Type I and Type II Herpes Simplex Virus IgG Antibody Detection Kit (ELISA)

Instruction Manual for Herpes Simplex Virus Type I and Type II IgG Antibody Detection Kit (Enzyme-Linked Immunosorbent Assay)
[Product Name]
Generic Name: Herpes Simplex Virus Type I and Type II IgG Antibody Detection Kit (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 IgG antibodies against herpes simplex virus types 1 and 2 in human serum or plasma samples.
This kit is suitable for the auxiliary diagnosis of herpes simplex virus type I and type II infections.
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‑type 1 is primarily spread through direct contact with skin or mucosal surfaces, whereas HSV‑type 2 is mainly transmitted through sexual contact. In the general population, HSV‑type 1 infection is more common and typically causes infections of the skin, mucosa, and various organs outside the genital region. HSV‑type 2 is predominantly associated with genital infections and neonatal infections, leading to conditions such as genital herpes and neonatal herpes. If a pregnant woman experiences primary HSV infection, the virus may cross the placenta and infect the fetus, resulting in congenital infection. Additionally, if a pregnant woman harbors primary or recurrent HSV infection in the birth canal, the newborn may become infected during delivery, leading 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 newborns; clinically, antibodies against both types are typically assessed simultaneously. This kit employs a mixed coating of HSV‑1 and HSV‑2 antigens to detect IgG antibodies against HSV‑1 and HSV‑2 in the sample.
[Principle of Assay]
Principle of the indirect ELISA: Microtiter plates are coated with purified type 1 and type 2 herpes simplex virus antigens. The serum or plasma to be tested is added to the wells; any type 1 and type 2 HSV‑specific IgG antibodies present in the sample bind specifically to the viral antigens immobilized on the plate surface, forming antigen–antibody immune complexes. After washing to remove unbound material, an enzyme‑conjugated anti‑human IgG 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 a microplate reader, allowing for the qualitative detection of type 1 and type 2 HSV‑specific IgG antibodies in the test sample.
[Main Ingredients]
1. Components included in this kit
Serial No. Component Name Main Ingredient Application Method Fill Volume
96 servings/box, 48 servings/box
1 Microtiter plate pre-coated with herpes simplex virus type I and type II antigens. The microtiter plate is pre-coated with natural antigens of herpes simplex virus types I and II. It can be separated into individual wells for direct use: 96-well and 48-well formats.
2 Anti-human IgG antibody enzyme conjugate: horseradish peroxidase (HRP)-labeled anti-human IgG 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 Positive Control for Herpes Simplex Virus Type I and Type II IgG Antibodies: Human plasma or serum positive for HSV‑I and HSV‑II IgG antibodies; borate buffer containing 0.02% thimerosal and a protein stabilizer. Ready to use. 1.0 mL × 1 vial, 1.0 mL × 1 vial.
4 Type I and Type II Herpes Simplex Virus IgG Antibody Negative Control: Human plasma or serum negative for Type I and Type II Herpes Simplex Virus IgG 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 Herpes Simplex Virus Type I and Type II IgG Antibody Critical Control: Positive human plasma or serum for HSV‑I and HSV‑II IgG 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 (IgG) Borate buffer, containing glycerol. For direct 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 Record Sheet 1 copy 1 copy
Note: Enzyme conjugates, positive controls, negative controls, and cut-off controls must be those supplied with the kit; products from other batch numbers may not be used. The sample diluent (IgG) is compatible with all of our ELISA kits for detecting IgG antibodies. Chromogen A, chromogen B, stop solution, and concentrated wash solution are universal reagents and can be used with all of our ELISA kits.
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 placed in a sealed bag and stored 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 after 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 the assay is performed after 8 hours, they 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 8 years. Minimize repeated freeze–thaw cycles, limiting them to no more than five. This assay is suitable for samples containing EDTA, sodium citrate, or sodium heparin as anticoagulants; however, there are currently no experimental data regarding the suitability of samples with other anticoagulant types. 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. 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 present 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 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 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 (IgG), 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. Use the positive, negative, and critical controls directly, 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 be > 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 ≥ 1.1
Suspicious (S): 0.9 < S/CO < 1.1
Negative (N): S/CO ≤ 0.9
[Interpretation of Test Results]
1. A reactive result indicates past infection with herpes simplex virus types 1 and 2. For indeterminate results, repeat testing or re-sampling after 1–2 weeks is recommended to monitor changes in antibody levels over time.
2. The IgG result should be interpreted in conjunction with the IgM antibody test findings.
3. An IgG-negative result generally indicates that the patient has no history of HSV‑1 or HSV‑2 infection; however, a negative result does not rule out the possibility of a recent infection. During the first two weeks of an acute infection, IgG may remain negative, and retesting is recommended after 1–2 weeks. A seroconversion to IgG positivity within a short time frame suggests either a recent infection or ongoing infection.
4. Serological antibody testing has limited reference value in patients with compromised immune function.
5. The test results are for clinical reference only; clinicians should integrate the patient’s medical history, symptoms, and other diagnostic findings to arrive at a diagnosis.
[Limitations of the Analytical Method]
Microbial contamination of samples or reagents, as well as cross-contamination between 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 98.0% and a specificity of 97.5%.
2. Precision
The within-run coefficient of variation (CV, %) shall not exceed 15% (n = 10).
3. 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.
4. 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 standard laboratory procedures.
3. All clinical specimens 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] 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 II 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 Requirement Number]
[Date of Approval and Date of Amendment of the Instruction Manual]