Type I and Type II Herpes Simplex Virus IgM Antibody Detection Kit (Enzyme-Linked Immunosorbent Assay)

Instruction Manual for Type I and Type II Herpes Simplex Virus IgM Antibody Detection Kit (Enzyme-Linked Immunosorbent Assay)
[Product Name]
Generic Name: IgM Antibody Detection Kit for Herpes Simplex Virus Types I and II (Enzyme-Linked Immunosorbent Assay)
[Packaging Specifications]
48 servings per box, 96 servings per box.
[Intended Use]
This kit is intended for the qualitative detection of IgM antibodies against herpes simplex virus types 1 and 2 in human serum or plasma samples.
This kit is intended for the auxiliary diagnosis of herpes simplex virus type 1 and type 2 infections. It is not to be used for prenatal screening in asymptomatic individuals, nor should its test results alone serve as the basis for terminating a pregnancy.
Herpes simplex virus (HSV) belongs to the subfamily Alphaherpesvirinae within the family Herpesviridae, with virion dimensions of approximately 180 nanometers. Based on antigenic differences, the virus is currently classified into type I and type II. HSV is widely prevalent in the human population, with humans serving as its sole host. Transmission occurs via multiple routes: HSV‑1 is primarily spread through direct contact with skin or mucosal surfaces, whereas HSV‑2 is mainly transmitted through sexual contact. HSV‑1 infection is more common in the general population and typically causes infections of the skin, mucosa, and various organs outside the genital region. In contrast, HSV‑2 is predominantly associated with genital infections and neonatal disease, leading to conditions such as genital herpes and neonatal herpes. If a pregnant woman experiences a primary HSV infection, the virus may cross the placenta and infect the fetus, resulting in congenital infection. Additionally, if a pregnant woman harbors a primary or recurrent HSV infection in her birth canal, the newborn may become infected during delivery, giving rise to neonatal herpes.
One week after viral infection, HSV IgM antibodies can already be detected in serum. HSV IgG antibodies appear 2 to 3 weeks following primary infection, persist for several months, and then begin to decline; in individuals with recurrent infections, IgG antibody titers typically do not show a significant increase. Detection of HSV IgG antibodies can be used to determine the presence or absence of HSV infection and to implement appropriate preventive measures in susceptible populations.
Both HSV‑1 and HSV‑2 can pose risks to fetuses or neonates. Clinically, antibodies against HSV‑1 and HSV‑2 are typically assessed simultaneously. This kit employs a mixed coating of HSV‑1 and HSV‑2 antigens to detect IgM antibodies against HSV‑1 and HSV‑2 in test samples.
[Principle of Assay]
Principle of the indirect ELISA: Microtiter plates are coated with type I and type II herpes simplex virus antigens. The serum or plasma to be tested is added to the wells; any type I or type II HSV‑IgM antibodies present in the sample specifically bind to the viral antigens immobilized on the plate surface, forming antigen–antibody immune complexes. After washing to remove excess material, an enzyme‑labeled anti‑human IgM antibody is added, resulting in an (antigen–antibody–secondary antibody–enzyme conjugate) immune complex. Following another wash to eliminate unbound reagents, a chromogenic substrate is introduced; under enzymatic catalysis, a color reaction develops. The intensity of this color change is measured with a microplate reader, allowing for the qualitative detection of type I and type II HSV‑IgM antibodies in the sample. The sample diluent contains goat 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 Package Quantity
96 servings per box / 48 servings per box
1 Microtiter plate pre-coated with herpes simplex virus type 1 and type 2 antigens. The microtiter plate is pre-coated with natural antigens of herpes simplex virus types 1 and 2. It can be separated into individual wells for direct use: 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 Type I and Type II Herpes Simplex Virus IgM Antibody Positive Control: Human plasma or serum positive for IgM antibodies against Type I and Type II Herpes Simplex Virus; borate buffer containing 0.02% thimerosal and a protein stabilizer. For direct use. 1.0 mL × 1 vial, 1.0 mL × 1 vial.
