Toxoplasma gondii Antibody (IgG) Detection Kit (ELISA)

Instruction Manual for Toxoplasma gondii Antibody (IgG) Detection Kit (ELISA)
[Product Name]
Generic Name: Toxoplasma gondii Antibody (IgG) Detection Kit (Enzyme-Linked Immunosorbent Assay)
[Packaging Specifications]
96 servings/box, 48 servings/box
[Intended Use]
This kit is intended for the in vitro qualitative detection of Toxoplasma gondii IgG antibodies in human serum or plasma samples.
This kit consists of a microtiter plate coated with Toxoplasma antigens, an enzyme‑labeled anti‑human IgG antibody, and other accompanying reagents, and is intended for the auxiliary diagnosis of toxoplasmosis.
Toxoplasmosis, also known as toxoplasma infection, is a zoonotic disease caused by Toxoplasma gondii. It is widespread worldwide. As a protozoan parasite, T. gondii can infect nearly all mammals and serves as a source of human infection. Among these hosts, cats and other felids play the most critical role in transmission, since they frequently shed large numbers of infectious oocysts in their feces, contaminating the external environment. Human infection can occur through ingestion of oocysts via contaminated hands, utensils, food, or water. The saliva of cats and dogs may also harbor T. gondii; contact with such saliva—particularly when playing with these animals—can lead to oral transmission. Additionally, pigs, cattle, and sheep can harbor tissue cysts in their muscles, and consumption of undercooked meat from these animals may result in oral infection. The prevalence of infection varies considerably across countries and regions, averaging approximately 25% to 50%. Foreign studies have reported natural infection rates ranging from 50% to 80% [1], whereas domestic data indicate rates between 0.1% and 47.3%, with an average of 5.17% [2,3].
Literature reports [4–6] indicate that the implementation of radiotherapy and chemotherapy for cancer often leads to immunosuppression in patients, which can be complicated by Toxoplasma gondii infection and even result in death. In some countries abroad, serological testing for Toxoplasma has been incorporated into routine screening for cancer patients [7]. When pregnant women are infected with Toxoplasma, the parasite can be transmitted vertically to the fetus via the placenta, impairing embryonic development and causing severe neurological malformations and intellectual disability, thereby reducing population health and, in extreme cases, leading to fetal death. Recent domestic studies have shown that Toxoplasma infection is relatively common among pregnant women in China, posing a serious threat to the health of future generations [8–9].
In general, following Toxoplasma gondii infection, anti‑Toxoplasma IgM antibodies rise first and can be detected approximately 6 days after infection; they then gradually increase and persist for several weeks to months before declining and eventually disappearing [10]. Therefore, measurement of Toxoplasma IgM is suitable for early diagnosis. In contrast, Toxoplasma IgG antibodies appear later than IgM, reaching their peak 2–3 months post‑infection and maintaining a longer duration, so they also disappear more slowly. A positive IgG result indicates prior exposure to Toxoplasma, typically reflecting chronic infection, though it may also signify an acute infection. If, upon retesting with the same method 2–3 weeks after the initial IgG assay, the antibody titer has increased fourfold or more, this constitutes diagnostic significance.
[Principle of Assay]
In the sample, the antibody to be detected forms an antigen–antibody complex with the Toxoplasma gondii antigen immobilized on the microtiter plate. An enzyme‑conjugated anti‑human IgG antibody is then added, resulting in the formation of an immune complex comprising (antigen–target antibody–enzyme‑conjugated anti‑human IgG antibody). A chromogenic substrate is subsequently added; under the catalytic action of the enzyme, a colorimetric reaction develops. The presence of Toxoplasma gondii IgG antibodies in the sample can be qualitatively determined by measuring the color development using a microplate reader.
[Main Ingredients]
1. The components included in this kit are:
Serial No. Component Name Main Ingredient Application Method Fill Volume
96 servings/box, 48 servings/box
1 Microtiter plate pre-coated with Toxoplasma antigens. The microtiter plate is pre-coated with native Toxoplasma antigens and can be separated into individual wells for direct use. Available in 96-well and 48-well formats.
