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Free Helicobacter pylori (Hp) ELISA Kit

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本试剂盒用于体外定量检测血清、血浆、组织、细胞上清及相关液体样本中 小鼠幽门螺旋杆菌 Hp )的含量。 l This kit is used for the quantitative detection of Helicobacter pylori ( Hp ) in serum, plasma, tissue, cell supernatant and related liquid samples in vitro .
有效期:6个月 l Validity: 6 months
保存条件: 2 -8℃ lStorage conditions: 2 -8 ℃
本试剂盒仅供体外研究使用,不用于临床诊断 l This kit is for in vitro research use only, not for clinical diagnosis
 
Experimental principle
The kit uses a double antibody one-step sandwich method for enzyme-linked immunosorbent assay (ELISA). 小鼠幽门螺旋杆菌 Hp )捕获抗体的包被微孔中,依次加入标本、标准品、HRP标记的检测抗体,经过温育并彻底洗涤。 The coated microwells pre-coated with the mouse H. pylori capture antibody were sequentially added with specimens, standards, and HRP-labeled detection antibodies, followed by incubation and thorough washing. The color is developed with the substrate TMB, and TMB is converted to blue by peroxidase catalysis, and is converted to the final yellow by the action of acid. 小鼠幽门螺旋杆菌 Hp )呈 正相关。 The shade of the color is positively correlated with the mouse H. pylori ( Hp ) in the sample . Measure the absorbance (OD value) at 450nm with a microplate reader to calculate the sample concentration.
Sample processing and requirements
血清 :全血标本请于室温放置2小时或4℃过夜后于1000g离心20分钟,取上清即可检测,或将标本放于-20℃或-80℃保存,但应避免反复冻融。 1. Serum : Whole blood samples should be left at room temperature for 2 hours or 4 C overnight. Centrifuge at 1000g for 20 minutes, and then take the supernatant to test. Or store the samples at -20 C or -80 C, but avoid repeated freezing. melt.
血浆 :可用EDTA或肝素作为抗凝剂,标本采集后30分钟内于2 - 8C 1000g离心 20 分钟,或将标本放于-20℃或-80℃保存,但应避免反复冻融。 2. Plasma : EDTA or heparin can be used as an anticoagulant. Centrifuge at 1000 g at 2-8 C for 20 minutes within 30 minutes after collection , or store the sample at -20 C or -80 C, but avoid repeated freeze-thaw cycles. .
组织匀浆 :用预冷的PBS (0.01M, pH=7.4)冲洗组织,去除残留血液(匀浆中裂解的红细胞会影响测量结果),称重后将组织剪碎。 3. Tissue homogenate : Rinse the tissue with pre-chilled PBS (0.01M, pH = 7.4) to remove residual blood (the lysed red blood cells in the homogenate will affect the measurement result), and cut the tissue after weighing. The cut tissue and the corresponding volume of PBS (generally based on a weight to volume ratio of 1: 9, such as 1g of tissue sample corresponds to 9mL of PBS, the specific volume can be adjusted according to the needs of the experiment, and make a record. It is recommended to add Protease inhibitors) were added to a glass homogenizer and ground thoroughly on ice. To further lyse the tissue cells, the homogenate can be sonicated or repeatedly freeze-thaw. Finally, the homogenate was centrifuged at 5000 g for 5-10 minutes, and the supernatant was taken for detection.
. 细胞培养物上清或其它生物标本 :1000g离心20分钟,取上清即可检测,或将标本放于-20℃或-80℃保存,但应避免反复冻融。 4. Cell culture supernatant or other biological specimens : Centrifuge at 1000g for 20 minutes, and take the supernatant for detection, or store the specimens at -20 C or -80 C, but avoid repeated freeze-thaw cycles.
Note: The hemolysis of the specimen will affect the final test results, so this test should not be performed on hemolyzed specimens.

Specimen processing
1. The company is only responsible for the kit itself, not for sample consumption caused by using the kit. Users are requested to fully consider the possible use of the sample before use, and reserve sufficient samples.
2. The content of the specimen should be predicted before the experiment. If the concentration of the specimen is too high, the specimen should be diluted so that the diluted specimen conforms to the detection range of the kit and then multiplied by the corresponding dilution factor during calculation. Specimens were diluted with 0.01mol / L PBS (PH = 7.0-7.2).
3. If the tested samples are not included in the samples listed in the instruction manual, it is recommended to conduct pre-experiments to verify their effectiveness, and pay attention to retaining the samples.