4 Type I and Type II Herpes Simplex Virus IgM Antibody Negative Control: Human plasma or serum negative for Type I and Type II Herpes Simplex 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 Type I and Type II Herpes Simplex Virus IgM Antibody Critical Control: Human plasma or serum positive for Type I and Type II Herpes Simplex 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 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 Recording Paper 1 copy 1 copy
Note: Enzyme conjugates, positive controls, negative controls, and cut-off controls must be used exclusively from the kit supplied; 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 this 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
Microporous 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 clinical blood‑collection guidelines. Samples stored at room temperature should not exceed 8 hours; if testing is performed after 8 hours, they must be kept at 2°C–8°C. For storage exceeding one week, samples should be frozen at −20°C or lower, and frozen specimens stored at −20°C or below may be kept for no more than 8 years. Repeated freeze–thaw cycles should be minimized, with no more than five cycles recommended. This assay is suitable for samples containing anticoagulants such as EDTA, sodium citrate, or sodium heparin; 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 test 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 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, disposable pipette tips should be used and not reused.
1.4 Before aliquoting the reference standard and the enzyme‑labeled reagent, 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 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 if dual-wavelength detection is used), one positive control well, one negative control well, and three critical‑control wells. The blank well contains no sample or enzyme conjugate; add only wash solution, developing reagent A, developing reagent B, and stop solution. 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 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.
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 ≥ 1.15
Suspicious (S): 0.85 < S/CO < 1.15
Negative (N): S/CO ≤ 0.85
[Interpretation of Test Results]
1. A reactive result indicates recent infection with herpes simplex virus types 1 and 2. For indeterminate results (0.85 < S/CO < 1.15), 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 very low IgM titers may result in a negative test outcome. 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 titer has risen significantly.
3. Positive IgM antibodies for HSV infection are observed not only in primary infections but also in secondary infections.
4. In patients with compromised immune function or those undergoing immunosuppressive therapy, the clinical utility of serum antibody testing is limited.
5. False-positive and false-negative results may have adverse clinical consequences; false negatives can lead to fetal or neonatal infections, while false positives may result in unnecessary antiviral treatment 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]
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 1,080 samples from diverse populations, demonstrating a sensitivity of 94.8% and a specificity of 93.1%.
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, cytomegalovirus, Chlamydia trachomatis, Treponema pallidum, human papillomavirus, Candida albicans, and bacterial vaginosis.
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. Batch-to-batch variation
The same replicate reference material was tested using three different kit batches, and the between-batch coefficient of variation (CV) among the three batches did not exceed 30% (n = 30).
[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 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] Levi M, Ruden U, Carlberg H, et al. The use of peptides from glycoproteins G-2 and G-1 for detecting herpes simplex virus type 2 and type-common antibodies [J]. J Clin Virol, 1999, 12(3): 243–252.
[2] Oladepo D, Klapper PE, Marsden HS. Peptide-based enzyme-linked immunoassays for the detection of anti-HSV-2 IgG in human sera [J]. J Virol Methods. 2000;87(1–2):63–70.
[3] Van Doornum GJ, Slomka MJ, Buimer M, et al. Comparison of a monoclonal antibody–blocking enzyme-linked immunoassay and a strip immunoblot assay for identifying type-specific herpes simplex virus type 2 serological responses [J]. Clin Diagn Lab Immunol, 2000, 7(4): 641–644.
[4] Hashido M, Lee FK, Inouye S, et al. Detection of herpes simplex virus type-specific antibodies by an enzyme-linked immunosorbent assay based on glycoprotein G [J]. J Med Virol, 1997, 53(4): 319–323.
[5] Prince HE, Ernst CE, Hogrefe WR. Evaluation of an enzyme immunoassay system for measuring herpes simplex virus (HSV) type 1-specific and type 2-specific IgG antibodies [J]. J Clin Lab Anal, 2000, 14(1): 13–16.
[6] Tunback P, Liljeqvist JA, Lowhagen GB, et al. Glycoprotein G of herpes simplex virus type 1: identification of type-specific epitopes by human antibodies [J]. J Gen Virol, 2000, 81: 1033–1040.
[7] Wittekindt C, Fleckenstein B, Wiesmuller K, et al. Detection of human serum antibodies against type-specifically reactive peptides from the N-terminus of glycoprotein B of herpes simplex virus type 1 and type 2 by surface plasmon resonance [J]. J Virol Methods, 2000, 87(1–2): 133–144.
[8] Pramod NP, Gopalakrishnan V, Mohan R., et al. Enhanced detection of herpes simplex virus from ocular specimens of herpetic keratitis patients [J]. Indian J Pathol Microbiol, 1998, 41(1): 49–53.

[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]