How 3 companies have teamed up to get you back to “Normal Life” With Covid-19 still on the loose, Prevenna and its partner, Welltok, have teamed up with Biohit to help you get back to work, back to school and back to life safely. How? Testing testing, testing. “What...
SARS-CoV-2 Antigen quantitative assay kit (Enzyme-linked immunoassay)
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This kit is used for quantitative detection of SARS-CoV-2 nucleocapsid protein (N protein) (hereinafter referred to as “SARS-CoV-2 N protein”) in human serum or plasma.
SARS-CoV-2 was discovered in novel coronavirus pneumonia (COVID-19) cases in 2019. It belongs to the coronavirus family of beta coronavirus, which is similar to that of SARS coronavirus in 2003 and MERS coronavirus in 2012. The genome of coronavirus encodes spike protein, envelope protein, membrane protein and nucleocapsid. In the process of viral assembly, N protein binds to viral RNA and leads to the formation of spiral nucleocapsid. N protein is a highly immunogenic phosphoprotein,which is related to viral genome replication and cell signaling. Because of the conserved sequence of N protein, quantitative detection of SARS-CoV-2 N protein is of great clinical significance.
This kit employs the principle of the double antibody sandwich method to detect the SARS-CoV-2 N protein in human serum or plasma. Microplates were coated with Anti-SARS-CoV-2 N protein antibody in advance to prepare the antibody-coated solid phase plates. After adding the sample, if the sample contains SARS-CoV-2 N protein, it will bind to the coated plate. Then it combined with the added biotin-labeled SARS-CoV-2 N protein antibody to form a solid-phase antibody-antigen-biotinylated antibody sandwich complex. Further, add horseradish peroxidase (HRP)-labeled streptavidin (SA) to combine with biotinylated antibody to form a complex, unbound conjugate is washed away and a solution containing 3,3’,5,5’-tetramethylbenzidine (TMB) and hydrogen peroxide is added to the wells. Wells with bound Conjugate develop a blue green colour which is converted to an orange colour which may be read at 450nm after the reaction has been stopped with stop solution and calculate the SARS-CoV-2 N protein content in the sample according to the protein concentration of SARS-CoV-2 calibrator.
|1||Coated microplate||96 wells ×1 plate|
|2||Conjugate 1||6mL/bottle ×1 bottle|
|3||Conjugate 2||12mL/bottle ×1 bottle|
|4||Calibrators (CAL1-CAL5)||0.5mL/bottle, 5 bottles in total|
|6||Substrate solution A||6mL/bottle ×1 bottle|
|7||Substrate solution B||6mL/bottle ×1 bottle|
|8||Stop solution||6mL/bottle ×1 bottle|
|9||Concentrated wash buffer 20×||30mL/bottle ×1 bottle|
|10||Sealing film||3 sheets|
|11||Instructions for use||1 copy|
4.1. Samples for human serum or plasma should be considered as potentially infectious. Operators should wear protective clothing, masks, gloves and take other appropriate safety precautions to avoid or reduce the risk of infection.
4.2. For professional use only.
4.3. This test should be performed at 18 to 30℃.Ensure that the kit is brought to operating temperature before performing testing.
4.4. Follow the instructions for use carefully. Reliability of assay results cannot be guaranteed if there is any deviation from the instructions inserted in this package.
4.5. Professionals must handle the potentially contaminated materials safely according to local requirements.
4.6. Do not smoke, drink, eat, or use cosmetics in the working area. Wear Personal Protective Equipment and disposable gloves when working with samples and reagents. Wash hands after operations.
4.7. Wipe and wash any splashed sample with highly effective disinfectant. Avoid splashing and nebulizing.
4.8. Use a new clean disposable sample tip for each sample to avoid cross contamination.
4.9. Decontaminate and dispose of all samples, reaction kits, and potentially contaminated materials as if they were infectious waste, in a biohazard waste container.
4.10. The microplate shall be dried to store. The unused microplate shall be immediately put into a clean plastic bag containing desiccant, sealed and moisture-proof. The remaining kits after use shall be put back to 2～8℃ for storage in time.
4.11. Do not use expired reagents.
4.12. The components in different lots of kits shall not be mixed.
4.13. Strong oxidants such as sodium hypochlorite disinfectant may cause the background of TMB substrate to rise, which may lead to misjudgment of the results.
