Rubella Virus IgM Antibody Detection Kit (ELISA)

Instructions for the Rubella Virus IgM Antibody Detection Kit (Enzyme-Linked Immunosorbent Assay)
[Product Name]
Generic Name: Rubella Virus IgM Antibody Detection Kit (Enzyme-Linked Immunosorbent Assay)
[Packaging Specifications]
48 servings/box, 96 servings/box.
[Intended Use]
The product is used for the qualitative detection of rubella virus IgM antibodies in human serum or plasma.
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 is transmitted 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, may lead to congenital rubella syndrome (CRS) in the fetus, characterized by multiorgan damage and congenital defects. The principal clinical manifestations include 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.
Following rubella virus infection, specific IgM antibodies appear early, rise rapidly, and persist for a short duration; they reach peak levels two weeks after the onset of rash and remain detectable for 1–2 months. Therefore, the detection of rubella-specific IgM antibodies is useful for diagnosing primary rubella infection.
[Principle of Assay]
Principle of the indirect ELISA: A microtiter plate is coated with recombinant rubella virus antigen. The serum or plasma to be tested is added to the wells, where rubella virus–specific IgM antibodies in the sample bind selectively to the antigen immobilized on the plate surface, forming an antigen–antibody immune complex. After washing to remove unbound material, an enzyme‑conjugated anti‑human IgM antibody is added, resulting in the formation of an (antigen–antibody–secondary antibody–enzyme conjugate) immune complex. Following another wash to eliminate excess bound reagents, a chromogenic substrate is introduced; under enzymatic catalysis, a colorimetric reaction develops. The intensity of this color change is measured with an ELISA reader, allowing for the qualitative detection of rubella virus–specific IgM antibodies in the sample. The sample diluent contains goat anti‑human IgG antibodies, which can bind to human IgG antibodies and rheumatoid factors present in the sample, thereby minimizing interference from specific IgG antibodies and rheumatoid factors.
[Main Ingredients]
1. The components included in this kit are:
Serial No. Component Name Main Ingredient Usage Method Fill Volume
96 servings/box 48 servings/box
1. Microtiter plate pre-coated with rubella virus antigen: The microtiter plate is pre-coated with recombinant rubella virus antigen. It can be separated into individual wells for direct use, available in 96-well and 48-well formats.
2 Anti-human IgM antibody enzyme conjugate: horseradish peroxidase (HRP)-labeled mouse anti-human IgM monoclonal antibody; borate buffer containing preservatives, stabilizers, and other additives. Ready-to-use. 6.5 ml × 1 vial, 3.5 ml × 1 vial.
3 Rubella Virus IgM Antibody Positive Control: Rubella virus IgM‑positive human plasma or serum; borate buffer containing 0.02% thimerosal, stabilizers, and other excipients. For direct use. 1.0 mL × 1 vial; 1.0 mL × 1 vial.
4 Rubella Virus IgM Antibody Negative Control: Rubella virus IgM‑negative human plasma or serum; borate buffer containing 0.02% thimerosal, stabilizers, etc. Ready to use. 1.0 mL × 1 vial; 1.0 mL × 1 vial.
5 Rubella Virus IgM Antibody Critical Control: Rubella virus IgM-positive human plasma or serum; borate buffer containing 0.02% thimerosal, stabilizers, etc. Ready-to-use. 1.8 mL × 1 vial. 1.8 mL × 1 vial.
6 Color-developing solution A: citric acid, hydrogen peroxide, etc. Use directly 6.5 ml × 1 bottle 3.5 ml × 1 bottle
7 Color-developing solution B: citric acid, tetramethylbenzidine (TMB), etc. Ready-to-use; 6.5 ml × 1 vial, 3.5 ml × 1 vial.
8 Concentrated Detergent Solution Phosphate-buffered solution, containing 0.5% Tween‑20, 10‑fold 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, goat anti-human IgG, etc. Ready-to-use. 50 mL × 2 bottles; 50 mL × 1 bottle.
10 Stop solution: 0.5 mol/L H2SO4 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: The enzyme conjugate, quality control (QC) sample, positive control, negative control, and sample diluent must be those provided in the kit; products from other batch numbers are not permitted. Chromogen A, chromogen B, stop solution, and concentrated wash solution are universal reagents and may be used with all ELISA kits manufactured by our company.
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
Microporous Oscillator
Microplate reader
A timer with a duration of 60 minutes or longer
[Storage Conditions and Shelf Life]
Store at 2°C to 8°C, protected from light; shelf life is 12 months.
