【Product name】
Generic name: New Coronavirus (SARS-CoV-2) IgG antibody titer Kit (enzyme-linked immunosorbent assay)
English Name: diagnostic kit for the quantitative determination tier of sars-cov-2 IgG antibodies (ELISA)
【Package specification】
96 copies / box
【Intended use】
In vitro qualitative and quantitative detection of New Coronavirus (SARS-CoV-2) IgG antibody in human serum or plasma.
New Coronavirus belongs to β The New Coronavirus of the genus has capsule, and its granules are round or oval, often polymorphic, with a diameter of 60-140nm. Its gene characteristics are significantly different from those of SARS CoV and mersr cov. The present research shows that the homology of SARS like coronavirus (bat-SL-CoVZC45) is more than 85%. In vitro, sars-cov-2 can be found in human respiratory epithelial cells in about 96 hours, while it takes about 6 days to isolate and culture in Vero E6 and Huh-7 cell lines. Based on the current epidemiological investigation, the incubation period is generally 3-14 days. The main manifestations are fever, fatigue and dry cough. A few patients are accompanied by nasal congestion, runny nose, diarrhea and other symptoms. Severe cases often have dyspnea after one week, and severe cases quickly progress to acute respiratory distress syndrome, septic shock, uncorrectable metabolic acidosis and coagulation dysfunction. It is worth noting that patients with severe and dangerous severe diseases can have moderate and low fever in the course of disease, or even no obvious fever. Some patients only showed low fever and slight fatigue, without pneumonia, and mostly recovered after 1 week.
In the early stage of the disease, the number of peripheral blood leukocytes was normal or decreased, the lymphocyte count decreased, and some patients increased liver enzymes, muscle enzymes and myoglobin. C-reactive protein (CRP) and ESR increased in most patients. Procalcitonin was normal. In severe cases, D dimer increased and peripheral blood lymphocytes decreased progressively. New Coronavirus nucleic acid can be detected in throat swabs, sputum lower respiratory tract secretions and blood samples. The quantitative detection of serum antibody is helpful to confirm the infection status of the case and clarify the negative infection, past infection and long-term latent infection of the infected person.
【Inspection principle】
The indirect method principle (ELISA) was used to quantitatively detect the New Coronavirus (SARS-CoV-2) IgG antibody in human serum or plasma. The recombinant protein (SARS-CoV-2) was prepackaged with the antibody associated with the sample on the enzyme strip and added to the specimen to incubate. The IgG antibody in the sample was combined with the washing plate to remove the substance which was not bound to the antigen coated with the enzyme. The enzyme was incubated with the enzyme reagent for the second time, and the temperature of the antibody was not increased to the same time. The IgG was also used for the first time. When the New Coronavirus (SARS-CoV-2) IgG antibody is present in the sample, a "inclusion antigen -IgG antibody anti human IgG enzyme complex" complex will be formed. After washing the board, the reagent will be added to the chromogenic agent, and then the HRP catalytic chromogenic agent on the complex will react to form the blue product, and then turn yellow after the reaction. If there is no sars-cov-2 virus IgG antibody in the sample, it will not develop color. The OD value was determined on the enzyme labelling system or enzyme immunoassay system, and the content of New Coronavirus (SARS-CoV-2) IgG was determined according to OD value and OD value.
【Main components】
The main components of each group were cov-2 antigen coated plate; Enzyme labeling reagent: horseradish peroxidase labeled anti human IgG antibody; Positive antibody titer serum: calibrated human serum containing anti-sars-cov-2; Sample diluent: buffer containing protein; Concentrated washing solution: containing 10% surfactant; Chromogenic agent A: containing no less than 0.3g/l peroxide; Developer B: containing TMB not less than 0.2g/l; Termination solution: containing sulfuric acid (concentration not higher than 2mol / L).
Note: the plate sealing membrane cannot be reused. Different batches of enzyme labeled plates, enzyme labeled reagents and reference materials cannot be mixed, and cannot be mixed with reagents of other manufacturers.
【Storage conditions and expiration date】
The kit is stored at 2-8 ℃ and is valid for 1 year. Please balance the kit to room temperature (about 30min) before use. Before the experiment, shake and mix the liquid reagent gently, and immediately seal it and put it back at 2-8 ℃ for storage after use. Unused enzyme labeled strips must be sealed with desiccant in a self sealing bag and stored at 2-8 ℃.
【Applicable instrument】
Sample feeder, incubator, plate washer, enzyme labeling instrument with wavelength of 450nm and 490nm.