4.14. In order to prevent sample evaporation and pollution, the reaction plate shall be placed in a closed and clean plastic bag or covered with a sealing film during warm cultivation.
4.15. When washing the microplate, the liquid injection volume of each well shall not be less than 350 μL, and the number of washing the microplate shall not be less than 5 times. Pay attention to check whether the liquid filling head is blocked. After washing the board, dry it on the clean absorbent paper. When washing the board by hand, each washing liquid shall be filled with micropores, and finally it shall be dried on clean water absorbent paper without paper scraps to prevent the accuracy of the test results from being affected.
4.16. The microplate cannot be reused.
The kit is stored at 2～8℃ and the shelf life is 12 months. After the coated microplate is opened, add desiccant and seal it in time, it is stable for 14 days at 2～8℃.
Liquid reagents such as conjugates, calibrators, substrate, etc. are stable for 60 days before the expire date if stored at 2 to 8℃ after opening. The stop solution and concentrated wash buffer are stable to the indicated expire date after opening.
Microplate EIA reader with 450nm wavelength
7.1 Applicable to human serum, heparin plasma, EDTA plasma and citrate plasma.
7.2 For serum and heparin plasma, EDTA plasma, or Citrate plasma samples, the samples shall be tested immediately after collection. Serum and heparin plasma, EDTA plasma or Citrate plasma samples can be stored for 5 days at 2～8℃. If long-term storage is required, it should be stored at -20℃. Serum or plasma specimens can be subjected to a maximum of 3 freezing/ thawing cycles.
7.3 Let the samples reach room temperature and mix well before testing. When there are visible particles in the sample, it should be centrifuged before the test to remove the precipitate.
7.4 If there is a lot of lipid (Triglyceride concentration over 37 mmol/L), hemolysis or turbidity in the sample, please do not use the sample to avoid affecting the result interpretation.
- Sample vortex mixer
- Adjustable or fixed range single channel and 8-channel pipettes that can meet the requirements of test sample adding amount
- Test tubes
- Sample collection tubes
- Microplate EIA reader
- Incubator: 37 ± 1℃
- Fresh distilled water or deionized water
- Water absorbent paper: strong, non-shedding fiber
Step 1: Preparation of wash buffer: dilute the concentrated wash buffer 20 times (30mL concentrated wash buffer + 570mL distilled water or deionized water) with fresh prepared distilled water or deionized water in a clean container, mix well for standby, and the diluted wash buffer can be kept at room temperature for 7 days.
Note: Please make sure that there is no crystallization in the concentrated wash buffer before use. If there is crystallization, it can be placed in a water bath pot for heating and dissolution, otherwise the washing effect will be affected.
Step 2: Preparation: take out the kit from the refrigerator, and balance it at room temperature (18～30℃) for 30 minutes. Take out the sample to be tested and let it reach room temperature. Mix the sample well before testing.
Step 3: Design: Take the microporous plate out of the sealed bag, make one well for the calibrator and one well for the quality control material, place the microporous plate on the plate frame according to the designed sample quantity, and mark the plate frame according to the experiment design.
Step 4: Conjugate 1: Add Conjugate 1 50 μL to each well.
Step 5:Sample: Add 50μL of sample, control and calibrators respectively：
Step 6: Incubation: Stick on the sealing film, shake well with a micro vibrator, incubate at 37℃ for 60 minutes.
Step 7: Washing: Wash each well with 350 μL of 20 times diluted wash buffer for 5 times.
Step 8: Conjugate 2: Add Conjugate 2 100 μL to each well.
Step 9: Stick on the sealing film, mix with micro vibrator for 5 seconds, and incubate at 37℃ for 30 minutes.
Step 10: Washing: Wash each well with 350 μL of 20 times diluted wash buffer for 5 times.
Step 11: Substrate solution: Add 50 μL substrate A to each well, and then add 50μL substrate B to each well.
Step 12: Stick on the sealing film, mix with micro vibrator for 5 seconds, and incubate at 37℃ for 15 minutes.
Step 13: Stop solution：Add 50 μL of stop solution into each well, shake gently and mix well.
Step 14: Reading: Set the wavelength of the Microplate EIA reader at 450nm, and measure the OD value of each well.
