TestLine TGE096 EIA Toxoplasma IgE Instruction Manual

TestLine TGE096 EIA Toxoplasma IgE

Product Information

• Product Name: EIA Toxoplasma IgE
• Product Code: TgE096
• Package Size: 96 tests
• Intended Use: The EIA Toxoplasma IgE kit is a professional diagnostic tool designed for the detection of Toxoplasma gondii infection using IgE antibodies in human serum or plasma. It is intended for use in a laboratory setting.

Product Usage Instructions

1. Introduction: Toxoplasmosis is an infectious disease caused by the parasite Toxoplasma gondii. It can be acquired through various routes, including ingestion of contaminated food or water, contact with infected animals, or transmission from mother to fetus during pregnancy. This kit is designed to aid in the diagnosis of Toxoplasma gondii infection.
2. Materials Provided: The kit includes the following materials:
   • Microtitre Plate: A 12 x 8 well plate coated with antibody
   • Negative Control: Solution containing no specific human antibodies
   • Cut-Off: Solution containing specific human antibodies in cutoff concentration
   • Positive Control: Solution containing specific human antibodies
   • Tracer: Buffer with protein stabilizers containing T. gondii antigen and conjugate
   • Sample Diluent 5: Buffer with protein stabilizers
   • TMB-Complete 2: Chromogenic substrate solution containing TMB/H2O2
   • Wash Solution: Solution for washing the plate
   • Stop Solution: Acid solution for stopping the reaction
3. Test Principle: The kit utilizes a purified and inactivated antigen of Toxoplasma gondii (RH strain) to detect IgE antibodies in human serum or plasma. The presence of these antibodies indicates a possible Toxoplasma gondii infection.
4. Other Materials Required: In addition to the materials provided in the kit, the following items are required for test performance:
   • Single- and multichannel pipettes
   • Disposable tips
   • Microplate washer
   • Timer
   • Incubator (37°C)
   • Microplate reader
5. Storage and Stability: The kit should be stored according to the specified conditions mentioned in the product packaging. Proper storage conditions ensure the stability and reliability of the kit components.

Document Records

Intended Purpose

The immunoenzymatic assay is intended for the diagnosis of Toxoplasma gondii infection using IgE antibodies in human serum or plasma in the general population. The semi-quantitative manual assay is designed for professional use in a laboratory.

Introduction

Toxoplasmosis is a widespread parasitic disease caused by protozoan Toxoplasma gondii – a parasite with a complicated life cycle consisting of several morphologically different stadia. Primary hosts are members of the feline family. Humans and most warm-blooded animals can be infected by either primarily infected food (insufficiently heat-treated meat) or by ingestion of oocysts (secondary contaminated food or contaminated fingers, objects, etc.). Acquired toxoplasmosis in immunocompetent individuals is usually asymptomatic or can manifest itself with flu-like symptoms (subfebrility, fatigue, lymphadenopathy, muscle aches), but without lasting ill effects. Severe life- threatening infections (encephalitis, hepatitis, chorioretinitis, myocarditis, generalized form of the disease) may develop in immunocompromised patients usually because of a reactivation of a latent infection. Congenital toxoplasmosis is caused by transmission of infection from mother to foetus. The mother has been primary infected with toxoplasmosis shortly before becoming pregnant or during pregnancy. Congenital toxoplasmosis might result in severe damages of the foetus (brain calcification, hydrocephalus, vision disorders, mental affections), still birth or abortion. Diagnosis of the disease is based on epidemiological anamnesis, clinical manifestation and laboratory tests. Direct detection of the parasite is not available for routine diagnostics. Serology is the most important tool for laboratory diagnostics of toxoplasmosis. Screening consists in determination of total antibodies by complement fixation test (CFT). Determination of specific IgA, IgE, IgM, IgG antibodies and IgG avidity is performed by ELISA and confirmation of results by immunoblot method. IgA, IgE and IgM antibodies are significant markers of acute toxoplasmosis. IgM antibodies are a highly sensitive marker of acute infection and they can persist more than 1 year. IgA antibodies can persist for 6–9 months from the beginning of infection. IgE antibodies as a highly specific marker of acute infection can persist up to 6 months from the beginning of infection. IgG antibodies reach maximum level in serum after 6 months from the beginning of infection and they can be detected for many years after past infection.

