Toxoplasma gondii IgM Antibody Detection Kit (ELISA)

Instruction Manual for Toxoplasma gondii IgM Antibody Detection Kit (ELISA)
[Product Name]
Generic Name: Toxoplasma gondii IgM Antibody Detection Kit (Enzyme-Linked Immunosorbent Assay)
[Packaging Specifications]
48 servings/box, 96 servings/box.
[Intended Use]
Qualitative detection of Toxoplasma gondii IgM antibodies in human serum or plasma samples.
For early auxiliary diagnosis of toxoplasmosis. The test shall not be used for prenatal screening in asymptomatic individuals; the results of this assay shall not, by themselves, serve as the sole basis for terminating a pregnancy.
Toxoplasmosis is a zoonotic disease caused by Toxoplasma gondii. It is transmitted through ingestion of contaminated food, via oocyst shedding, direct transmission from livestock, or intrauterine infection. Following infection, specific IgM antibodies can be detected approximately 6 days after exposure, persist for several weeks to months, and then gradually decline until they disappear. In contrast, IgG antibodies appear later than IgM, reaching their peak 2–3 months post‑infection and maintaining a longer duration. Testing for Toxoplasma IgM antibodies is useful for diagnosing recent infections, whereas Toxoplasma IgG antibody testing can confirm past exposure and also serve as an adjunctive tool for identifying recent infections. If, after the initial detection of Toxoplasma IgG antibodies, a follow‑up sample taken 2–3 weeks later shows a fourfold or greater rise in IgG titer, this indicates a recent infection.
[Principle of Assay]
This product employs the indirect enzyme-linked immunosorbent assay (ELISA) principle to detect Toxoplasma gondii IgM antibodies in human serum or plasma samples. In the sample, the antibody of interest forms an antigen–antibody complex with the Toxoplasma antigen immobilized on the surface of the microtiter plate. An enzyme‑labeled anti‑human IgM antibody is then added, resulting in the formation of an immune complex comprising antigen–target antibody–enzyme‑labeled anti‑human IgM antibody. Upon addition of a chromogenic substrate, an enzymatic colorimetric reaction occurs; the intensity of the color is measured using a microplate reader, enabling qualitative detection of Toxoplasma gondii IgM antibodies in the sample.
[Main Ingredients]
1. The components included in this kit are:
Serial No. Component Name Main Ingredient Usage Method Package Quantity
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 IgM antibody enzyme conjugate: horseradish peroxidase (HRP)-labeled anti-human IgM antibody; borate buffer containing 0.002% phenol red and a protein stabilizer. Ready to use. 6.5 mL × 1 vial, 3.5 mL × 1 vial.
3 Toxoplasma IgM Antibody Positive Control: Human serum or plasma positive for Toxoplasma IgM; borate buffer containing 0.02% thimerosal and a protein stabilizer. For direct use. 1.0 mL × 1 vial, 1.0 mL × 1 vial.
4 Toxoplasma IgM Antibody Negative Control: Human serum or plasma negative for Toxoplasma IgM; borate buffer containing 0.02% thimerosal and a protein stabilizer. For direct use. 1.8 mL × 1 vial, 1.8 mL × 1 vial.
5 Color-developing solution A contains citric acid and hydrogen peroxide; use directly. 6.5 mL × 1 bottle, 3.5 mL × 1 bottle.
6 Color Development Solution B contains citric acid and tetramethylbenzidine (TMB). Ready to use. 6.5 mL × 1 bottle, 3.5 mL × 1 bottle.
7 Concentrated Detergent Solution
(10×) Phosphate-buffered saline, containing Tween‑20; 10× concentrated. Dilute with distilled water to a 10× working solution. 50 mL × 1 bottle 50 mL × 1 bottle
8 Sample Diluent
(IgM) Phosphate-buffered saline, containing glycerol, with goat anti-human IgG antibody. Ready-to-use. 50 mL × 2 vials, 50 mL × 1 vial.
9 Stop solution Sulfuric acid solution Ready-to-use 6.5 mL × 1 bottle 3.5 mL × 1 bottle
10 zip-lock bags 1 piece 1 piece
11 Sealing film 1 piece 1 piece
12 Recording paper 1 copy 1 copy

2. Materials required but not yet provided:
Distilled water or deionized water
Microplate washer
10 μL to 100 μL, 1 mL pipettes with disposable tips
37°C constant-temperature equipment
Micro-pore oscillator
Microplate reader
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 the label for the production date and expiration date.
[Applicable Instruments]
A microplate reader with wavelengths of 450 nm and 630 nm.
