Novel Coronavirus N-Protein Detection Kit (ELISA)

Instruction Manual for the Novel Coronavirus N-Protein Detection Kit (ELISA)
[Product Name]
Generic Name: Novel Coronavirus N-Protein Detection Kit (ELISA)
[Packaging Specifications]
96 servings per box
[Intended Use]
This product is intended for the qualitative detection of novel coronavirus nucleocapsid (N) antigen in serum or plasma samples.
Infectious atypical pneumonia, also known as Severe Acute Respiratory Syndrome (SARS), is a newly emerging infectious disease [1]. On April 16, 2003, the World Health Organization identified the causative agent of SARS as a novel coronavirus, designated SARS‑CoV [2]. Since its first outbreak in Guangdong Province, China, in November 2002, SARS has spread to more than 30 countries and regions, resulting in 8,422 confirmed cases and 916 deaths (as of August 7, 2003) [3]. According to available data [4–6], the disease is primarily transmitted through respiratory droplets, bodily fluids, and contaminated surfaces over short distances, with clear patterns of familial and hospital‑acquired clusters. Its main clinical features include an abrupt onset, fever, cough, pulmonary infiltrates, and either normal or decreased peripheral white blood cell counts. Despite significant advances in pathogen research, breakthroughs remain elusive in areas such as the pathogen’s biological characteristics, disease mechanisms, early and reliable diagnostic assays, and specific therapeutic strategies, leaving many critical issues unresolved. In particular, early diagnosis of SARS poses a major challenge for clinicians seeking timely detection, isolation, and treatment, as the same viral infection may present with diverse clinical manifestations in different individuals, while distinct viral infections can share identical symptoms. Consequently, relying solely on clinical signs and epidemiological information often proves insufficient to identify the true causative agent; definitive diagnosis typically requires laboratory testing, yet no fully established, objective laboratory criteria currently exist. Given that most viral diseases still lack specific antiviral therapies, laboratory diagnostics have become a crucial tool for controlling the spread and prevalence of viral illnesses. The immediate priority is to rapidly develop effective diagnostic reagents, enabling early diagnosis, prompt isolation, interruption of transmission routes, and containment of the epidemic.
At present, laboratory diagnosis of SARS coronavirus infection primarily encompasses the following approaches: molecular diagnostics based on PCR and gene‑chip technologies; serological diagnostics relying on antibody monitoring; and pathogen‑based diagnostics grounded in in vitro virus culture.
At present, early laboratory diagnosis of SARS‑CoV infection primarily relies on PCR‑based viral nucleic acid detection. Existing experimental data indicate that the sensitivity of viral nucleic acid testing is approximately 70% within about 14 days of symptom onset [7, 8]. However, because viral RNA is prone to degradation, test results are susceptible to variations in sample collection and processing, and sample contamination can lead to false‑positive outcomes. Consequently, rigorous quality control and expertly trained personnel are essential. Moreover, specialized equipment is required, and such instruments are costly. Given these factors, many hospitals still lack the capacity to implement this diagnostic approach.
Serological diagnosis based on the detection of specific antibodies currently relies on two methods: enzyme-linked immunosorbent assay (ELISA) and immunofluorescence. A seroconversion from negative to positive, or a fourfold or greater increase in antibody titer between the acute phase and the convalescent phase, indicates that the infection occurred recently. Because specific antibodies typically begin to appear 7 to 10 days after symptom onset, serological testing is not suitable for the early diagnosis of SARS.
Pathogen‑based diagnostics relying on in vitro virus culture, which involve isolating SARS‑CoV from patient specimens, represent the “gold standard” for laboratory diagnosis. However, due to limitations in experimental conditions and other factors, this approach is generally not employed as a routine method. Moreover, it is technically demanding, operationally complex, time‑consuming, and requires sophisticated equipment, making it unsuitable for use in clinical settings.