4. Tissue homogenates or cell extracts prepared using chemical lysates may cause deviations in ELISA results due to the introduction of certain chemicals.
5. If the sample is a cell culture supernatant, it may not be detected because there are many interference factors in this type of sample, such as: cell status, cell number, and sampling time.
6. Certain natural or recombinant proteins, including prokaryotic and eukaryotic recombinant proteins, may not be detected because they do not match the detection and capture antibodies used in this product.
7. It is recommended to use fresh samples. Excessive storage time may result in protein degradation or denaturation, which may lead to deviations in experimental results.
Required and not supplied reagents and equipment
酶标仪(450nm) 1. Microplate reader (450nm)
高精度加样器及枪头:0.5-10uL、2-20uL、20-200uL、200-1000uL 2. High-precision sampler and pipette tip: 0.5-10uL, 2-20uL, 20-200uL, 200-1000uL
37℃恒温箱 3. 37 ℃ constant temperature box
蒸馏水或去离子水 4. Distilled or deionized water
Kit composition
name
96-well configuration
48-hole configuration
Remark
Microtiter plate
12 8 holes 12
6 8 holes 6
no
Standard
3 mL*6管 0.3 mL * 6 tube
3 mL*6管 0.3 mL * 6 tube
no
Sample diluent
6mL
3mL
no
Detection antibody-HRP
10mL
5mL
no
20 washing buffer
25mL
15mL
Dilute according to instructions
Substrate A
6mL
3mL
no
Substrate B
6mL
3mL
no
Stop solution
6mL
3mL
no
Sealing film
2 pieces
2 pieces
no
Manual
1 serving
1 serving
no
Ziplock bag
1
1
no
Remarks :
标准品浓度依次为: 120 、60、30、15、7.5、0 U/mL 1. The standard concentrations are: 120 , 60, 30, 15, 7.5, 0 U / mL
μL 样本上样即可。 2. After a large number of normal specimens are tested, the normal concentration values of the specimens are within the detection range provided by the kit, and 50 μL samples can be directly loaded during the experiment . When some sample values exceed the maximum standard concentration, the sample dilution solution can be used to properly dilute the sample before the experiment.
Precautions
严格按照规定的时间和温度进行温育以保证准确结果。 1. Incubate strictly according to the prescribed time and temperature to ensure accurate results. All reagents must reach room temperature 20-25 C before use. Keep reagents refrigerated immediately after use.
洗板不正确可以导致不准确的结果。 2. Improper washing can lead to inaccurate results. Be sure to suck up as much liquid as possible before adding the substrate. Do not allow microwells to dry out during incubation.
消除板底残留的液体和手指印,否则影响OD值。 3. Eliminate the residual liquid and fingerprints on the bottom of the board, otherwise it will affect the OD value.
底物显色液应呈无色或很浅的颜色,已经变蓝的底物液不能使用。 4. The substrate coloring solution should be colorless or very light. The substrate liquid that has turned blue cannot be used.
避免试剂和标本的交叉污染以免造成错误结果。 5. Avoid cross-contamination of reagents and specimens to avoid erroneous results.
在储存和温育时避免强光直接照射。 6. Avoid direct light exposure during storage and incubation.
平衡至室温后再打开密封袋以防水滴凝聚在冷板条上。 7. After equilibrating to room temperature, open the sealed bag to prevent water droplets from condensing on the cold slat.
任何反应试剂不能接触漂白溶剂或漂白溶剂所散发的强烈气体。 8. No reaction reagent should come into contact with bleaching solvents or strong gases emitted by bleaching solvents. Any bleaching ingredient will destroy the biological activity of the reagents in the kit.
不能使用过期产品。 9. Do not use expired products.
如果可能传播疾病,所有的样品都应管理好,按照规定的程序处理样品和检测装置 10. If the disease is likely to be transmitted, all samples should be managed, and the samples and testing equipment should be handled in accordance with the prescribed procedures .
Reagent preparation
Remove the kit from the refrigerated environment and allow it to equilibrate at room temperature before use.
0洗涤缓冲液的稀释:蒸馏水按1:20稀释,即1份20洗涤缓冲液加19份蒸馏水。 20 washing buffer dilution: Distilled water is diluted 1:20, ie 1 part of 20 washing buffer plus 19 parts of distilled water.
Steps
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℃。 1. Take out the required slat from the aluminum foil bag after equilibrating at room temperature for 20 minutes, and seal the remaining slat with ziplock bag and return to 4 C.