[Sample Requirements]
This assay is performed using serum or plasma, with a sample volume of 10 μL. Serum or plasma should be collected according to standard procedures; samples stored at room temperature should not exceed 8 hours. If the assay is conducted after 8 hours, samples must be refrigerated at 2°C–8°C; for storage exceeding one week, they should be frozen at −20°C or lower. This assay is applicable to samples anticoagulated with ethylenediaminetetraacetic acid (EDTA), sodium citrate, sodium heparin, or sodium oxalate; however, there are currently no experimental data regarding the suitability of samples containing other types of 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 visible before use. Any unused coated plates after opening 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 residual wash solution remains in any well, thereby preventing foam formation. If an automated plate washer is used, 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 when using dual-wavelength detection), one positive control well, and three negative control wells. The blank well should contain no sample or enzyme conjugate; add only wash solution, developing solutions A and B, and stop solution. Use the positive and negative controls directly, 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), discard the solution, and blot dry; repeat 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 Zero the instrument using the blank well, and measure the absorbance (A) at 450 nm with a microplate reader.
4. Quality Control
Zero the instrument using a blank well, and measure the absorbance (A) at 450 nm. The average A value of the positive control should be ≥0.25, while the average A value of the negative control should be ≤0.20, thereby confirming that the assay is valid.
[Positive Judgment Value]
If the negative control is <0.07, calculate using 0.07;
Calculate the S/N ratio, where S/N ratio = Sample A value ÷ Mean of negative control A values.
If the S/N ratio is ≥3.0, the result is considered positive.
An S/N ratio of less than 2.43 is considered negative.
If the S/N ratio is 2.43 ≤ S/N < 3.0, or if the A value falls between 0.17 and 0.21 (when the negative control is < 0.07), the result is considered suspect and a repeat test is required.
[Interpretation of Test Results]
This product can be used for the qualitative detection of Toxoplasma gondii IgM antibodies. A positive result in this assay indicates an acute or recent Toxoplasma infection; results are intended for clinical reference only. For positive and indeterminate results, it is recommended to repeat sampling and testing 1–2 weeks later to monitor changes in antibody levels over time. For confirmatory or repeat‑positive results, a blocking assay or other methods should be employed for definitive confirmation.
[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. Specificity, sensitivity
This product was tested on 1,327 samples from diverse populations, demonstrating a sensitivity of 100% and a specificity of 99.9%.
2. Repetitiveness
When measured using precision reference materials, the coefficient of variation (CV) shall not exceed 15% (n = 10).
[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 plate should be stored in a sealed bag.
4. Any serum that is contaminated with bacteria, exhibits severe hemolysis, jaundice, or hyperlipidemia may yield erroneous results and should be re‑collected for repeat testing.
5. A positive result in this assay indicates an acute or recent 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. For positive results from testing or retesting, it is recommended to perform a blocking assay or use other methods for confirmation.
7. During the preparation of this kit, the serum or plasma used as raw materials (negative and positive controls) has been tested negative for hepatitis B surface antigen (HBsAg), hepatitis C virus (HCV) antibodies, Treponema pallidum antibodies, and human immunodeficiency virus (HIV) antibodies. The Toxoplasma gondii antigen has been completely disrupted and inactivated by ultrasonic treatment, rendering it non‑infectious. All other components of the kit are likewise free from biological infectivity. However, since it is impossible to guarantee the absence of all potential infectious agents in 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.
8. All clinical specimens should be treated as infectious materials.
9. Color-developing solution 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] Gan Shaobo, et al. Chinese Journal of Zoonoses, 1991, 7(5):61.
[2] Yu Enshu, et al. Toxoplasmosis, 1st ed. Fujian Science and Technology Press, pp. 159–160.
[3] Li Piping, Wang Qiming, Ma Yiwu, et al. A preliminary investigation of leukemia complicated by toxoplasmosis, Journal of Suzhou Medical College, 1993, 13(4):282.
[4] 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.
[5] 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 Road No. 4, Gangwan Avenue, Tangjiawan Town, Zhuhai City
Contact Information: Phone Number: 0756-3890858
Fax number: 0756-3890848
Name of After-Sales Service Provider: Zhuhai Special Economic Zone Haitai Biopharmaceutical Co., Ltd.
Contact Information: Phone Number: 0756-3890858
Production Address: No. 18, Innovation Road 4, Gangwan Avenue, Tangjiawan Town, Zhuhai City
Production License No.: Yue Food and Drug Administration Medical Device Production License No. 20040941
[Medical Device Registration Certificate Number/Product Technical Requirements Number] National Medical Device Registration No. 20163402549
[Date of Approval and Amendment of the Instruction Manual] December 29, 2016