Polio IgG ELISA Kit
(Germany IBL imported original, article number: RE56921)
Germany IBL China exclusive agent "Shenzhen Kerunda Biological Engineering Co., Ltd." dedicated to serve you
1 , the scope of application
The polio IgG antibody ELISA kit can be used for the quantitative detection of anti-polio-specific IgG antibodies in serum and plasma. This kit can also be used to detect other body fluids, and you can get technical support from IBL. This kit is for in vitro diagnostic use only.
The results of the experiment can never be used as the sole basis for medical reports. Medical reports must also consider the patient's medical history and conduct further tests.
2 , the preface
Polio is an infectious disease caused by enteroviruses, which is widespread in the world and often causes paralysis and death. There are three types of human pathogenic polio known to humans:
Class 1 (Brunhilde): usually has serious symptoms.
Class 2 (Lansing): mild symptoms appear.
Category 3 (Leon): Less, but with severe symptoms.
The main proliferation of poliovirus spreads in the lymph nodes of the intestines and is excreted through the feces. The throat may also be infected with poliovirus, and the virus will remain in the human mouth. After human infection with polio, the virus can be distributed to other lymph nodes through monocytes, and then multiplied into various polioviruses in the lymph nodes. In the second viremic phase, the virus stays in all organs and other organs of the central nervous system. More than 90% of patients infected with polio do not experience any subjective symptoms. In other remaining cases, non-specific diseases with low fever, headache and throat irritation, dysentery, nausea and vomiting may occur. Classic paralysis with muscle pain and brain nerves is very rare. The re-rehabilitation phase lasts for two years and often results in lasting damage. There is currently no treatment for polio, and symptomatic treatment with aspirin and gymnastics alone will have some effect. Poliomyelitis is still endemic in many countries in Asia, but WHO has begun a huge project to eradicate the disease. There are cases of polio in Europe that are brought in by tourists and are sometimes fatal. During the viral incubation period, polio can be diagnosed by detecting infectious agents in the stool or throat rinse or by detecting antibodies in the blood. The detection of blood antibodies is usually carried out by neutralization test, and the double serum test has a difference in titer, so the three types of poliovirus must be detected separately. Recently, polio IgG ELISA experiments have been improved using methods similar to diphtheria and whooping cough, which can simultaneously detect three types of poliovirus. This serological response is caused by previous infections or immunizations.
3 , the principle of experiment
The polio IgG antibody detection reagent uses the principle of enzyme-linked immunosorbent assay. The polio antigen is coated on the microwells of the coated strips. The diluted patient serum or the ready-to-use standard is added to the microwell of the coated plate, and then the IgG antibody in the serum is bound to the solid phase polio antigen. After incubating for 1 hour at room temperature, the plate was washed with a washing solution to remove unbound material. Peroxygenase-labeled ready-to-use anti-human IgG was then added and incubated for 30 minutes. The plate was then washed, the TMB substrate solution was added after the plate was washed and incubated for 20 minutes, at which time the solution turned blue. The enzyme solution was terminated by the addition of a stop solution, and the solution turned blue again to yellow, and the OD value was measured at 450 nm. The concentration of the IgG antibody is proportional to the intensity of the color displayed by the solution.
4 , storage and stability
The kit was stored at 2-8 ° C and the shelf life (marked on the label) was stable. All ingredients were equilibrated to room temperature (18-25 ° C) before use. The unused slats should be resealed, the lid of the reagent bottle should be covered, and stored at 2-8 °C. The opened kit must be used within three months.
5 , sample collection and storage
Serum, plasma (EDTA, heparin)
Samples are collected according to routine precautions for venipuncture, and blood samples must be chemically intact from collection to testing. Do not use significant hemolysis, jaundice, and lipemia samples. The turbid sample should be centrifuged before the start of the experiment to remove all particulate matter. | |||
store | 2-8 ° C | ≤-20°C | Avoid direct sunlight and avoid repeated freezing and thawing. |
stability | 48 hours | >48 hours | |
6 , kit components
4×2ml | CAL AD | Standard Ad: Human serum diluted in PBS containing anti-poliovirus IgG antibody, 0.01% Methylisothiazolone and 0.01% Bromonitrodioxane, ready to use. |
1×14ml | ENZCONJ IgG | Enzymes: horseradish peroxidase-labeled anti-human IgG and protein buffer. Another Methylisothiazolone containing 0.01% and 0.01% Bromonitrodioxane and 5mg / l of Proclin TM, i.e. use. |
1×12×8 | MTP | Coated plates: 12 8-hole coated plates coated with polio antigen (a vaccine for purified viral material mixed with type 1, 2, and 3 viruses), ready to use. |
1×14ml | TMB SUBS | TMB substrate solution containing TMB, ready to use. |
1×14ml | TMB STOP | TMB stop solution, 0.5 M sulfuric acid, ready to use. |
1×60ml | DILBUF | Dilution buffer, PBS/BSA buffer containing 0.095% sodium azide, ready to use. |
1×60ml | WASHBUF CON | Wash buffer, 10 times concentrated, PBS + Tween 20. |
2× | FOIL | A viscous metal plate used for cover during incubation. |
1× | BAG | A plastic bag that can be resealed to store unused slats. |
7. Materials required for the experiment but the kit does not provide
1) 5ul, 100ul and 500ul multichannel pipettes.
