犬細(xì)小病毒（CPV）抗體酶聯(lián)免疫（ELISA）

試劑盒使用說明書

本試劑盒僅供研究使用。本公司專業(yè)供應(yīng)Elisa試劑盒，*，*，可免費提供代測服務(wù)，咨詢：，  :

藥品名稱：

通用名：犬細(xì)小病毒（cPV）抗體酶聯(lián)免疫分析試劑盒

使用目的：

本試劑盒定性測定犬血液、或其它相關(guān)組織中細(xì)小病毒（CPV）抗體

實驗原理：

本試劑盒采用雙抗體夾心酶聯(lián)免疫法（ELISA）測定標(biāo)本中犬細(xì)小病毒（CPV） 抗體。用純化的犬細(xì)小病毒（CPV）抗原包被微孔板，制成固相抗原，可與樣品中細(xì)小病毒（CPV） 抗體相結(jié)合，經(jīng)洗滌除去未結(jié)合的抗體和其他成分后再與HRP標(biāo)記的細(xì)小病毒（CPV）抗原結(jié)合，形成抗原-抗體-酶標(biāo)抗原復(fù)合物，經(jīng)過*洗滌后加底物TMB顯色。TMB在HRP酶的催化下轉(zhuǎn)化成藍(lán)色，并在酸的作用下轉(zhuǎn)化成zui終的黃色。用酶標(biāo)儀在450nm波長下測定吸光度（OD值），與CUTOFF值相比較，從而判定標(biāo)本中犬細(xì)小病毒（CPV）抗體的存在與否。

試劑盒組成：

標(biāo)本要求：

1．標(biāo)本處理：血清、血漿標(biāo)本可直接檢測

2．標(biāo)本按要求制備后盡早進(jìn)行實驗。如不能及時檢測，可將標(biāo)本在-20℃保存一個月，但應(yīng)避免反復(fù)凍融。

3．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(yīng)微孔按序編號，每板應(yīng)設(shè)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(biāo)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50μl。然后在待測樣品孔先加樣品稀釋液40μl，然后再加待測樣品10μl。加樣將樣品加于酶標(biāo)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20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(fù)5次，拍干。

6.         加酶：每孔加入酶標(biāo)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(yīng)（此時藍(lán)色立轉(zhuǎn)黃色）。

11.     測定：以空白空調(diào)零，450nm波長依序測量各孔的吸光度（OD值）。 測定應(yīng)在加終止液后15分鐘以內(nèi)進(jìn)行。

結(jié)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≤0.10

  臨界值（CUT OFF）計算：臨界值=陰性對照孔平均值+0.15

  陰性判定：樣品OD值< 臨界值（CUT OFF）者為犬細(xì)小病毒（PPV）抗體陰性

  陽性判定：樣品OD值≥ 臨界值（CUT OFF）者為犬細(xì)小病毒（PPV）抗體陽性

注意事項

1．操作嚴(yán)格按照說明書進(jìn)行，本試劑不同批號組分不得混用。

2．試劑盒從冷藏環(huán)境中取出應(yīng)在室溫平衡15-30分鐘后方可使用，酶標(biāo)包被板開封后如未用完，板條應(yīng)裝入密封袋中保存。

3．濃洗滌液可能會有結(jié)晶析出，稀釋時可在水浴中加溫助溶，洗滌時不影響結(jié)果。

4．  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(jié)果判定必須以酶標(biāo)儀讀數(shù)為準(zhǔn)，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(yīng)按傳染物處理。終止液為2M的硫酸，使用時必須注意安全。

規(guī)格：

     48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

上海逸峰生物科技有限公司代理不同品牌價格檔次的ELISA試劑盒。數(shù)萬種抗體產(chǎn)品等, 品種多，質(zhì)量好，靈敏度高，價格實惠，并且還提供免費代檢測服務(wù)。

訂貨：          

  網(wǎng)   站：              ：yfswbio@

     canine parvovirus

Drug Names

Generic Name：CPV ELISA Kit.

Purpose

This kit allows for the determination of CPV concentrations in canine serum, and other biological fluids.

Principle of the assay

The kit assay CPV level in the sample，use Purified CPV antibody to coat microtiter plate wells, make solid-phase antibody, then add CPV to wells, Combined With CPV, after washing and removing non-combinative antibody and other components ,then Combined CPV antibody which with HRP labeled become antibody - antigen - enzyme-antibody complex, 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 CPV 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 50μl to the Positive and Negative well . add Sample dilution 40μl to testing sample well, then add testing sample 10μl. 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 ≤0.10.

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

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

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