Rubella Virus IgG Antibody Test Kit (Fluorescent Immunochromatographic Assay)_NMPA Approval in February 2018
Product Name: Rubella Virus IgG Antibody Test Kit (Fluorescent Immunochromatographic Assay)
Category: Domestic Class III
Registration Pathway: Priority
NMPA Approval Time: February 2018
Product Structure and Composition: The kit consists of a rubella virus IgG antibody test card, ID chip, centrifuge tube, and buffer solution. The nitrocellulose membrane of the rubella virus IgG antibody test card is coated with anti-human IgG antibody (mouse-derived, monoclonal antibody) and biotin. The buffer solution contains fluorescently labeled measles virus antigen (recombinant antigen) and fluorescently labeled avidin.
Scope of Application: This kit is used for qualitative detection of rubella virus IgG antibodies in human serum or plasma. It is clinically used as an auxiliary diagnostic tool for rubella virus infection.
Packing Specification: Card type (individually packaged in aluminum foil bags): 1 person/box, 10 people/box, 20 people/box, 25 people/box, 40 people/box, 50 people/box
Test Principle: The kit utilizes an indirect method and fluorescent immunochromatography principle to detect rubella virus IgG antibodies in human serum or plasma. When the concentration of rubella virus IgG antibodies in the sample is equal to or higher than the minimum detection limit, the antibodies bind with the fluorescent latex-labeled rubella virus antigen (RV-Ag) in the buffer solution to form a reaction complex. The mixed sample is then added to the sample well of the test card, and due to chromatographic action, the reaction complex moves forward along the nitrocellulose membrane, captured by the anti-human IgG antibodies in the detection zone (T), forming an "anti-human IgG-RV-IgG-RV Ag-Flu" immune complex. The higher the amount captured, the stronger the positive result indicated by the signal value scanned by the fluorescence immunoassay analyzer. A lack of complex accumulation in the detection zone or minimal accumulation indicates a negative result. The control zone (C) is coated with biotin, reacting with the fluorescent latex-labeled avidin to form a reaction line with a certain fluorescence signal value as a control.
Main Raw Materials: 1. The main raw materials of this product include rubella virus antigen and anti-human IgG antibody. The rubella virus antigen is a recombinant antigen, and the anti-human IgG antibody is a mouse-derived monoclonal antibody. 2. The main raw materials are externally sourced, and after evaluation of concentration, purity, potency, and functional tests, the best materials and suppliers are selected. The applicant has established quality requirements for each main raw material, which have been inspected and approved. 3. Reference Material Setting: The applicant has designed a complete set of reference materials, including positive reference materials, negative reference materials, minimum detection limit reference materials, and precision reference materials. These reference materials are prepared from clinical samples, with positive reference materials sourced from 10 different RV IgG positive samples. The negative control sample is derived from one RV IgG negative sample and one each of CMV IgG positive, HSV1 IgG positive, HSV2 IgG positive, TOXO IgG positive, and another RV IgG negative sample. The minimum detection limit reference materials are prepared by serial dilution of RV IgG positive samples, labeled as L1 (1:4), L2 (1:8), and L3 (1:16), with the 1:4 dilution sample (R) also used as a precision reference material. Confirmation of the positivity and negativity of relevant antibodies in the clinical samples is done using two commercially available similar products. The comprehensive use of these reference materials is for evaluating product sensitivity, specificity, and repeatability.
Research on Production Processes and Reaction Systems:
By conducting functional experiments such as reference product testing, the optimal production processes and reaction systems are determined. This includes parameters such as coating concentration for the test line (T-line) and control line (C-line, film spraying parameters for the T-line, film spraying parameters for the C-line, drying time for the coating film, preparation parameters for fluorescent markers (amount of fluorescent latex-labeled rubella virus antigen, amount of fluorescent latex-labeled affinity substance, ratio of fluorescently labeled rubella virus antigen to PBS buffer, ratio of fluorescently labeled affinity substance to PBS buffer). Additionally, the applicant determines the optimal sample size and mixing sample addition amount through clinical sample testing, along with observation time for sample addition and luminous stability.
Analytical Performance Research:
The analytical performance of this product includes reference material conformity, serum and plasma sample applicability, minimum detection limit, specificity (cross-reactivity, interference test), accuracy, precision, and the hook effect. The applicant submitted performance evaluation data for three batches of products (W656D501A, W656D502A, W656D503A) on the applicable instrument models. Using 10 positive reference materials, 10 negative reference materials, 3 minimum detection limit reference materials, and 1 precision reference material for testing this product, all results met the requirements.
