6. 人工處理：以標(biāo)準(zhǔn)濃度取log值為橫坐標(biāo)，對應(yīng)的logit值為縱坐標(biāo)在普通坐標(biāo)紙上或以標(biāo)準(zhǔn)濃度為橫坐標(biāo)，對應(yīng)的B/B0為縱坐標(biāo)在logit-log坐標(biāo)紙上畫出標(biāo)準(zhǔn)曲線（理想化時是一條直線）。根據(jù)待測樣品的B/B0可以從坐標(biāo)紙上查出樣品的濃度值。如果使用普通坐標(biāo)紙，查出的數(shù)值應(yīng)取反對數(shù)才是zui后的濃度值。
7. 自動處理：使用logit-log或四參數(shù)數(shù)據(jù)處理模式，由電腦自動計算得出結(jié)果。
8. 敏感度：0.01ng/ml；
9. 圖例
Rat Thromboxanes B2 ELISA Kit
48 Tests
Catalogue Number: E02T0003
Store all reagents at 2-8°C
Collect sample: serum or blood plasma
FOR LABORATORY RESEARCH USE ONLY. NOT FOR THERAPEUTIC OR DIAGNOSTIC APPLICATIONS!PLEASE READ THROUGH ENTIRE PROCEDURE BEFORE BEGINNING!
INTENDED USE
This B.G TXB2 ELISA kit is intended Laboratory for Research use only and is not for use in diagnostic or therapeutic procedures. The Stop Solution changes the color from blue to yellow and the intensity of the color is measured at 450nm using a spectrophotometer. In order to measure the concentration of TXB2 in the sample, this TXB2 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 TXB2 concentration. The concentration of TXB2 in the samples is then determined by comparing the O.D. of the samples to the standard curve.
PRINCIPLE OF THE ASSAY
The coated well immunoenzymatic assay for the quantitative measurement of serum TXB2 utilizes a monoclonal anti-TXB2 and a TXB2-HRP conjugate. The assay sample and buffer are incubated together with anti-TXB2 antibody coated plate for sixty and washed. The diluted TXB2-HRP conjugate is then added to each well and incubated. After the incubation period, the wells are decanted and washed three times. The wells are then incubated with a substrate for the enzyme. The product of the enzyme-substrate reaction forms a blue colored complex. Finally, a stopping solution is added to stop the reaction, which will then turn the solution yellow. The intensity of color is measured spectrophotometrically at 450nm in a microplate reader. The intensity of the color is inversely proportional to the TXB2 concentration since TXB2 from samples and TXB2-HRP conjugate compete for the anti-TXB2 antibody binding site. Since the number of sites is limited, as more sites are occupied by TXB2 from the sample, fewer sites are left to bind TXB2-HRPconjugate. Standards of known TXB2 concentrations are run concurrently with the samples being assayed and a standard curve is plotted relating the intensity of the color （Optical Density） to the concentration of TXB2. The unknown TXB2
