Rubella Virus IgM Antibody Detection Kit (Enzyme-Linked Immunosorbent Assay Capture Method)

Instructions for the Rubella Virus IgM Antibody Detection Kit (Enzyme-Linked Immunosorbent Assay Capture Method)
[Product Name]
Chinese Name: Rubella Virus IgM Antibody Detection Kit (Enzyme-Linked Immunosorbent Capture Assay)
[Packaging Specifications]
48 servings per box, 96 servings per box.
[Intended Use]
Qualitative detection of rubella virus IgM antibodies in human serum or plasma samples.
It is intended for auxiliary diagnosis of rubella virus infection. The test shall not be used for prenatal screening in asymptomatic individuals, nor shall the results of this assay alone serve as the basis for terminating a pregnancy.
Rubella[1–3] belongs to the genus Rubivirus within the family Togaviridae and is the pathogen responsible for rubella; it is an RNA virus. The rubella virus (RV) spreads via the respiratory tract, with an incubation period of approximately 2–3 weeks. Following viral replication, it enters the bloodstream, leading to viremia. Clinically, rubella resembles measles but is milder, with a shorter course; its symptoms can be difficult to distinguish from those of measles, necessitating serological testing for definitive diagnosis. In the mid-1960s, the development of a rubella vaccine made congenital rubella infection a preventable condition. Rubella virus is highly prevalent in the population; in developed countries and regions, widespread vaccination of preschool children, adolescents, and women of childbearing age has resulted in very low incidence rates[4,5]. However, in many parts of China, outbreaks and endemic transmission persist[4,6–8]. During pregnancy—particularly in the first trimester—rubella virus infection, whether overt or asymptomatic, can lead to congenital rubella syndrome (CRS), characterized by multiorgan damage and congenital defects. The principal clinical manifestations include neonatal congenital heart disease, cataracts, congenital deafness, intellectual disability, developmental delay, and microcephaly. The extent of these impairments correlates with the timing of maternal infection: the earlier the infection occurs during gestation, the higher the rate of malformations and the more severe the resulting damage.
Because IgM antibodies serve as an early indicator of infection and can have a profound impact on the fetus, their detection has attracted considerable attention; the measurement of specific IgM antibodies in the placenta provides a reliable basis for diagnosing intrauterine fetal infection.
[Principle of Assay]
Principle of the enzyme‑linked immunosorbent capture assay: A microtiter plate is coated with a monoclonal antibody against the human IgM μ chain. The serum or plasma to be tested is added to the wells, where any IgM antibodies present in the sample bind to the anti‑human IgM μ chain antibodies immobilized on the plate, forming an antibody–secondary antibody complex. After washing, a rubella virus antigen conjugated to an enzyme is added, resulting in the formation of an (antibody–secondary antibody–enzyme‑conjugated antigen) immune complex. Following another wash, a chromogenic substrate is introduced; under the catalytic action of the enzyme, a colorimetric reaction develops. The intensity of this color reaction is measured with an ELISA reader, allowing for the qualitative detection of rubella virus‑specific IgM antibodies in the sample.
[Main Ingredients]
1. The components included in this kit are:
Serial No. Component Name Main Ingredient Usage Instructions Quantity per Box
96 servings/box, 48 servings/box
1 Microtiter plate pre-coated with anti-human IgM-μ chain antibody. The microtiter plate is pre-coated with mouse monoclonal antibody against the human IgM-μ chain. Ready to use; can be separated into individual wells. 96-well and 48-well formats available.
2 Rubella virus antigen conjugate: Rubella virus antigen labeled with horseradish peroxidase (HRP); borate buffer, containing preservatives and protein stabilizers. For direct use. 6.5 mL × 1 vial; 3.5 mL × 1 vial.
3 Rubella Virus IgM Antibody Positive Control: Human serum or plasma positive for rubella virus IgM antibodies; borate buffer containing 0.02% thimerosal and a protein stabilizer. For direct use. 1.0 mL × 1 vial, 1.0 mL × 1 vial.
4 Rubella Virus IgM Antibody Negative Control: Human serum or plasma negative for rubella virus IgM antibodies; borate buffer containing 0.02% thimerosal and a protein stabilizer. For direct use. 1.0 mL × 1 vial, 1.0 mL × 1 vial.
5 Rubella Virus IgM Antibody Critical Control: Human serum or plasma positive for rubella virus IgM antibodies; borate buffer containing 0.02% thimerosal and a protein stabilizer. For direct use. 1.8 mL × 1 vial, 1.8 mL × 1 vial.
