ELISA/assay

Toxoplasma IgG ELISA Kit

DEIA-JY2121

96T

Toxoplasma IgG ELISA Kit

Toxoplasma

Toxoplasma IgG ELISA Kit

ELISA

Toxoplasma

Human

Quantitative and Qualitative

Serum and Plasma

96T

The kit is stored at 2 - 8°C (Avoid Direct Light), and not be frozen or thawed. The product is valid for 12 months. After opening, store at 2 - 8°C, it can be stable for 6 months, avoid contamination.

To determine detection of cross-reactive antibodies directed against different parameters sera were analyzed with CD ELISA classic Toxoplasma gondii IgG and a commercially available anti-Toxoplasma gondii IgG ELISA. Positive sera (10 sera each) for Cytomegalovirus IgG, Epstein-Barr Virus IgG, Herpes Simplex Virus 1 IgG, Measles Virus IgG, Parvovirus B19 IgG and Varicella Zoster Virus IgG have been tested as well as sera positive for rheumatoid factor (RF) and anti-nuclear antibodies (ANA). Within this internal evaluation potential cross-reactivities with seven RF and four ANA positive serum samples have been observed. All reactivities have been confirmed by positive or borderline results in the reference assay. The positive rate can be explained by the seroprevalence in the healthy population. Other cross-reactivities cannot be ruled out in general.

Human

Toxoplasma; Toxoplasma gondii; T. gondii

The Toxoplasma gondii IgG test is quantitative and qualitative immunoassays for the detection of human antibodies in serum or plasma directed against Toxoplasma gondii. The Toxoplasma gondii IgG test allows for the determination of immune status as well as for the detection of intrathecally synthesized antibodies for CSF diagnostics and, by using the corresponding avidity reagent, of IgG antibody avidity determination in order to differentiate acute from past infections.

Optimum results can only be achieved if the instructions are strictly followed. Only use ELISA reagents when using ELISA immunoassays. The components must not be exchanged for reagents of other manufacturers. Standard and control sera of ELISA immunoassays are defined exclusively for the test kit to be used and must not be used in other lots. Washing solution, substrate and stop solution can be used for all ELISA immunoassays irrespective of the lot and the test. Each ELISA test contains a ready-to-use sample dilution buffer. In some cases the use of special dilution buffers is necessary to guarantee consistent quality and reliable results. The dilution buffers can be used irrespective of the lots. There are three different conjugate concentrations for each immunoglobulin class (IgA, IgG, IgM), indicated on the label as + (low), ++ (medium) and +++ (high). Conjugates with the same concentration and of the same immunoglobulin class are interchangeable and can be used for other ELISA immunoassays irrespective of the lot and the test. Dilution or alteration of the reagents may result in a loss of sensitivity. Use aseptic techniques when removing aliquots from the reagent tubes to avoid contamination. Reproducibility of test results is dependent on thorough mixing of the reagents. Agitate the flasks containing control sera before use and also all samples after dilution (e.g. by using a vortex mixer). Be sure to pipette carefully and comply with the given incubation times and temperatures. Significant time differences between pipetting the first and last well of the microtiter plate when dispensing samples and control sera, conjugate or substrate can result in different pre-incubation times, which may influence the precision and reproducibility of the results. Avoid exposure of reagents to strong light during storage and incubation. Adequate washing avoids test unspecificities. Therefore, the washing procedure should be carried out carefully. All of the flat bottom wells should be filled with equal volumes of washing buffer. At the end of the procedure ensure that the wells are free of all washing buffer in order to avoid uncontrolled dilution effects. Avoid foaming! Reagents must be tightly closed after use to avoid evaporation and contamination. Take care not to mix-up the caps of the bottles and/or vials. The ELISA immunoassay is only valid if the lot-specific validation criteria on the quality control certificate are fulfilled.

The ELISA (Enzyme Linked Immunosorbent Assay) is an immunoassay, which is particularly suited to the determination of antibodies in the field of infectious serology. The reaction is based on the specific interaction of antibodies with their corresponding antigen. The test strips of the microtiter plate are coated with specific antigens of the pathogen of interest. If antibodies in the patient´s serum sample are present, they bind to the fixed antigen. A secondary antibody, which has been conjugated with the enzyme alkaline phosphatase, detects and binds to the immune complex. The colourless substrate p-nitrophenylphosphate is then converted into the coloured product p-nitrophenol. The signal intensity of this reaction product is proportional to the concentration of the analyte in the sample and is measured photometrically.