Test Principle

The kit is intended for detection of specific IgE antibodies to Toxoplasma gondii in a sample by means of a capture type of the EIA method (i.e. a solid phase coated with animal antibody to human IgE immunoglobulins – antibody from the analysed sample – tracer, i.e. specific antigen with labelled antibody). The labelled antibody is a mouse monoclonal antibody to p30 surface protein of Toxoplasma gondii conjugated with horseradish peroxidase. Peroxidase activity is determined in the test by a substrate containing TMB. Positivity is indicated when blue colour appears; after stopping solution has been added, blue changes to yellow. The yellow colour intensity is measured by a photometer at 450 nm, and it is proportional to the concentration of specific antibodies in the sample.
Antigen Used
Purified and inactivated antigen of Toxoplasma gondii (RH strain)

Materials Provided

coated with antibody, 12 x 8 wells in bag with desiccant
CONTROL   –|  |  | Negative Control| 1 × 2 ml
Solution containing no specific human antibodies, ready to use
CUTOFF|  |  | CUT-OFF| 1 × 3 ml
Solution containing specific human antibodies in cut- off concentration, ready to use
CONTROL   +|  |  | Positive Control| 1 × 2 ml
Solution containing specific human antibodies, ready to use
TRACER|  |  | Tracer| 1 × 14 ml
Buffer with protein stabilisers containing T. gondii

antigen and conjugate, ready to use

DILUENT 5|  |  | Sample Diluent 5| 1 × 105 ml
Buffer with protein stabilisers, ready to use
SUBSTRATE 2|  |  | TMB-Complete 2| 1 × 15 ml
Chromogenic substrate solution containing TMB/H2O2, ready to use
WASH 20x|  |  | Wash Solution| 1 × 75 ml
 |  |  | 20× concentrated buffer|
STOP|  |  | Stop Solution| 1 × 15 ml
 |  |  | Acid solution, ready to use|
 |  |  | Instructions for use| 1 pc

Other Material Required for Test Performance

• Single- and multichannel pipettes
• Disposable tips
• Microplate washer
• Timer
• Incubator (37C)
• Microplate reader

Storage and Stability
Store the kit at +2 °C to +8 °C. Do not freeze. If the kit is stored as described, the labelled expiration date is valid. The expiration date is indicated on the package. The opened kit should be used within three months.

Sample Preparation and Storage

Samples listed in the intended use may be used for the examination. The following human body liquids can be used for testing: serum and citrate plasma. Anticoagulants in the plasma (except for citrate) as well as bacterially contaminated, haemolytic or chylous samples can affect the test results. A coagulating blood collection tube is recommended for serum collection. A citrated plasma collection bag is recommended for plasma collection. Other types of plasma (EDTA, heparin) may be used but they are not recommended since anticoagulants may affect the test result.
Follow the manufacturer’s instructions when using commercial or other specially modified samples. Clinical samples collected within standard medical procedures into standardized tubes are ready for immediate use. Centrifugation or other separations are not required.
The examined samples can be stored at +2 °C to +8 °C for a maximum of 1 week.

Preparation of Reagents
Dilute the Wash Solution 1:20 (1 part of solution and 19 parts of distilled water); e.g. 75 ml of the concentrated the Wash Solution + 1425 ml of distilled water. Salt crystals might develop in the bottle with the concentrated the Wash Solution. Prior to use, it is necessary to dissolve the crystals by warming the bottle in a water bath. The diluted Wash Solution is stable at +2 °C to +8 °C for one week. The Controls (positive, negative and CUT-OFF) and Tracer are supplied ready to use, do not dilute further! TMB- Complete is a one-component chromogenic substrate solution ready to use, do not dilute further!

Interchangeability of reagents
The Sample Diluent, TMB-Complete and the Avidity Solution are interchangeable in EIA kits of TestLine Clinical Diagnostics s.r.o., provided they have the identical numeric marking (e.g. Sample Diluent 2, Sample Diluent 3, etc.). The Stop Solution and the Wash Solution are universal in all kits.

Preparation of Samples

• Mix gently the Sample Diluent prior to use.
• Dilution of sera and plasma samples
• Dilute well mixed samples 1:101 with the Sample Diluent: e.g.: 10 μl of sample + 1 ml of the Sample Diluent Mix well.
• Dilute foetal and neonatal sera 1:21 with the Sample Diluent: e.g.: 50 μl of serum + 1 ml of the Sample Diluent Mix well.

