人幽门螺旋杆菌（HP）酶联免疫分析试剂盒使用说明书

人幽门螺旋杆菌（HP）酶联免疫分析试剂盒使用说明书
本试剂盒仅供研究使用。
96T
使用目的：
本试剂盒用于测定人血清、血浆及相关液体样本中幽门螺旋杆菌（HP）表达。
实验原理
本试剂盒应用双抗体夹心法测定标本中人幽门螺旋杆菌（HP）表达。用纯化的人幽门螺旋
杆菌（HP）抗体包被微孔板，制成固相抗体，可与样品中幽门螺旋杆菌（HP）相结合，经
洗涤除去未结合的抗原和其他成分后再与HRP 标记的幽门螺旋杆菌（HP）抗体结合，形成
抗体-抗原-酶标抗体复合物，经过*洗涤后加底物TMB 显色。TMB 在HRP 酶的催化下
转化成蓝色，并在酸的作用下转化成zui终的黄色。用酶标仪在450nm 波长下测定吸光度（OD
值），与CUTOFF 值相比较，从而判定标本中幽门螺旋杆菌（HP）的存在与否。
试剂盒组成
1 30 倍浓缩洗涤液20ml×1 瓶7 终止液6ml×1 瓶
2 酶标试剂6ml×1 瓶8 阳性对照0.5ml×1 瓶
3 酶标包被板12 孔×8 条9 阴性对照0.5ml×1 瓶
4 样品稀释液6ml×1 瓶10 说明书1 份
5 显色剂A 液6ml×1 瓶11 封板膜2 张
6 显色剂B 液6ml×1/瓶12 密封袋1 个
标本要求
1．标本采集后尽早进行提取，提取按相关文献进行，提取后应尽快进行实验。若不能
马上进行试验，可将标本放于-20℃保存，但应避免反复冻融
2．不能检测含NaN3 的样品，因NaN3 抑制辣根过氧化物酶的（HRP）活性。
操作步骤
1. 编号：将样品对应微孔按序编号，每板应设阴性对照2 孔、阳性对照2 孔、空白对照1
孔（空白对照孔不加样品及酶标试剂，其余各步操作相同）
2. 加样：分别在阴、阳性对照孔中加入阴性对照、阳性对照50μl。然后在待测样品孔先
加样品稀释液40μl，然后再加待测样品10μl。加样将样品加于酶标板孔底部，尽量不
触及孔壁，轻轻晃动混匀，
3. 温育：用封板膜封板后置37℃温育30 分钟。
4. 配液：将30 倍浓缩洗涤液用蒸馏水30 倍稀释后备用
5. 洗涤：小心揭掉封板膜，弃去液体，甩干，每孔加满洗涤液，静置30 秒后弃去，如此
重复5 次，拍干。
6. 加酶：每孔加入酶标试剂50μl，空白孔除外。
7. 温育：操作同3。
8. 洗涤：操作同5。
9. 显色：每孔先加入显色剂A50μl，再加入显色剂B50μl，轻轻震荡混匀，37℃避光显色
15 分钟.
10. 终止：每孔加终止液50μl，终止反应（此时蓝色立转黄色）。
11. 测定：以空白空调零，450nm 波长依序测量各孔的吸光度（OD 值）。测定应在加终止
液后15 分钟以内进行。
计算和结果判定：
试验有效性：阳性对照孔平均值≥1.00; 阴性对照平均值≤0.10
临界值（CUT OFF）计算：临界值=阴性对照孔平均值+0.15
阴性判定：样品OD 值< 临界值（CUT OFF）者为幽门螺旋杆菌（HP）阴性
阳性判定：样品OD 值≥ 临界值（CUT OFF）者为幽门螺旋杆菌（HP）阳性
。
注意事项
1．操作严格按照说明书进行，本试剂不同批号组分不得混用。
2．试剂盒从冷藏环境中取出应在室温平衡15-30 分钟后方可使用，酶标包被板开封后如未
用完，板条应装入密封袋中保存。
3．浓洗涤液可能会有结晶析出，稀释时可在水浴中加温助溶，洗涤时不影响结果。
4． 封板膜只限一次性使用，以避免交叉污染。
5．底物请避光保存。
6．试验结果判定必须以酶标仪读数为准，使用双波长检测时，参考波长为630nm
7．所有样品，洗涤液和各种废弃物都应按传染物处理。终止液为2M 的硫酸，使用时必须
注意安全。
保存条件及有效期
1．试剂盒保存：；2-8℃。
2．有效期：6 个月
Human HP
FOR RESEARCH USE ONLY
96 determinations
Purpose
This kit allows for the determination of HP concentrations in Human serum, and other
biological fluids.
Principle of the assay
The kit assay HP level in the sample，use Purified HP antibody to coat microtiter plate
wells, make solid-phase antibody, then add HP to wells, Combined With HP, after washing and
removing non-combinative antibody and other components ,then Combined HP 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 HP exist in the sample or not.
Materials provided with the kit
1 wash solution 20ml× 1bottle 7 Stopp Solution 6ml× 1 bottle
2 HRP-Conjugate reagent 6ml× 1 bottle 8 Positive control 0.5ml× 1 bottle
3 Microelisa stripplate 12well× 8strips 9 Negative control 0.5ml× 1bottle
4 Sample diluent 6ml× 1 bottle 10 Instruction 1
5 Chromogen Solution A 6ml× 1 bottle 11
Closure plate
membrane
2
6 Chromogen Solution B 6ml× 1 bottle 12 Sealed bags 1
Specimen requirements
RD
1. 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.
2. 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 HP Negative control.
Positive control: ample OD≥ Calculate Critical(CUT OFF) is HP 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.