[Sample dilution procedure]
When the SARS-CoV-2 N protein is more than 347.25pg/mL, it should be marked as "＞347.25pg /mL", which can be further tested after diluting the sample. The dilution method is as follows:
- It is recommended to dilute at a dilution ratio of 1:10: for example, 20 μL sample+180 μL normal saline;
- If the diluted sample test is still more than 25pg/mL, conduct 1:10 dilution again;
- When calculating the sample concentration, it is necessary to input dilution factor to calculate the final sample concentration.
- The concentration of the sample to be tested can be calculated as follows：
- Data processing procedure: subtract the absorbance value of S1 from each test mean, take point 0 as the starting point, the absorbance value of standard solution is y-axis, and the given value of standard solution is X-axis for binomial fitting, and calculate the corresponding concentration value according to the absorbance value of sample and control after subtracting.
10.1. Cutoff value
According to the test results of clinical samples, ROC curve method was used to determine the cutoff value of 10pg/mL.
10.2. Quality control standard
(1) Check the OD value of calibrator (S5). When S5 ≤1.000, the test is invalid, and the test shall be repeated.
(2) Check the OD value of the calibrator (S1). When S1>0.1000, the test is invalid and should be retested.
(3) The correlation coefficient r of the calibration curve should be ≥0.9900, otherwise the experiment will be invalid, and the experiment should be repeated.
(4) The test results of quality control products should be within ± 20% of the target value, otherwise the experiment is invalid, and the experiment should be repeated.
10.3. SARS-CoV-2 was determined to be positive in the range of 10pg/mL-347.25pg/mL, and the concentration results were reported directly;.
10.4. When the test concentration is ＞347.25pg/mL, it is judged to be positive and reported as "＞347.25pg/mL"; if it is necessary to further determine the sample concentration, it can refer to the method in the sample dilution procedure to test the sample after dilution.
10.5. Due to the complex structure of bioactive substances in samples and the difference of antigen antibody specificity, the possibility of false positive results can not be completely ruled out by using this kit. If the test results are inconsistent with the clinical indications, other appropriate test methods should be used for confirmation.
10.6. SARS-CoV-2 N protein novel coronavirus nucleocapsid protein, N protein positive is an important sign of SARS-CoV-2 infection, indicating that there is SARS-CoV-2 infection. But the negative result of SARS-CoV-2 N protein can not completely exclude the infection of SARS-CoV-2. The reason is that when the content of SARS-CoV-2 N protein in the sample is below the conventional detection limit, or anti-N protein antibody has been produced in the serum, SARS-CoV-2 N protein can not be detected.
10.7. When the specific antibody appears in the blood, SARS-CoV-2 N protein is neutralized, which leads to the decrease of N protein content in the serum to the degree that it can not be detected.
10.8. The test results of this kit are only used as the basis of auxiliary diagnosis. Clinical diagnosis should be combined with clinical symptoms and other diagnostic methods.
10.9. Each laboratory can establish its own reference range according to the actual situation.
11.1. Hyperlipidemia, hemolysis, microbial contamination, repeated freeze-thaw for more than three times or after heat inactivation may affect the accuracy of the test and lead to wrong results.
11.2. Samples with severe jaundice or serious pollution will lead to wrong results.
11.3. Sodium azide contained in the sample will affect the experimental results, and it cannot be used as sample preservative.
11.4. If the microplate is not washed sufficiently or there is residual liquid, it may cause unreliable results.
11.5. If the time interval of sample adding is too long, it may cause the deviation of test results; if more than one microplate is used, each microplate shall be provided with calibrator.
LOB: Test with blank solution as sample, repeat 20 times, LOB should be less than 1.0pg/mL.
In the concentration range of 5.75-347.25pg/mL, the correlation coefficient (r) is not less than 0.9900.
Recovery test is used to evaluate the accuracy of the kit. The recovery rate should be in the range of 85%～115%.
For determination of intra-assay variation, four serum samples and four plasma samples were tested for a total of 40 times（assayed in duplicate）over 20 days for each level of the test material. In the inter-assay variation for serum samples, the range for the means was from 12.48 pg/mL to 319.1 pg/mL，and the %CV from 1.56% to 13.08%. For EDTA-plasma samples, the ranges and %CV were from 12.54 pg/mL to 319.58 pg/mL and from 1.5% to 11.4%，respectively.
For determination of inter-assay variation, four serum samples were tested 10 times by three lots of kits. The range for the means and the %CV was from 12.01 pg/mL to 318.21 pg/mL，and the %CV from 2.27% to 12.96%，respectively.