Pathogen‑based diagnosis using specific antibodies is an early laboratory diagnostic approach for SARS, involving the detection of viral antigens in samples with well‑characterized, specific antibodies. The technical challenge of developing antigen‑detection kits based on such antibodies lies in obtaining monoclonal or polyclonal antibodies with exceptionally high specificity. Mature hybridoma technology has already played a pivotal role in the immunological diagnosis of numerous diseases; owing to the highly specific immunological properties of monoclonal antibodies, it is possible to construct assays with outstanding sensitivity and specificity, a capability that has been demonstrated by previously developed antigen‑detection reagents for various viruses [9–11].
Since SARS-CoV has been shown to be a positive-sense single-stranded RNA virus, genomic structural analysis indicates that its gene‑encoded structural proteins comprise the spike glycoprotein (S), the small envelope protein (E), the membrane protein (M), and the nucleocapsid protein (N). Although the functions of these structural proteins remain unclear, studies of coronavirus biology have revealed that the proteins on the viral envelope are prone to mutation, whereas the nucleocapsid protein is relatively stable. Moreover, the nucleocapsid protein exhibits stronger antigenicity than other structural proteins and serves as the virus’s primary antigenic determinant. Accordingly, by obtaining highly purified, highly specific viral N antigens, immunizing animals, and generating monoclonal antibodies with high affinity and specificity as well as high‑titer polyclonal sera, a diagnostic kit for detecting SARS‑CoV antigens has been developed.
[Principle of Assay]
This kit is composed of a microtiter plate coated with purified anti‑SARS‑CoV‑2 antibodies (three monoclonal antibodies against the recombinant N protein), rabbit polyclonal anti‑N protein antigen, horseradish peroxidase (HRP)‑labeled goat anti‑rabbit IgG, negative and positive controls, chromogenic substrate, and other necessary auxiliary reagents. It employs a double‑sandwich ELISA principle: the antigen to be tested binds to the antibody immobilized on the solid‑phase carrier, forming an antigen–antibody complex; subsequent addition of rabbit anti‑N protein antibody generates a sandwich immune complex; followed by the addition of HRP‑labeled goat anti‑rabbit antibody, resulting in an immune complex of (antibody–N antigen–secondary antibody–enzyme‑labeled secondary antibody). Upon addition of the substrate, an enzymatic colorimetric reaction occurs; the intensity of this reaction is measured by an instrument, enabling qualitative detection of the N protein in the sample under test.
[Main Ingredients]
1. The components included in this kit are:
Serial Number Component Name Main Ingredient Package Quantity
1 Microtiter plate pre-coated with monoclonal antibodies against the novel coronavirus N protein. The microtiter plate is pre-coated with three monoclonal antibodies that recognize the recombinant N protein. 96 wells.
2 Rabbit anti-SARS-CoV N-protein antibody: Rabbit immune serum against the novel coronavirus N-protein, in Tris–NaCl buffer, containing 0.1% Proclin‑300, 0.001% phenol red, and a protein stabilizer. 7.0 mL × 2 vials.
3 HRP‑labeled goat anti‑rabbit IgG: horseradish peroxidase (HRP)‑conjugated goat anti‑rabbit IgG antibody, in borate buffer containing 0.1% Proclin‑300, 0.001% phenol red, and a protein stabilizer. 7.0 mL × 2 vials.
4 Positive Control: Recombinant SARS-CoV‑2 N protein, in borate buffer, containing 0.02% thimerosal and a protein stabilizer. 1.0 mL × 1 vial
5 Negative Control: Normal human serum or plasma, borate buffer, containing 0.02% thimerosal and a protein stabilizer. 1.8 mL × 1 vial
6 Color-developing solution A: citric acid, hydrogen peroxide. 6.5 mL × 1 bottle
7 Color-developing solution B: citric acid, tetramethylbenzidine (TMB). 6.5 mL × 1 bottle
8 Concentrated Detergent (10×) Phosphate-buffered solution, containing Tween‑20, 10× concentrated. 50 mL × 2 bottles
9 Stop solution: 0.5 mol/L H2SO4 solution. 6.5 mL × 1 bottle
10 zip-lock bags, 1 piece
11 Sealing films, 4 pieces
12 Recording paper, 1 copy
Note: Rabbit anti‑SARS‑CoV N‑protein antibody, enzyme conjugate, positive control, and negative control must be used exclusively from the kit; do not mix reagents from different lots. Chromogen A, chromogen B, stop solution, and concentrated wash solution are universal reagents and may be interchanged between batches.
