豬流行性腹瀉病毒抗體（PEDV）酶聯免疫分析（ELISA）

試劑盒使用說明書

本試劑僅供研究使用       目的：本試劑盒用于測定豬血清，血漿及相關液體樣本中流行性腹瀉病毒抗體（PEDV）水平。

實驗原理：

  本試劑盒采用雙抗原夾心法測定血清或血漿中豬流行性腹瀉病毒抗體（PEDV）。用純化的豬流行性腹瀉病毒抗體（PEDV）抗原包被微孔板，制成固相抗原，可與樣品中流行性腹瀉病毒抗體（PEDV）相結合，經洗滌除去未結合的抗體和其他成分后再與HRP標記的流行性腹瀉病毒抗體（PEDV）抗原結合，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。用酶標儀在450nm波長下測定吸光度（OD值），與CUTOFF值相比較，從而判定標本中豬流行性腹瀉病毒抗體（PEDV）的存在與否。

試劑盒組成：

樣本處理及要求：

1. 血清：室溫血液自然凝固10-20分鐘，離心20分鐘左右（2000-3000轉/分）。仔細收集上清，保存過程中如出現沉淀，應再次離心。

2. 血漿：應根據標本的要求選擇EDTA或檸檬酸鈉作為抗凝劑，混合10-20分鐘后，離心20分鐘左右（2000-3000轉/分）。仔細收集上清，保存過程中如有沉淀形成，應該再次離心。

3. 尿液：用無菌管收集，離心20分鐘左右（2000-3000轉/分）。仔細收集上清，保存過程中如有沉淀形成，應再次離心。胸腹水、腦脊液參照實行。

4. 細胞培養上清：檢測分泌性的成份時，用無菌管收集。離心20分鐘左右（2000-3000轉/分）。仔細收集上清。檢測細胞內的成份時，用PBS（PH7.2-7.4）稀釋細胞懸液，細胞濃度達到100萬/ml左右。通過反復凍融，以使細胞破壞并放出細胞內成份。離心20分鐘左右（2000-3000轉/分）。仔細收集上清。保存過程中如有沉淀形成，應再次離心。

5. 組織標本：切割標本后，稱取重量。加入一定量的PBS，PH7.4。用液氮迅速冷凍保存備用。標本融化后仍然保持2-8℃的溫度。加入一定量的PBS（PH7.4），用手工或勻漿器將標本勻漿充分。離心20分鐘左右（2000-3000轉/分）。仔細收集上清。分裝后一份待檢測，其余冷凍備用。

6. 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.

7. 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. 編號：將樣品對應微孔按序編號，每板應設陰性對照2孔、陽性對照2孔、空白對照1孔（空白對照孔不加樣品及酶標試劑，其余各步操作相同）
2. 加樣：分別在陰、陽性對照孔中加入陰性對照、陽性對照（標準品）100μl。然后在待測樣品孔先加樣品稀釋液50μl，然后再加待測樣品50μl。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（48T的20倍）倍濃縮洗滌液加蒸餾水至600ml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(或一滴)，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A 50μl(或一滴)，再加入顯色劑B 50μl(或一滴)，輕輕震蕩混勻，37℃避光顯色15分鐘
10. 終止：每孔加終止液50μl(或一滴)，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。

結果判定：

  試驗有效性：陽性對照孔平均值≥1.00; 陰性對照平均值≤0.15

  臨界值（CUT OFF）計算：臨界值=陰性對照孔平均值+0.10（陰性對照平均值低于0.05按0.05計算）

  陰性判定：樣品OD值< 臨界值（CUT OFF）者為豬流行性腹瀉病毒抗體（PEDV）陰性

  陽性判定：樣品OD值≥ 臨界值（CUT OFF）者為豬流行性腹瀉病毒抗體（PEDV）陽性

注意事項

1．操作嚴格按照說明書進行，本試劑不同批號組分不得混用。

2．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。

3．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。

1. 封板膜只限一次性使用，以避免交叉污染。

5．底物請避光保存。

6．試驗結果判定必須以酶標儀讀數為準，使用雙波長檢測時，參考波長為630nm

7．所有樣品，洗滌液和各種廢棄物都應按傳染物處理。終止液為2M的硫酸，使用時必須注意安全。

保存條件及有效期

1．試劑盒保存：；2-8℃。

2．有效期：6個月

FOR RESEARCH USE ONLY

Drug Names

Generic Name： PEDV ELISA Kit.