Cross-reactivity of the SARS-CoV-2 Antigen quantitative assay kit was evaluated by using clinical samples containing antibody in the underlying conditions listed below. Potential cross-reacting substances were spiked into serum samples negative for SARS-CoV-2 antigen. Five samples with antigen or antibody to the underlying conditions 1 to16 were tested in singlicate with three lots of SARS-CoV-2 Antigen quantitative assay kit. None of the antigen in the listed underlying conditions cross-reacted with SARS-CoV-2 antigen quantitative assay kit by generating false positive results.
|Number||Name of potential cross-reactants|
|1||2009-H1N1 influenza virus IgG|
|2||Seasonal H1N1 influenza virus IgG|
|3||H3N2 influenza virus IgG|
|4||H5N1 influenza virus HA protein|
|5||H7N9 influenza virus HA protein|
|6||Influenza B virus IgG|
|7||Respiratory syncytial virus IgG|
|9||EB virus IgG|
|11||Hepatitis B virus|
|12||Hepatitis C virus|
|13||229E (alpha coronavirus) IgG|
|14||NL63 (alpha coronavirus) IgG|
|15||OC43 (beta coronavirus) IgG|
|16||HKU1 (beta coronavirus) IgG|
SARS-CoV-2 antigen positive serum and SARS-CoV-2 antigen negative serum were spiked with one of the following substances to specified concentrations and tested in multiple replicates. No false positivity or false negativity results were found with the following:
|4||Rheumatoid factor||1000 IU/mL|
|5||Mouse IgG||1.2 mg/mL|
|7||Anti-mitochondrial antibody||80 U/mL|
|8||α - interferon||9×106 IU/mL|
|21||Histamine hydrochloride||30 mg/L|
Positive Percent Agreement (PPA) statistics
Methods: A retrospective study was conducted on 74 samples from a local center for Disease Control and prevention in Anhui Province,74 sera collected on the day of diagnosis confirmed (ie, nucleic acid test positive day) from 74 patients with novel coronavirus pneumonia (COVID-19), Among these samples, 30 samples were sampled from the onset time ≤3 days, 26 samples were sampled from 4 to 7 days from the onset time, and 18 samples were sampled from 8 to 14 days from the onset time.
|Group||Days from onset of symptoms||Number of antigen positive samples||Total number of samples||*PPA|
*PPA= Positive Percent Agreement.
Negative Percent Agreement
Methods: a retrospective study was carried out with 633 samples from an Anhui local Tier 3 hospital, including 369 samples of other respiratory tract infections (The serum samples were collected in the Clinical Laboratory of the local hospital from February to May 2020, the patients were confirmed negative by PCR), 100 samples were pregnancy test samples (The serum samples were collected in the Clinical Laboratory of the local hospital from February to May 2020, the patients were confirmed negative by PCR), 119 abnormal samples of rheumatoid factor (The serum samples were collected in the Clinical Laboratory of the local hospital from May 2020, the patients were confirmed negative by PCR), and 45 samples of physical examination samples (The serum samples were collected in the Clinical Laboratory of the local hospital on May 2020, the patients were confirmed negative by PCR).
Antigen Test Results Statistics
|Group no.||Research samples||No. of positive samples||No. of samples||*NPA|
|1||Infection by other respiratory pathogens||0||369||100%|
|3||Elevated rheumatoid factor||0||119||100%|
|4||Health examiner Hemolysis sample||0||45||100%|
*NPA=Negative Percent Agreement statistics
Antigen clinical NPA=100%×633/633=100%.
13.1. Read this manual carefully before testing the kit.
13.2. It needs to be tested in a laboratory with proper testing conditions. All samples and materials in the testing process shall be handled according to the operation specifications of infectious diseases laboratory.
13.3. This kit is an in vitro diagnostic reagent. The operation requires a certain degree of professionalism, and the operators shall receive professional training.
13.4. All reagents and samples should reach room temperature (18-30℃) before use.
13.5. Do not use lipemic samples.
13.6. Do not use hemolytic samples.
13.7. Do not use turbid contaminated samples.
13.8. Do not store this kit in frozen condition.
13.9. The interpretation of the test results must be carried out in strict accordance with this manual.
13.10. This kit is limited to quantitative detection of SARS-CoV-2 antigen in human serum, heparin plasma, EDTA plasma or citrate plasma.
13.11. False negative results will be caused when the antegen titer in the sample is lower than the minimum detection limit of the test.
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