本试剂盒只能用于科学研究，不得用于医学诊断
猪（Porcine）流行性腹泻病毒抗体（PEDV）ELISA检测试剂盒使用说明书
检测原理
试剂盒采用双抗一步夹心法酶联免疫吸附试验（ELISA）。往预先
包被抗原的包被微孔中，依次加入标本、HRP标记的检测抗原，经过
温育并彻底洗涤。用底物TMB显色，TMB在过氧化物酶的催化下转化
成蓝色，并在酸的作用下转化成最终的黄色。用酶标仪在450nm波长
下测定吸光度（OD 值），与CUT OFF值相比较，从而判定标本中猪流
行性腹泻病毒抗体（PEDV）的存在与否。
样品收集、处理及保存方法
1. 血清：使用不含热原和内毒素的试管，操作过程中避免任何细胞
刺激，收集血液后，3000 转离心10 分钟将血清和红细胞迅速小心地
分离。
2. 血浆：EDTA、柠檬酸盐或肝素抗凝。3000 转离心30 分钟取上清。
3. 细胞上清液：3000 转离心10 分钟去除颗粒和聚合物。
4. 组织匀浆：将组织加入适量生理盐水捣碎。3000 转离心10 分钟
取上清。
5. 保存：如果样本收集后不及时检测，请按一次用量分装，冻存于
-20℃，避免反复冻融，在室温下解冻并确保样品均匀地充分解冻。
自备物品
1. 酶标仪（450nm）
2. 高精度加样器及枪头：0.5-10uL、2-20uL、20-200uL、200-1000uL
3. 37℃恒温箱
操作注意事项
1. 试剂盒保存在2-8℃，使用前室温平衡20 分钟。从冰箱取出的
浓缩洗涤液会有结晶，这属于正常现象，水浴加热使结晶完全溶解
后再使用。
2. 实验中不用的板条应立即放回自封袋中，密封（低温干燥）保存。3. 严格按照操作步骤中的说明稀释样本，以达到最佳检测效果。
4. 严格按照说明书中标明的时间、加液量及顺序进行温育操作。
5. 所有液体组分使用前充分摇匀。
试剂盒组成
名称96孔配置48孔配置备注
微孔酶标板12 孔×8 条12 孔×4 条无
样本稀释液6mL 3mL 无
阴性对照0.5mL 0.5mL 无
阳性对照0.5mL 0.5mL 无
检测抗原-HRP 10mL 5mL 无
20×洗涤缓冲液25mL 15mL 按说明书进行稀释
底物A 6mL 3mL 无
底物B 6mL 3mL 无
终止液6mL 3mL 无
封板膜2 张2 张无
说明书1 份1 份无
自封袋1 个1 个无
试剂的准备
20×洗涤缓冲液的稀释：蒸馏水按1：20 稀释，即1 份的20×洗涤缓
冲液加19 份的蒸馏水。
洗板方法
1. 手工洗板：甩尽孔内液体，每孔加满洗涤液，静置1min 后甩尽
孔内液体，在吸水纸上拍干，如此洗板5 次。
2. 自动洗板机：每孔注入洗液350μL，浸泡1min，洗板5 次。
操作步骤
1. 从室温平衡20min 后的铝箔袋中取出所需板条，剩余板条用自封
袋密封放回4℃。
2. 设置阴、阳性对照孔和样本孔，阴、阳性对照孔中加入阴性对照、
阳性对照各50μL；
3. 样本孔先加入待测样本10μL，再加入40μL 样本稀释液。
4. 随后阴、阳性对照孔和样本孔中每孔加入辣根过氧化物酶（HRP）
标记的检测抗原100μL，用封板膜封住反应孔，37℃水浴锅或恒温
箱温育60min。
5. 弃去液体，吸水纸上拍干，每孔加满洗涤液，静置1min，甩去
洗涤液，吸水纸上拍干，如此重复洗板5 次（也可用洗板机洗板）。
6. 每孔加入底物A、B 各50μL，37℃避光孵育15min。7. 每孔加入终止液50μL，15min 内，在450nm 波长处测定各孔的
OD 值。
结果判断
1. 试验有效性：阳性对照孔OD 值平均值≥1.00；
阴性对照孔OD 值平均值≤0.15。
2. 临界值（Cut off）计算：临界值=阴性对照孔平均值+0.15
3. 阴性判断：样品OD 值＜临界值（Cut off），样品为阴性
4. 阳性判断：样品OD 值＞临界值（Cut off），样品为阳性
试剂盒性能
1. 准确性：阳性对照孔OD 值平均值≥1.00；阴性对照孔OD 值平均
值≤0.15，说明试验结果有效。
2. 特异性：不与其它可溶性结构类似物交叉反应。
3. 重复性：板内、板间变异系数均小于15%。
4. 贮藏：2-8℃，避光防潮保存。
5. 有效期：6 个月
免责声明
1. 试剂盒仅供研究使用，不得用于临床实验或人体实验，否则所
产生的一切后果，由实验者承担，本公司概不负责。
2. 严格按照说明书操作，实验者违反说明书操作，后果由实验者
承担。FOR RESEARCH USE ONLY.
