Toxoplasma gondii IgM Antibody Detection Kit (ELISA)

Instruction Manual for Toxoplasma gondii IgM Antibody Detection Kit (ELISA)
[Product Name]
Chinese Name: Toxoplasma gondii IgM Antibody Detection Kit (ELISA)
[Packaging Specifications]
48 servings/box, 96 servings/box.
[Intended Use]
Qualitative detection of Toxoplasma gondii IgM antibodies in human serum or plasma samples.
For auxiliary diagnosis of Toxoplasma gondii infection. 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, 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 shed large numbers of infectious oocysts in their feces, contaminating the environment. Humans can become infected 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 can 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 remain elevated 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 peak levels 2–3 months post‑infection and persisting for a prolonged period, with a slower decline. A positive IgG result indicates prior exposure to Toxoplasma, typically reflecting chronic infection, though it may also signify an acute episode. If, upon repeat testing 2–3 weeks after the initial IgG assay, the antibody titer has increased fourfold or more, this finding is diagnostically significant.
When detecting IgM antibodies by indirect ELISA or IFA, the presence of rheumatoid factor (RF) and antinuclear antibodies (ANA) can lead to false‑positive results, while high‑titer IgG may cause false‑negative outcomes. The ELISA capture assay, which measures IgM, overcomes these limitations in terms of both sensitivity and specificity, making it the preferred method for detecting IgM antibodies.
[Principle of Assay]
Principle of the enzyme‑linked immunosorbent capture assay: A microtiter plate is coated with a monoclonal antibody against the human IgM μ chain. The serum or plasma to be tested is added to the wells, where any IgM antibodies present in the sample specifically bind to the anti‑human IgM μ chain antibody immobilized on the plate surface, forming an antibody–secondary antibody complex. An enzyme‑labeled anti‑Toxoplasma antibody and Toxoplasma antigen are then added, resulting in the formation of an immune complex consisting of (antibody–secondary antibody–antigen–enzyme‑labeled anti‑Toxoplasma reagent). Upon addition of a substrate, an enzymatic colorimetric reaction occurs; the intensity of the color development is measured using a microplate reader, allowing for the qualitative detection of Toxoplasma 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 anti-human IgM-μ chain antibody. The microtiter plate is pre-coated with mouse monoclonal anti-human IgM-μ chain antibody. Ready to use; can be separated into individual wells. Available in 96-well and 48-well formats.
2 Toxoplasma antibody enzyme label: horseradish peroxidase (HRP)-labeled anti-Toxoplasma monoclonal antibody; borate buffer containing Toxoplasma antigen, preservative, and 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 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 Toxoplasma gondii IgM antibody negative control: human serum or plasma negative for Toxoplasma gondii IgM antibodies; borate buffer containing 0.02% thimerosal and a protein stabilizer. Ready to use. 1.0 mL × 1 vial, 1.0 mL × 1 vial.
5 Toxoplasma gondii IgM antibody critical control: human serum or plasma positive for Toxoplasma gondii 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
(10×) Phosphate-buffered saline, containing 0.5% Tween‑20, 10× 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. Ready-to-use. 50 mL × 2 vials, 50 mL × 1 vial.
10 Stop solution: 0.5 mol/L sulfuric acid solution. Use directly: 6.5 mL × 1 bottle, 3.5 mL × 1 bottle.
11 Sealed bags 1 piece 1 piece
12 sealing films 1 piece 4 pieces
13 Recording Paper 1 copy 1 copy
Note: Enzyme conjugates, quality control samples, positive controls, and negative controls must be those provided with the kit; products from other batch numbers may not be used. The sample diluent (IgM) is compatible with our company’s ELISA kits for IgM antibody detection. 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
10 μL to 100 μL, 1 mL pipettes with disposable tips
37°C constant-temperature equipment
Microporous 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.
After opening, any unused microtiter plates should be stored in a sealed bag at 2–8°C. Liquid components should be tightly capped and stored at 2–8°C. All components must be used within one month of opening.
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]
Serum or plasma samples should be collected in accordance with the clinical blood‑collection technical guidelines. Samples stored at room temperature should not exceed 8 hours; if testing 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 avoid repeated freeze–thaw cycles. This assay is suitable for samples containing ethylenediaminetetraacetic acid (EDTA), sodium citrate, sodium heparin, sodium oxalate, or double‑oxalate anticoagulants; however, there are currently no experimental data regarding the suitability of samples containing other types of anticoagulants. Although bilirubin, hemoglobin, and triglycerides do not significantly interfere with the assay 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, allow them to equilibrate at room temperature 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 conjugate, 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, one positive control well, one negative control well, and three critical‑control wells per plate. The blank well contains no sample or enzyme conjugate; it is supplemented only with wash buffer, 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 remaining wells. Incubate at 37°C for 60 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). After a 1‑minute incubation, discard the solution and blot dry; repeat this step three times. Except for the blank well, add 50 μL of enzyme‑labeled reagent to each well and incubate at 37°C for 60 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 at 37°C in the dark for 10–15 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 read the absorbance (A) at 450 nm with a microplate reader. Alternatively, calculate the absorbance using dual wavelengths (A450 nm – A630 nm).
4. Quality Control
If the positive control A-value is ≥0.30, the negative control A-value is ≤0.15, and the mean A-value of the critical control is ≥0.15, the assay is considered valid; otherwise, the results are invalid and the assay must be repeated.
[Positive Judgment Value]
A sample is considered negative if its A value is less than the mean of the critical control values; it is considered positive if its A value is greater than or equal to the mean of the critical control values.
[Interpretation of Test Results]
1. A positive result indicates an acute or recent Toxoplasma gondii infection. For such cases, it is recommended to repeat sampling and testing 1–2 weeks later to dynamically monitor changes in antibody levels.
2. 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]
1. Microbial contamination of samples or reagents, as well as cross-contamination between reagents, may lead to erroneous results.
2. The experimental results indicate that this product exhibits no cross-reactivity with the interfering factors listed in the cross-reactivity panel; however, no experimental data are currently available regarding cross-reactivity with other interfering substances.
[Product Performance Specifications]
1. Sensitivity, specificity
In a clinical study involving 1,122 cases, this product demonstrated a sensitivity of 96.4% and a specificity of 99.7% when compared with a comparable imported reagent that has obtained CE certification.
2. Repetitiveness
When measured using a precision reference material, the coefficient of variation (CV, %) shall not exceed 15% (n = 10).
3. Cross-reactivity
No cross-reactivity was observed with rheumatoid factor or with IgM‑positive sera against the following pathogens: Schistosoma, rubella virus, measles virus, herpes simplex virus type 1, herpes simplex virus type 2, and cytomegalovirus.
4. Interference Test
4.1 Anticoagulants that come into contact with the sample (EDTA, sodium citrate, sodium heparin, sodium oxalate, and double oxalate) do not significantly interfere with the assay results of this product.
4.2 Endogenous substances—bilirubin, hemoglobin, and triglycerides—do not significantly interfere with the assay results of this product.
4.3 Fibrinogen does not significantly interfere with the assay results of this product; therefore, this product can be used to analyze human serum or plasma samples.
[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 kit have been tested negative for hepatitis B surface antigen (HBsAg), hepatitis C virus (HCV) antibodies, Treponema pallidum antibodies, and human immunodeficiency virus (HIV) antibodies. All other components included in the kit are non‑infectious. However, since it cannot be guaranteed that all infectious agents have been detected in each component, this kit should be treated as potentially infectious material, and all personnel handling it must follow standard laboratory procedures.
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] 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 counselling[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. 20163402554
[Date of Approval and Amendment of the Instruction Manual] December 29, 2016