Assay Procedure

Allow all reagents to come to room temperature and mix well. If you do not use a whole microplate, return unnecessary strips into the bag with desiccant. Seal the bag tightly and store at +2 °C to +8 °C. Keep dry!

1. Dispense the controls and the diluted samples according to the working schedule.
   • Leave A1 well empty (blank).
   • Pipette 100 μl of the Negative Control into 1 well.
   • Pipette 100 μl of CUT-OFF into 2 wells.
   • Pipette 100 μl of the Positive Control into 1 well.
   • Pipette 100 μl of the diluted samples (see Chapter Preparation of Samples) into the other wells.
2. Cover the microplate with the lid and incubate at 37 °C for 60 minutes.
3. Aspirate the content of the wells and wash 5× with the working strength Wash Solution. Fill the wells up to the edge. Finally, tap the inverted microplate thoroughly on an absorbent paper to remove solution remnants.
4. Pipette 100 μl of the Tracer into all wells except A1 well.
5. Cover the microplate with the lid and incubate at 37 °C for 60 minutes.
6. Aspirate the content of the wells and wash 5× with the working strength Wash Solution. Fill the wells up to the edge. Finally, tap the inverted microplate thoroughly on an absorbent paper to remove solution remnants.
7. Pipette 100 μl of TMB-Complete into all wells. Avoid contamination – see Chapter Procedural Notes.
8. Cover the microplate with the lid and incubate at 37 °C for 20 minutes. Keep out of light.
9. Stop the reaction by adding 100 μl of the Stop Solution in the same order and intervals as the substrate was added.
10. Read the colour intensity in wells against blank (A1 well) using photometer set to 450 nm. The absorbance should be read within 30 minutes after stopping the reaction.

Working Schedule

Quality Control

Test is valid if: The absorbance of blank is lower than 0.150.

BLANK< 0.150
The absorbance of the Negative Control is lower than half of the mean absorbance of CUT‒OFF.
The mean absorbance of CUT-OFF is within a range of 0.200 – 1.000.
The absorbance of the Positive Control is 1.5-fold higher than the mean absorbance of CUT‒OFF.

Results Interpretation

Calculation of Index of Positivity (IP)
Divide the absorbance of a tested sample by the mean absorbance of CUT-OFF measured in the same test run:

Interpretation of the test results is described in the table (Table 1).
Table 1 Interpretation of test results

Examination of borderline samples, i.e. samples with Index of Positivity from 0.9 to 1.1, should be repeated from a new sample collected after 2 to 6 weeks regarding to the disease specifics. Serological finding can be interpreted only in the context of results of other laboratory tests and patient clinical picture.

Analytical Performance

• Specificity and Sensitivity
  Specificity was determined in the panel of negative samples. Sensitivity was determined in the panel of positive samples. The number of samples tested and the results obtained are described in the table (Table 2).

• Trueness (bias)
  Trueness is the closeness of agreement between the average value obtained from a large number of measurement results and the reference value. Its measure is bias. The nature of the method does not allow quantitative determination of bias (and thus the trueness). The trueness of the method is ensured by clinical parameters such as sensitivity and specificity, comparison with the reference method and batch continuity. The obtained results are described in the table (Table 2).

• Precision: Repeatability – Intra-assay (within run)
  The precision is defined as the closeness of agreement between measured values obtained by replicate measurements on the same object under specified conditions. The Intra-assay repeatability is expressed as agreement level among sample replicates within a run of the assay (in one batch). The obtained results are described in the table (Table 2).

• Precision: Reproducibility – Inter-assay (between-run)
  Reproducibility is a measure of precision under a defined set of conditions which include the inter-assay, expressed as agreement level among sample replicates within runs of the assay in one batch. The obtained results are described in the table (Table 2).

• Accuracy
  Accuracy is defined as the closeness of agreement between the measured value and the reference value. It is expressed as an achievable measure of the combined uncertainty. The obtained results are described in the table (Table 2).

• Analytical sensitivity – limits of detection and quantitation
  The analytical sensitivity is the maximum binary dilution of CUT-OFF or international standard samples, respectively, giving absorbance significantly different from the background. The value is expressed as an index of positivity and/or a concentration in units. This value is a minimum limit of detection and quantification. The obtained results are described in the table (Table 2).

