

 

1. Add 100μl of Sta-n-dard, 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 a-n-d mix gently until solution appears uniform.

4. Aspirate each well a-n-d wash, repeating the process three times for a

total of three washes. Wash: Fill each well with Wash Buffer (200μl) a-n-d let it sta-n-d 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 a-n-d 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 a-n-d 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 sta-n-dards 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.

 

Average the duplicate readings for each sta-n-dard, control, a-n-d sample a-n-d subtract the average zero sta-n-dard optical density. Create a sta-n-dard curve by reducing the data using computer software capable of generating a four parameter logistic (4-PL) curve-fit. As an alternative, construct a sta-n-dard curve by plotting the mean absorbance for each sta-n-dard on the x-axis against the concentration on the y-axis a-n-d draw a best fit curve through the points on the graph. The data may be linearized by plotting the log of the MIF concentrations versus the log of the O.D. a-n-d 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 sta-n-dard 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           It is important that the Sta-n-dard Diluent selected for the sta-n-dard curve be consistent with the samples being assayed.

4           If samples generate values higher than the highest sta-n-dard, dilute the samples with the appropriate Sta-n-dard Diluent a-n-d repeat the assay.

5           Any variation in Sta-n-dard Diluent, operator, pipetting technique, washing technique, incubation time or temperature, a-n-d kit age can cause variation in binding.

6         ? This assay is designed to eliminate interference by soluble receptors, binding proteins, a-n-d other factors present in biological samples. Until all factors have been tested in the Immunoassay, the possibility of interference cannot be excluded. TECHNICAL HINTS

7           Centrifuge vials before opening to collect contents.

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

9           To avoid cross-contamination, change pipette tips between additions of each sta-n-dard level, between sample additions, a-n-d between reagent additions. Also, use separate reservoirs for each reagent.

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

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

12       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.

13       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.

 

豬巨噬細(xì)胞遷移抑制因子 (糖基化抑制因子 )(MIF)

酶聯(lián)免疫分析試劑盒使用說明書本試劑盒僅供研究使用產(chǎn)品編號：CSB-EL013826PI檢測范圍：1.25 ng/ml-80 ng/mlzui低檢測限：0.312 ng/ml特異性：本試劑盒可檢測豬 MIF，且與其他相關(guān)蛋白無交叉反應(yīng)。有效期：6個月預(yù)期應(yīng)用：ELISA法定量測定豬血清、血漿中 MIF含量。說明

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 MIF抗體的微孔中依次加入標(biāo)本或標(biāo)準(zhǔn)品、生物素化的抗 MIF抗體、HRP標(biāo)記的親和素，經(jīng)過*洗滌后用底物 TMB顯色。TMB在過氧化物酶的催化下轉(zhuǎn)化成藍(lán)色，并在酸的作用下轉(zhuǎn)化成zui終的黃色。顏色的深淺和樣品中的 MIF呈正相關(guān)。用酶標(biāo)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(lián)板 (Assay plate )：一塊( 96孔)。

2.標(biāo)準(zhǔn)品(Sta-n-dard)： 2瓶(凍干品)。

3.樣品稀釋液 (Sample Diluent)： 1×20ml/瓶。

4.生物素標(biāo)記抗體稀釋液( Biotin-antibody Diluent)： 1×10ml/瓶。

5.辣根過氧化物酶標(biāo)記親和素稀釋液( HRP-avidin Diluent) 1×10ml/瓶。

6.生物素標(biāo)記抗體(Biotin-antibody)： 1×120μl/瓶(1：100)。

7.辣根過氧化物酶標(biāo)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(biāo)準(zhǔn)規(guī)格酶標(biāo)儀

2.高速離心機(jī)

3.電熱恒溫培養(yǎng)箱

4.干凈的試管和 Eppendof管

5.系列可調(diào)節(jié)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(biāo)本的采集及保存

1.血清：全血標(biāo)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(jìn)行分裝，并將標(biāo)本放于 -20℃或 -80℃保存，但應(yīng)避免反復(fù)凍融。解凍后的樣品應(yīng)再次離心，然后檢測。

2.血漿：可用 EDTA或肝素作為抗凝劑，標(biāo)本采集后 30分鐘內(nèi)于 2 -8°C 1000 g離心 15分鐘，取上清即可立即檢測；或進(jìn)行分裝，并將標(biāo)本放于 -20℃或 -80℃保存，但應(yīng)避免反復(fù)凍融。解凍后的樣品應(yīng)再次離心，然后檢測。

 

注：標(biāo)本溶血會影響zui后檢測結(jié)果，因此溶血標(biāo)本不宜進(jìn)行此項檢測。標(biāo)準(zhǔn)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(fù)顛倒 /搓動以助溶解，其濃度為 80 ng/ml，做系列倍比稀釋后，分別稀釋 80 ng/ml, 40 ng/ml, 20 ng/ml, 10 ng/ml, 5 ng/ml, 2.5 ng/ml,1.25 ng/ml，樣品稀釋液直接作為標(biāo)準(zhǔn)濃度 0 ng/ml，臨用前 15分鐘內(nèi)配制，用完丟棄，下次檢測使用新鮮配置的標(biāo)準(zhǔn)品。

如配制 40 ng/ml標(biāo)準(zhǔn)品：取 0.5ml(不要少于 0.5ml)80 ng/ml的上述標(biāo)準(zhǔn)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。