【Sample requirements】
*Type of plasma: human serum.
Sample collection: the collection and detection of blood samples should be carried out in accordance with the guidelines issued by the national health and Health Commission (New Coronavirus infection laboratory guidelines for pneumonia) (Third Edition). "Sample preservation: blood samples should be collected and samples should be separated in time for testing: if the samples can not be detected in time, they should be stored in accordance with the requirements issued by the national health and Health Commission (New Coronavirus infection laboratory guidelines for the detection of pneumonia)" (Third Edition).
Sample safety: all samples are regarded as potentially infectious items, which shall be implemented in strict accordance with relevant national standards and guidelines.
Please balance the sample at room temperature for more than 30min before use. Thaw and mix the frozen sample before experiment.
【Inspection method】
Reagent preparation: all reagents shall be balanced at room temperature (10-30 ℃) for 30 minutes before use, and it shall be confirmed that the surface moisture has dried out before use.
Coated board: can be used directly. Only after the coated plate is balanced to room temperature can the outer packaging aluminum bag be opened to prevent the strip from absorbing water vapor in the air. Please put the remaining lath back into the aluminum bag (or plastic bag) containing desiccant immediately and seal it.
Negative and positive control: it can be used directly and fully mixed before use.
Enzyme working solution: it can be used directly and fully mixed before use. 20 times concentrated washing solution: draw the required amount from the bottle with a clean straw, dilute it with purified water at 1:19 to become washing solution for use. For example, take IML concentrated washing solution and dilute it with 19mL purified water.
Buffer after dilution: stable for one week at 2-8 ℃. If the 20 times concentrated washing solution crystallizes, it shall be heated to 379c, fully dissolved and mixed before dilution.
Medium substrate solution a, B: can be used directly. As the substrate solution is sensitive to light, the bottle cap should be covered immediately after use and stored away from light as far as possible. Termination solution: it can be used directly.
Sealing glue: used directly. Sealant is only - used once to avoid cross contamination.
【Detection procedure】
1. Solution preparation: dilute the concentrated washing solution 20 times with distilled water or deionized water.
2. Number: number the enzyme labeled plates corresponding to the samples in sequence. Each plate shall be provided with a reference hole and a blank control hole (no blank control hole can be provided for dual wavelength detection).
3. Positive standard, sample dilution: 20 times, 40 times, 80 times, 160 times, 320 times.
4. Sample adding: diluted sample 100 μ L / well, at least two holes are parallel (the sample to be tested can also be tested by gradient dilution).
5. Plate washing and manual washing operation: add 300ul washing solution into each hole, leave it for 5-10 seconds, and then discard it. After repeated washing for 5 times, pat it dry; Operation of plate washer: add 300-350ul washing liquid into each hole, wash every 5.10 seconds, repeat washing for 5 times and pat dry.
6. Add enzyme working solution for incubation, add 100ul enzyme working solution to each well, stick sealing glue, and incubate at 37C for 20 minutes.
7. Plate washing and manual washing operation: add 300ul washing solution into each hole, leave it for 5-10 seconds, and then discard it. After repeated washing for 5 times, pat it dry; Operation of plate washer: add 300-350ul per hole Wash the lotion at an interval of 5.10 seconds. Repeat washing for 5 times and pat dry.
50 μ l of the substrate was added and the reaction solution was gently patted for 10 minutes to avoid light, and 50 μ l of the substrate was added.
9. Stop the reaction. After the color development is completed, add 5oul of termination solution to each hole and pat it gently for mixing
10. Read the results, determine the results within 10 minutes after terminating the reaction, and measure the a value of each hole after placing it at the wavelength of 450nm and 490nm of the microplate reader.
For quality control, each test shall meet the OD value of positive control > 0.50 and the OD value of negative control S0 10; Otherwise, the test results shall be deemed invalid.
【Result judgment】
The OD value of the tested sample is greater than the critical value. It should be identified as New Coronavirus (SARS-CoV-2) lgG antibody positive, less than the critical value is negative, the value near the threshold is recommended to be re tested, the positive test is positive, otherwise it is negative. For weakly positive samples, other methods shall be used to eliminate false positives.
[positive judgment value]
Calculation of critical value:
Cut off = 0.10 + mean od of negative control
(mean od of negative control, calculated as 0.05 if less than 0.05)
The normal population should be negative for this index. If positive, it may be New Coronavirus (SARS-CoV-2) infection. It should be confirmed by clinical symptoms and other diagnostic methods.