• Measuring Range
  The measuring range of the kit lies between values where the lower limit is determined by the analytical sensitivity value and the upper limit depends on the measuring capability of the equipment used.

• Linearity
  The linearity is the ability of the method to obtain final values proportional (directly or after mathematical transformation) to the concentration of analyte in the sample; it is expressed as the range in which the method provides linear results. The obtained results are described in the table (Table 2).

• Hook effect
  Hook effect is an immunological phenomenon that causes falsely low results in the presence of an excess amount of analyte. Its presence is detected by serial dilution of a highly positive sample (Table 2).

• Comparison with the reference method
  Comparison with the reference method was performed. The results of both methods are comparable, considering the differences of both methods and completely meeting the requirements if the agreement in the classification of the samples is at least 90% (Table 2).

Table 2 Analytical Performance

N/A – not applicable

Interference
Two samples (one negative plasma pool and one positive plasma pool) were spiked with potentially interfering substances. Results of interference testing are shown in the table (Table 3).
Table 3 Interference Results

Interfering substance

| The result was not affected up to concentration:
---|---
Bilirubin| 0.4 mg/ml
Triacylglycerols| 20 mg/ml
Haemoglobin| 5 mg/ml
Biotin| 3500 ng/ml

Cross-reactivity
The assay was evaluated for potential cross-reactivity using samples positive for selected pathogens and factors. Results of testing are shown in the table (Table 4).
Table 4 Results of Cross-Reacting Pathogens or Factors

Clinical Performance

• Diagnostic specificity and diagnostic sensitivity
  Diagnostic specificity was determined in the panel of negative samples. Diagnostic sensitivity was determined in the panel of positive samples. The number of samples tested and the results obtained are shown in the table (Table 5).

• Positive and negative predictive value
  A positive predictive value is probability that a person is actually affected by infection if the result was positive. A negative predictive value is probability that a person is actually healthy if the result was negative. The results obtained are shown in the table (Table 5).

• Likelihood ratio of the kit
  The likelihood ratio of the kit for a positive test is the ratio of probability that an individual from affected population is diagnosed as positive by the test and probability that a healthy individual is misdiagnosed as positive. The likelihood ratio of the kit for a negative test is the ratio of probability that an individual from affected population is misdiagnosed as negative by the test and probability that a healthy individual is diagnosed as negative. The results obtained are shown in the table (Table 5).

• Expected values in population
  Expected values in population are established based on the value results in a file of samples declared as negative and a file of samples declared as positive for the presence of specific antibodies. The results obtained are shown in the table (Table 5).

Table 5 Clinical performance

 | Parameter| Value| 95% Confidence Interval (CI)|
---|---|---|---|---
 | Diagnostic sensitivity (n 40)| 97.50%| 86.84–99.94%|
 | Diagnostic specificity (n 50)| 99.99%| 92.89–100.00%|
 | Positive predictive value (n 40)| 99.99%| 90.97–100.00%|
 | Negative predictive value (n 50)| 98.04%| 89.55–99.95%|
 | Likelihood ratio of the kit for a positive test| >100| –|
 | Likelihood ratio of the kit for a negative test| 0.025| –|
 | Expected values in healthy population| IP 0.15| IP 0.14–0.16|
 | Expected values in affected population| IP 2.90| IP 2.55–3.25|

Safety Precautions

The kit is intended for in vitro diagnostic use only. The sera used for controls were tested and found to be negative for HIV 1 and HIV 2, HBsAg, HCV, TPHA. In spite of this fact, they still need to be handled as potentially infectious materials. Some reagents contain the toxic component sodium azide or gentamicin, but in very low concentrations. Avoid contact with skin. The Stop Solution contains diluted acid solution. Avoid contact with eyes and skin. It is necessary to observe the local safety rules and regulations.
First aid
In case of contact with eyes, flush with copious amount of water and seek medical assistance. In case of contact with skin and clothing, remove all the contaminated clothes. Wash the skin with soap and plenty of running water. In case of contact with solutions containing plasma or clinical samples, disinfect the skin. In case of accidental ingestion, flush the mouth with drinking water and seek medical assistance.
Remnants disposal
All the materials used for performing the test must be treated as potentially infectious due to the contact with biological materials. Therefore they need to be disposed together with biological waste.
Expired kit disposal
Disassemble the kit and dispose the components as biological material. Discard the packaging material as required by local regulations.

