Porcine PV  ELISA Kit

For the qualitative in vitro d

 serum - plasma - tissue homogenates - other biological fluids

FOR LABORATORY RESEARCH USE ONLY.

NOT FOR USE IN DIAGNOSTIC PROCEDURES.

This package insert must be read in its entirety before using this product.

ELISA

ENZYME linkED IMMUNOSORBENT ASSAY

INTENDED USE AND TEST PRINCIPLE

This PV  ELISA kit is intended Laboratory for Research use only and is not for use in diagnostic or therapeutic procedures. In order to determine whether contains PV  in the sample, this PV  ELISA Kit includes Negative control and Positive control. The Stop Solution changes the color from blue to yellow and the intensity of the color is measured at 450 nm using a spectrophotometer. The color depth was positively correlated with the PV  in the sample .

SAMPLE COLLECTION AND STORAGES

Serum - Use a serum separator tube and allow samples to clot for two hours at room temperature or overnight at 4℃ before centrifugation for 20 minutes at approximately 1000×g. Assay freshly prepared serum immediately or store samples in aliquot at -20℃ or -80℃ for later use. Avoid repeated freeze/thaw cycles.

Plasma - Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples for 15 minutes at 1000×g at 2-8℃ within 30 minutes of collection. Remove plasma and assay immediately or store samples in aliquot at -20℃ or -80℃ for later use. Avoid repeated freeze/thaw cycles.

Tissue homogenates - For general information, hemolysis blood may affect the result, so you should rinse the tissues with ice-cold PBS (0.01M, pH=7.4) to remove excess blood thoroughly. Tissue pieces should be weighed and then minced to small pieces which will be homogenized in PBS (the volume depends on the weight of the tissue. 9mL PBS would be appropriate to 1 gram tissue pieces. Some protease inhibitor is recommended to add into the PBS.) with a glass homogenizer on ice. To further break the cells, you can sonicate the suspension with an ultrasonic cell disrupter or subject it to freeze-thaw cycles. The homogenates are then centrifugated for 5minutes at 5000×g to get the supernate.

Cell culture supernates and other biological fluids - Centrifuge samples for 20 minutes at 1000×g. Remove particulates and assay immediately or store samples in aliquot at -20℃ or -80℃ for later use. Avoid repeated freeze/thaw cycles.

Note:  The samples shoule be centrifugated dequately and no hemolysis or granule was allowed.

MATERIALS REQUIRED BUT NOT SUPPLIED

1.  37 ℃ incubator

2.  Standard microplate reader capable of measuring absorbance at 450 nm

3.  Precision pipettes, disposable pipette tips and Absorbent paper

4.  Distilled or deionized water

REAGENTS PROVIDED

All reagents provided are stored at 2-8°C. Refer to the expiration date on the label.

PRECAUTIONS

1.        Do not substitute reagents from one kit lot to another. Standard, conjugate and microtiter plates are matched for optimal performance. Use only the reagents supplied by manufacturer.

2.        Allow kit reagents and materials to reach room temperature (20-25°C) before use. Do not use water baths to thaw samples or reagents.

3.        Do not use kit components beyond their expiration date.

4.        Use only deionized or distilled water to dilute reagents.

5.        Do not remove microtiter plate from the storage bag until needed. Unused strips should be stored at 2-8°C in their pouch with the desiccant provided.

6.        Use fresh disposable pipette tips for each transfer to avoid contamination.

7.        Do not mix acid and sodium hypochlorite solutions.

8.        Serum and plasma should be handled as potentially hazardous and capable of transmitting disease. Disposable gloves must be worn during the assay procedure, since no known test method can offer complete assurance that products derived from Rat blood will not transmit infectious agents. Therefore, all blood derivatives should be considered potentially infectious and good laboratory practices should be followed.

9.        All samples should be disposed of in a manner that will inactivate viruses.

10.    Liquid Waste: Add sodium hypochlorite to a final concentration of 1.0%. The waste should be allowed to stand for a minimum of 30 minutes to inactivate the viruses before disposal.

11.    Substrate Solution is easily contaminated. If bluish prior to use, do not use.

12.    Substrate B contain 20% acetone, keep this reagent away from sources of heat or flame.

13.    Remove all kit reagents from refrigerator and allow them to reach room temperature ( 20-25°C).

REAGENT PREPARATION AND STORAGE

Wash Solution (1X) - Dilute 1 volume of Wash solution (20X) with 19 volumes of deionized or distilled water. Wash Solution is stable for 1 month at 2-8°C.