For the applicable sample types, the applicant conducted comparative research experiments using homologous serum and plasma samples, including 10 positive and 5 negative cases. The results showed complete consistency between serum and plasma test results. Additionally, by examining the effects of different anticoagulants on sample testing through the addition of anticoagulants, the results showed that five types of anticoagulants, including heparin, did not interfere with the testing. In the evaluation of the lowest detection limit, the applicant used three positive samples for gradient dilution, conducted 20 repeated tests on samples of various concentrations, and determined the maximum dilution factor with ≥95% positive detection rate as the lowest detection limit. The final determined lowest detection limit level for the product was a 1:8 dilution concentration of the three samples mentioned above. Comparatively, the lowest detection limit level was similar to that of ELISA products.
In the cross-reactivity assessment, the applicant evaluated cross-reactivity using positive samples of antibodies against various pathogens such as Toxoplasma (TOXO), Measles Virus (MV), Cytomegalovirus (CMV), Herpes Simplex Virus (HSV), Mycoplasma pneumoniae (MP), Chlamydia pneumoniae (CP), EB Virus, Respiratory Syncytial Virus (RSV), Coxsackie Virus, Hepatitis A Virus, Hepatitis B Virus, Parainfluenza Virus, Varicella-Zoster Virus IgG antibodies, and Measles Virus-specific IgM antibodies. The results showed that the antibodies against the mentioned pathogens did not produce cross-reactivity.
In the interference test, the applicant selected one negative and one weakly positive sample for rubella virus IgG, and evaluated various potential endogenous and exogenous interfering substances using a recovery test method. The results indicated that endogenous interfering substances such as bilirubin, hemoglobin, triglycerides, rheumatoid factor, total IgG, total IgM, antinuclear antibodies (ANA), and anti-mitochondrial antibodies (AMA) at concentrations of 350 μmol/L, 9g/L, 10 mmol/L, 80 IU/mL, 36g/L, 6.4g/L, 80U/mL, and (titer) 1:240 did not interfere with this test. Exogenous drugs such as acetaminophen, amoxicillin, cefaclor, vitamin C, aspirin, and ibuprofen at concentrations of 5.0mg/mL, 5.0mg/mL, 10.0mg/mL, 0.250mg/mL, 5.0mg/mL, and 6.0mg/mL respectively did not interfere with this test.
In the precision study, the applicant evaluated intra-batch, inter-batch, intra-day, inter-day, and inter-operator precision using positive reference samples for rubella virus IgG antibodies (P1), the lowest detection limit reference samples (L2), and negative reference samples (N1). The results showed a 100% conformity rate for detection results, indicating good precision within batches, between batches, within days, between days, and between different operators, demonstrating stable test performance.
In the accuracy study, the applicant compared this product with a similar ELISA method already on the market. They tested 219 clinical positive samples and 46 clinical negative samples, showing a positive agreement rate of 99.09%, negative agreement rate of 100%, and an overall agreement rate of 99.25%. The Kappa consistency analysis yielded k=0.974, indicating good consistency between the two methods.
For the hook effect assessment, high-concentration rubella virus IgG positive samples were diluted and tested, showing no hook effect for samples with a rubella virus IgG antibody titer of 1:64. This product includes six different packaging sizes.
The assessment of diagnostic sensitivity was conducted using the Rubella-Version Rubella IgM and IgG Seroconversion Panel from the German company BIOMEX. This panel consists of 20 plasma samples from the same donor collected at different time points, demonstrating the complete process of Rubella virus IgM and IgG seroconversion. The test results of this product are consistent with those from two other manufacturers, Abbott Labs' EIA Rubazyme-M (IgG) Index and Gull Labs ELISA Rubella IgG.
The applicant evaluated these sizes (1 person/box, 10 people/box, 20 people/box, 25 people/box, 40 people/box, 50 people/box) using a full set of enterprise positive reference samples, 10 clinical positive samples, and 10 clinical negative samples. The results indicated that the different packaging sizes only differed in the quantity of reagent packaging, with consistent test results across all sizes.
Research on Positive Judgment Value:
This product utilizes the immunochromatographic method, where the positive judgment value represents the lowest detection limit concentration of the product. Based on the performance evaluation above, it is evident that the lowest detection limit of this product is comparable to similar products already on the market, meeting the requirements. Furthermore, the applicant conducted further validation of the applicability of the positive judgment value using clinical samples (including 219 positive samples and 46 negative samples), and the results demonstrate a good concordance between this product and the comparative reagent test results.
Stability Research:
A systematic study was conducted on the real-time stability after storage under actual conditions until the end of the product shelf life, 50°C accelerated stability, -20°C storage stability, and stability after opening the packaging to determine the effective storage time of the reagents under various conditions. The reagent batches used include W656D501A, W656D502A, W656D503A.