溫（20-25℃）內(nèi)進(jìn)行。
2. 取出酶標(biāo)板，按照標(biāo)準(zhǔn)品的次序分別加入100μl的標(biāo)準(zhǔn)品溶液于空白微孔中。
3. 空白微孔中加入100μl的樣品，空白對照加入100μl的蒸餾水；
4. 在各孔中加入50μl的酶標(biāo)記溶液；（不含空白對照孔）
5. 將酶標(biāo)板用封口膠密封后，37℃孵育反應(yīng) 1小時；（在孵育箱中保持穩(wěn)定的溫度與濕度）
6. 充分清洗酶標(biāo)板3-5次，保持各孔有充足的水壓；（濃縮洗滌液以1：100的比例與蒸餾水稀釋）
7. 酶標(biāo)板洗滌后用吸水紙*拍干；
8. 各孔加入顯色劑A、B液各50μl；（不含空白對照孔）
9. 20-25℃下避光反應(yīng)15分鐘；
10. 各孔加入50μl終止液，終止反應(yīng)；
結(jié)果判斷
1. 30分鐘內(nèi)在波長450nm的酶標(biāo)儀上讀取各孔的OD值；
2. 百分結(jié)合率計算：設(shè)S0管計數(shù)為B0，各標(biāo)準(zhǔn)管或樣品管計數(shù)為B，非特異管計數(shù)為NSB，則百分結(jié)合率計算公式如下：B/ B0=（B－NSB）/（ B0－NSB）×100%
3. logit計算:各標(biāo)準(zhǔn)點(diǎn)或樣品管的logit值計算公式如下:logit=ln（B/ B0）/（1－B/ B0）
4. 將標(biāo)準(zhǔn)品的OD均值與標(biāo)準(zhǔn)品0點(diǎn)的OD均相除，為標(biāo)準(zhǔn)點(diǎn)的百分結(jié)合率，在log-logit坐標(biāo)紙上繪圖。
5. Log-logit雙對數(shù)標(biāo)準(zhǔn)曲線：坐標(biāo)紙上橫軸從左至右*個1-9表示為*個10進(jìn)位，第二個1-9表示為第二個10進(jìn)位。第三個1-9表示為第三個10進(jìn)位。坐標(biāo)紙縱軸為百分比（1-99），即各標(biāo)準(zhǔn)吸光值的百分結(jié)合率。取一條通過各點(diǎn)的直線。要求盡可能多的點(diǎn)在線上，同時剩余的點(diǎn)均勻分布在直線的兩邊。樣品也同樣由吸光值計算百分結(jié)合率，再從縱軸上的相應(yīng)結(jié)合率找到直線上的點(diǎn)，此點(diǎn)對應(yīng)的橫坐標(biāo)濃度即為樣品的濃度，無須換算。
1、表達(dá)上清（非融合性蛋白）
直接將培養(yǎng)物和上清吸出，10000rmp x10min，取上清，-20℃或4℃保存，作為標(biāo)本進(jìn)行檢測，如有需要可進(jìn)行透析或純化處理；
2、菌體裂解物（融合性蛋白）
直接將培養(yǎng)物和上清吸出，10000rmp x10min，棄上清，沉淀用PBS洗一遍，500-800rmp x10min，棄上清，DDW重懸沉淀，冰水浴中用超聲波裂解儀進(jìn)行裂解，10000rmp x10min，取上清，-20℃或4℃保存，作為標(biāo)本進(jìn)行檢測，如有需要可進(jìn)行透析或純化處理；
注意事項
1. 試劑準(zhǔn)備：所有試劑都必須在使用前達(dá)到室溫，使用后請立即按照說明書要求保存。實驗操作中必須使用一次性吸頭，避免交叉污染。
2. 加樣：加樣時，要控制加樣速度，避免*孔與zui后一孔之見的時間間隔過大，否則將會導(dǎo)致不同的預(yù)孵育時間，從而影響實驗的準(zhǔn)確性以及重復(fù)性；一般加樣時間控制在10分鐘內(nèi)，如果樣本數(shù)量過多，可使用多道移液器。
3. 孵育：樣品要在密閉的容器內(nèi)進(jìn)行孵育，嚴(yán)格按照說明書上規(guī)定的孵育時間和溫度進(jìn)行。
4. 洗滌：洗滌過程中反應(yīng)孔中的殘留的洗滌液應(yīng)在濾紙上充分拍干，同時要消除板底殘留的液體和手指痕跡，避免影響zui后的酶標(biāo)儀讀數(shù)。
5. 反應(yīng)時間的控制：加入底物后請定時觀察反應(yīng)孔的顏色變化（比如，10分鐘左右），如果顏色較深，請?zhí)崆凹尤虢K止液終止反應(yīng)。
6. 建議實驗前預(yù)測樣品含量，如樣品濃度過高，應(yīng)對樣品進(jìn)行稀釋，計算結(jié)果時乘以相應(yīng)的稀釋倍數(shù)。
7. 建議使用本試劑盒時先做預(yù)實驗（即先做標(biāo)準(zhǔn)曲線，試用幾個標(biāo)本），如果對本試劑盒有任何疑問，可和所購經(jīng)銷商，如果因運(yùn)輸過程導(dǎo)致試劑盒失效，可要求調(diào)換，但概不承擔(dān)產(chǎn)品本身以外的任何損失。
操作步驟
1. 取出試劑盒，于室溫（20-25℃）放置15-30分鐘。實驗過程應(yīng)在室
concentration in each sample is interpolated from this curve.
