法氏囊病毒（BDV）抗體ELISA試劑盒說(shuō)明書

本試劑僅供研究使用       目的：本試劑盒用于測(cè)定雞血清，血漿及相關(guān)液體樣本中法氏囊病毒（BDV）抗體的含量。

實(shí)驗(yàn)原理：

    本試劑盒應(yīng)用雙抗原夾心法測(cè)定標(biāo)本中雞法氏囊病毒（BDV）抗體水平。用純化的雞法氏囊病毒（BDV）抗原包被微孔板，制成固相抗原，往包被抗原的微孔中依次加入法氏囊病毒（BDV）抗體，再與HRP標(biāo)記的法氏囊病毒（BDV）抗原結(jié)合，形成抗原-抗體-酶標(biāo)抗體原復(fù)合物，經(jīng)過(guò)*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。顏色的深淺和樣品中的法氏囊病毒（BDV）抗體呈正相關(guān)。用酶標(biāo)儀在450nm波長(zhǎng)下測(cè)定吸光度（OD值），通過(guò)標(biāo)準(zhǔn)曲線計(jì)算樣品中雞法氏囊病毒（BDV）抗體濃度。

試劑盒組成：

注意事項(xiàng)：

1．  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。

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

3．  各步加樣均應(yīng)使用加樣器，并經(jīng)常校對(duì)其準(zhǔn)確性，以避免試驗(yàn)誤差。一次加樣時(shí)間控制在5分鐘內(nèi)，如標(biāo)本數(shù)量多，推薦使用排槍加樣。

4．  請(qǐng)每次測(cè)定的同時(shí)做標(biāo)準(zhǔn)曲線，做復(fù)孔。如標(biāo)本中待測(cè)物質(zhì)含量過(guò)高（樣本OD值大于標(biāo)準(zhǔn)品孔*孔的OD值），請(qǐng)先用樣品稀釋液稀釋一定倍數(shù)（n倍）后再測(cè)定，計(jì)算時(shí)請(qǐng)zui后乘以總稀釋倍數(shù)（×n×5）。

5．  封板膜只限一次性使用，以避免交叉污染。

6．  底物請(qǐng)避光保存。

7．  嚴(yán)格按照說(shuō)明書的操作進(jìn)行，試驗(yàn)結(jié)果判定必須以酶標(biāo)儀讀數(shù)為準(zhǔn).

8．  所有樣品，洗滌液和各種廢棄物都應(yīng)按傳染物處理。

9．  本試劑不同批號(hào)組分不得混用。

10. 如與英文說(shuō)明書有異，以英文說(shuō)明書為準(zhǔn)。

計(jì)算：

以標(biāo)準(zhǔn)物的濃度為橫坐標(biāo)，OD值為縱坐標(biāo)，   

在坐標(biāo)紙上繪出標(biāo)準(zhǔn)曲線，根據(jù)樣品的OD     

值由標(biāo)準(zhǔn)曲線查出相應(yīng)的濃度；再乘以稀釋      

倍數(shù)；或用標(biāo)準(zhǔn)物的濃度與OD值計(jì)算出標(biāo)      

準(zhǔn)曲線的直線回歸方程式，將樣品的OD值      

代入方程式，計(jì)算出樣品濃度，再乘以稀釋      

倍數(shù)，即為樣品的實(shí)際濃度。

試劑盒性能：

1.樣品線性回歸與預(yù)期濃度相關(guān)系數(shù)R值為0.92以上。

2.批內(nèi)與批見應(yīng)分別小于9%和15%

保存條件及有效期：

1.試劑盒保存：2-8℃。

2．有效期：6個(gè)月

FOR RESEARCH USE ONLY

Drug Names

Generic Name：chicken Bursal Disease Virus antibody （Bdv-Ab） ELISA Kit.

Purpose

This kit allows for the determination of Bdv-Ab concentrations in chicken serum, blood plasma, and other biological fluids.

Principle of the assay

The kit assay chicken Bdv-Ab level in the sample，use Purified chicken Bdv-Ab antigen to coat microtiter plate wells, make solid-phase antigen, then add Bdv-Ab to wells, Combined Bdv-Ab antigen which With HRP labeled , become antigen - antibody - 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. The concentration of Bdv-Ab in the samples is then determined by comparing the O.D. of the samples to the standard curve.

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.Dilute and add sample to Standard: set 10 Standard wells on the ELISA plates coated, add Standard 100μl to the first and the second well, then add Standard dilution 50μl to the first and the second well, mix; take out 100μl form the first and the second well then add it to the third and the forth well separay. then add Standard dilution 50μl to the third and the forth well ,mix ; then take out 50μl from the third and the forth well discard, add 50μl to the fifth and the sixth well ,then add Standard dilution 50μl to the fifth and the sixth well, mix ; take out 50μl from the fifth and the sixth well and add to the seventh and the eighth well, then add Standard dilution 50μl to the seventh and the eighth well ,mix ; take out 50μl from the seventh and the eighth well and add to the ninth and the tenth well, add Standard dilution 50μl to the ninth and the tenth well, mix , take out 50μl from the ninth and the tenth well discard（add Sample 50μl to each well after Diluting ,（density: 12ng/L,8ng/L ,4ng/L,2ng/L, 1ng/L）

2.add sample：Set blank wells separay （blank comparison wells don’t add sample and HRP-Conjugate reagent, other each step operation is same）. testing sample well. add Sample dilution 50μl to testing sample well, then add testing sample 10μl （sample final dilution is 5-fold）, add sample to wells , 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 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 50μl to each well, except  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.

Important notes

1.       The kit takes out from the refrigeration environment should be balanced 15-30 minutes in the room temperature, 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 mins, if the number of sample is much , recommend to use Volley .

4.       if the testing material content is excessively higher （The sample OD is bigger than the first standard well ）,please dilute Sample （n-fold）, Please diluente and multiplied by the dilution factor.（×n×5）.

5.       Closure plate membrane only limits the disposable use, to avoid cross-contamination.

6.       The substrate evade the light preservation.

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

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

9.       Do not mix reagents with those from other lots.

Storage and validity

1．Storage：  2-8℃.

2．validity： six months.