ASSAY PROCEDURE

1.  Prepare all reagents before starting assay procedure. It is recommended that all Standards and Samples be added in duplicate to the Microtiter plate.

2.  Add 50μl of Negative control，Positive control and test sample to the appropriate wells. Blank well doesn’t add anyting.

3.  Add 100μl of Enzymeconjugate to standard wells and sample wells except the blank well, cover with an adhesive strip and incubate for 60 minutes at 37°C.

4.  Wash the Microtiter Plate 4 times.

Manual Washing - Remove incubation mixture by aspirating contents of the plate into a sink or proper waste container. Using a squirt bottle, fill each well completely with Wash Solution (1X), then aspirate contents of the plate into a sink or proper waste container. Repeat this procedure for a total of four times. After final wash, invert plate, and blot dry by hitting plate onto absorbent paper or paper towels until no moisture appears. Note: Hold the sides of the plate frame firmly when washing the plate to assure that all strips remain securely in frame.

Automated Washing - Aspirate all wells, then wash plates four times using Wash Buffer (1X). Always adjust your washer to aspirate as much liquid as possible and set fill volume at 350μL/well/wash. After final wash, invert plate, and blot dry by hitting plate onto absorbent paper or paper towels until no moisture appears.

5.  Add Substrate A 50μl and Substrate B 50μl to each well. Gently mix and incubate for 15 minutes at 37°C. Protect from light.

6.  Add 50μl Stop Solution to each well. The color in the wells should change from blue to yellow. If the color in the wells is green or the color change does not appear uniform, gently tap the plate to ensure thorough mixing.

7.  Read the Optical Density (O.D.) at 450 nm using a microtiter plate reader within 15 minutes.

DETERMINE THE RESULT

1.        Test validity: the average of Positive control well≥0.8; the average of Negative control well ≤0.2.

2.        Calculate Critical (CUT OFF): Critical= the average of Negative control well + 0.15.

3.        Negative Result: sample OD< Calculate Critical (CUT OFF) is Negative.

4.        Positive Result: sample OD≥ Calculate Critical (CUT OFF) is Positive.

CALCULATION OF RESULTS

1.        This standard curve is used to determine the amount in an unknown sample. The standard curve is generated by plotting the average O.D. (450 nm) obtained for each of the six standard concentrations on the vertical (X) axis versus the corresponding concentration on the horizontal (Y) axis.

2.        First, calculate the mean O.D. value for each standard and sample. All O.D. Values are subtracted by the mean value of the balnk well before result interpretation. Construct the standard curve using graph paper or statistical software.

3.        To determine the amount in each sample, first locate the O.D. value on the Y-axis and extend a horizontal line to the standard curve. At the point of intersection, draw a vertical line to the X-axis and read the corresponding concentration.

4.        Any variation in operator, pipetting and washing technique, incubation time or temperature, and kit age can cause variation in result. Each user should obtain their own standard curve.

5.        Intra-assay CV(%) and Inter-assay CV（％）are less than 15%.

6.        Cross-reactivity: No significant cross-reactivity or interference was observed.

7.        Storage: 2-8℃ (Use frequently); six months (-20℃)。

FOR RESEARCH USE ONLY; NOT FOR THERAPEUTIC OR DIAGNOSTIC APPLICATIONS! PLEASE READ THROUGH ENTIRE PROCEDURE BEFORE BEGINNING!
豬細小病毒（PV ）試劑盒（ELISA）

使用說明書

l  本試劑盒用于體外定性檢測血清、血漿、組織勻漿及相關液體樣本中豬細小病毒（PV ）。

l  有效期：6個月

l  保存條件：2-8℃

l  本試劑盒僅供科研使用，不得用于臨床診斷

實驗原理

試劑盒采用雙抗體夾心法酶聯免疫吸附試驗（ELISA）。往預先包被豬細小病毒（PV ）捕獲抗體的包被微孔中，依次加入標本、陰性和陽性對照、HRP標記的檢測抗體，經過溫育并徹底洗滌。用底物TMB顯色，TMB在過氧化物酶的催化下轉化成藍色，并在酸的作用下轉化成Z終的黃色。顏色的深淺和樣品中的豬細小病毒（PV ）呈正相關。用酶標儀在450nm 波長下測定吸光度（OD 值），判定陰陽性。