2. Materials required but not yet provided:
Distilled water or deionized water
Microplate washer
5 μL–40 μL, 40 μL–200 μL, and 200 μL–1 mL pipettes with disposable tips
37°C constant-temperature equipment
Micro-pore oscillator
Microplate reader
A timer with a duration of 60 minutes or longer
[Storage Conditions and Shelf Life]
Store protected from light at 2°C to 8°C; shelf life is 6 months.
After opening, any unused microtiter plate should be placed in a sealed bag and stored at 2°C to 8°C. Each component, once opened, should be used within one month.
Production date and expiration date: See label for details.
[Applicable Instruments]
A microplate reader with a 450 nm wavelength
[Sample Requirements]
Sampling precautions: Due to the high transmissibility of the SARS coronavirus, which can spread through close, face-to-face contact, thorough safety precautions must be taken during sample collection. Samples should be transported in dedicated tube racks. Immediately seal and submit the samples for testing after collection. Such specimens must be stored under strict isolation by designated personnel in a separate cabinet. Clearly label each specimen and ensure it is not mixed with samples from other pathogens. The sampling procedure must be carried out in strict accordance with the “Technical Guidelines for Laboratory Specimen Collection in Cases of Infectious Atypical Pneumonia.”
After venous blood collection, allow the sample to stand at room temperature for 1–2 hours, then centrifuge to separate the serum. If plasma is required, the sample can be directly centrifuged to obtain plasma after collection. For use within 24 hours, store at 2°C–8°C; for long-term storage, keep below −20°C and avoid repeated freeze–thaw cycles. The impact of samples with high concentrations of lipids, bilirubin, or hemolysis, as well as microbial contamination, on assay results has not yet been established.
Inactivation of serum or plasma samples at 56°C for 30 minutes does not affect the assay results; the impact of other inactivation conditions remains undetermined.
[Testing Method]
1. Precautions During the Experiment
1.1 When the kit components are removed from refrigeration, allow them to equilibrate at room temperature until no condensation is present before use. Do not mix components from different batches. After opening, any unused microtiter plates should be stored in a sealed bag.
1.2 Do not leave the kit at room temperature for extended periods; store it at 2°C to 8°C, avoid freezing, and use it within the expiration date. During storage and incubation, protect the kit from direct exposure to strong light. Ensure that all reagent vials are tightly capped to prevent evaporation and contamination.
1.3 To prevent microbial contamination and cross-contamination of reagents or samples, disposable pipette tips should be used and not reused.
1.4 Before aliquoting the reference standard, rabbit anti‑SARS‑CoV N‑protein antibody, and enzyme conjugate, gently shake each reagent to ensure thorough mixing; prior to sample addition, use a microplate shaker to thoroughly mix the samples.
1.5 Strictly adhere to the specified incubation time and temperature. If the interval between adding samples to the first well and the last well is too long, it will result in varying “pre-incubation” times, thereby compromising the accuracy and reproducibility of the measurements.
1.6 At the end of the washing step, invert the microplate onto a paper towel and gently tap it to ensure that no wash solution remains in any well, thereby preventing foam formation. If using an automated plate washer, be sure to operate it correctly.
1.7 Each assay must include negative and positive controls.
1.8 Experimental procedures shall be conducted in strict compliance with the “Laboratory Biosafety Guidelines for Infectious Atypical Pneumonia.”
2. Experimental Preparation
2.1 Preparation of Reagent Kit Reaction Reagents
2.1.1 Microporous Strip
Secure the required number of microplate strips to the plate holder, and store the remaining strips in a sealed bag.