生物素標(biāo)記抗體的稀釋原則：

打開瓶蓋前請離心，收集瓶壁上的溶液。臨用前以生物素標(biāo)記抗體稀釋液稀釋，稀釋前根據(jù)預(yù)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(yīng)多配制 0.1-0.2ml。如 10μl生物素標(biāo)記抗體加 990μl生物素標(biāo)記抗體稀釋液的比例配制，輕輕混勻，在使用前一小時內(nèi)配制。

辣根過氧化物酶標(biāo)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(biāo)記親和素稀釋液稀釋，稀釋前根據(jù)預(yù)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(yīng)多配制 0.1-0.2ml。如 10μl辣根過氧化物酶標(biāo)記親和素加 990μl辣根過氧化物酶標(biāo)記親和素稀釋液的比例配制，輕輕混勻，在使用前一小時內(nèi)配制。

操作步驟

實驗開始前，請?zhí)崆芭渲煤盟性噭?，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(yīng)該做標(biāo)準(zhǔn)曲線。如樣品濃度過高時，用樣品稀釋液進(jìn)行稀釋，以使樣品符合試劑盒的檢測范圍。加樣時，槍頭應(yīng)直接對準(zhǔn)液面，切勿沿孔壁加樣。

加樣：分別設(shè)空白孔、標(biāo)準(zhǔn)孔、待測樣品孔?？瞻卓准訕悠废♂屢?100μl，

余孔分別加標(biāo)準(zhǔn)品或待測樣品 100μl，注意不要有氣泡，加樣將樣品加于

酶標(biāo)板孔底部，盡量不觸及孔壁，輕輕晃動混勻，酶標(biāo)板加上蓋或覆膜，

37℃反應(yīng) 120分鐘。

為保證實驗結(jié)果有效性，每次實驗請使用新的標(biāo)準(zhǔn)品溶液。

2棄去液體，甩干，不用洗滌。每孔加生物素標(biāo)記抗體工作液 100μl(取 1μl生物素標(biāo)記抗體加 99μl生物素標(biāo)記抗體稀釋液的比例配制，輕輕混勻，在使用前一小時內(nèi)配制)，37℃,60分鐘。

3溫育 60分鐘后，棄去孔內(nèi)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(biāo)記親和素工作液(同生物素標(biāo)記抗體工作液) 100μl，37℃， 60分鐘。 5溫育 60分鐘后，棄去孔內(nèi)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(nèi)，此時肉眼可見標(biāo)準(zhǔn)品的前 3-4孔有明顯的梯度藍(lán)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(yīng)(此時藍(lán)色立轉(zhuǎn)黃色)。終止液的加入順序應(yīng)盡量與底物液的加入順序相同。為了保證實驗結(jié)果的準(zhǔn)確性，底物反應(yīng)時間到后應(yīng)盡快加入終止液。

8用酶聯(lián)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(nèi)進(jìn)行檢測。

實驗備注

1          用戶在初次使用試劑盒時，應(yīng)將各種試劑管離心數(shù)分鐘，以便試劑集中到管底。

2          為防止樣品蒸發(fā)，試驗時將反應(yīng)板放于鋪有濕布的密閉盒內(nèi)，酶標(biāo)板加上蓋或覆膜。

3          未使用完的酶標(biāo)板或者試劑，請于 2-8℃保存。標(biāo)準(zhǔn)品、生物素標(biāo)記抗體工作液、辣根過氧化物酶標(biāo)記親和素工作液請依據(jù)所需的量配置使用。請勿重復(fù)使用已稀釋過的標(biāo)準(zhǔn)品、生物素標(biāo)記抗體工作液、或辣根過氧化物酶標(biāo)記親和素工作液。

4          建議檢測樣品時均設(shè)雙孔測定，以保證檢測結(jié)果的準(zhǔn)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(biāo)板內(nèi)的液體；在實驗臺上鋪墊幾層吸水紙，酶標(biāo)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(nèi)，浸泡 1-2分鐘。根據(jù)需要，重復(fù)此過程數(shù)次。自動洗板：如果有自動洗板機(jī)，應(yīng)在熟練使用后再用到正式實驗過程中。

計算

請從我們的下載專業(yè)軟件"Curve Exert 1.3"，并根據(jù)提示制作標(biāo)準(zhǔn)曲線。以標(biāo)準(zhǔn)物的濃度為縱坐標(biāo)(對數(shù)坐標(biāo))，OD值為橫坐標(biāo)(普通坐標(biāo))，在半對數(shù)坐標(biāo)紙上繪出標(biāo)準(zhǔn)曲線，根據(jù)樣品的 OD值由標(biāo)準(zhǔn)曲線查出相應(yīng)的濃度；再乘以稀釋倍數(shù)；或用標(biāo)準(zhǔn)物的濃度與 OD值計算出標(biāo)準(zhǔn)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(shù)，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(lián)板、標(biāo)準(zhǔn)品、生物素標(biāo)記抗體及辣根過氧化物酶標(biāo)記親和素減半。

2          當(dāng)混合蛋白溶液時應(yīng)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(nèi)，如標(biāo)本數(shù)量多，推薦使用排槍加樣。

5          請每次測定的同時做標(biāo)準(zhǔn)曲線，做復(fù)孔。

6          如標(biāo)本中待測物質(zhì)含量過高，請先稀釋后再測定，計算時請zui后乘以稀釋倍數(shù)。

7          在配制標(biāo)準(zhǔn)品、檢測溶液工作液時，請以相應(yīng)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(chǎn)廠家的試劑替換試劑盒中的試劑。