2 Anti-human IgG antibody enzyme conjugate: horseradish peroxidase (HRP)-labeled anti-human IgG antibody (goat-derived); borate buffer, containing preservatives and stabilizers. Ready-to-use. 6.5 mL × 1 vial; 3.5 mL × 1 vial.
3 Toxoplasma gondii IgG antibody positive control: human serum or plasma positive for Toxoplasma gondii IgG antibodies; borate buffer containing 0.02% thimerosal and a stabilizer. For direct use. 1.0 mL × 1 vial, 1.0 mL × 1 vial.
4 Toxoplasma gondii IgG antibody negative control: human serum or plasma negative for Toxoplasma gondii IgG antibodies; borate buffer containing 0.02% thimerosal and a stabilizer. For direct use. 1.0 mL × 1 vial, 1.0 mL × 1 vial.
5 Toxoplasma gondii IgG antibody cutoff control: human serum or plasma positive for Toxoplasma gondii IgG antibodies; borate buffer containing 0.02% thimerosal and a stabilizer. Ready to use. 1.8 mL × 1 vial, 1.8 mL × 1 vial.
6 Color-developing solution A contains citric acid and hydrogen peroxide; use directly. 6.5 mL × 1 bottle, 3.5 mL × 1 bottle.
7 Color-developing solution B contains citric acid and 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. 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 Record Sheets 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. 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.
[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. All components, once opened, should be used within one month.
[Sample Requirements]
This assay is performed using serum or plasma, with a sample volume of 10 μL. Do not use samples that are contaminated with bacteria, lipemic, or hemolyzed. Collect serum according to standard procedures and store at room temperature for no more than 8 hours; if the assay is conducted after 8 hours, maintain the sample at 2°C–8°C. For storage exceeding one week, keep the sample at −20°C or lower, and minimize repeated freeze–thaw cycles, limiting them to no more than five. This assay can be applied to samples anticoagulated with sodium citrate, EDTA‑K2, sodium oxalate, or sodium heparin; however, there are currently no experimental data regarding the suitability of samples containing other anticoagulants.
[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 compromising 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 no wash solution remains in any well, thereby preventing 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 a blank well is not required), 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 at 100 µL per well. Add 100 µL of diluted sample to all remaining 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 wells dry, and repeat this step three times. Except for the blank well, add 50 µL (1 drop) 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 (1 drop) of developing reagent A and 50 µL (1 drop) of developing reagent B to each well. Incubate in the dark at 37°C ± 1°C for 15 minutes, then add 50 µL (1 drop) 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: Measure the absorbance (A) at 450 nm and 630 nm using a microplate reader, and calculate the final A value as A450 nm – A630 nm.
4. Quality Control
If the mean A value of the positive control is ≥0.50, the mean A value of the negative control is ≤0.10, and the mean A value of the cutoff control falls between 0.12 and 0.35, the assay is considered valid; otherwise, the results are invalid and the assay must be repeated.
5. Result Interpretation
Sample A value ≥ the average of the critical control A values is considered positive.
Sample A value < average of the critical control A values is judged as negative.
[Interpretation of Test Results]
This product can perform a qualitative assay for Toxoplasma gondii IgG antibodies. A positive result in this test indicates a past infection with Toxoplasma; the results are intended for clinical reference only. For positive or indeterminate results, it is recommended to repeat sampling and testing 1–2 weeks later to monitor changes in antibody levels over time, and to make a comprehensive clinical assessment in conjunction with other diagnostic information.
[Limitations of the Analytical Method]
1. Sample or reagent contamination by microorganisms, cross-contamination among reagents, and thermal inactivation can all lead to erroneous results.
2. A diagnosis should not be based solely on a single test result. Accurate diagnosis requires consideration of the clinical history, symptoms, and serological tests; moreover, serological findings have limited diagnostic value in immunocompromised patients.
[Product Performance Specifications]
1. Appearance
Labels shall be clear and easily identifiable. All components of the kit must be present and intact, with no leakage of liquids.
2. Concordance rate of negative reference materials
The test results for the 15 negative enterprise reference samples must show no false positives (15/15).
3. Concordance rate of positive reference materials
The test results for the 15 positive enterprise reference samples must not include any false negatives (15/15).
4. Limit of Detection
The assay is performed using the limit-of-detection reference standard, and the result shall not exceed 1.0 IU/mL.