REAGENTS PROVIDED
All reagents provided are stored at 2-8° C. Refer to the expiration date on the label.
1. MICROTITER PLATE 48 wells
2. ENZYME CONJUGATE 6.0 mL 1 vial
3. STANDARD.1 0ng/ml 1 vial
4. STANDARD.2 0.5ng/ml 1 vial
5. STANDARD.3 1.0ng/ml 1 vial
6. STANDARD.4 2.5ng/ml 1 vial
7. STANDARD.5 5.0ng/ml 1 vial
8. STANDARD.6 10ng/ml 1 vial
9. SUBSTRATE A 6.0 mL 1 vial
10. SUBSTRATE B 6.0 mL 1 vial
11. STOP SOLUTION 6.0 mL 1 vial
12. WASH SOLUTION x100 10 mL 1 vial
13. Instruction 1
SAMPLE COLLECTION AND STORAGE
Serum- Use a serum separator tube（SST） and allow samples to clot for 30minutes before centrifugation for 15minutes at approximay 1000xg. Remove serum and assay immediay or aliquot and store samples at -20 °C or -80°C.
Plasma - Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples for 15 minutes at 1000 x g at 2-8°C within 30minutes of collection. Store samples at -20°C or -80°C.Avoid repeated freeze-thaw cycles.
Cell culture fluid and other biological fluids - Remove particulates by centrifugation and assay immediay or aliquot and store samples at -20°C or -80°C.Avoid repeated freeze-thaw cycles.
NOTE: Serum, plasma, and cell culture fluid samples to be used within 7 days may be stored at 2-8°C, otherwise samples must stored at -20°C（≤2months） or -80°C（≤6months） to avoid loss of bioactivity and contamination. Avoid freeze-thaw cycles .When performing the assay slowly bring samples to room temperature.
DO NOT USE HEAT-TREATED SPECIMENS.
MATERIALS REQUIRED BUT NOT SUPPLIED
1. Microplate reader capable of measuring absorbance at 450 nm.
2. Precision pipettes to deliver 2 ml to 1 ml volumes.
3. Adjustable 10ml -100ml pipettes for reagent preparation.
4. Adjustable 10ml -100ml pipettes for reagent preparation.
5. 100 ml and 1 liter graduated cylinders.
6. Calibrated adjustable precision pipettes, preferably with disposable plastic tips. （A manifold multi-channel pipette is desirable for large assays.）
7. Absorbent paper.
8. 37°C incubator.
9. Distilled or deionized water.
10. Data analysis and graphing software. Graph paper: linear （Cartesian）, log-log or semi-log, or log-logit as desired.
11. Tubes to prepare standard or sample dilutions.
PRECAUTIONS
1. Do not substitute reagents from one kit lot to another. Standard, conjugate and microtiter plates are matched for optimal performance. Use only the reagents supplied by manufacturer.
2. Allow kit reagents and materials to reach room temperature （20-25°C） before use. Do not use water baths to thaw samples or reagents.