NOT FOR USE IN DIAGNOSTIC PROCEDURES.
Porcine epidemic diarrhea virus antibody (PEDV)
ELISAKit instruction
Intended use
This PEDV ELISA kit is intended Laboratory for Research use only and is not
for use in diagnostic or therapeutic procedures.The kit assay PEDV level in the
sample, use Purified antigen to coat microtiter plate wells, make solid-phase
antigen, then add PEDV to wells, Combined With PEDV, then Combined antigen
which with HRP labeled become antigen – antibody - enzyme- antigen complex,
after washing Completely, 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.
Sample collection and storages
Serum - Use a serum separator tube and allow samples to clot for 30 minutes
before centrifugation for 10 minutes at approximately 3000×g. Remove serum and
assay immediately or aliquot and store samples at -20℃ or -80℃.Avoid repeated
freeze-thaw cycles
Plasma - Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge
samples for 30 minutes at 3000×g at 2-8℃ within 30 minutes of collection. Store
samples at -20℃or -80℃. Avoid repeated freeze-thaw cycles.
Cell culture supernates and other biological fluids - Remove particulates
by centrifugation and assay immediately or aliquot and store samples at -20℃or
-80℃. 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. Standard microplate reader(450nm)
2. Precision pipettes and Disposable pipette tips.
3. 37 ℃ incubator
Precautions
1. Do not substitute reagents from one kit to another. Standard, conjugate and
microplates are matched for optimal performance. Use only the reagents supplied by
manufacturer.2. Do not remove microplate from the storage bag until needed. Unused strips
should be stored at 2-8°C in their pouch with the desiccant provided.
3. Mix all reagents before using.
Remove all kit reagents from refrigerator and allow them to reach room temperature
( 20-25°C)
Materials supplied
Name 96 determinations 48 determinations
Microelisa stripplate 12*8strips 12*4strips
Sample Diluent 6.0ml 3.0ml
Negative control 0.5ml 0.5ml
Positive control 0.5ml 0.5ml
HRP-Conjugate reagent 10.0ml 5.0ml
20X Wash solution 25ml 15ml
Chromogen Solution A 6.0ml 3.0ml
Chromogen Solution B 6.0ml 3.0ml
Stop Solution 6.0ml 3.0ml
Closure plate membrane 2 2
User manual 1 1
Sealed bags 1 1
Reagent preparation
20×wash solution:Dilute with Distilled or deionized water 1:20.
Assay procedure
1. Prepare all r e a g e n t s before starting assay procedure. It is recommended that
all Positive control, Negative control and Samples be added in duplicate to the
Microelisa Stripplate.
2. Separately add Positive control and Negative control 50μl to the Positive and
Negative well. Add testing sample 10μl then add Sample Diluent 40μl to testing
sample well.
3. Add 100μl of HRP-conjugate reagent to each well, cover with an adhesive strip
and incubate for 60 minutes at 37°C.
4. Aspirate each well and wash, repeating the process four times for a total of five
washes. Wash by filling each well with Wash Solution (400μl) using a squirt bottle,
manifold dispenser or autowasher. Complete removal of liquid at each step is
essential to good performance. After the last wash, remove any remaining Wash
Solution by aspirating or decanting. Invert the plate and blot it against clean paper
towels.
5. Add chromogen solution A 50μl and chromogen solution 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 readerwithin 15 minutes.
Determine the result
1. Test validity: the average of Positive control well≥1.00; the average of
Negative control well ≤0.15.
2. Calculate Critical (CUT OFF): Critical= the average of Negative control
well + 0.15.
Negative Result: sample OD< Calculate Critical (CUT OFF) is Negative.
Positive Result: sample OD≥ Calculate Critical (CUT OFF) is Positive.
Storage and validity
Storage： 2-8℃.
validity： six months.
FOR RESEARCH USE ONLY;
NOT FOR THERAPEUTIC OR DIAGNOSTICAPPLICATIONS!
PLEASE READ THROUGH ENTIRE PROCEDURE BEFORE
BEGINNING!