After opening, any unused microtiter plates should be stored in a sealed bag at 2°C to 8°C, and 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. Although bilirubin, hemoglobin, and triglycerides do not significantly interfere with the assay results of this kit, samples exhibiting jaundice, hemolysis, or hyperlipidemia should still be avoided. Clearly contaminated samples are unsuitable 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 (20°C–25°C) 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‑labeled reagents, gently shake them to ensure thorough mixing. Prior to sample addition, use a microplate shaker to thoroughly mix the diluted samples.
1.5 Strictly adhere to the prescribed 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 significantly affecting 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 per plate (no blank well is required when using dual-wavelength detection), one positive control well, one negative control well, and three critical‑control wells. The blank well should contain no sample or enzyme conjugate; add only wash solution, developing reagent A, developing reagent B, and stop solution. Use the positive, negative, and critical controls directly, 100 µL per well. Add 100 µL of diluted sample to all other wells. Incubate at 37°C ± 1°C for 30 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 step three times. Except for the blank well, add 50 μl of enzyme‑conjugate to each well and incubate at 37°C ± 1°C for 30 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 ± 1°C in the dark for 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: Read the absorbance values at 450 nm and 630 nm using a microplate reader, and calculate the corrected absorbance as A450nm – A630nm.
4. Quality Control
The negative control A-value must be ≤0.15; the average A-value of the critical control must be between 0.20 and 0.80; the ratio of the critical control’s average A-value to the negative control’s average A-value must be ≥2.0; and the positive control A-value must exceed the critical control’s average A-value. Only when these criteria are met is the assay considered valid; otherwise, the results are invalid and the assay must be repeated.
[Positive Judgment Value]
Calculate the S/CO value as follows: S/CO = Sample A value / Mean of the critical control A values.
Reactivity (R): S/CO ≥ 1.1
Suspicious (S): 0.9 < S/CO < 1.1
Negative (N): S/CO ≤ 0.9
[Interpretation of Test Results]
1. A reactive result indicates a recent rubella virus infection. For reactive or indeterminate results (0.9 < S/CO < 1.1), it is recommended that the patient undergo repeat sampling within one week, along with rubella IgG antibody testing, to monitor changes in antibody levels over time; alternatively, other methods may be used to confirm the result.
2. In the early stages of infection, the absence of IgM or a very low IgM titer may yield a negative result. Suspected cases should undergo repeat testing within 7 to 14 days, with concurrent parallel analysis of the previously collected specimen to confirm whether seroconversion has occurred or if the antibody titer has significantly increased.
3. In patients with compromised immune function or those undergoing immunosuppressive therapy, the clinical utility of serum antibody testing is limited.
4. IgM antibody positivity occurs not only in primary infections but may also be observed in secondary infections.
5. False-positive and false-negative results may have adverse clinical consequences; false negatives may increase the risk of a fetus being born with congenital rubella syndrome, while false positives may lead to unnecessary antiviral treatment in pregnant women or newborns.
6. 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]
Microbial contamination of samples or reagents, as well as cross-contamination among reagents, may lead to erroneous results.
[Product Performance Specifications]
1. Sensitivity, specificity
This product was evaluated using 1,020 samples from diverse populations, demonstrating a sensitivity of 96.7% and a specificity of 97.1%.
2. Precision
Intra-assay imprecision, expressed as CV (%), shall not exceed 15% (n = 10).
3. Cross-reactivity
No cross-reactivity was observed with antinuclear antibody–positive serum, RF‑positive serum, or IgM‑positive serum for the following pathogens: measles virus, cytomegalovirus, herpes simplex virus, Toxoplasma gondii, Epstein–Barr virus, Coxsackievirus, mumps virus, and human papillomavirus. No experimental data are currently available regarding cross-reactivity with other pathogens.
4. Interference Test
Common anticoagulants that come into contact with the sample (EDTA, sodium citrate, and sodium heparin) do not significantly interfere with the assay results of this product.
Endogenous substances within a certain concentration range (bilirubin 0.1–0.4 mg/mL, hemoglobin 5–50 mg/mL, triglycerides 2–8 mg/mL) do not significantly interfere with the assay results of this product.
[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 product have been tested negative for HBsAg, HCV antibodies, Treponema pallidum antibodies, and HIV antibodies, and all other components included in the kit are non‑infectious. However, since it cannot be guaranteed that all potential infectious agents have been detected in each component, this product should be treated as potentially infectious material, and all personnel handling the kit must follow laboratory safety protocols.
3. All clinical samples should 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 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] Dai Bin, Chang Huilan, Han Xiujuan, et al. 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 in women during 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 Fourth Road, Gangwan Avenue, Tangjiawan Town, Zhuhai City Postal Code: 519085
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. 20163402227
[Date of Approval and Amendment of the Instruction Manual] November 1, 2016