本試劑盒只能用于科學研究，不得用于醫(yī)學診斷。

人甲型流感H5N1血凝素蛋白（HA ）定量檢測試劑盒（ELISA）

使用說明書

【試劑盒名稱】

人甲型流感H5N1血凝素蛋白（HA ）定量檢測試劑盒（ELISA）

【試劑盒用途】

定量檢測人血清、血漿及相關液體樣本中甲型流感H5N1血凝素蛋白（HA ）的含量。

【檢測原理】

本試劑盒采用雙抗體一步夾心酶聯(lián)免疫吸附法（ELISA）。將標準品、待測樣本和酶標工作液加入到預先包被人甲型流感H5N1血凝素蛋白（HA ）抗體的透明酶標包被板中，溫育足夠時間后，洗滌除去未結合的成分。依次加入顯色劑A、B液，顯色劑（TMB）在辣根過氧化物酶（HRP）催化下轉化為藍色產物，在酸的作用下變成黃色，顏色的深淺與樣品中人甲型流感H5N1血凝素蛋白（HA ）濃度呈正相關，450nm波長下測定OD值，根據(jù)標準品和樣品的OD值，計算樣本中人甲型流感H5N1血凝素蛋白（HA ）含量。

【試劑盒組成】

備注：標準品用標準品稀釋液依次稀釋為：32、16、8、4、2、1 ng/mL

【需要而未提供的試劑和器材】

1、37℃恒溫箱

2、標準規(guī)格酶標儀

3、精密移液器及一次性吸頭

4、蒸餾水

5、一次性試管

6、吸水紙

【操作步驟】

1、準備：從冰箱取出試劑盒，室溫復溫平衡30分鐘。

2、配液：用蒸餾水將20倍濃縮洗滌液稀釋成原倍的洗滌液。

3、加標準品和待測樣本：取足夠數(shù)量的酶標包被板，固定于框架上，分別設置標準品孔、待測樣本孔和空白對照孔，記錄各孔位置，在標準品孔中加入標準品50μL；待測樣本孔中先加入待測樣本10μL，再加*40μL（即樣本稀釋5倍）；每孔再加入酶標工作液100μL；空白對照孔不加。

4、溫育：37℃水浴鍋或恒溫箱溫育60min。

5、洗板：棄去液體，吸水紙上拍干，每孔加滿洗滌液，靜置1min，甩去洗滌液，吸水紙上拍干，如此重復洗板5次（也可用洗板機按說明書操作洗板）。

6、顯色：每孔先加入顯色劑A 液50μL，再加入顯色劑B液 50μL，37℃避光顯色15min。

7、終止：取出酶標板，每孔加終止液50μL，終止反應（顏色由藍色立轉黃色）。

8、測定：以空白孔調零，在終止后15分鐘內，用450nm波長測量各孔的吸光值（OD值）。

9、計算：根據(jù)標準品的濃度及對應的OD值，計算出標準曲線的直線回歸方程，再根據(jù)樣本的OD值，在回歸方程上計算出對應的樣品濃度，也可以使用各種應用軟件來計算。zui終濃度為實際測定濃度乘以稀釋倍數(shù)。

【樣本要求】

1、樣本不能含疊氮鈉（NaN₃），因為疊氮鈉（NaN₃）是辣根過氧化物酶（HRP）的抑制劑。

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。若不能立即試驗，可將標本放于-20℃保存，但應避免反復凍融。

3、樣本應充分離心，不得有溶血及顆粒。

【注意事項】

1、實驗嚴格按照說明書的操作進行，實驗結果判定必須以酶標儀讀數(shù)為準。

2、酶標包被板開封后如未用完，應立即裝入密封袋中加干燥劑保存。

3、建議所有的標準品、樣本和空白對照都做雙份檢測，取平均值，以減小實驗誤差。

4、若顯色過淺，可適當延長底物溫育時間。

5、為避免交叉污染，標準品、樣本和空白對照每加一個就要更換一次吸頭；酶標工作液、*和底物等公共組分，要懸臂加樣，不得碰到微孔；不得重復使用封板膜。

6、試劑盒保質期內使用，不同批號的試劑不得混用。

7、底物B對光敏感，避免長時間暴露于光下。

【操作程序總結】

準備試劑，樣品和標準品

加入準備好的樣品、標準品和酶標工作液，37℃反應60分鐘

洗板5次，加入顯色液A、B，37℃顯色15分鐘

加入終止液

15分鐘之內讀OD值

計算

【檢測范圍】

1-32ng/mL

【規(guī)格】

96人份/盒

【貯藏】

2-8℃，避光防潮保存。

【有效期】

6個月

FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES.