樣本處理及要求

1.  血清：將收集于血清分離管的全血標本在室溫放置2小時或4℃過夜，然后1000×g離心20 分鐘，取上清即可，或將上清置于-20℃或-80℃保存，但應避免反復凍融。
2.  血漿：用EDTA或肝素作為抗凝劑采集標本，并將標本在采集后的30分鐘內于2-8℃ 1000×g離心15分鐘，取上清即可檢測，或將上清置于-20℃或-80℃保存，但應避免反復凍融。
3.  組織勻漿：用預冷的PBS (0.01M, pH=7.4)沖洗組織，去除殘留血液（勻漿中裂解的紅細胞會影響測量結果），稱重后將組織剪碎。將剪碎的組織與對應體積的PBS（一般按1:9的重量體積比，比如1g的組織樣品對應9mL的PBS，具體體積可根據實驗需要適當調整，并做好記錄。推薦在PBS中加入蛋白酶抑制劑）加入玻璃勻漿器中，于冰上充分研磨。為了進一步裂解組織細胞，可以對勻漿液進行超聲破碎，或反復凍融。Z后將勻漿液于5000×g離心5~10分鐘，取上清檢測。

4.  細胞培養物上清或其它生物標本：請1000×g離心20分鐘，取上清即可檢測，或將上清置于-20℃或-80℃保存，但應避免反復凍融。

注：標本溶血會影響Z后檢測結果，因此溶血標本不宜進行此項檢測。

需要而未提供的試劑和器材

1.       酶標儀（450nm）

2.       加樣器及槍頭：0.5-10uL、2-20uL、20-200uL、200-1000uL

3.       37℃恒溫箱

4.  蒸餾水或去離子水

試劑盒組成

注意事項

1.        嚴格按照規定的時間和溫度進行溫育以保證準確結果。所有試劑都必須在使用前達到室溫20-25℃。使用后立即冷藏保存試劑。

2.        洗板不正確可以導致不準確的結果。在加入底物前確保盡量吸干孔內液體。溫育過程中不要讓微孔干燥掉。

3.        消除板底殘留的液體和手指印，否則影響OD值。

4.        底物顯色液應呈無色或很淺的顏色，已經變藍的底物液不能使用。

5.        避免試劑和標本的交叉污染以免造成錯誤結果。

6.        在儲存和溫育時避免強光直接照射。

7.        平衡至室溫后再打開密封袋以防水滴凝聚在冷板條上。

8.        任何反應試劑不能接觸漂白溶劑或漂白溶劑所散發的強烈氣體。任何漂白成分都會破壞試劑盒中反應試劑的生物活性。

9.        不能使用過期產品。

10.    如果可能傳播疾病，所有的樣品都應管理好，按照規定的程序處理樣品和檢測裝置。

試劑準備

試劑盒從冷藏環境中取出應在室溫平衡后方可使用。

20×洗滌緩沖液的稀釋：蒸餾水按1：20稀釋，即1份20×洗滌緩沖液加19份蒸餾水。

操作步驟

1.  從室溫平衡20min后的鋁箔袋中取出所需板條，剩余板條用自封袋密封放回4℃。

2.  設置陰性對照孔、陽性對照孔和樣本孔，陰性、陽新對照孔各加50μL對照品；

3.  樣本孔中加入待測樣本50μL；空白孔不加。

4.  除空白孔外，標準品孔和樣本孔中每孔加入辣根過氧化物酶（HRP）標記的檢測抗體100μL，用封板膜封住反應孔，37℃水浴鍋或恒溫箱溫育60min。

5.  棄去液體，吸水紙上拍干，每孔加滿洗滌液（350μL），靜置1min，甩去洗滌液，吸水紙上拍干，如此重復洗板5次（也可用洗板機洗板）。

6.  每孔加入底物A、B各50μL，37℃避光孵育15min。

7.  每孔加入終止液50μL，15min內，在450nm波長處測定各孔的OD值。

實驗結果計算

1. 陰性對照OD值：小于0.2。

2. 陽性對照OD值：大于0.8。

3、陽性判斷（Cut-Off值）：陰性對照OD值+0.15，樣本OD值大于閾值，判定為陽性，反之，為陰性。