Cytomegalovirus IgM Antibody Detection Kit (ELISA)

Instructions for the Cytomegalovirus IgM Antibody Detection Kit (ELISA)
[Product Name]
Generic Name: Cytomegalovirus IgM Antibody Detection Kit (Enzyme-Linked Immunosorbent Assay)
[Packaging Specifications]
48 servings/box, 96 servings/box.
[Intended Use]
This kit is intended for the in vitro qualitative detection of cytomegalovirus IgM antibodies in human serum or plasma samples.
This kit is intended for the auxiliary diagnosis of cytomegalovirus infection and shall not be used for prenatal screening in asymptomatic individuals. The test results obtained with this kit must not be used as the sole basis for terminating a pregnancy.
Cytomegalovirus (CMV) belongs to the β‑herpesvirus subfamily. Its primary modes of transmission are direct or indirect contact, including exposure to the saliva, genital secretions, urine, and breast milk of infected individuals; it can also be transmitted transplacentally or via iatrogenic routes. Following infection, CMV IgM antibodies remain detectable in serum for 4–6 months, and in some cases may persist for several years, whereas CMV IgG antibodies generally persist lifelong. Detection of CMV IgM antibodies is useful for diagnosing acute CMV infection but cannot distinguish between primary and secondary infections. Detection of CMV IgG antibodies helps determine whether a person has been exposed to CMV and enables appropriate interventions for susceptible populations. Testing for CMV antibodies is particularly important for immunocompromised patients, pregnant women, organ transplant recipients and donors, as well as blood donors.
[Principle of Assay]
Principle of the indirect ELISA: A microtiter plate is coated with recombinant cytomegalovirus antigen. The serum or plasma to be tested is added to the wells, where any cytomegalovirus IgM antibodies present in the sample specifically bind to the antigen immobilized on the plate surface, forming an antigen–antibody immune complex. After washing to remove excess material, an enzyme‑labeled 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 unbound reagents, 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 cytomegalovirus IgM antibodies in the sample. The sample diluent contains sheep anti‑human IgG antibodies, which can bind to human IgG antibodies and rheumatoid factors in the sample, thereby minimizing interference from specific IgG antibodies and rheumatoid factors.
[Main Ingredients]
1. Components included in this kit
Serial No. Component Name Main Ingredient Usage Method Fill Volume
96 servings per box, 48 servings per box
1. Microwell plate pre-coated with cytomegalovirus antigen: The microwell plate is pre-coated with a recombinant cytomegalovirus 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 anti-human IgM monoclonal antibody (mouse-derived); borate buffer containing 0.1% Proclin‑300, 0.002% phenol red, and a protein stabilizer. Ready to use. 6.5 mL × 1 vial; 3.5 mL × 1 vial.
3 Cytomegalovirus IgM Antibody Positive Control: Cytomegalovirus IgM‑positive human plasma or serum; borate buffer containing 0.02% thimerosal and a protein stabilizer. For direct use. 1.0 mL × 1 vial, 1.0 mL × 1 vial.
4 Cytomegalovirus IgM Antibody Negative Control: Cytomegalovirus IgM‑negative human plasma or serum; borate buffer containing 0.02% thimerosal and a protein stabilizer. For direct use. 1.0 mL × 1 vial, 1.0 mL × 1 vial.
5 Cytomegalovirus IgM Antibody Cut‑Off Control: Cytomegalovirus IgM‑positive human plasma or serum; 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 bottle, 3.5 mL × 1 bottle.
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 and goat anti-human IgG. For direct use. 50 mL × 2 vials, 50 mL × 1 vial.
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 Record Sheet 1 copy 1 copy
Note: Enzyme conjugates, positive controls, negative controls, and cutoff controls must be used exclusively from the kit supplied; products from other batch numbers are not permitted. Chromogenic substrate A, chromogenic substrate 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
(5–40) μL, (40–200) μL, and 200 μL–1 mL pipettes with disposable tips
37°C constant-temperature equipment
Micro-pore oscillator
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. See label for the date of manufacture and the expiration date.
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.
[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 at −20°C or lower, and freeze at −20°C or below for no more than 5 years. Repeated freeze–thaw cycles should be minimized, with a maximum of 5 cycles. This assay is suitable for samples containing EDTA, sodium citrate, or sodium heparin anticoagulants; however, there are currently no experimental data regarding the suitability of samples containing other types of anticoagulants. Although bilirubin (0.4 mg/mL), hemoglobin (50 mg/mL), and triglycerides (8 mg/mL) do not significantly interfere with the kit’s results, samples exhibiting jaundice, hemolysis, or hyperlipidemia should still be avoided. Clearly contaminated samples are not suitable for testing.