3. Do not use kit components beyond their expiration date.
空腹采集；
3、尿液的采集方式基本和唾液一樣的，即現(xiàn)采現(xiàn)用，但是一般隔夜尿不采集；
4、組織提取液如肺泡灌洗液等，采集后離心分離，800-1000rmp x 5min，取上清，-20℃或4℃保存?zhèn)溆茫?/span>
三．糞便以及天然孔排泄物：采集后一般現(xiàn)用PBS/生理鹽水溶解，充分混勻，800-1000rmp x 5min分離，取上清，-20℃或4℃保存?zhèn)溆茫?/span>
四．活檢組織一般進(jìn)行穿刺檢測，zui常見的就是肝活檢，像病毒性肝炎、脂肪肝、各種類型的肝硬化等進(jìn)行活檢簡單方便、準(zhǔn)確。
三、表達(dá)產(chǎn)物的檢測
（一）真核表達(dá)產(chǎn)物
1、 細(xì)胞上清（分泌性蛋白）
1.2、貼壁細(xì)胞或半貼壁，直接將細(xì)胞培養(yǎng)上清吸出，10000rmp x10min，取上清，-20℃或4℃保存，作為標(biāo)本進(jìn)行檢測，如有需要可進(jìn)行透析或純化處理；
1.2、不貼壁細(xì)胞，將培養(yǎng)基和細(xì)胞轉(zhuǎn)移至10ml離心管，500-800rmp x10min，吸取上清至另一10ml離心管中，10000rmp x10min， -20℃或4℃保存，作為標(biāo)本進(jìn)行檢測，如有需要可進(jìn)行透析或純化處理；
2、 細(xì)胞裂解物（非分泌性蛋白）
2.1、貼壁細(xì)胞或半貼壁，直接將細(xì)胞培養(yǎng)上清吸出，然后用0.01M PBS（pH 7.4）將細(xì)胞吹下至10ml離心管，500-800rmp x10min，棄上清，沉淀轉(zhuǎn)移至1.5ml離心管中， DDW重懸，置冰水浴中用超聲波裂解儀進(jìn)行裂解，10000rmp x10min，取上清，-20℃或4℃保存，作為標(biāo)本進(jìn)行檢測，如有需要可進(jìn)行透析或純化處理；
2.2、不貼壁細(xì)胞，將培養(yǎng)基和細(xì)胞轉(zhuǎn)移至10ml離心管，500-800rmp x10min，棄上清，沉淀移至另一10ml離心管中，001M PBS（pH 7.4）重懸，500-800rmp x10min，棄上清，沉淀移至1.5ml離心管中， DDW重懸，置冰水浴中用超聲波裂解儀進(jìn)行裂解，10000rmp x10min，取上清， -20℃或4℃保存，作為標(biāo)本進(jìn)行檢測，如有需要可進(jìn)行透析或純化處理；
（二）原核（大腸桿菌表達(dá)系統(tǒng)）表達(dá)產(chǎn)物
自備物品
1.酶標(biāo)儀（盡量提前預(yù)熱）
2.微量加液器、吸頭
3.蒸餾水或去離子水以及濾紙
樣本的采集及保存
一般的生物標(biāo)本包括有：
血液、體液、內(nèi)臟器官、糞便、胃液、活檢組織、組織提取液、天然孔分泌物及各種表達(dá)系統(tǒng)的表達(dá)產(chǎn)物等
一般為無菌操作，低溫保存（-80℃、液氮）
一．如果采集的實質(zhì)器官則要求：
1、 器官實質(zhì)不能太小
2、 以采集實質(zhì)性病灶區(qū)為佳：如淋巴結(jié)、心臟、非、肺、脾以及腸管等病毒、病菌聚集的地方為佳
3、 同一病例裝入同一容器，并做好標(biāo)記
4、 應(yīng)在0-8℃的低溫儲存和運(yùn)輸
5、 實質(zhì)性器官的處理：
5.1、取實質(zhì)性器官約0.5mg左右，充分剪碎或研磨
5.2、加入約500ul的PBS/生理鹽水，充分混勻
5.3、離心5000rmp x10min，去上清
5.4、-20℃保存，作為待檢標(biāo)本（切勿反復(fù)凍融）
二．體液（常見的包括血清、血漿、唾液以及尿液等）的處理方式
1、血液包括血漿和血清，它們的主要區(qū)別就是：血清凝血而血漿不凝血，所以它們的處理方式也就不一樣
1.1、采集血清時一般不加抗凝劑（主要為枸櫞酸鹽和檸檬酸鹽），采集后立即離心800-1000rmp x 5min分離，取上清，-20℃或4℃保存?zhèn)溆茫?/span>
1.2、如要采集血漿，一般要加入總體積1%的抗凝劑，采集后先室溫或4℃靜置半小時后，800-1000rmp x 5min分離，取上清，-20℃或4℃保存?zhèn)溆茫?/span>
2、胃液和唾液的采集方式一般現(xiàn)采現(xiàn)用，但是一般飯后半小時內(nèi)不易采集，因為剛進(jìn)食的唾液中富含豐富的唾液*，的采集的時間時
4. Use only deionized or distilled water to dilute reagents.
5. Do not remove microtiter plate from the storage bag until needed. Unused strips should be stored at 2-8°C in their pouch with the desiccant provided.