设置标准品孔和样本孔,标准品孔各加不同浓度的标准品50μL; 2. Set up standard wells and sample wells, and add 50 μL of standards at different concentrations to each well;
样本孔 待测样本 5 0μL;空白孔不加。 3. Add 50 μL of sample to be tested into the sample well; blank wells are not added.
除空白孔外,标准品孔和样本孔中每孔加入辣根过氧化物酶(HRP)标记的检测抗体100μL,用封板膜封住反应孔,37℃水浴锅或恒温箱温育60min。 4. In addition to blank wells, add 100 μL of horseradish peroxidase (HRP) labeled detection antibody to each well of the standard and sample wells, seal the reaction well with a sealing plate membrane, and incubate in a 37 C water bath or incubator 60min.
弃去液体,吸水纸上拍干,每孔加满洗涤液 (350 μL ,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)。 5. Discard the liquid, pat dry on absorbent paper, fill each well with washing solution (350 μL ) , let it stand for 1 minute, shake off the washing solution, pat dry on absorbent paper, and repeat washing the plate 5 times (also use a washing machine) Wash plate).
每孔加入底物A、B各50μL,37℃避光孵育15min。 6. Add 50 μL of substrate A and B to each well and incubate at 37 C in the dark for 15 min.
每孔加入终止液50μL,15min内,在450nm波长处测定各孔的OD值。 7. Add 50 μL of stop solution to each well, and measure the OD value of each well at a wavelength of 450 nm within 15 minutes.
Calculation of experimental results
所测标准品的OD值 为横坐标, 标准品的浓度 值为纵坐标,在坐标纸上 或用相关软件绘制 标准曲线 ,并得到 直线回归方程 将样品的OD值代入方程,计算出样品 浓度 Take the measured OD value of the standard product as the abscissa, and the concentration value of the standard product as the ordinate, draw a standard curve on the coordinate paper or use related software , and obtain a linear regression equation . Substitute the OD value of the sample into the equation to calculate the sample. The concentration .
Kit performance
检测范围 3.75 U/mL 120 U/mL 1. Detection range : 3.75 U / mL 120 U / mL .
灵敏度:最低检测浓度小于 0.1 U/mL 2. Sensitivity: The minimum detection concentration is less than 0.1 U / mL .
特异性:不与其它可溶性结构类似物交叉反应。 3. Specificity: Does not cross-react with other soluble structural analogs.
重复性:板内变异系数小于 10 % 板间变异系数小于1 5 % 4. Repeatability: the coefficient of variation within the plate is less than 10 % , and the coefficient of variation between the plates is less than 15 % .
Description
1. Due to the current conditions and scientific and technological level, it is not yet possible to conduct a comprehensive identification and analysis of all raw materials provided by all suppliers, this product may have certain quality and technical risks.
2. The final experimental results are closely related to the effectiveness of the reagents, the related operations of the experimenter, and the experimental environment at the time. Be sure to prepare sufficient specimen backups.
3. There may be slight differences in the same product in different batches, such as detection limit, sensitivity, and color development time, etc. Please perform experiments according to the instructions in the kit, and the electronic version of the website is for reference only.
4. Only the reagents used in this kit can guarantee the detection effect. Do not mix products from other manufacturers. Only by strictly following the experimental instructions of this kit will the best test results be obtained.
Q & A
If the experimental results are not good, please take photos of the color development results in a timely manner, keep the used slats and unused reagents, and save them properly, then contact our company's technical support to solve the problem for you. You can also refer to the following materials:
problem
Possible Causes
solution
Standard curve deviation
Insufficient pipetting and washing
Sufficient pipetting and washing
Inaccurate pipetting
Check and correct the pipette
Low precision
Insufficient washing
Wash and soak as required
Insufficient mixing and insufficient pipetting reagents
Mix well and pipette reagents
Reuse of tips, containers and films
Use a pipettor to replace a new tip, use a new container, and cover
Inaccurate loading
Check and correct the pipette
Low OD value
Inaccurate reagent volume per well
Calibrate the pipette for precise reagent addition
Incubation time is incorrect
Guarantee sufficient incubation time
Incubation temperature is incorrect
Reagents are equilibrated to room temperature and ensure accurate incubation temperature
Enzyme label or substrate is invalid
By mixing enzyme labels and substrates, the color should appear quickly to check
No stop solution was added
Add the stop solution according to the instructions in the experimental operation steps
Out of reading time readings
Read within the reading time recommended in the instructions
Sample value
Incorrect sample storage method
Store samples properly and experiment with fresh samples
Incorrect sample collection and processing methods
Take correct sample collection and processing methods
The test substance is low in the sample
Use fresh samples and repeat the experiment
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