2.1.2 Washing Solution
Dilute the concentrated detergent in a clean graduated cylinder. Add 50 mL of concentrated detergent to distilled or deionized water up to a final volume of 500 mL, and mix thoroughly.
2.1.3 Other Liquid Components
Use directly; shake gently before use to ensure thorough mixing.
2.2 Sample Preparation
The samples to be tested do not require dilution and should be thoroughly mixed before use. When analyzing clinical specimens, strict adherence to the “Interim Measures for the Administration of Research Laboratories on the SARS Virus,” promulgated by the Ministry of Health, the Ministry of Science and Technology, and the State Food and Drug Administration, is mandatory.
3. Testing Procedure
3.1 For each assay, include one blank well, two positive control wells, and three negative control wells per plate. The blank well should contain no sample, rabbit anti‑N protein antibody, or enzyme conjugate; only wash solution, developing solutions A and B, and stop solution are added. The positive and negative controls are used directly at 100 µL per well. All other wells receive 100 µL of sample.
3.2 Add the sealing plate membrane and incubate at 37°C for 60 minutes.
3.3 Aspirate the liquid from the wells, wash the plate five times, allowing each wash to stand for 30 seconds before discarding and blotting dry.
3.4 Except for the blank control, add 100 µL of rabbit anti-N protein antibody to each well, seal with a plate cover film, and incubate at 37°C for 60 minutes.
3.5 Aspirate the liquid from the wells, wash the plate five times, allowing each wash to stand for 30 seconds before discarding and blotting dry.
3.6 Except for the blank control, add 100 µL of enzyme‑labeled goat anti‑rabbit IgG antibody to each well, seal with a plate cover, and incubate at 37°C for 60 minutes.
3.7 Aspirate the liquid from the wells, wash the plate 5 times, allowing each wash to stand for 30 seconds before discarding and blotting dry.
3.8 After blotting dry, add 50 µL of developing solution A and 50 µL of developing solution B to each well. Incubate at 37°C in the dark for 10 minutes, then immediately add 50 µL of stop solution to each well and gently shake the microplate to ensure thorough mixing.
3.9 Zero the instrument using a blank well, then measure the absorbance (A) at 450 nm with a microplate reader.
4. Quality Control
The average A value of the positive control is ≥0.50, and the average A value of the negative control is ≤0.20, demonstrating that the assay is valid.
[Positive Judgment Value]
Cut-off value = 0.09 + mean A-value of the negative control (if the mean A-value of the negative control is less than 0.10, use 0.10; if it is greater than or equal to 0.10, use the actual value). The results are interpreted as follows:
Sample A value < critical value × 0.8 Negative
Critical value × 0.8 ≤ Sample A value < Critical value Suspect
Sample A value ≥ cutoff value Positive
[Interpretation of Test Results]
1. The cutoff value of this kit was determined based on the test results obtained from 1,297 healthy individuals and clinical validation studies.
2. The test results obtained with this kit do not constitute the sole definitive criterion for clinical diagnosis; they must be interpreted in conjunction with clinical indications and other laboratory findings through comprehensive analysis.
3.可疑 and positive samples should be retested in a second assay, with duplicate wells analyzed.
4. A positive test result indicates only the presence of novel coronavirus proteins in the sample; it does not confirm the presence of viable virus or that the viral load is sufficient to infect others.
5. Clinical evaluation results indicate that the assay outcomes for this product may vary depending on the timing of sample collection; therefore, samples testing negative cannot rule out infection with the novel coronavirus.
6. In addition to repeat testing of suspicious samples, high‑density serial sampling (e.g., once daily) should be conducted for the case, with antigen testing performed promptly to determine infection as early as possible. When feasible, earlier serum specimens should also be tracked and tested for antigen to rule out the possibility that the antigen peak has already been missed. For cases presenting more than 10 days after symptom onset, antibody testing should be performed concurrently.