6 Color-developing solution A: citric acid, hydrogen peroxide. Use directly: 6.5 mL × 1 bottle, 3.5 mL × 1 bottle.
7 Color-developing solution B: citric acid, tetramethylbenzidine (TMB). Ready to use. 6.5 mL × 1 vial, 3.5 mL × 1 vial.
8 Concentrated Detergent
(10×) Phosphate-buffered saline, containing 0.5% Tween‑20, 10× concentrated. Dilute 10-fold with distilled or deionized water before use. 50 mL × 1 bottle 50 mL × 1 bottle
9 Sample Diluent (IgM): Borate buffer containing glycerol. For direct use. 50 mL × 2 vials, 50 mL × 1 vial.
10 Stop solution: 0.5 mol/L sulfuric acid solution. Use directly: 6.5 mL × 1 bottle, 3.5 mL × 1 bottle.
11 Sealed bags 1 piece 1 piece
12 sealing films, 4 pieces, 4 pieces
13 Recording Paper 1 copy 1 copy
Note: Enzyme conjugates, quality control samples, positive controls, and negative controls must be used exclusively from the kit supplied; products from other batch numbers are not permitted. The sample diluent (IgM) is compatible with our company’s ELISA kits for IgM antibody detection. Chromogen A, chromogen B, stop solution, and concentrated wash solution are universal reagents and may be used with all of our ELISA kits.
2. Materials required but not yet provided:
Distilled water or deionized water
Microplate washer
10 μL to 100 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 12 months.
After opening, any unused microtiter plates should be stored in a sealed bag at 2°C to 8°C. Liquid components should be tightly capped and stored at 2°C to 8°C. All components must be used within one month of opening.
See the label for the production date and expiration date.
[Applicable Instruments]
A microplate reader with wavelengths of 450 nm and 630 nm.
[Sample Requirements]
Serum or plasma samples should be collected in accordance with the clinical blood‑collection technical guidelines. Samples stored at room temperature should not exceed 8 hours; if testing is performed after 8 hours, samples must be kept at 2°C–8°C. For storage exceeding one week, maintain samples at –20°C or lower and avoid repeated freeze–thaw cycles. This assay is suitable for samples containing anticoagulants such as ethylenediaminetetraacetic acid (EDTA), sodium citrate, sodium heparin, sodium oxalate, or double oxalate; however, there are currently no experimental data regarding the suitability of samples containing other types of anticoagulants. Although bilirubin, hemoglobin, and triglycerides do not significantly interfere with the assay results, samples exhibiting jaundice, hemolysis, or hyperlipidemia should still be avoided. Samples that are clearly contaminated must not be used for testing.
[Testing Method]
1. Precautions During the Experiment
1.1 When removing the kit components from refrigeration, allow them to equilibrate at room temperature until no condensation is visible before use. After opening, any unused microtiter plates should be stored in a sealed bag.
1.2 During storage and incubation, direct exposure to strong light should be avoided. All reagent vials must be 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 standard and enzyme conjugate, gently shake to ensure thorough mixing; prior to sample addition, use a microplate shaker to thoroughly mix the diluted samples.
1.5 Strictly adhere to the specified incubation time and temperature. If the time 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 all wells are free of wash buffer and to prevent foam formation. If using an automated plate washer, be sure to operate it correctly.
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
Sample 1:100 dilution: Pipette 10 μL of the sample to be tested into a small test tube containing 1 mL of sample diluent (IgM), and mix thoroughly.
3. Testing Procedure
3.1 For each assay, include one blank well (unless using dual-wavelength detection, in which case no blank well is required), one positive control well, one negative control well, and three critical‑control wells. The blank well should contain only wash buffer, developing reagent A, developing reagent B, and stop solution; the positive, negative, and critical controls should be used directly at 100 μL per well. Add 100 μL of the diluted sample to all other wells. Incubate at 37°C for 60 minutes.
3.2 Discard the liquid from the wells, then add washing solution to each well until it is completely filled (approximately 300 μL per well). Remove the excess by tapping the plate gently and blotting dry, repeating this process three times. Except for the blank well, add 50 μL of enzyme‑labeled reagent to each well and incubate at 37°C for 60 minutes.
3.3 Discard the liquid from the wells, wash the plate three times as described above, and after blotting dry, add 50 μL of developing reagent A and 50 μL of developing reagent B to each well. Incubate at 37°C in the dark for 10–15 minutes, then add 50 μL of stop solution to each well and gently shake the microplate to ensure thorough mixing.
3.4 Single-wavelength detection: Zero the instrument using the blank well, then measure the absorbance (A) at 450 nm with a microplate reader.
Dual-wavelength detection: Measure absorbance at 450 nm and 630 nm using a microplate reader, and calculate the net absorbance as A450nm – A630nm.
4. Quality Control
The negative control value should fall between 0.0 and 0.15, the positive control value between 0.3 and 1.8, and the mean of the critical control value between 0.15 and 0.8; only under these conditions is the assay considered valid. Otherwise, the results are invalid and the assay must be repeated.
[Positive Judgment Value]
Sample A value < the average of the critical control A values is considered negative.
