人瘦素（LEP）酶聯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

0.3μg/L -12μg/L

使用目的：

本試劑盒用于測定人血清、血漿及相關液體樣本中瘦素（LEP）含量。

實驗原理

本試劑盒應用雙抗體夾心法測定標本中人瘦素（LEP）水平。用純化的人瘦素（LEP）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瘦素（LEP），再與HRP標記的瘦素（LEP）抗體結合，形成抗體-抗原-酶標抗體復合物，經過*洗滌后加底物TMB顯色。TMB在HRP酶的催化下轉化成藍色，并在酸的作用下轉化成終的黃色。顏色的深淺和樣品中的瘦素（LEP）呈正相關。用酶標儀在450nm波長下測定吸光度（OD值），通過標準曲線計算樣品中人瘦素（LEP）濃度。 

試劑盒組成 

標本要求 

1．標本采集后盡早進行提取，提取按相關文獻進行，提取后應盡快進行實驗。若不能馬上進行試驗，可將標本放于-20℃保存，但應避免反復凍融

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

2.加樣：分別設空白孔（空白對照孔不加樣品及酶標試劑，其余各步操作相同）、標準孔、待測樣品孔。在酶標包被板上標準品準確加樣50μl，待測樣品孔中先加樣品稀釋液40μl，然后再加待測樣品10μl（樣品終稀釋度為5倍）。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。

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5次，拍干。

6.加酶：每孔加入酶標試劑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10分鐘.

10.終止：每孔加終止液50μl，終止反應（此時藍色立轉黃色）。

11.測定：以空白空調零，450nm波長依序測量各孔的吸光度（OD值）。 測定應在加終止液后15分鐘以內進行。

操作程序總結：

計算

  以標準物的濃度為橫坐標，OD值為縱坐標，在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．試劑盒從冷藏環境中取出應在室溫平衡15-30分鐘后方可使用，酶標包被板開封后如未用完，板條應裝入密封袋中保存。

2．濃洗滌液可能會有結晶析出，稀釋時可在水浴中加溫助溶，洗滌時不影響結果。

3．各步加樣均應使用加樣器，并經常校對其準確性，以避免試驗誤差。一次加樣時間控制在5分鐘內，如標本數量多，*使用排槍加樣。

4．請每次測定的同時做標準曲線，做復孔。如標本中待測物質含量過高（樣本OD值大于標準品孔孔的OD值），請先用樣品稀釋液稀釋一定倍數（n倍）后再測定，計算時請后乘以總稀釋倍數（×n×5）。

5．封板膜只限一次性使用，以避免交叉污染。

6．底物請避光保存。

7．嚴格按照說明書的操作進行，試驗結果判定必須以酶標儀讀數為準.

8．所有樣品，洗滌液和各種廢棄物都應按傳染物處理。

9．本試劑不同批號組分不得混用。

保存條件及有效期

1．試劑盒保存：2-8℃。

2．有效期：6個月

Human leptin

FOR RESEARCH USE ONLY

Assay range：0.3μg/L -12μg/L                96 determinations

Purpose

This kit allows for the determination of leptin concentrations in Human serum, cell culture supernates and other biological fluids

Principle of the assay

The kit assay Human leptin level in the sample, use Purified Human leptin antibody to coat microtiter plate wells, make solid-phase antibody, then add leptin to wells, Combined leptin antibody which With HRP labeled, become antibody - antigen - enzyme-antibody complex, after washing Completely, 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 Human leptin in 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 Stop Solution 6ml×1 bottle

2 HRP-Conjugate reagent 6ml×1 bottle 8 Standard（24μg/L） 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.extract as 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:

12μg/L 5 Standard 150μl Original density Standard+150μl Standard diluent

6μg/L 4 Standard 150μl 5 Standard+150μl Standard diluent

3μg/L 3 Standard 150μl 4 Standard+150μl Standard diluent

1.5μg/L 2 Standard 150μl 3 Standard +150μl Standard diluent

0.75μg/L 1 Standard 150μl 2 Standard +150μl Standard diluent

2.add sample：Set blank wells separately (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, discard Liquid, 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 50ul to each well, evade the light preservation for 10 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

Standard, Sample diluent

Add Standard, Sample diluent, incubate for 30 min at 37℃.

Wash 5 time,Add HRP-Conjugate reagent, incubate for 30 min at 37℃.

Wash 5 times,Add Chromogen Solution A and B, incubate for 10 min at 37℃.

Add Stopp Solution

Read absorbance at 450nm within 15 min

calculate

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 diluente and 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