大鼠極低密度脂蛋白（VLDL）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。
檢測范圍： 96T
3μg/ml -120μg/ml
使用目的：
本試劑盒用于測定大鼠血清、血漿及相關(guān)液體樣本中極低密度脂蛋白（VLDL）含量。
實(shí)驗(yàn)原理
本試劑盒應(yīng)用雙抗體夾心法測定標(biāo)本中大鼠極低密度脂蛋白（VLDL）水平。用純化的大鼠極低密度脂蛋白（VLDL）抗體包被微孔板，制成固相抗體，往包被單抗的微孔中依次加入極低密度脂蛋白（VLDL），再與HRP標(biāo)記的極低密度脂蛋白（VLDL）抗體結(jié)合，形成抗體-抗原-酶標(biāo)抗體復(fù)合物，經(jīng)過*洗滌后加底物TMB顯色。TMB在HRP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極低密度脂蛋白（VLDL）呈正相關(guān)。用酶標(biāo)儀在450nm波長下測定吸光度（OD值），通過標(biāo)準(zhǔn)曲線計(jì)算樣品中大鼠極低密度脂蛋白（VLDL）濃度。
試劑盒組成
標(biāo)本要求
1．標(biāo)本采集后盡早進(jìn)行提取，提取按相關(guān)文獻(xiàn)進(jìn)行，提取后應(yīng)盡快進(jìn)行實(shí)驗(yàn)。若不能馬上進(jìn)行試驗(yàn)，可將標(biāo)本放于-20℃保存，但應(yīng)避免反復(fù)凍融
2．不能檢測含NaN3的樣品，因NaN3抑制辣根過氧化物酶的（HRP）活性。
操作步驟
1. 標(biāo)準(zhǔn)品的稀釋：本試劑盒提供原倍標(biāo)準(zhǔn)品一支，用戶可按照下列圖表在小試管中進(jìn)行稀釋。
2. 加樣：分別設(shè)空白孔（空白對(duì)照孔不加樣品及酶標(biāo)試劑，其余各步操作相同）、標(biāo)準(zhǔn)孔、待測樣品孔。在酶標(biāo)包被板上標(biāo)準(zhǔn)品準(zhǔn)確加樣50μl，待測樣品孔中先加樣品稀釋液40μl，然后再加待測樣品10μl（樣品zui終稀釋度為5倍）。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(dòng)混勻。
3. 溫育：用封板膜封板后置37℃溫育30分鐘。
4. 配液：將30倍濃縮洗滌液用蒸餾水30倍稀釋后備用
5. 洗滌：小心揭掉封板膜，棄去液體，甩干，每孔加滿洗滌液，靜置30秒后棄去，如此重復(fù)5次，拍干。
6. 加酶：每孔加入酶標(biāo)試劑50μl，空白孔除外。
7. 溫育：操作同3。
8. 洗滌：操作同5。
9. 顯色：每孔先加入顯色劑A50μl，再加入顯色劑B50μl，輕輕震蕩混勻，37℃避光顯色15分鐘.
10. 終止：每孔加終止液50μl，終止反應(yīng)（此時(shí)藍(lán)色立轉(zhuǎn)黃色）。
11. 測定：以空白空調(diào)零，450nm波長依序測量各孔的吸光度（OD值）。 測定應(yīng)在加終止液后15分鐘以內(nèi)進(jìn)行。
操作程序總結(jié)：
計(jì)算
以標(biāo)準(zhǔn)物的濃度為橫坐標(biāo)，OD值為縱坐標(biāo)，在坐標(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(jì)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(jì)算出樣品濃度，再乘以稀釋倍數(shù)，即為樣品的實(shí)際濃度。
注意事項(xiàng)
1．試劑盒從冷藏環(huán)境中取出應(yīng)在室溫平衡15-30分鐘后方可使用，酶標(biāo)包被板開封后如未用完，板條應(yīng)裝入密封袋中保存。
2．濃洗滌液可能會(huì)有結(jié)晶析出，稀釋時(shí)可在水浴中加溫助溶，洗滌時(shí)不影響結(jié)果。
3．各步加樣均應(yīng)使用加樣器，并經(jīng)常校對(duì)其準(zhǔn)確性，以避免試驗(yàn)誤差。一次加樣時(shí)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。
4． 請(qǐng)每次測定的同時(shí)做標(biāo)準(zhǔn)曲線，做復(fù)孔。如標(biāo)本中待測物質(zhì)含量過高（樣本OD值大于標(biāo)準(zhǔn)品孔*孔的OD值），請(qǐng)先用樣品稀釋液稀釋一定倍數(shù)（n倍）后再測定，計(jì)算時(shí)請(qǐng)zui后乘以總稀釋倍數(shù)（×n×5）。
5． 封板膜只限一次性使用，以避免交叉污染。
6．底物請(qǐng)避光保存。
7．嚴(yán)格按照說明書的操作進(jìn)行，試驗(yàn)結(jié)果判定必須以酶標(biāo)儀讀數(shù)為準(zhǔn).