Sample A value ≥ the average of the critical control A values is considered positive.
[Interpretation of Test Results]
1. A positive result indicates an acute or recent rubella virus infection. For both positive and indeterminate results, it is recommended to collect a second sample for testing 1–2 weeks later to dynamically monitor changes in antibody levels.
2. The test results are for clinical reference only; clinicians should integrate the patient’s medical history, symptoms, and other laboratory findings to arrive at a diagnosis.
[Limitations of the Analytical Method]
1. Sample or reagent contamination by microorganisms, as well as cross-contamination between reagents, may lead to erroneous results.
2. The experimental results indicate that this product exhibits no cross-reactivity with IgM‑positive sera from the pathogens listed in the cross‑reactivity panel, nor with rheumatoid factor; however, no experimental data are currently available regarding cross‑reactivity with other potential interfering factors.
[Product Performance Specifications]
1. Sensitivity, specificity
In a clinical study involving 1,079 cases, this product demonstrated a sensitivity of 98.3% and a specificity of 98.9% when compared with a CE‑certified comparable imported reagent.
2. Precision
The intra-assay coefficient of variation (CV, %) shall not exceed 15% (n = 10).
3. Cross-reactivity
No cross-reactivity was observed between this product and IgM‑positive sera against rheumatoid factor or the following pathogens: Toxoplasma gondii, measles virus, cytomegalovirus, herpes simplex virus type 1, and herpes simplex virus type 2.
4. Interference Test
4.1 Anticoagulants that come into contact with the sample (EDTA, sodium citrate, sodium heparin, sodium oxalate, and double oxalate) do not significantly interfere with the assay results of this product.
4.2 Fibrinogen does not significantly interfere with the assay results of this product; therefore, human serum or plasma samples may be used for testing.
[Precautions]
1. This product is intended for in vitro diagnostic use only and is for single-use.
2. The serum or plasma used to prepare the negative control, positive control, and cut-off control for this kit have been tested negative for hepatitis B surface antigen (HBsAg), hepatitis C virus (HCV) antibodies, Treponema pallidum antibodies, and human immunodeficiency virus (HIV) antibodies. All other components of the kit are non‑infectious. However, since it cannot be guaranteed that all infectious agents have been detected in each component, this kit should be treated as potentially infectious material, and all personnel handling it must follow laboratory safety protocols.
3. All clinical specimens shall be treated as infectious materials.
4. Samples awaiting testing and other potentially infectious materials shall be disinfected after testing.
5. Take care to avoid direct contact between the reagents or samples and skin or mucous membranes. The color-developing solution contains TMB, and the stop solution contains sulfuric acid. Reagents containing TMB may trigger unintended reactions during the assay, while sulfuric-acid–containing reagents 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] Dai Bin, Chang Huilan, Han Xiujuan, et al. A study on the rubella immune status of the general population in China. Chinese Journal of Microbiology and Immunology, 1981, 1(6): 411.
[2] Waxham MN. Immunochemical identification of rubella virus hemagglutinin. Virology, 1983, 126: 194.
[3] Ho M. Laboratory diagnosis of congenital and perinatal infection in Ed . New York: Plenum Medical Book Company, 1982,172.
[4] Geng Guanyi. Epidemiology (Volume 2). 2nd ed. Beijing: People’s Medical Publishing House, 1996. pp. 541–551.
[5] Pau lD, Park man K. Making vaccination policy: the experience with rubella. Clin
Infec Dis, 1999, 28: 140-146.
[6] Xu Fugeng, Huang Chengxiao, Yao Huaifang, et al. Rubella antibody screening and immunization study among individuals aged 1–29 years in Hangzhou. Chinese Journal of Epidemiology, 1995, 16: 94–96.
[7] Dai Bin, Fang Hanhua, Han Xiujuan, et al. A study on the incidence of primary rubella infection among women in early pregnancy. Chinese Journal of Preventive Medicine, 1992, 269: 197–199.
[8] Jin Qi, editor-in-chief. Medical Molecular Virology. Beijing: People’s Medical Publishing House, 2001. pp. 489–493.
[Basic Information]
Registrant/Manufacturer Name: Zhuhai Special Economic Zone Haitai Biopharmaceutical Co., Ltd.
Address: No. 18, Innovation Road No. 4, Gangwan Avenue, Tangjiawan Town, Zhuhai City
Contact Information: Phone Number: 0756-3890858
Fax number: 0756-3890848
Name of After-Sales Service Provider: Zhuhai Haitai Biopharmaceutical Co., Ltd.
Contact Information: Phone Number: 0756-3890858
Production Address: No. 18, Innovation Road 4, Gangwan Avenue, Tangjiawan Town, Zhuhai City
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. 20163402550
[Date of Approval and Amendment of the Instruction Manual] December 29, 2016