Real-time stability research: Three batches of reagents were stored at 4-30°C (with buffer stored at 2-8°C) conditions, and appearance checks, physical checks, positive reference material compliance rate, negative reference material compliance rate, lowest detection limit, and precision detection were conducted at 1, 6, 9, 12, 15, 17, 18, 19, 20, 21 months to determine that the reagents can be stably stored for 18 months at 4-30°C (with buffer stored at 2-8°C). Reagent 50°C accelerated stability study: Three batches of reagents were stored at 50°C (with buffer stored at 2-8°C) conditions, and appearance checks, physical checks, positive reference material compliance rate, negative reference material compliance rate, lowest detection limit, and precision detection were conducted at 1, 7, 14, 21, 24, 26, 28, 29, 30 days to determine that the reagents can be stably stored for 30 days at 50°C (with buffer stored at 2-8°C).
Buffer 37°C accelerated stability research: Three batches of buffer were stored at 37°C (with reagents stored at 4-30°C) conditions, and appearance checks, physical checks, positive reference material compliance rate, negative reference material compliance rate, lowest detection limit, and precision detection were conducted at 1, 3, 4, 5, 6, 7, 8, 9, 10 days to determine that the buffer can be stably stored for 10 days at 37°C (with reagents stored at 4-30°C).
Reagent -20°C storage stability research: Three batches of reagents were stored at -20°C (with buffer stored at 2-8°C) conditions, and appearance checks, physical checks, positive reference material compliance rate, negative reference material compliance rate, lowest detection limit, and precision detection were conducted at 1, 7, 14, 21, 24, 26, 28, 29, 30 days to determine that the reagents can be stably stored for 30 days at -20°C (with buffer stored at 2-8°C).
Buffer -20°C storage stability research: Three batches of buffer were stored at -20°C (with reagents stored at 4-30°C) conditions, and appearance checks, physical checks, positive reference material compliance rate, negative reference material compliance rate, lowest detection limit, and precision detection were conducted at 1, 7, 14, 21, 24, 26, 28, 29, 30 days to determine that the buffer can be stably stored for 30 days at -20°C (with reagents stored at 4-30°C).
Reagent stability after opening the packaging: Three batches of reagents were stored in opened minimum packaging bags (aluminum foil bags) and stored at high temperature (40°C, 50% humidity), high humidity (25°C, 90% humidity), and room temperature with normal humidity (25°C, 50% humidity) conditions, and subjected to appearance checks, physical checks, positive reference material compliance rate, negative reference material compliance rate, lowest detection limit, and precision detection after 1-2 hours to determine that the quality can be stably maintained for 1.5 hours after opening the aluminum foil packaging.
Clinical Evaluation:
Clinical trials were conducted at three institutions including Guangzhou Women and Children's Medical Center, The First Affiliated Hospital of Jinan University, and Henan Provincial Hospital for Infectious Diseases. The study compared the investigational in vitro diagnostic reagents with similar products already on the market to validate the clinical performance of this product.
The study included samples from suspected or confirmed rubella virus-infected patients, samples from patients infected with other pathogens that could be easily confused (including Toxoplasma gondii, cytomegalovirus, herpes simplex virus, adenovirus, Epstein-Barr virus, Mycoplasma pneumoniae, hepatitis B virus, hepatitis C virus, parainfluenza virus, measles virus etc.), samples containing interfering substances (such as samples from patients with bilirubin, hemoglobin, triglycerides, rheumatoid factor, and systemic lupus erythematosus. etc.), and a small number of samples from healthy subjects, totaling 1050 cases, including 775 positive cases and 275 negative cases.
The comparator reagent selected was the Rubella Virus IgG Antibody Test Kit (Colloidal Gold Method) from Beijing Zhongjian Antai Diagnostic Technology Co., Ltd.
The trial results showed that this product had a positive coincidence rate of 98.84%, a negative coincidence rate of 97.10%, and an overall coincidence rate of 98.38% compared to the comparator reagent in the detection of clinical samples. Statistical analysis using the Kappa test method yielded a Kappa value of 0.958, indicating statistically significant consistency between the results of the two tests.
This product underwent third-party verification using similar products from the Institut Virion\Serion GmbH to verify samples that showed inconsistencies with the comparison reagents. Based on the third-party verification results, the applicant conducted a detailed analysis of the reasons for the inconsistencies.
Based on the results of the third-party verification, the applicant conducted a detailed analysis of the reasons for the inconsistent results. Additionally, the applicant conducted a comparative test on 200 subjects' matched serum and plasma samples (150 positive cases, 50 negative cases), showing a 100% concordance rate in detecting both sample types. In conclusion, the clinical trial results demonstrate the equivalence of this product to similar products already on the market.