羊戊型肝炎病毒IgG（HEV-IgG）酶聯免疫分析（ELISA）

試劑盒使用說明書

本試劑盒僅供研究使用。

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戊型肝炎病毒IgG（HEV-IgG）的含量。

實驗原理

本試劑盒應用間接法測定標本中人戊型肝炎病毒IgG（HEV-IgG）水平。用純化的抗原包被微孔板，制成固相抗原，往包被單抗的微孔中依次加入已知濃度的戊型肝炎病毒IgG（HEV-IgG）標準品和未知濃度的布魯氏桿菌IgG抗體（Brucella IgG）待檢樣品，溫育后，加入*標記的抗IgG抗體，再與鏈霉親和素-HRP結合，形成免疫復合物，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zui終的黃色。顏色的深淺和樣品中的戊型肝炎病毒IgG（HEV-IgG）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戊型肝炎病毒IgG（HEV-IgG）濃度。

試劑盒組成

標本要求

1．不能檢測含NaN3的樣品，因NaN3抑制辣根過氧化物酶的（HRP）活性。

2．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

操作步驟

1.       根據待測樣品數量加上標準品的數量決定所需的板條數。每個標準品和空白孔建議做復孔。每個樣品根據自己的數量來定，能使用復孔的盡量做復孔。

2.       加樣：分別設空白孔（空白對照孔不加樣品，其余各步操作相同）、標準品孔、待測樣品孔。然后在標準品孔中加入標準品50μl，在樣本反應孔中加入50微升樣本，蓋上封板膜，輕輕振蕩混勻，37℃溫育45分鐘。

3.       配液：將30倍濃縮洗滌液用蒸餾水30倍稀釋后備用

4.       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4次，拍干。

5.       加*標記的抗-IgG抗體：每孔加入*標記的抗-IgG抗體50μl。37℃溫育30分鐘

6.       洗滌：操作同4。

7.       加鏈霉親和素-HRP：每孔加入50μl的鏈酶親和素-HRP，輕輕振蕩混勻，37℃溫育30分鐘。

8.       洗滌：操作同4。

9.       顯色：每孔先加入顯色劑A 50μl，再加入顯色劑B 50μl，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（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。

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值待入方程式，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，推薦使用排槍加樣。

4．  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），請先將樣本稀釋一定倍數（n倍）后再測定，計算時請zui后乘以稀釋倍數（×5×n）。

5．  封板膜只限一次性使用，以避免交叉污染。

6．本試劑不同批號組分不得混用。顯色劑B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. 如與英文說明書有異，以英文說明書為準。

線形范圍：

0.7ng/L - 20ng/L

規格：

96人份/盒

保存條件及有效期

1．試劑盒保存：；2-8℃。

2．有效期：6個月

Goat HEV-IgG

Package size: 96 determinations.

Drug Names

Generic Name：Goat HEV-IgG ELISA Kit.

Purpose

This kit allows for the determination of HEV-IgG concentrations in Goat serum, blood plasma, and other biological fluids.

Principle of the assay

The kit assay Goat HEV-IgG level in the sample，use Purified antigen to coat microtiter plate wells, make solid-phase antigen, Samples which including standards of known HEV-IgG concentrations and unknowns are pipetted into coated microtiter wells, after Incubating ,add Biotinylated anti-IgG,and Combined Streptavidin-HRP, become Immune complex, after washing Compley, Add TMB substrate solution, TMB substrate becomes blue color At HRP enzyme-catalyzed, And at the effect of acid the color finally become yellow, The intensity of this coloured product is directly proportional to the concentration of AFP present in the samples. measure the optical densit （OD） at 450 nm with microtiter plate reader, calculate Goat HEV-IgG concentration by standard curv.

Materials provided with the kit

Specimen requirements

1.       Can’t detect the sample which contain NaN3, because NaN3 inhibits HRP active.

2.       extract as soon as possible after Specimen collection, Extracted according to the relevant literature, and should be experiment as soon as possible after the extraction. If it can not be tested immediay, specimen can be kept in -20 ℃ to preserve, Avoid repeated freeze-thaw cycles.

Assay procedure

1.         Determine the number of microwell strips required to test the desired number of samples,. Each sample, standard and blank should be assayed in duplicate.

2.         add sample：Set blank wells separay （blank comparison wells don’t add sample and ELISA reagent, other each step operation is same）. Add 50 μL of Standards or Samples to the appropriate well of the antibody pre-coated Microtiter Plate, and Gently mix. Incubate for 45 min at 37℃

3.         Configurate liquid: 20 times of wash solution diluted 20 times with distilled water and reserve.

4.         washing：remove Liquid, dry by swing, add washing buffer to every well, still for 30 second then remove, repeat 4 times.

5.         add Biotinylated anti-IgG: Add diluted Biotinylated anti-IgG 50ul to all wells, Incubate for 30 min at 37℃

6.         washing：Operation with 4.

7.         add streptavidin-HRP : Add streptavidin-HRP 50ul to all wells, Gently mix Incubate for 15 min at 37℃

8.         washing：Operation with 4.

9.         color：Add Chromogen Solution A 50ul and Chromogen Solution B to each well,Incubate 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 Immediay）.

11.     assay：take blank well as zero , measure the optical densit （OD） at 450 nm after Adding Stop Solution and within 15min.

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 multiple, 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  then use, 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.       Please make specification curve when you assay, had better make duplicate well, if in the sample the testing material content is excessively high （The sample OD is bigger than the first standard well OD ）,please use Sample dilution to dilute certain multiple （n times）,then assay. Please multiply total Dilution Times when calculate（×n×5）.

5.       Please according to use instruction strictly, The test result determination must take the microtiter plate reader as a standard.

6.       This reagent which different batch number component do not mix. Chromogen Solution B evade the light preservation.

7.       Closure plate membrane only limits the disposable use, in order to avoid the overlapping pollution.

8.       All samples, washing buffer and each kind of reject should according to infective material process.

Assay range

0.7ng/L - 20ng/L

Storage and validity

1．Storage：  2-8℃.

2．validity： six months.