8．所有樣品，洗滌液和各種廢棄物都應(yīng)按傳染物處理。
9．本試劑不同批號(hào)組分不得混用。
10. 如與英文說明書有異，以英文說明書為準(zhǔn)。
保存條件及有效期
1．試劑盒保存：；2-8℃。
2．有效期：6個(gè)月
Rat VLDL
FOR RESEARCH USE ONLY
Assay range：3μg/mL – 120μg/mL 96determinations
Purpose
This kit allows for the determination ofVLDL concentrations in Ratserum, cellculture supernates and other biological fluids
Principle of the assay
The kit assay RatVLDLlevel in the sample，use Purified RatVLDLantibody to coat microtiter plate wells, make solid-phase antibody, then addVLDLto wells,CombinedVLDL antibody which With HRP labeled,become antibody - antigen - enzyme-antibody complex, after washing Compley,Add TMB substrate solution,TMB substrate becomes blue color At HRP enzyme-catalyzed,reaction is terminated by the addition of a sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450 nm. The concentration of RatVLDLin the samples is then determined by comparing the O.D. of the samples to the standard curve.
Materials provided with the kit
| 1 | wash solution | 20ml×1bottle | 7 | Stopp Solution | 6ml×1 bottle |
| 2 | HRP-Conjugate reagent | 6ml×1 bottle | 8 | Standard（240μg/ml） | 0.5ml×1 bottle |
| 3 | Microelisa stripplate | 12well×8strips | 9 | Standard diluent | 1.5ml×1bottle |
| 4 | Sample diluent | 6ml×1 bottle | 10 | Instruction | 1 |
| 5 | Chromogen Solution A | 6ml×1 bottle | 11 | Closure plate membrane | 2 |
| 6 | Chromogen Solution B | 6ml×1 bottle | 12 | Sealed bags | 1 |
Specimen requirements
1. extractas soon as possible after Specimen collection,and according to the relevant literature, and should be experiment as soon as possible after the extraction. If it can’t, specimen can be kept in -20 ℃ to preserve, Avoid repeated freeze-thaw cycles.
2. Can’t detect the sample which contain NaN3, because NaN3 inhibits HRP active.
Assay procedure
1. Dilute and add sample:Dilute Original density Standard as follow table:
| 120μg/ml | 5 Standard | 150μl Original density Standard+150μl Standard diluent |
| 80μg/mL | 4 Standard | 150μl 5 Standard+150μl Standard diluent |
| 40μg/mL | 3 Standard | 150μl 4 Standard+150μl Standard diluent |
| 20μg/mL | 2 Standard | 150μl 3 Standard +150μl Standard diluent |
| 10μg/mL | 1 Standard | 150μl 2 Standard +150μl Standard diluent |
2.add sample：Set blank wells separay （blank comparison wells don’t add sample and HRP-Conjugate reagent, other each step operation is same）. testing sample well. add Sample dilution 40μl to testing sample well, then add testing sample 10μl （sample final dilution is 5-fold）, add sample to wells , don’t touch the well wall as far as possible, and Gently mix.
3.Incubate: After closing plate with Closure plate membrane ,incubate for 30 min at 37℃.
4.Configurate liquid: 30-fold（or 20-fold） wash solution diluted 30-fold （or 20-fold） with distilled water and reserve.
5.washing：Uncover Closure plate membrane, discardLiquid, dry by swing, add washing buffer to every well, still for 30s then drain,repeat 5 times, dry by pat.
6.add enzyme：Add HRP-Conjugate reagent 50μl to each well, except blank well.
7.incubate：Operation with 3.
8.washing：Operation with 5.
9.color：Add Chromogen Solution A 50ul and Chromogen Solution B to each well, evade the light preservation for 15 min at 37℃
10.Stop the reaction：Add Stop Solution50μl to each well, Stop the reaction（the blue color change to yellow color）.
11.assay：take blank well as zero , Read absorbance at 450nm after Adding Stop Solution and within 15min.
Steps description
| AddStandard, Sample diluent, incubate for 30 min at 37℃. |
| Wash 5 time,AddHRP-Conjugate reagent, incubate for 30 min at 37℃. |
| Wash 5 times,Add Chromogen Solution A and B, incubate for 30 min at 37℃. |
| Read absorbance at 450nm within 15 min |
Calculate
Take the standard density as the horizontal, the OD value for the vertical ,draw the standard curve on graph paper, Find out the corresponding density according to the sample OD value by the Sample curve, multiplied by the dilution multiple, or calculate the straight line regression equation of the standard curve with the standard density and the OD value ,with the sample OD value in the equation, calculate the sample density, multiplied by the dilution factor, the result is the sample actual density.
Important notes
1. The kit takes out from the refrigeration environment should be balanced 15-30 minutes in the room temperature, ELISA plates coated if has not use up after opened, the plate should be stored in Sealed bag.
2. washing buffer will Crystallization separation, it can be heated the water helps dissolve when dilute . Washing does not affect the result.
3. add Sample with sampler Each step, And proofread its accuracy frequently, avoids the experimental error. add sample within 5 min, if the number of sample is much , recommend to use Volley .
4. if the testing material content is excessively higher （The sample OD is bigger than the first standard well ）,please dilute Sample （n-fold）, Please diluenteand multiplied by the dilution factor.（×n×5）.
5. Closure plate membrane only limits the disposable use, to avoid cross-contamination.
6. The substrate evade the light preservation.
7. Please according to use instruction strictly, The test result determination must take the microtiter plate reader as a standard.
8. All samples, washing buffer and each kind of reject should according to infective material process.
9. Do not mix reagents with those from other lots.
Storage and validity
1．Storage： 2-8℃.
2．validity： six months