5. Repetitiveness
The assay was performed in duplicate using a reference material, and the coefficient of variation (CV) did not exceed 15% (n = 10).
6. Stability
After incubation at 37°C for 6 days, the assay met the requirements of 2–5 for all validation parameters, including negative reference‑material conformity, positive reference‑material conformity, limit of detection, and repeatability.
7. Biological Safety
The reference standards in the kit must test negative for HBsAg, HIV‑Ab, HCV‑Ab, and Treponema pallidum antibodies.
8. Testing Capability for National Reference Materials
This product was tested against the national reference standard (230023-200901): all 15 negative reference samples yielded negative results, and all 9 positive reference samples yielded positive results. Precision was ≤15% (n=10), and the limit of detection did not exceed 5 IU/mL, both in full compliance with the requirements of the national reference standard.
[Precautions]
1. This product is intended for in vitro diagnostic use only; reagent components from different lot numbers must not be mixed.
2. Operate strictly in accordance with the instructions; before use, allow the kit to equilibrate to room temperature, and ensure that reaction temperature and incubation time are rigorously controlled during the procedure.
3. After opening, any unused microtiter plates should be stored in a sealed bag.
4. Any serum that is contaminated with bacteria, exhibits severe hemolysis, jaundice, or hyperlipidemia may yield inaccurate results and should be re‑collected for repeat testing.
5. A positive result in this assay indicates a past Toxoplasma gondii infection; clinicians should integrate the patient’s medical history and clinical findings to arrive at a comprehensive diagnosis. To assess clinical significance, serial monitoring is recommended: patients with positive or indeterminate results should undergo follow-up testing 1–2 weeks later to evaluate changes in antibody levels.
6. During the preparation of this kit, the serum or plasma used as raw materials (negative, positive, and critical control) has been tested and found to be negative for HBsAg, HCV antibodies, Treponema pallidum antibodies, and HIV antibodies. The Toxoplasma antigen has been completely disrupted by ultrasonic treatment and is non‑infectious. All other components of the kit are likewise free from biological infectivity. As it cannot be guaranteed that all infectious agents have been eliminated from the test materials, this kit should be handled as potentially infectious material, and all personnel who come into contact with it must follow standard laboratory practices.
7. All clinical specimens shall be treated as infectious materials.
8. Color development reagent B contains TMB, which may trigger additional reactions during the assay; the stop solution contains sulfuric acid and is corrosive. If these liquids come into contact with 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] Mao SB. Chin Med. J. 1997, 100:445.
[2] Gan Shaobo, et al. Chinese Journal of Zoonoses, 1991, 7(5):61.
[3] Yu Enshu, et al. Toxoplasmosis, 1st ed. Fujian Science and Technology Press, pp. 159–160.
[4] Lu Shen, Zong Dayuan. Two cases of Toxoplasma infection in patients with malignant tumors, Journal of Practical Internal Medicine, 1990, 10(3):144.
[5] Wang Chonggong, Zhu Yiwen, Wang Jianwu, et al. A case of toxoplasmosis misdiagnosed as a malignant tumor. Chinese Journal of Infectious Diseases, 1994, 12(3):140.
[6] Li Piping, Wang Qiming, Ma Yiwu, et al. A preliminary study on leukemia complicated by toxoplasmosis, Journal of Suzhou Medical College, 1993, 13(4):282.
[7] Li Xujiong, Wu Shaozhi, Li Fue. An investigation into the relationship between tumors and Toxoplasma gondii infection, Chinese Journal of Oncology, 1991, 13(2):125.
[8] Lü Yuancong. Incidence and therapeutic advances of toxoplasmosis in children in China [J]. Chinese Journal of Practical Pediatrics, 1995, 6:367.
[9]. Ye Huifang. Human Toxoplasmosis and Adverse Pregnancy Outcomes [J]. Chinese Journal of Obstetrics and Gynecology, 1987, 6:360.
[10] Dunn D, WaLLon M, Perron F, et al. Mother-to-child transmission of toxoplasmosis: risk estimates for clinical counseling [J]. Lancet, 1999, 353:1829–1835.
[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
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 Amendment of the Instruction Manual]