6. Use fresh disposable pipette tips for each transfer to avoid contamination.
7. Do not mix acid and sodium hypochlorite solutions.
8. Serum and plasma should be handled as potentially hazardous and capable of transmitting disease. Disposable gloves must be worn during the assay procedure, since no known test method can offer complete assurance that products derived from human blood will not transmit infectious agents. Therefore, all blood derivatives should be considered potentially infectious and good laboratory practices should be followed.
9. All samples should be disposed of in a manner that will inactivate viruses.
10. Solid Waste: Autoclave 60 min. at 121°C.
11. Liquid Waste: Add sodium hypochlorite to a final concentration of 1.0%. The waste should be allowed to stand for a minimum of 30 minutes to inactivate the viruses before disposal.
12. Substrate Solution is easily contaminated. If bluish prior to use, do not use.
13. Substrate B contains 20% acetone, keep this reagent away from sources of heat or flame.
14. Remove all kit reagents from refrigerator and allow them to reach room temperature （ 20-25°C）.
ASSAY PROCEDURE
1. Prepare all Standards before starting assay procedure （see Preparation Reagents）. It is recommended that all Standards and Samples be added in duplicate to the Microtiter Plate.
2. First, secure the desired number of coated wells in the holder, then add 100 μL of Standards or Samples to the appropriate well of the antibody pre-coated Microtiter Plate.
3. Add 50 μL of Conjugate to each well. Mix well. Complete mixing in this step is important. Cover and incubate for 1 hours at 37°C.
4. Prepare Substrate Solution no more than 15 minutes before end of incubation （see Preparation of Reagents）.
5. Wash the Microtiter Plate using one of the specified methods indicated below:
6. Manual Washing: Remove incubation mixture by aspirating contents of the plate into a sink or proper waste container. Using a squirt bottle, fill each well compley with distilled or de-ionized water, then aspirate contents of the plate into a sink or proper waste container. Repeat this procedure four more times for a total of FIVE washes. After final wash, invert plate, and blot dry by hitting plate onto absorbent paper or paper towels until no moisture appears. Note: Hold the sides of the plate frame firmly hen washing the plate to assure that all strips remain securely in frame.
7. Automated Washing: Aspirate all wells, then wash plate FIVE times using distilled or de-ionized water. Always adjust your washer to aspirate as much liquid as possible and set fill volume at 350 μL/well/wash （range: 350-400 μL）. After final wash, invert plate, and blot dry by hitting plate onto absorbent paper or paper towels until no moisture appears. It is recommended that the washer be set for a soaking time of 10 seconds or shaking time of 5 seconds between washes.
8. Add 50 μL Substrate A&B to each well. Cover and incubate for 15 minutes at 20-25°C.
9. Add 50 μL of Stop Solution to each well. Mix well.
大鼠血栓戊烷TXB2
標(biāo)本：血清或血漿
使用說明書
本試劑盒僅供科研使用，不得用于臨床及診斷使用！
預(yù)期應(yīng)用
ELISA法定量檢測大鼠血清或者血漿中血栓戊烷TXB2的含量。
試劑組成
| 名稱 | 規(guī)格 | 數(shù)量 | 保存 |
| 包被平底微孔板 | 48孔 | 1可拆卸板 | 2-8℃密封冷藏 |
| 酶結(jié)合物 | 6毫升 | 1瓶 | 2-8℃冷藏 |
| 標(biāo)準(zhǔn)品 | 1毫升 | 6瓶 | 2-8℃冷藏 |
| 顯色劑A | 6毫升 | 1瓶 | 2-8℃冷藏 |
| 顯色劑B | 6毫升 | 1瓶 | 2-8℃避光冷藏 |
| 終止液 | 6毫升 | 1瓶 | 2-8℃冷藏 |
| 濃縮洗滌液（100倍稀釋） | 10毫升 | 1瓶 | 2-8℃冷藏 |
| 使用說明書 | | 1份 | |
13. All residual wash liquid must be drained from the wells by efficient
aspiration or by decantation followed by tapping the plate forcefully on absorbent paper. Never insert absorbent paper directly into the wells.