1. ELISA Microplate: 12 × 8 Break apart microtiter test strips each with eight antigen coated single wells. The coating material is inactivated. 2. Standard Serum (ready-to-use) 2 × 2 ml Human serum in protein containing phosphate buffer; negative for anti-HIV Ab, HBsAg (Hepatitis B-Virus surface antigen) and anti-HCV Ab; preservative: 3. Negative Control Serum (ready-to-use) 2 ml Human serum in protein containing phosphate buffer; negative for anti-HIV Ab, HBsAg (Hepatitis B-Virus surface antigen) and anti-HCV Ab; preservative: 4. Anti-Human IgG Conjugate (ready-to-use) 13 ml Anti-human IgG polyclonal antibody, conjugated to alkaline phosphatase, stabilised with protein stabilisation solution; preservative: 5. Washing Solution Concentrate (sufficient for 1000 ml) 33.3 ml Sodium chloride solution with Tween 20 and 30 mM Tris/HCl, pH 7.4; preservative: 6. Dilution Buffer (ready-to-use) 2 × 50 ml Protein containing phosphate buffer with Tween 20; preservative: 7. Stopping Solution (ready-to-use) 15 ml 8. Substrate (ready-to-use) 13 ml Para-nitrophenylphosphate in solvent free buffer; preservative: 9. Quality control certificate with standard curve and evaluation table 2 pages (quantification of antibodies in IU/ml or U/ml)

1.Common laboratory equipment 2.Photometer for microtiter plates with filter, wavelength 405 nm, recommended reference wavelength 620 nm - 690 nm (e.g. 650 nm) 3.Microtiter plate washer 4.Incubator 37°C 5.Moist chamber 6.Distilled water 7.Click-Clips 8.Optional: control

Lipaemic, hemolytic or icteric samples (serum or plasma) should only be tested with caution. Obviously contaminated samples should not be tested. Serum or plasma (EDTA, citrate, heparin) collected according to standard laboratory methods are suitable samples. Samples must not be thermally inactivated. Dilution of Samples Before running the test, patient samples (V1) must be diluted in dilution buffer (V2) as follows: Sample Storage The patient's samples should not be stored for more than 7 days at 2 – 8 °C. Extended storage is possible at  -20 °C. Avoid repeated freezing and thawing of samples. Diluted samples can be stored at 2 – 8 °C for one week.

Bring all reagents to room temperature before testing. 1. Microtiter Test Strips The microtiter test strips labeled with abreviations for pathogen and immunoglobulin class are packed with a desiccant in an aluminum bag. To open the aluminum bag of the microtiter plate please cut off the top of the marked side only, in order to guarantee proper resealing. Take unrequired cavities out of the frame and put them back into the aluminum bag. Close bag carefully to ensure airtight conditions. Do not use the strips if the aluminum bag is damaged or if the bag with remaining strips and desiccant was not properly resealed. 2. Negative control Sera / Standard Sera (ready-to-use) Negative control and standard sera are ready-to-use and must not be diluted any further. For each test run - independent of the number of microtiter test strips to be used – negative control and standard sera must be included. Standard sera should be set up in duplicate. 3. Anti-human IgG AP-Conjugate (ready-to-use) The required conjugate concentration (+, ++, +++) is indicated on the quality control certificate. Please refer also to the specification on the label. Avoid contamination. 4. Washing Solution (Concentrate) Dilute washing buffer concentrate (V1) 1:30 with aqua dest. to a final volume of V2. Bottles used for the working dilution should be cleaned regularly. Discard cloudy solutions. Example: 5. Dilution Buffer for Samples (ready-to-use) Discard cloudy solutions. 6. Substrate (ready-to-use) Substrate in unopened bottle may have a slightly yellow coloring, which does not reduce the quality of the product! Avoid contamination. 7. Stopping Solution (ready-to-use)