[Limitations of the Detection Method]
1. The antibodies used in this product are directed against the nucleocapsid protein of the novel coronavirus. Although their sensitivity and specificity have been validated through relevant laboratory and clinical studies, given that fundamental research on the various stages of SARS‑CoV‑2 pathogenesis—particularly regarding the virus’s distribution and persistence across different tissues and body sites—remains insufficient, further investigation and validation of these parameters are still required.
2. Although the test results indicate that this assay system exhibits no cross-reactivity with the viral cultures of the pathogens listed in the specificity panel, clinical evaluation using samples infected with these viruses has not been performed. Furthermore, no experimental data are currently available regarding cross-reactivity with other pathogens (i.e., those not included in the specificity panel).
3. This reagent utilizes mouse monoclonal antibodies and rabbit polyclonal antibodies; therefore, a positive result may also be attributable to the presence of high-titer human anti-mouse or human anti-rabbit antibodies in certain patients, such as those who have received mouse‑derived monoclonal antibody preparations during diagnosis or treatment.
4. Continuous monitoring of clinically diagnosed SARS patients using this product indicates that it exhibits a relatively high positive detection rate only during the early phase of the illness (days 1–10), whereas in the later stages, most test results are negative.
5. For samples that test negative, the presence of the novel coronavirus in the sample cannot be completely ruled out. The following factors may all contribute to a negative result:
5.1 The patient was not infected with the novel coronavirus; rather, the illness was caused by another pathogen that has not yet been identified, such as a virus, bacterium, or fungus.
5.2 False-negative results may occur due to limitations in assay sensitivity.
5.3 The virus or viral proteins in the sample may be present only during a specific time period, and the sample was not collected when the virus or viral proteins were actually present.
6. At present, whether high-titer antibodies in serum affect antigen test results remains to be further investigated.
[Product Performance Specifications]
1. Appearance
The packaging box should be neat and tidy, with the product batch number and expiration date clearly marked and accurate. The liquid components should be clear and free of sediment or flocculent particles.
2. Concordance rate of negative reference materials
The test results for the 16 negative reference samples must all be negative (16/16).
3. Concordance rate of positive reference materials
The test results for the 16 positive reference samples must show no false negatives (16/16).
4. Limit of detection
A total of 7 samples are required, with at least 4 testing positive; moreover, the matrix serum must be negative, i.e., 4 out of 7.
5. Precision
When tested using precision reference materials, the coefficient of variation (CV, %) shall not exceed 15% (n = 10).
6. Stability Testing
Prior to shipment, the product shall be stored at 37°C for 3 days, after which it shall be tested. The positive reference material conformity rate, negative reference material conformity rate, limit of detection, and precision must all meet the specified requirements.
7. Biosafety
The reference standards in the kit must test negative for HBsAg, HIV‑Ab, HCV‑Ab, and Treponema pallidum antibodies.
[Precautions]
1. All reagents in this product are non-infectious; however, they must still be handled as infectious materials.
2. All clinical specimens shall be treated as infectious materials. All experiments must be conducted in accordance with the relevant provisions of the “Interim Measures for the Management of Laboratories Conducting Research on the SARS Virus” and the “Interim Measures for the Preservation, Use, and Animal Model Development Involving the SARS Virus,” jointly issued by the Ministry of Science and Technology, the Ministry of Health, the State Food and Drug Administration, and the State Environmental Protection Administration.
3. The sample collection method poses certain risks to the sampling personnel; therefore, they must place utmost emphasis on safety precautions.
4. All types of waste generated in the laboratory must be treated as infectious materials. Used pipette tips should be disposed of directly into a waste container containing 1% sodium hypochlorite, and together with other waste, they must be incinerated or otherwise destroyed at the designated location and in accordance with the prescribed procedures.
5. The workbench should be cleaned immediately after the experiment is completed. The workbench and all experimental materials should be regularly disinfected with 1% sodium hypochlorite, 75% ethanol, or a UV lamp.