14. Because Stabilized Chromogen is light sensitive, avoid prolonged exposure to light. Also avoid contact between Stabilized Chromogen and metal, or color may develop.
15. Incomplete washing will adversely affect the test outcome. All washing must be performed with Wash Buffer provided.
16. Washing can be performed manually as follows: compley aspirate the liquid from all wells by gently lowering anaspiration tip （aspiration device） into the bottom of each well. Take care not to scratch the inside of the well.
17. After aspiration, fill the wells with at least 0.4 mL of diluted wash solution. Let soak for 15 to 30 seconds, then aspirate the liquid. Repeat as directed under ASSAY METHOD. After the washing procedure, the plate is inverted and tapped dry on absorbent tissue.
18. Alternatively, the wash solution may be put into a squirt bottle. If a squirt bottle is used, flood the plate with wash buffer, compley filling all wells. After the washing procedure, the plate is inverted and tapped dry on absorbent tissue.
19. If using an automated washer, the operating instructions for washing equipment should be carefully followed.
20. Assay Procedure Preliminary notes: Do not mix reagents from different lots. It is recommended that assays be performed in duplicate .Standards and samples must be assayed at the same time. Avoid exposing the substrate to direct sunlight.
10. Read the Optical Density （O.D.） at 450 nm using a microtiter plate reader within 30 minutes.
B.G CALCULATION OF RESULTS
1. Calculate the mean absorbance value A450 for each set of reference standards and samples.
2. Divide the average A450 value for each standard, control and test sample by the average A450 of standard0 and multiply by 100 to obtain %B/B0 for each sample.
3. Prepare a standard curve by plotting the average absorbance of each standard versus the corresponding concentrations of the standards on linear-log graph paper or the %B/B0 value for each standard versus the corresponding concentration of the standard on linear-log or logit-log graph paper. logit=ln（B/ B0）/（1－B/ B0）
4. Any values obtained for diluted samples must be further converted by applying the appropriate dilution factor in the calculations.
5. The standard density is a X, the B/ B0 is a Y, sitting to mark the paper in the logit- log up draw a standard curve.According to the B/ B0 that need to be measured the sample can from sit to mark the density value that the paper looks up the sample up.
6. The sensitivity by this assay is 0.01ng/ml
7. The standard curve presented here is an example of the data typically produced with this kit; however, your results will not be identical to these.
8. Standard curve
Disposal Note Safety
1. This kit contains materials with small quantities of sodium azide. Sodium azide reacts with lead and copper plumbing to form explosive metal azides. Upon disposal, flush drains with a large volume of water to prevent azide accumulation. Avoid ingestion and contact with eyes, skin and mucous membranes. In case of contact, rinse affected area with plenty of water. Observe all federal, state and local regulations for disposal.
2. All blood components and biological materials should be handled as potentially hazardous. Follow universal precautions as established by the Centers for Disease Control and Prevention and by the Occupational Safety and Health Administration when handling and disposing of infectious agents.
Additional Materials Required Quality Control
1. When not in use, kit components should be refrigerated. All reagents should be warmed to room temperature before use.
2. Microtiter plates should be allowed to come to room temperature before opening the foil bags. Once the desired number of strips has been removed, immediay reseal the bag and store at 2 - 8°C to maintain plate integrity.
3. Samples should be collected in pyrogen/endotoxin-free tubes.
4. Samples should be frozen if not analyzed shortly after collection. Avoid multiple freeze-thaw cycles of frozen samples. Thaw compley and mix well prior to analysis.
5. When possible, avoid use of badly hemolyzed or lipemic sera. If large amounts of particulate matter are present, centrifuge or filter prior to analysis.
6. It is recommended that all standards, controls and samples be run in duplicate.
7. When pipetting reagents, maintain a consistent order of addition from well-to-well. This ensures equal incubation times for all wells.
8. Cover or cap all reagents when not in use.
9. Do not mix or interchange different reagent lots from various kit lots.
10. Do not use reagents after the kit expiration date.
11. Read absorbances within 2 hours of assay completion.
12. The provided controls should be run with every assay. If control values fall outside pre-established ranges, the accuracy of the assay is suspect.
