大鼠透明質(zhì)酸(HA)ELISA說明書

1、大鼠透明質(zhì)酸(HA)酶聯(lián)免疫分析ELISA試劑盒使用說明書本試劑盒僅供研究使用北京奇松生物科技有限公司 產(chǎn)品編號：QS42052預(yù)期應(yīng)用ELISA法定量測定大鼠血清、血漿以及蛋清和蛋黃中透明質(zhì)酸（HA）含量。實驗原理本試劑盒應(yīng)用雙抗體夾心酶標(biāo)免疫分析法測定標(biāo)本中透明質(zhì)酸水平。用純化的透明質(zhì)酸結(jié)合蛋白(HABP)包被微孔板，制成固相抗體，往包被單抗的微孔中依次加入透明質(zhì)酸、生物素化的抗大鼠透明質(zhì)酸結(jié)合蛋白（HABP）、HRP標(biāo)記的親和素，經(jīng)過徹底洗滌后用底物TMB顯色。TMB在過氧化物酶的催化下轉(zhuǎn)化成藍色，并在酸的作用下轉(zhuǎn)化成最終的黃色。顏色的深淺和樣品中的透明質(zhì)酸呈正相關(guān)。用酶標(biāo)儀在450nm

2、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：一塊（96孔） 標(biāo)準(zhǔn)品（凍干品）：2瓶，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100 nmol/L，做系列倍比稀釋后，分別稀釋成100 nmol/L，50 nmol/L ，25 nmol/L，12.5 nmol/L，6.25 nmol/L，3.12 nmol/L，1.56 nmol/L，其原液直接作為最高標(biāo)準(zhǔn)濃度，樣品稀釋液直接作為標(biāo)準(zhǔn)濃度0 nmol/L，臨用前15分鐘內(nèi)配制。如配制50 nmol/L標(biāo)準(zhǔn)品：取ml 100 nmol/Lml樣品稀釋液的Ep

3、pendorf管中，混勻即可，其余濃度以此類推。2. 樣品稀釋液：120ml/瓶。3. 檢測稀釋液A：110ml/瓶。4. 檢測稀釋液B：110ml/瓶。5. 檢測溶液A：1120ul/瓶（1：100）臨用前以檢測稀釋液A 1：100稀釋ml。如1ul檢測溶液A加99ul檢測稀釋液A的比例配制，輕輕混勻，在使用前一小時內(nèi)配制。6. 檢測溶液B：1120ul/瓶（1：100）臨用前以檢測稀釋液B1：100稀釋。稀釋方法同檢測溶液A。7. 底物溶液：110ml/瓶。 8. 濃洗滌液：130ml/瓶，使用時每瓶用蒸餾水稀釋25倍。 9. 終止液：110ml/瓶（2N H2SO4）。 標(biāo)本的采集及保存

4、 1血清：標(biāo)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?qū)?biāo)本放于-20保存，但應(yīng)避免反復(fù)凍融。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 x g離心15分鐘，或?qū)?biāo)本放于-20保存，但應(yīng)避免反復(fù)凍融。3. 大鼠蛋：分離蛋清和蛋黃，處理后的標(biāo)本放于-20保存，但應(yīng)避免反復(fù)凍融。，注：標(biāo)本溶血會影響最后檢測結(jié)果，因此溶血標(biāo)本不宜進行此項檢測。操作步驟實驗開始前，請?zhí)崆芭渲煤盟性噭噭┗驑悠废♂寱r，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，用樣品稀釋液進行稀釋，以使樣品符合試劑盒的檢測

5、范圍。1. 加樣：分別設(shè)空白孔、標(biāo)準(zhǔn)孔、待測樣品孔。空白孔加樣品稀釋液100ul，余孔分別加標(biāo)準(zhǔn)品或待測樣品100ul，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。每孔加檢測溶液A工作液 100ul（取1ul檢測溶液A加99ul檢測稀釋液A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，350ul/每孔，甩干。 4. 每孔加檢測溶液B工作液（同檢測A工作液）