6. Take care to avoid direct contact between the reagents or samples and skin or mucous membranes. The chromogenic solution contains TMB, and the stop solution contains sulfuric acid. Reagents containing TMB may trigger unintended reactions during the assay, while those containing sulfuric acid are corrosive. If these liquids come into contact with skin or mucous membranes, rinse thoroughly with water immediately.
[Explanation of the Logo]
Packaging label
The logo
Meaning of the label: Temperature limit; In vitro diagnostic medical device; Refer to the instructions for use; Do not reuse.
[References]
[1] Drosten C, Gunther S, Preiser W, van der Werf S, Brodt HR, Becker S, et al. Identification of a novel coronavirus in patients with severe acute respiratory syndrome. N Engl J Med 2003;348:1967–76.
[2] World Health Organization. Coronavirus never before seen in humans is the cause of SARS. April 16, 2003. http://www.who.int/entity/csr/sarsarchive/2003_04_16/en
[3] World Health Organization. Summary table of SARS cases by country, 1 November 2002-7 August 2003. http://www.who.int/csr/sars/country/en/country2003_08_15.pdf
[4] Peiris JSM, Lai ST, Poon LLM, Guan Y, Yam LY, Lim W, et al. Coronavirus as a possible cause of severe acute respiratory syndrome. Lancet 2003;361:1319–25.
[5] Tsang KW, Ho PL, Ooi GC, Yee WK, Wang T, Chan-Yeumg M, et al. A cluster of cases of severe acute respiratory syndrome in Hong Kong. N Engl J Med 2003;348:1977–85.
[6] Booth CM, Matukas LM, TomLinson GA, Rachlis AR, Rose DB, Dwosh HA, et al. Clinical features and short-term outcomes of 144 patients with SARS in the greater Toronto area. JAMA 2003;289:2801–9.
[7] Peiris JS, Chu CM, Cheng VC, et al. Clinical progression and viral load in a community outbreak of coronavirus-associated SARS pneumonia: a prospective study. Lancet 2003; 361:1767-72.
[8] Chan KH, Poon LLLM, Cheng VCC, et al. Detection of SARS Coronavirus in Patients with Suspected SARS. Emerg Infect Dis [serial online] 2004 Feb[date cited]. Available from: URL: http://www.cdc.gov/ncidod/EID/vol10no2/03-0610.htm
[9] Daginakatte GC, Chard-Bergstrom C, Andrews GA, et al. Production, characterization, and uses of monoclonal antibodies against recombinant nucleoprotein of ELK coronavirus[J]. Clin Diagn Lab Immunol. 1999, 6(3): 341-4.
[10] Yousif AY, Fahad AM, Cindy CB, Anderson J, et al. Development, characterization, and diagnostic applications of monoclonal antibodies against bovine rotavirus. Clin. Diagn. Lab. Immunol. 2000,7:288-292.
[11] Souza CM, Rocha FRT, Martins NRS, et al. Production of monoclonal antibodies against conserved components of infectious bronchitis virus. Arq. Bras. Med. Vet. 2001, 53:523-530.
[Basic Information]
Registrant/Manufacturer Name: Zhuhai Special Economic Zone Haitai Biopharmaceutical Co., Ltd.
Address: No. 18, Innovation Fourth Road, Gangwan Avenue, Tangjiawan Town, Zhuhai City Postal Code: 519085
Contact Information: Telephone: 0756-3890858 Fax: 0756-3890848
Name of After-Sales Service Provider: Zhuhai Special Economic Zone Haitai Biopharmaceutical Co., Ltd.
Contact Information: Telephone: 0756-3890858 Fax: 0756-3890848
Production Address: No. 18, Innovation Road 4, Gangwan Avenue, Tangjiawan Town, Zhuhai City Postal Code: 519085
Production License No.: Yue Food and Drug Administration Medical Device Production License No. 20040941
[Medical Device Registration Certificate Number/Product Technical Requirements Number] National Medical Device Registration No. 20173401143
[Date of Approval and Amendment of the Instruction Manual] June 28, 2017