Manual Test Procedure 1.Place the required number of cavities in the frame and prepare a protocol sheet. 2.Add each 100 μl of diluted sample or ready-to-use negative control/standard sera into the appropriate wells of microtiter test strips. Spare one well for substrate blank, e.g.: 3.Sample incubation for 60 minutes (+/- 5 min.) at 37°C (+/- 1°C) in moist chamber 4.After incubation wash all wells with washing solution (by automated washer or manually): - aspirate or shake out the incubation solution - fill each well with 300 μl washing solution - aspirate or shake out the washing buffer - repeat the washing procedure 3 times (altogether 4 times!) - dry by tapping the microtiter plate on a paper towel 5.Addition of conjugate Add 100 μl of the ready-to-use IgG conjugate to the appropriate wells (except substrate blank) 6.Conjugate incubation for 30 minutes (+/- 1 min.) at 37°C (+/- 1°C) in moist chamber. 7.After incubation wash all wells with washing solution (see above). 8.Addition of substrate Add 100 μl of ready-to-use substrate solution to each well (including well for substrate blank!) 9.Substrate incubation for 30 minutes (+/- 1 min.) at 37°C (+/- 1°C) in moist chamber. Ensure dark incubation. 10.Stopping of the reaction Add 100 μl stopping solution to each well, shake microtiter plate gently to mix. 11.Read extinction Read optical density (OD) within 60 minutes at 405 nm against substrate blank, reference wave length between 620 nm and 690 nm (e.g. 650 nm). Automated Test Procedure CD ELISA are validated for use with Immunomat (using the following consumables: VT124, VT111, VT112) and suited for processing on similar analyzers. For processing on the Immunomat the current software version including reagent check has to be used. The automated processing is performed analogous to manual use. Please note, that under special working-conditions internal laboratory adaptations of the substrate incubation times may be necessary.

ELISA controls (external Positive Control / Accuracy Control) For the periodic verification of the test method, in order to fulfil the requirements of laboratory internal quality management systems, we recommend using CD ELISA controls to determine precision and accuracy of CD ELISA classic test runs. CD ELISA controls are separately available and the usage is described in specific instruction manuals. CD ELISA controls are not available in all countries and the customer should consult the local distributor.

The mathematical curve fitting for antibody quantification with CD ELISA classic immunoassays is based on the 4-parameter logistic (4 PL) function. The 4 parameters A, B, C, and D are representative for the exact shape of the standard curve: Parameter A: Lower asymptote (OD) Parameter B: Slope of the curve Parameter C: Inflection point Parameter D: Upper asymptote (OD) Institut CD establishes a lot-specific 4 PL standard curve for each CD ELISA classic immunoassay in multiple test runs under optimal test conditions. The four parameters are indicated on the quality control certificate of each individual CD classic test. For the adaption of the test level to the given 4 PL standard curve the correction factor F is calculated by dividing the standard reference OD value indicated on the quality control certificate with the measured, and consequently test run-specific, standard OD value. By multiplying the OD values obtained from patient samples with the correction factor F, the level of each individual test run is adjusted to the given 4 PL standard curve. Thereby, interassay deviations are compensated for and antibody activities can be directly evaluated from the 4 PL standard curve. After subtraction of the substrate blank from all measured OD values and calculation of the mean OD value of the standard serum (STD), tested in duplicate, the evaluation of antibody activities from the optical measurement signals (OD) of patient samples can be performed with the 4PL function presented above. The determination of antibody activity in IU/ml (International Units/ml) for the CD ELISA classic Toxoplasma gondii IgG test is referenced to the second International Standard for Toxoplasma gondii (1980) with 2000 IU per vial. Qualitative Evaluation For the CD ELISA classic test evaluation a lot-specific quality control certificate with standard curve and an evaluation table is included in the test kit so that the obtained OD values may be assigned to the corresponding antibody activities. The substrate blank must be subtracted from all OD values prior to evaluation. Mean OD value of the standard serum (STD), tested in duplicate, has to be used. Method 1: In the first line of the table, several ranges of OD values for the standard serum are depicted covering the whole standard validity range. According to the measured mean OD value of the standard serum, the corresponding column can be chosen. This column contains the information of upper and lower cut-off OD values to allow evaluation of the patient sample. OD values below the lower cut-off are evaluated negative and values above the upper cut-off are evaluated positive. Implementation of the correction factor F is not necessary in the context of the evaluation table. Method 2: Qualitative Evaluation To fix the cut-off ranges multiply the mean value of the measured standard OD with the numerical data of the quality control certificate (see special case formulas), e.g.: OD = 0.502 x MW(STD) with upper cut-off OD = 0.352 x MW(STD) with lower cut-off If the measured mean absorbance value of the standard serum is 0.64 OD, the range of the cut-off is in between 0.225-0.321 OD. Calculation example: Standard serum mean OD = 0.64 Upper cut-off: OD = 0.502 x 0.64 = 0.321 Lower cut-off: OD = 0.352 x 0.64 = 0.225 Criteria of Validity 1. The substrate blank must be 2. The negative control must be negative. 3. The mean OD value (after subtraction of the substrate blank!) of the standard serum must be within the validity range, which is given on the lot specific quality control certificate. 4. The variation of OD values of the standard serum must not be higher than 20 %. 5. If these criteria are not met, the test is not valid and must be repeated.

