小蒼蘭條斑病毒（FSV）酶��(lián)免疫分析（ELISA��

【資料簡(jiǎn)介��

小蒼蘭條斑病毒（FSV）酶��(lián)免疫分析��ELISA��

試劑盒使用說(shuō)明書

本試劑僅供研究使��       目的：本試劑盒用于檢��(cè)植物相關(guān)樣本��小蒼蘭條斑病毒（FSV��水平��

��(shí)��(yàn)原理��

  本試劑盒采用雙抗體夾心酶��(lián)免疫法（ELISA）測(cè)定標(biāo)本中小蒼蘭條斑病毒（FSV）。用純化��小蒼蘭條斑病����FSV��抗體包被微孔板，制成固相抗體，可與樣品中小蒼蘭條斑病毒（FSV��相結(jié)������(jīng)洗滌除去未結(jié)合的抗體和其他成分后再與HRP��(biāo)記的小蒼蘭條斑病毒（FSV��抗體��(jié)合，形成抗體-抗原-酶標(biāo)抗體��(fù)合物，經(jīng)��(guò)*洗滌后加底物TMB顯色��TMB��HRP酶的催化下轉(zhuǎn)化成��(lán)色，并在酸的作用下轉(zhuǎn)化成zui終的黃色。用酶標(biāo)儀��450nm波長(zhǎng)下測(cè)定吸光度��OD值），與CUTOFF值相比較，從而判定標(biāo)本中小蒼蘭條斑病毒（FSV��的存在與否��

 

試劑盒組����

試劑盒組��	48孔配��	96孔配��	保存
��(shuō)明書	1��	1��
封板��	2片（48��	2片（96��
密封��	1��(gè)	1��(gè)
酶標(biāo)包被��	1×48	1×96	2-8��保存
陰性對(duì)��	0.5ml×1��	0.5ml×1��	2-8��保存
��(yáng)性對(duì)��	0.5ml×1��	0.5ml×1��	2-8��保存
酶標(biāo)試劑	3 ml×1��	6 ml×1��	2-8��保存
樣品稀釋液	3 ml×1��	6 ml×1��	2-8��保存
顯色��A��	3 ml×1��	6 ml×1��	2-8��保存
顯色��B��	3 ml×1��	6 ml×1��	2-8��保存
終止��	3ml×1��	6ml×1��	2-8��保存
濃縮洗滌��	��20ml×20倍）×1��	��20ml×30倍）×1��	2-8��保存

 

樣本處理及要����

1. 血清：室溫血液自然凝��10-20分鐘，離��20分鐘左右��2000-3000��(zhuǎn)/分）。仔��(xì)收集上清，保存過(guò)程中如出��(xiàn)沉淀，應(yīng)再次離心��

2. 血漿：��(yīng)根據(jù)��(biāo)本的要求選擇EDTA或檸檬酸鈉作為抗凝劑，混��10-20分鐘后，離心20分鐘左右��2000-3000��(zhuǎn)/分）。仔��(xì)收集上清，保存過(guò)程中如有沉淀形成，應(yīng)該再次離心��

3. 尿液：用��(wú)菌管收集，離��20分鐘左右��2000-3000��(zhuǎn)/分）。仔��(xì)收集上清，保存過(guò)程中如有沉淀形成，應(yīng)再次離心。胸腹水、腦脊液參照��(shí)行��

4. ��(xì)胞培��(yǎng)上清：檢��(cè)分泌性的成份��(shí)，用��(wú)菌管收集。離��20分鐘左右��2000-3000��(zhuǎn)/分）。仔��(xì)收集上清。檢��(cè)��(xì)胞內(nèi)的成份時(shí)，用PBS��PH7.2-7.4）稀釋細(xì)胞懸液，��(xì)胞濃度達(dá)��100��(wàn)/ml左右。通過(guò)反復(fù)凍融，以使細(xì)胞破壞并放出��(xì)胞內(nèi)成份。離��20分鐘左右��2000-3000��(zhuǎn)/分）。仔��(xì)收集上清。保存過(guò)程中如有沉淀形成，應(yīng)再次離心��

5. 組織��(biāo)本：切割��(biāo)本后，稱取重量。加入一定量��PBS��PH7.4。用液氮迅速冷凍保��?zhèn)溆谩��?biāo)本融化后仍然保持2-8��的溫度。加入一定量��PBS��PH7.4），用手工或勻漿器將��(biāo)本勻漿充分。離��20分鐘左右��2000-3000��(zhuǎn)/分）。仔��(xì)收集上清。分裝后一份待檢測(cè)，其余冷凍備用��

6. ��(biāo)本采集后盡早��(jìn)行提取，提取按相��(guān)文獻(xiàn)��(jìn)行，提取后應(yīng)盡快��(jìn)行實(shí)��(yàn)。若不能馬上��(jìn)行試��(yàn)，可將標(biāo)本放��-20��保存，但��(yīng)避免反復(fù)凍融.

