Hydatidosis IgG Antibody Detection Kit (ELISA)

Instruction Manual for the Echinococcus IgG Antibody Detection Kit (ELISA)
[Product Name]
Generic Name: Echinococcus IgG Antibody Detection Kit (ELISA)
[Packaging Specifications]
48 servings/box, 96 servings/box
[Intended Use]
This kit is intended for the in vitro qualitative detection of echinococcus IgG antibodies in human serum samples.
This product is manufactured using microtiter plates coated with echinococcus antigens, enzyme‑labeled anti‑human IgG monoclonal antibodies, and other reagents, and is intended for the clinical auxiliary diagnosis of echinococcosis.
Hydatid disease is the common name for echinococcosis, a zoonotic parasitic disease caused by the larval stages of tapeworms of the genus Echinococcus that infect humans. The principal causative agents are: the larva of Echinococcus granulosus, which gives rise to cystic hydatid disease, and the larva of Echinococcus multilocularis, which causes alveolar hydatid disease. Domestic dogs, as well as wolves, jackals, foxes, and other carnivores, serve as reservoirs of these tapeworms, with domestic dogs being the primary source of infection. When infected dogs, as definitive hosts, shed mature proglottids and large numbers of eggs, they contaminate pastures, water sources, and household environments; herbivores and humans can become infected by ingesting these eggs. In the early stages, patients with hydatid disease may be entirely asymptomatic and are often detected incidentally during routine medical examinations. The main clinical manifestations result from compression, irritation, or rupture of the hydatid cyst, leading to a spectrum of symptoms. Hydatid disease can affect multiple organs throughout the body, most commonly the liver and lungs. Alveolar hydatid disease poses greater harm than cystic hydatid disease, as it can infiltrate and spread into surrounding tissues through exophytic growth and may also disseminate via the lymphatic or circulatory systems to other organs, resembling a malignant neoplasm; consequently, many patients present at an advanced stage.
The serum of patients with echinococcosis contains specific anti‑Echinococcus IgG antibodies, circulating antigens, or immune complexes. Detection of anti‑Echinococcus IgG antibodies in human serum samples can serve as an auxiliary diagnostic tool for echinococcosis.
[Principle of Assay]
Principle of the indirect ELISA: The microtiter plate is coated with echinococcus antigen. The serum from the sample to be tested is added to the wells, where echinococcus IgG antibodies specifically bind to the antigen immobilized on the plate surface, forming an antigen–antibody immune complex. After washing to remove excess material, an enzyme‑conjugated anti‑human IgG antibody is added, resulting in an (antigen–antibody–secondary antibody–enzyme conjugate) immune complex. Following another wash to eliminate unbound components, 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 echinococcus IgG antibodies in the sample.
[Main Ingredients]
1. Components included in this kit
Serial No. Component Name Main Ingredient Usage Method Package Quantity
96 servings/box 48 servings/box
1 Microtiter plate pre-coated with echinococcus antigen. The microtiter plate is pre-coated with native echinococcus antigen. 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 preservatives and stabilizers. Ready-to-use. 6.5 mL × 1 vial; 3.5 mL × 1 vial.
3. Echinococcus IgG Antibody Positive Control: Echinococcus IgG-positive human serum; borate buffer containing 0.02% thimerosal and a stabilizer. For direct use. 1.0 mL × 1 vial, 1.0 mL × 1 vial.
4. Echinococcus IgG Antibody Negative Control: Echinococcus IgG‑negative human serum; borate buffer containing 0.02% thimerosal and a stabilizer. For direct use. 1.0 mL × 1 vial, 1.0 mL × 1 vial.
5. Echinococcus IgG Antibody Critical Control: Echinococcus IgG‑positive human serum; borate buffer containing 0.02% thimerosal and a 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, glycerol. Ready-to-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 Sheets 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 our company.
2. Materials required but not yet provided:
Microplate washer
(5–40) μL, (40–200) μL, and 200 μL–1 mL pipettes
37°C constant-temperature equipment
Micro-pore oscillator
[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 plate should be stored in a sealed bag at 2°C to 8°C. All components, once opened, should be used within one month.
[Applicable Instruments]
A microplate reader with wavelengths of 450 nm and 630 nm.
[Sample Requirements]
Serum should be collected in accordance with the clinical blood‑collection technical guidelines. Samples stored at room temperature should not exceed 8 hours; if the assay 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 minimize repeated freeze–thaw cycles, limiting them to no more than five. 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 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 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 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: Take 10 µL of the sample to be tested and add it to a small test tube containing 1 mL of sample diluent (IgG), then 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; do not add sample or enzyme conjugate. Use the positive, negative, and critical controls directly at 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 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 cut‑off control must be between 0.20 and 0.80; the ratio of the cut‑off 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 cut‑off 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 suggests echinococcosis; for reactive or indeterminate results, imaging studies are recommended.
2. The test results are for clinical reference only; clinicians should integrate the patient’s medical history, symptoms, and imaging 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. Studies have shown that this product exhibits no cross-reactivity with sera positive for IgG antibodies against Toxoplasma gondii, Paragonimus westermani, Trichinella spiralis, or Schistosoma spp.; however, it does show some cross-reactivity with sera positive for cysticercus IgG antibodies, and such cases should be differentiated in conjunction with medical history, clinical symptoms, imaging findings, and epidemiological investigations. No experimental data are currently available regarding cross-reactivity with other pathogens.
[Product Performance Specifications]
1. Sensitivity, specificity
This product was evaluated in 1,080 samples from both endemic and non-endemic populations; compared with imaging-based diagnostics, it demonstrated a sensitivity of 91.0% and a specificity of 94.7%.
2. Precision
The within-run coefficient of variation (CV, %) shall not exceed 15% (n = 10).
3. Cross-reactivity
No cross-reactivity was observed with IgG‑positive sera against Toxoplasma gondii, Paragonimus westermani, Trichinella spiralis, Schistosoma spp., and other pathogens; however, cross-reactivity was detected with some IgG‑positive sera against cysticercus.
No cross-reactivity was observed with sera positive for hepatitis A virus, hepatitis B virus, hepatitis C virus, human immunodeficiency virus (HIV-1/2), hepatocellular carcinoma, hepatic cysts, bacterial liver abscesses, gallbladder hydrops, or pulmonary tuberculosis.
4. Endogenous Substance Interference Test
Within the concentration ranges of 0.1–0.4 mg/mL for bilirubin, 5–50 mg/mL for hemoglobin, and 2–8 mg/mL for triglycerides, no significant interference was observed in the assay results of this product.
5. Testing Capability for National Reference Materials
This product was tested against the national reference material (230029-201001): all eight negative reference samples yielded negative results, and all eight positive reference samples yielded positive results. Precision was ≤15% (n=10), and the limit of detection was no greater than 1:16, both in compliance with the specifications of the national reference material.
[Precautions]
1. This product is intended for in vitro diagnostic use only and is for single-use.
2. The sera 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 is not possible to guarantee the absence of all potential infectious agents 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] Hygienic Standard of the People’s Republic of China (WS257-2006): “Diagnostic Criteria for Echinococcosis.”
[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 Amendment of the Instruction Manual]