2) A microplate reader that can read at 450 nm.
3) Washing machine
4) Test tubes for serum dilution
5) distilled water
8. Preparation instructions before the experiment
Preparation of ingredients 8.1
Dilution/dissolution | ingredient | Thinner | proportion | Remarks | store | stability | |
60ml | detergent | Add 540ml | Distilled water | 1:10 | If necessary, heat to 37 ° C to dissolve all crystals, mix well | 2-8 ° C | 8 weeks |
8.2 dilution of the sample
sample | dilution | Thinner | proportion | Remarks |
Serum/plasma | General regular dilution | Dilution buffer | 1:101 | 5ul+500ul |
If the IgG concentration in the sample is higher than the highest standard, the sample must be further diluted.
11 , experimental steps
1 | Prepare enough slats for standards, controls, and samples (dual test), leaving one hole for a blank control. |
2 | Add 100 ul of each diluted sample, ready-to-use standard and control product to the corresponding micropores, leaving no blank holes for adding the substrate solution. |
3 | The cover was incubated for 60 minutes at room temperature. |
4 | The reaction solution in the well was discarded, and 300 ul of the diluted washing liquid was added, and the plate was repeatedly washed three times, and the inside of the well was discarded, and then patted on a blotting paper to remove residual liquid. |
5 | Add 100 ul of the ready-to-use enzyme conjugate to each well, leaving no blanks. |
6 | Cover plate and incubate for 30 minutes at room temperature. |
7 | The reaction solution in the well was discarded, and 300 ul of the diluted washing liquid was added, and the plate was repeatedly washed three times, and the inside of the well was discarded, and then patted on a blotting paper to remove residual liquid. |
8 | Add 100 ul of the ready-to-use substrate solution to each well, leaving no blanks. |
9 | Cover plate, incubate at room temperature for 20 minutes in the dark. |
10 | 100 ul of the ready-to-use stop solution was added to each well to terminate the substrate reaction, and the blank well was also added with a substrate solution. |
11 | Fully mixed, the OD value was read at 450 nm, and the color displayed by the solution was stable within 60 min. |
12 , quality control
The experimental results in strict accordance with the experimental steps are effective. Users must strictly adhere to experimental guidelines or other experimental standards. As stated on the reagent bottle label: all controls must be built within an acceptable range. If the standard does not meet the requirements, then the experiment is invalid and must be redone. In order to be able to conduct further research, each laboratory must know all the samples. If the experiment deviates from the actual results, the following parameters should be checked: reagent expiration date, storage conditions, sampler, experimental equipment, incubation time, cleaning method.
13 , evaluation of results
The average OD value is the average value after the OD value is subtracted from the blank hole value, and the difference between the two holes cannot exceed 10%.
OD value | Corrected OD value | Average OD value | |
Blank hole | 0.019 | ||
Standard A (female control) | 0.112/0.134 | 0.103/0.115 | 0.109 |
Standard B (critical quality control) | 0.410/0.472 | 0.391/0.453 | 0.421 |
Standard C (weak positive control) | 1.069/1.121 | 1.050/1.102 | 1.076 |
Standard D (positive control) | 2.186/2.220 | 2.167/2.201 | 2.184 |
The above table is just an example. All values ​​are obtained under the current temperature and conditions, so the above values ​​cannot be used as reference values. The reference values ​​must be obtained from other laboratories.
13.1 Quantitative evaluation
The ready-to-use standards and controls for the poliovirus IgG antibody test kit are defined in a specific unit (U/ml). If the results of the experiment are accurate and can be repeated quantitatively, the results may be used as controls for subsequent patients. See the reagent bottle label for the concentration of controls and standards. For quantitative detection, the concentration of the standard and the control product is plotted on the coordinate paper, and then the concentration value of each patient sample can be quantified from the reference curve using its OD value. We can use the computer program to automatically calculate the record. When the concentration is calculated, the dilution factor of the sample should be reconsidered.
14 , characteristics
The intra-plate difference of the poliovirus IgG kit can be obtained by repeating the detection of the weak positive property for 10 times, and the intraplate difference is less than 10%.
This translation is for reference only, please refer to the original for details.
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