7. 不能檢測(cè)含NaN3的樣��，因NaN3抑制辣根��(guò)氧化物酶的（HRP）活性��

 

操作步驟��

1.         編號(hào)：將樣品��(duì)��(yīng)微孔按序編號(hào)，每板應(yīng)��(shè)陰性對(duì)��2孔����(yáng)性對(duì)��2孔、空白對(duì)��1孔（空白��(duì)照孔不加樣品及酶��(biāo)試劑，其余各步操作相同）

2.         加樣：分別在陰、陽(yáng)性對(duì)照孔中加入陰性對(duì)照、陽(yáng)性對(duì)��50μl。然后在待測(cè)樣品孔先加樣品稀釋液40μl，然后再加待��(cè)樣品10μl��加樣將樣品加于酶��(biāo)板孔底部，盡量不觸及孔壁，輕輕晃��(dòng)混勻��

3.         溫育：用封板膜封板后��37��溫育30分鐘��  

4.         配液：將30��48T��20倍）倍濃縮洗滌液加蒸餾水��600ml后備��

5.         洗滌：小心揭掉封板膜，棄去液體，甩干，每孔加滿洗滌液，靜��30秒后棄去，如此重��(fù)5次，拍干��

6.         加酶：每孔加入酶��(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��A 50μl，再加入顯色��B 50μl，輕輕震蕩混勻，37��避光顯色15分鐘

10.     終止：每孔加終止��50μl，終止反��(yīng)（此��(shí)��(lán)色立��(zhuǎn)黃色）��

11.     ��(cè)定：以空白空��(diào)零，450nm波長(zhǎng)依序��(cè)量各孔的吸光度（OD值）�� ��(cè)定應(yīng)在加終止液后15分鐘以內(nèi)��(jìn)行��

 

��(jié)果判定：

  試驗(yàn)有效性：��(yáng)性對(duì)照孔平均��≥1.00; 陰性對(duì)照平均��≤0.10

  臨界值（CUT OFF）計(jì)算：臨界��=陰性對(duì)照孔平均��+0.15

  陰性判定：樣品OD��< 臨界值（CUT OFF）者為小蒼蘭條斑病毒（FSV��陰��

  ��(yáng)性判定：樣品OD��≥ 臨界值（CUT OFF）者為小蒼蘭條斑病毒（FSV����(yáng)��

注意事項(xiàng)

1．操作嚴(yán)格按照說(shuō)明書��(jìn)行，本試劑不同批��(hào)組分不得混用��

2．試劑盒從冷藏環(huán)境中取出��(yīng)在室溫平��15-30分鐘后方可使用，酶標(biāo)包被板開(kāi)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(huì)有結(jié)晶析出，稀釋時(shí)可在水浴中加溫助溶，洗滌��(shí)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(qǐng)避光保存��

6．試��(yàn)��(jié)果判定必須以酶標(biāo)儀讀��(shù)為準(zhǔn)，使用雙波長(zhǎng)檢測(cè)��(shí)，參考波��(zhǎng)��630nm

7．所有樣品，洗滌液和各種廢棄物都��(yīng)按傳染物處理。終止液��2M的硫酸，使用��(shí)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��(gè)��

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Freesia streak virus（FSV��                                   FOR RESEARCH USE ONLY

 

Drug Names

Generic Name��Freesia streak virus（FSV）ELISA Kit.

Purpose

This kit allows for the determination of FSV concentrations in Freesia.

Principle of the assay

The kit assay FSV level in the sample��use Purified FSV antibody to coat microtiter plate wells, make solid-phase antibody, then add FSV to wells, Combined With FSV, after washing and removing non-combinative antibody and other components ,then Combined FSV 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 FSV exist in the sample or not.

 

 

 

 

 

 

 

Materials provided with the kit

Materials provided with the kit	48determinations	96 determinations	Storage
User manual	1	1
Closure plate membrane	2	2
Sealed bags	1	1
Microelisa stripplate	1	1	2-8��
Negative control	0.5ml×1 bottle	0.5ml×1 bottle	2-8��
Positive control	0.5ml×1 bottle	0.5ml×1 bottle	2-8��
HRP-Conjugate reagent	3ml×1 bottle	6ml×1 bottle	2-8��
Sample diluent	3ml×1 bottle	6ml×1 bottle	2-8��
Chromogen Solution A	3ml×1 bottle	6ml×1 bottle	2-8��
Chromogen Solution B	3ml×1 bottle	6ml×1 bottle	2-8��
Stop Solution	3ml×1 bottle	6ml×1 bottle	2-8��
wash  solution	��20ml×20 fold�� ×1bottle	��20ml×30 fold�� ×1bottle	2-8��

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 FSV Negative control.

Positive control: ample OD≥ Calculate Critical（CUT OFF�� is FSV 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.

 

 

 /