【Interpretation of test results】
Take the content of sars-cov-2 antibody in standard serum (0.63u/ml ~ 20.00u/ml) and its corresponding absorbance a value as a curve, and calculate the four parameter regression equation. Substitute the absorbance a value of the sample to be tested into the regression equation, and then multiply it by the dilution coefficient of the sample to be tested to obtain the anti sars-cov-2-igg titer (U / ml) of the sample to be tested. 1U / ml is equivalent to 1iu / ml.
For example, taking the content of sars-cov-2-igg as the independent variable (x) and its corresponding absorbance as the dependent variable (y), the four parameter regression equation is: y = -2.321 / (1 + (x / 8.325) 1.069) + 2.322.
The data and schematic diagram are as follows:
Note: according to this kit system, the potency of all test samples is the calculated value through the standard curve multiplied by the dilution coefficient.
【matters needing attention】
1. This product is only used for in vitro diagnosis, and the operation should be carried out strictly according to the instructions. The plate sealing membrane cannot be reused. Enzyme labeled plates of different batches, enzyme labeled reagents and reference materials cannot be mixed, and cannot be mixed with reagents of other manufacturers.
2. Avoid operating in the environment of volatile substances and hypochlorous acid disinfectants (such as 84 disinfectant).
3. Before use, please balance the reagent to room temperature (balance for 30min), shake and mix the reagent gently before the experiment, and put it back to 2 ~ 8 ℃ immediately after use. The unused microporous lath and desiccant shall be sealed with self sealing bag and stored at 2 ~ 8 ℃. Do not use expired reagents.
4. The sample feeder must be used when adding liquid, and the accuracy of the sample feeder must be checked frequently. When adding different samples or different reagent components, the suction head and sampling tank of the sampler shall be replaced to prevent cross contamination.
5. When washing, each hole shall be filled with washing liquid to prevent free enzyme in the hole from being washed. After washing the plate, the next step must be carried out immediately, and the enzyme label plate cannot be dried. Avoid interrupting the experimental steps for a long time to ensure the uniformity of the experimental conditions of each hole.
6. The result judgment must be based on the reading of enzyme labeling instrument. When reading the results, the bottom of the enzyme label plate should be wiped dry, and there should be no bubbles in the hole. Do not touch the outer wall at the bottom of the hole. Fingerprints or scratches may affect the reading value of the plate hole.
7. The reference substance of this product has been inactivated, and HBsAg, anti HIV, anti HCV and anti TP are negative, but it cannot be guaranteed that it does not have the infectivity of potential viruses and other microorganisms. The samples for yin-yang control and detection should be operated in strict accordance with the relevant provisions of Biosafety, follow the routine provisions of laboratory operation, and be used and treated as the samples with potential biological infectivity. All samples, waste liquid and wastes used shall be treated as infectious substances. The termination solution is sulfuric acid, so you must pay attention to safety when using it.
8. When developing color, add developer a solution first and then developer B solution to avoid too low color.
9. Each experiment shall be carried with a positive standard, and the experimental results must be obtained with the current standard curve, otherwise the error of quantitative results may be too large.
10. The abnormal points in the standard curve may cause the deviation of the whole plate experimental results. Therefore, it is recommended to detect the reference material with two holes to improve the experimental accuracy.
11. When the goodness of fit R2 of the standard curve is ≥ 0.99, the experiment is considered effective, otherwise the experiment is invalid
12. The sample dilution ratio of this test method is large, so the sample dilution has an impact on the results. It is recommended that the sample be diluted by gradient, not more than 5 times in each step, and the dilution volume shall not be less than 0.5ml.
[Limitations of inspection methods]
1. According to the basic theory of antibody production after infection, after the body is infected by virus, specific IgM antibody is produced earlier and lasts for a short time: IgG antibody is produced later and lasts longer than IgM antibody. In addition, it takes a certain time from virus infection to the production of specific antibodies, and there are individual differences in the intensity of antibodies, which is related to the amount of infected antigens and the antigenic intensity of antigens. Therefore, the LGG antibody test results, IgM antibody test results, sampling time, clinical indications and occurrence time of this product should be considered comprehensively. If the antibody test is positive, it should also be judged in combination with other clinical indications.
2. This kit can only be used for the determination of serum or plasma samples, not other body fluid samples.
【manufacturing enterprise】
Company name: Beijing qianzhaoxinye Biotechnology Co., Ltd