[Testing Method]
1. Precautions During the Experiment
1.1 When the kit components are removed from refrigeration, they should be allowed 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, use disposable pipette tips and do not reuse them.
1.4 Before aliquoting the standard and enzyme conjugate, gently shake to ensure thorough mixing; prior to sample addition, use a microplate shaker to agitate.
Aliquot the diluted sample.
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 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 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: Take 10 µL of the sample to be tested and add it to a small test tube containing 1 mL of sample diluent (IgM), then mix thoroughly.
3. Testing Procedure
3.1 For each assay, include one blank well (unless using dual-wavelength detection, in which case a blank well is not 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; no sample or enzyme conjugate is added. The positive, negative, and critical controls are used directly at 100 μL per well. Add 100 μL of the 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 process three times. Except for the blank well, add 50 μL of enzyme‑labeled reagent 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 in the dark at 37°C ± 1°C 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. Reference Range
The negative control A-value must be ≤0.15; the average A-value of the cutoff control must be between 0.20 and 0.80; the ratio of the cutoff 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 cutoff 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.
5. Result Interpretation
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 an acute or recent cytomegalovirus infection. For indeterminate results (0.9 < S/CO < 1.1), it is recommended that the patient be retested within one week or confirmed using an alternative assay.
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 7–14 days, with concurrent parallel analysis of the previously collected specimen to confirm whether seroconversion has occurred or 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. Positive IgM antibodies for CMV infection occur not only in primary infections but also in secondary infections.
5. False-positive and false-negative results may have adverse clinical consequences; false negatives can lead to infections in fetuses, newborns, or organ transplant recipients, while false positives may result in unnecessary antiviral therapy for 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]
1. Sample or reagent contamination by microorganisms, as well as cross-contamination among reagents, may lead to erroneous results.
2. The experimental results indicate that this product shows no cross-reactivity with IgM‑positive sera from the pathogens listed in the cross‑reactivity panel; however, no experimental data are currently available regarding cross‑reactivity with other pathogens.
[Product Performance Specifications]
1. Sensitivity, specificity
This product was evaluated using 1,080 samples from diverse populations, demonstrating a sensitivity of 94.1% and a specificity of 94.8%.
2. Precision
The within-run coefficient of variation (CV, %) shall not exceed 15% (n = 10).
3. Cross-reactivity
No cross-reactivity was observed with IgM‑positive sera against the following pathogens: Toxoplasma gondii, measles virus, rubella virus, herpes simplex virus types 1 and 2, Treponema pallidum, Epstein–Barr virus, mumps virus, parvovirus B19, and Coxsackievirus.
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.
5. Testing Capability for National Reference Standards
This product was tested against the national reference material (310016-200901): all nine negative reference samples yielded negative results, and all five positive reference samples yielded positive results. Precision was ≤15% (n=10). Among the six minimum detection limit reference samples, S1 through S5 were positive, while S6 was negative, with all results conforming to the specifications of the national reference material.
6. Batch-to-batch variation
Using three different lot numbers of the kit to test the same replicate reference material, the between‑lot coefficient of variation (CV) among the three lots did not exceed 30% (n = 30).
[Precautions]
1. This product is intended for in vitro diagnostic use only and is for single-use only.
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 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 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 the skin or eyes, 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] Griffiths PD. Molecular biology and immunology of cytomegalovirus. Biochem J, 1978, 241:313.
[2] Infection and Gastroenterology Group of the Chinese Society of Pediatrics, Editorial Board of the Chinese Journal of Pediatrics. Proceedings of the National Symposium on Cytomegalovirus Infection in Children. Chinese Journal of Pediatrics, 1995, 33(1):7.
[3] Xu Guilin, Chen Jiahua, Zhang Su, et al. Analysis of cytomegalovirus infection in 199 parturients and newborns. Chinese Journal of Obstetrics and Gynecology, 1989, 24(3):130.
[4] Surresh B. Boppana, Robert F. Pass, Willian J. Britt. Virus-specific antibody responses in mothers and their infants cytomegalovirus infection. The Journal Infection Diseases, 1993,167(1):72.
[5] Stagno S. In: Remington JS, Klein JO editors. Infectious Diseases of the Fetus and Newborn Infant [J]. Philadelphia: WB Saunders Co., 1995, 312–353.
[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:
Name of After-Sales Service Provider:
Contact Information:
Production Address: No. 18, Innovation Road 4, Gangwan Avenue, Tangjiawan Town, Zhuhai City
Production License No.:
[Medical Device Registration Certificate Number/Product Technical Requirements Number]
[Date of Approval and Date of Amendment of the Instruction Manual]