Human Influenza A H5N1 （ Avian Flu ） Hemagglutinin ELISA Kit instruction

Kit name

Human Influenza A H5N1 （ Avian Flu ） Hemagglutinin ELISA Kit

Intended use

The kit is used to assay the content of Human Influenza A H5N1 （ Avian Flu ） Hemagglutinin in Human serum，blood plasma and other related tissue liquid.

Test principle

The kit uses a double-antibody sandwich enzyme-linked immunosorbent assay （ELISA） to assay the level of Human Influenza A H5N1 （ Avian Flu ） Hemagglutinin in samples. Add Influenza A H5N1 （ Avian Flu ） Hemagglutinin  to pre-coated Influenza A H5N1 （ Avian Flu ） Hemagglutinin monoclonal antibody microelisa well, incubation; washing. Add HRP tagged Influenza A H5N1 （ Avian Flu ） Hemagglutinin antibodies. After another incubation and washing, remove the unbound enzyme, add Chromogen Solution A and B, the color of the liquid change into blue, and the color finally become yellow at the effect of acid. The depth of the color is positively correlated with concentration of the Human Influenza A H5N1 （ Avian Flu ） Hemagglutinin in samples.

Materials supplied

Note: Standard was diluent with Standard diluent followed by: 32、16、8、4、2、1 ng/mL

Materials required but not supplied

1.         37 ℃ incubator

2.         Standard microplate reader

3.         Precision pipettes and Disposable pipette tips

4.         Distilled water

5.         Disposable tubes for sample dilution

6.         Absorbent paper

Assay procedure

1.        Prepare: The kit takeing out from the environment of 2-8℃ should be balanced 30 minutes at less in the room temperature before using.

2.        Diluent: Diluent the 20×wash solution.

3.        Add standard and Sample: Set Standard wells, testing sample wells and blank wells. Add Diluted standard 50μl to standard well; Add Sample dilution 40μl to testing sample well which on Assay plate, then add testing sample 10μl （sample final dilute degree is 5 times）, Add HRP-conjugate reagent 100μl to each well, except the blank well.

4.        Incubation: Incubate 60 minutes at 37 ℃ in incubator.

5.        Wash: Discard Liquid, drying, filling in diluted washing liquid to each well, oscillation for 1 min, discard the washing liquid with absorbent paper Pat dry. Repeat four times, Pat dry.

6.        Add chromogen solution A and B: Add chromogen solution A 50μl and chromogen solution B 50μl to each well. Gently mix, incubate for 15 min at 37℃.

7.        Add Stop Solution: Add Stop Solution 50μl to each well, Stop the reaction（the blue color change to yellow immediay）.

8.        Take blank well as zero, measure the optical densit （OD） at 450 nm after adding Stop Solution and within 15 min.

9.        According to standard concentration and the corresponding OD values calculated standard curve linear regression equation, then the OD values according to the sample on the regression equation to calculate the corresponding sample concentration. It should be remembered that the sample has been diluted and its actual concentration should be multiplied by the total dilution.

Specimen requirements

1.      Can’t detect the samples which contain NaN3, because NaN3 inhibits HRP activity of the horseradish peroxidase.

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, but repeated freezing and thawing should be avoided.

3.      The samples shoule be centrifugated dequay and no hemolysis or granule was allowed.

Important notes

1.         The operation should be carried out in strict accordance with instructions and test results must be based on microplate reader to determine readings shall prevail.

2.         If the microelisa stripplate has not used up after open, it should be stored in the sealed bag.

3.         Recommended that all standard materials, test samples are doing double to minish the Experimental error.

4.         Please multiply total dilution times when calculation. 5 times is the best dilute time according to this ELISA Kit design.

5.         If the testing material content in the sample is excessively high, please use Special dilution to dilute certain multiple, then assay.

6.         If the color too shallow, It may be appropriate to extend the substrate incubation time.

7.         Add Sample with sampler each step and proofread its accuracy frequently to avoid the experimental error. In order to avoid cross-contamination, avoid to reusing the suction head and closure plate membrane.

8.         Use the kit in validity, not mix the reagents of different batches.

9.         Chromogen Solution B is light-sensitive, avoid prolonged exposure to light,

Summary procedures

Preparing reagents, samples and standard

Add prepared sample and standard, adding HRP-Conjugate Reagent incubated 60 minutes at 37 ℃

Plate washed five times, adding Chromogen Solution A and B incubated 15 minutes at 37 ℃

Add stop solution

Measure within 15min

Calculation

Assay range：1-32ng/mL

Package size: 96 determinations

Storage：  2-8℃.

validity： six months.