Procedural Notes

In order to obtain reliable results, it is necessary to strictly follow the Instructions for Use. Always use clean preferably disposable tips and glassware. Microtitre Plate–in order to prevent water condensation on the surface of the microplate, always allow the bag with the microplate to warm up to room temperature before opening. Wash Solution–use high quality distilled water for preparing the working strength Wash Solution. Washing procedure–keep to the prescribed number of wash cycles and fill the wells to the upper edge. The soak time (i.e. interval between two different wash cycles during which the wells stay filled up with the Wash Solution) should be approx. 30-60 seconds. TMB-Complete–the vessel used for multichannel pipetting should not be used for other reagents. Do not return the surplus TMB-Complete from the pipetting vessel into the vial.
Non-reproducible results might be caused by improper methodology as following:

• insufficient mixing of reagents and samples before use
• improper replacement of vial caps
• using the same tip for pipetting different reagents
• reagent exposure to excessive temperature; bacterial or chemical contamination
• insufficient washing or filling of the wells (the wells should be filled to the upper edge), improper aspiration of Wash Solution remnants
• contamination of the well edges with Conjugate or samples
• using reagents from different kit lots
• contact of reagents with oxidants, heavy metals and their salts

Technical limitation of samples

Materials of a human origin from donor population listed in the intended use were used for manufacture and development of the kit. Kits are intended for use in general population, unless otherwise stated. When using samples from other specific populations (comorbid, immunocompromised, pregnant, paediatric population), the risk of a specific effect on the result of the applied test due to e.g. interference or cross-reactivity should be considered in the context of expert knowledge and current  scientific knowledge.

Other notes

• The kit might be used for sequential examinations. When preparing working strength solutions, use only the amount of reagents needed for the analysis.
• The kit might be used in all types of automatic EIA analysers. If necessary, TestLine Clinical Diagnostics s.r.o. can offer a certified modification of the Instructions for Use for the specific type of analyser.
• The producer cannot guarantee that the kit will function properly if the assay procedure instructions are not strictly adhered to.

References

1. Henriquez SA, Brett R, Alexander J, Pratt J, Roberts CW. Neuropsychiatric Disease and Toxoplasma gondii Infection. Neuroimmunomodulation. 2009. 16 (2):122–133.
2. Hill D, Dubey JP. Toxoplasma gondii: transmission, diagnosis and prevention. Clin Microbiol Infect. 2002. 8 (10): 634–640.
3. Howe DK, Sibley LD. Toxoplasma gondii Comprises Three Clonal Lineages: Correlation of Parasite Genotype with Human Disease. J Infect Dis. 1995. 172 (6): 1561–1566.
4. Johnson AM, Roberts H, Tenter AM. Evaluation of a recombinant antigen ELISA for the diagnosis of acute toxoplasmosis and comparison with traditional antigen ELISAs. J Med Microbiol. 1992. 37 (6): 404–409.
5. Naot Y, Remington JS. An Enzyme-Linked Immunosorbent Assay for Detection of IgM Antibodies to Toxoplasma gondii: Use for Diagnosis of Acute Acquired Toxoplasmosis. J Infect Dis. 1980. 142 (5): 757–766.
6. Saadatnia G, Golkar M. A review on human toxoplasmosis. Scand J Infect Dis. 2012. 44 (11): 805–814.
7. Sensini A. Toxoplasma gondii infection in pregnancy: opp ortunities and pitfalls of serological diagnosis. Clin Microbiol Infect. 2006. 12 (6): 504–512.
8. Teshager D, Getachew T, Mebratu A, Tesfaye S. Toxoplasmosis: Epidemiology with the emphasis of its public health importance. Merit Res J Med Med Sci. 2014. 2 (4): 97–108.

IFU Symbols

Summary of EIA Toxoplasma IgE Protocol

CONTACT

• TestLine Clinical Diagnostics s.r.o.
• Krizikova 68, 612 00 Brno, Czech Republic
• Tel.: +420 541 248 311
• FAX: +420 541 243 390
• E-mail: info@testlinecd.com.
• www.testlinecd.cz.
• www.testlinecd.com.

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