6、 100ul，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，350ul/每孔，甩干。6. 依序每孔加底物溶液90ul，37避光顯色（30分鐘內(nèi),此時肉眼可見標(biāo)準(zhǔn)品的前3-4孔有明顯的梯度蘭色，后3-4孔梯度不明顯，即可終止）。7. 依序每孔加終止溶液50ul，終止反應(yīng)（此時藍色立轉(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)進行檢測。注：1 每次實驗留一孔作為空白調(diào)零孔，該孔不加任何試劑，只是最后

7、加底物溶液及2NH2SO4。測量時先用此孔調(diào)OD值至零。 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、檢測溶液A工作液、檢測溶液B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、檢測溶液A工作液或檢測溶液B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0.3ml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，應(yīng)在

8、熟練使用后再用到正式實驗過程中。特異性 本試劑盒可同時檢測重組或天然的大鼠透明質(zhì)酸，且與其它相關(guān)蛋白無交叉反應(yīng)。計算 以標(biāo)準(zhǔn)物的濃度為橫坐標(biāo)（對數(shù)坐標(biāo)），OD值為縱坐標(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 洗滌過程非常重要，不充分的洗滌易造成假陽性。2 一次加樣時間最好控制在5分鐘內(nèi)，如標(biāo)本數(shù)量多，推薦使用排槍加樣。3 請每次測定的同時做標(biāo)準(zhǔn)曲線，最好做復(fù)孔。4 如標(biāo)本中待測物質(zhì)含量過

9、高，請先稀釋后再測定，計算時請最后乘以稀釋倍數(shù)。5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。6底物請避光保存。檢測范圍：1.56 nmol/L -100 nmol/L說明 1試劑盒保存：-20（較長時間不用時）；2-8（頻繁使用時）。2有效期：6個月3濃洗滌液會有鹽析出，稀釋時可在水浴中加溫助溶。 4剛開啟的酶聯(lián)板孔中可能會含有少許水樣物質(zhì)，此為正?，F(xiàn)象，不會對實驗結(jié)果造成任何影響。 5中、英文說明書可能會有不一致之處，請以英文說明書為準(zhǔn)。英文版Rat Hyaluronic acid (HA) ELISA kitCatalog No. QS42052(96 tests)I

10、ntended useThis immunoassay kit allows for the specific measurement of Rat HA concentrations in serum and plasma.IntroductionHyaluronic Acid (HA), also called hyaluronate or hyaluronan, is a mucopolysaccharide widely distributed throughout the body. HA is produced mainly by fibroblasts and other spe

11、cialized connective tissue cells. As a free molecule, HA can be found in the plasma and synovial fluid. HA is quickly removed from circulation by specific receptors present in sinusoidal cells (SEC) of the liver; the estimated half-life in plasma is 5-6 minutes.Test principleThis assay employs the q

12、uantitative sandwich enzyme immunoassay technique. A Hyaluronic Acid binding protein (HABP) specific for HA has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and any HA present is bound. An enzyme-linked HABP specific for HA is added to the wells. Following a w

13、ash to remove any unbound antibody-enzyme reagent, a substrate solution is added to the wells and color develops in proportion to the amount of HA bound in the initial step. The color development is stopped and the intensity of the color is measured.Materials and componentsReagent QuantityAssay plat

14、e 1Standard 2Sample Diluent1 x 20mlAssay Diluent A1 x 10mlAssay Diluent B1 x 10mlDetection Reagent A1 x 120ulDetection Reagent B1 x 120ulWash Buffer1 x 30ml(25 x concentrate)Substrate1 x 10mlStop Solution1 x 10mlSample collection and storageSerum - Use a serum separator tube (SST) and allow samples

15、to clot for 30 minutes beforecentrifugation for 15 minutes at approximately 1000 x g. Remove serum and assay immediately or aliquot and store samples at -20 C.Plasma - Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples for15 minutes at 1000 x g at 2 - 8 C within 30 minutes