Purpose

This kit allows for the determination of PEDV in Porcine serum, and other biological fluids.

Principle of the assay

The kit assay PEDV level in the sample，use Purified Porcine PEDV antigen to coat microtiter plate wells, make solid-phase antigen, then add PEDV to wells, Combined With PEDV, after washing and removing non-combinative antibody and other components ,then Combined PEDV antigen which With HRP labeled, 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. Compared with the CUTOFF value, according to this to judge PEDV exist in the sample or not.

Materials provided with the kit

Specimen requirements

1. serum- coagulation at room temperature 10-20 mins，centrifugation 20-min at the speed of 2000-3000 r.p.m. remove supernatant, If precipitation appeared, Centrifugal again.
2. plasma-use suited EDTA or citrate plasma as an anticoagulant,mix 10-20 mins ,centrifugation 20-min at the speed of 2000-3000 r.p.m. remove supernatant, If precipitation appeared, Centrifugal again.
3. Urine-collect sue a sterile container, centrifugation 20-min at the speed of 2000-3000 r.p.m. remove supernatant, If precipitation appeared, Centrifugal again. The Operation of Hydrothorax and cerebrospinal fluid Reference to it.
4. cell culture supernatant-detect secretory components, collect sue a sterile container, centrifugation 20-min at the speed of 2000-3000 r.p.m. remove supernatant,detect the composition of cells, Dilut cell suspension with PBS（PH7.2-7.4）, Cell concentration reached 1 million / ml, repeated freeze-thaw cycles, damage cells and release of intracellular components, centrifugation 20-min at the speed of 2000-3000 r.p.m. remove supernatant, If precipitation appeared, Centrifugal again.
5. Tissue samples- After cutting samples, check the weight,add PBS（PH7.2-7.4）, Rapidly frozen with liquid nitrogen, maintain samples at 2-8℃ after melting,add PBS（PH7.4）, Homogenized by hand or Grinders, centrifugation 20-min at the speed of 2000-3000 r.p.m. remove supernatant.
6. 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.
7. Can’t detect the sample which contain NaN3, because NaN3 inhibits HRP active.

Assay procedure

1.Number: to sample correspond microtitration well and Number Sequence, each plate should be set feminine comparison 2 wells, masculine comparison 2 wells, blank comparison 1 well(don’t add sample and HRP-Conjugate reagent to blank comparison well, other each step the operation are same).

2.add sample：separay add Positive control and Negative control 100μl to the Positive and Negative well . add Sample dilution 50μl to testing sample well, then add testing sample 50μl. add sample to the bottom of ELISA plates coated well , 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 until 600ml,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 50μlto each well, except the 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.

Determine the result

Test validity: the average of Positive control well≥1.00; the average of Negative control well ≤0.15.

Calculate Critical(CUT OFF) : Critical= the average of Negative control well + 0.10.

Negative control: sample OD< Calculate Critical(CUT OFF) is PEDV Negative control.

Positive control: ample OD≥ Calculate Critical(CUT OFF) is PEDV Positive control.

Important notes

1.Please according to use instruction strictly, Do not mix reagents with those from other lots.

2.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.

3.washing buffer will Crystallization separation, it can be heated the water helps dissolve when dilute . Washing does not affect the result.

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

5.The substrate please evade the light preservation.

6.The test result determination must take the microtiter plate reader as a standard, when use dual-wavelength to assay, Reference wavelength is 630nm.

7.All samples, washing buffer and each kind of reject should according to infective material process. Stopp Solution is 2M sulphuric acid. You must pay attention to safe when use .

Storage and validity

1．Storage：  2-8℃.

2．validity： six months.