To determine the performance characteristics of the CD ELISA classic Toxoplasma gondii IgG test, an external comparison study, utilizing sera from 450 patients and healthy individuals, was performed in parallel with an indirect immunofluorescence test. The results indicate a sensitivity of 98.2 % and specificity of 99.4 %. An internal study was also carried out using sera from 994 pregnant women. An indirect immunofluorescence test was also used as the reference test and discrepant results were further tested in the Sabin-Feldman-Test to determine their true status. The results indicate a sensitivity of 98.2 % and specificity of 99.8 %.

The borderline ranges of the CD ELISA classic Toxoplasma gondii IgG tests are specified on the quality control certificates and indicate the range of borderline test results. Values below this range indicate a negative test result; values above the borderline range are interpreted positive. The limits of quantification are specified on the quality control certificate of the CD ELISA classic Toxoplasma gondii IgG. The linearity of dilution within this range has been demonstrated in comprehensive evaluation studies. In case a patient sample shows a test result above the upper limit of quantification, the sample may be tested at a higher dilution. The resulting antibody activity must then be multiplied by the additional dilution factor. 5 - 500 IU/mL

To determine the influence of interfering substances, sera with different reactivities were analyzed with CD ELISA classic Toxoplasma gondii IgG/IgM. No interferences have been detected for sera with concentrations up to 2.00 g/L Hämoglobin, 11.50 g/L Lipemia/Triglyceride or 0.201 g/L Bilirubin (conjugated and unconjugated).

The Toxoplasma gondii IgG test is quantitative and qualitative immunoassays for the detection of human antibodies in serum or plasma directed against Toxoplasma gondii. The Toxoplasma gondii IgG test allows for the determination of immune status as well as for the detection of intrathecally synthesized antibodies for CSF diagnostics and, by using the corresponding avidity reagent, of IgG antibody avidity determination in order to differentiate acute from past infections.

1. ELISA Microplate: 12 × 8 Break apart microtiter test strips each with eight antigen coated single wells. The coating material is inactivated. 2. Standard Serum (ready-to-use) 2 × 2 ml Human serum in protein containing phosphate buffer; negative for anti-HIV Ab, HBsAg (Hepatitis B-Virus surface antigen) and anti-HCV Ab; preservative: 3. Negative Control Serum (ready-to-use) 2 ml Human serum in protein containing phosphate buffer; negative for anti-HIV Ab, HBsAg (Hepatitis B-Virus surface antigen) and anti-HCV Ab; preservative: 4. Anti-Human IgG Conjugate (ready-to-use) 13 ml Anti-human IgG polyclonal antibody, conjugated to alkaline phosphatase, stabilised with protein stabilisation solution; preservative: 5. Washing Solution Concentrate (sufficient for 1000 ml) 33.3 ml Sodium chloride solution with Tween 20 and 30 mM Tris/HCl, pH 7.4; preservative: 6. Dilution Buffer (ready-to-use) 2 × 50 ml Protein containing phosphate buffer with Tween 20; preservative: 7. Stopping Solution (ready-to-use) 15 ml 8. Substrate (ready-to-use) 13 ml Para-nitrophenylphosphate in solvent free buffer; preservative: 9. Quality control certificate with standard curve and evaluation table 2 pages (quantification of antibodies in IU/ml or U/ml)

See the individual product datasheet

See the individual product datasheet

See the individual product datasheet

See the individual product datasheet

See the individual product datasheet