16、of collection. Store samples at -20 C. Avoid repeated freeze-thaw cycles.Note: Citrate plasma has not been validated for use in this assay.Limitations of the procedureFOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES.1. The kit should not be used beyond the expiration date on the kit label

17、.2. Do not mix or substitute reagents with those from other lots or sources.3. If samples generate values higher than the highest standard, further dilute the sampleswith the Assay Diluent and repeat the assay. Any variation in standard diluent, operator, pipetting technique, washing technique,incub

18、ation time or temperature, and kit age can cause variation in binding.4. This assay is designed to eliminate interference by soluble receptors, ligands, binding proteins, and other factors present in biological samples. Until all factors have been tested in the Quantikine Immunoassay, the possibilit

19、y of interference cannot be excluded.Reagent preparationBring all reagents to room temperature before use.Wash Buffer - If crystals have formed in the concentrate, warm to room temperature and mix gently until the crystals have completely dissolved. Dilute 20 mL of Wash Buffer Concentrate into deion

20、ized or distilled water to prepare 500 ml of Wash Buffer.Standard - Reconstitute the Standard with 1.0 mL of Sample Diluent. This reconstitution produces a stock solution of 100 nmol/L. Allow the standard to sit for a minimum of 15 minutes with gentle agitation prior to making serial dilutions. The

21、undiluted standard serves as the high standard (100 nmol/L). The Sample Diluent serves as the zero standard (0 nmol/L).Detection Reagent A and B - Dilute to the working concentration specified on the vial label using Assay Diluent A and B (1:100), respectively.Assay procedureAllow all reagents to re

22、ach room temperature. Arrange and label required number of strips.1. Prepare all reagents, working standards and samples as directed in the previous sections.2. Add 100 uL of Standard, Control, or sample* per well. Cover with the adhesive strip. Incubate for 2 hours at 37 C. 3. Remove the liquid of

23、each well, dont wash. 4. Add 100 uL of Detection Reagent A to each well. Incubate for 1 hour at 37C. Detection Reagent A may appear cloudy. Warm to room temperature and mix gently until solution appears uniform.5. Aspirate each well and wash, repeating the process three times for a total of three wa

24、shes. Wash by filling each well with Wash Buffer (350 uL) 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

25、 the plate and blot it against clean paper towels.6. Add 100 uL of Detection Reagent B to each well. Cover with a new adhesive strip.Incubate for 1 hours at 37 C.7. Repeat the aspiration/wash as in step 5.8. Add 90 uL of Substrate Solution to each well. Incubate for 30 minutes at room temperature. P

26、rotect from light.9. Add 50 uL of Stop Solution to each well. If color change does not appear uniform, gently tap the plate to ensure thorough mixing.10. Determine the optical density of each well within 30 minutes, using a microplate reader setto 450 nm. SpecificityThis assay recognizes recombinant

27、 and natural Rat HA. No significant cross-reactivity or interference was observed.Important Note:1. The wash procedure is critical. Insufficient washing will result in poor precision and falsely elevated absorbance readings.2. It is recommended that no more than 32 wells be used for each assay run i

28、f manual pipetting is used since pipetting of all standards, specimens and controls should be completed within 5 minutes. A full plate of 96 wells may be used if automated pipetting is available.3. Duplication of all standards and specimens, although not required, is recommended.4. When mixing or re

29、constituting protein solutions, always avoid foaming.5. 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.6. To ensure accurate results, proper adhesion o

30、f plate sealers during incubation steps is necessary.Calculation of resultsAverage 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 p

31、arameter logistic (4-PL) curve-fit. As an alternative, construct a standard curve by plotting the mean absorbance for each standard on the y-axis againstthe concentration on the x-axis and draw a best fit curve through the points on the graph. The data may be linearized by plotting the log of the HA concentrations versus the log of the O.D. and the best fit line can be