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| Catalog # | Availability | Size | Quantity | Unit Price | Save For Later Wish List | |
|---|---|---|---|---|---|---|
| EKFT18485RAT-96 | 2 weeks | 96T | $600.00 |
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| EKFT18485RAT-48 | 2 weeks | 48T | $420.00 |
Product Overview | |||||||||||||||||||||||||||
| Name | Rat Toxoplasma-Immunoglobulin G (TOX-IgG) ELISA Kit | ||||||||||||||||||||||||||
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| Reactivity | Rat | ||||||||||||||||||||||||||
| Sensitivity | Qualitative | ||||||||||||||||||||||||||
| Assay Range | Qualitative | ||||||||||||||||||||||||||
| Detection Method | Indirect ELISA | ||||||||||||||||||||||||||
| Sample type(s) | Serum, Plasma, Cell Culture Supernatant, cell or tissue lysate, Other liquid samples | ||||||||||||||||||||||||||
| Principle of the Assay | |||||||||||||||||||||||||||
| This kit was based on indirect enzyme-linked immune-sorbent assay technology. TOX-Ag was pre-coated onto 96-well plates. The test samples were added to the wells, unbound conjugates were washed away with wash buffer. Then added HRP- Anti-Rat IgG Antibody, if there were any TOX-IgG in the samples, it would form a TOX-Ag- TOX-IgG- HRP- Anti-Rat IgG Antibody complex.The substrate TMB was added for color development, and the presence or absence of TOX-IgG antibody was determined according to OD value after colorimetry on the enzyme label. | |||||||||||||||||||||||||||
| Sample Collection and Storage | |||||||||||||||||||||||||||
| Isolate the test samples soon after collecting, then, analyze immediately (within 2 hours). Or aliquot and store at -20℃ for long term. Avoid multiple freeze-thaw cycles. Serum: Coagulate the serum at room temperature (about 1 hours). Centrifuge at approximately 1000 × g for 15 min. Analyze the serum immediately or aliquot and store at -20℃. Note: Samples to be used within 5 days may be stored at 2-8℃, otherwise samples must be stored at -20℃ (≤1 month) or -80℃(≤2 months) to avoid loss of bioactivity and contamination. Hemolyzed samples are not suitable for use in this assay. Wash Buffer Preparation: Dilute 50mL of Concentrated Wash Buffer into 950 mL of Wash Buffer with deionized or distilled water. | |||||||||||||||||||||||||||
| Kit Components | |||||||||||||||||||||||||||
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| Precautions for Use | |||||||||||||||||||||||||||
| To inspect the validity of experiment operation and the appropriateness of sample dilution proportion, pilot experiment using standards and a small number of samples is recommended. After opening and before using, keep plate dry. Before using the kit, spin tubes and bring down all components to the bottom of tubes. Storage TMB reagents avoid light. Washing process is very important, not fully wash easily cause a false positive and high background. Duplicate well assay is recommended for both standard and sample testing. Don’t let microplate dry at the assay, for dry plate will inactivate active components on plate. Don’t reuse tips and tubes to avoid cross contamination. Please do not mix the reagents in different kits of our company. Do not mix reagents from other manufacturers. To ensure accurate results, proper adhesion of plate sealers during incubation steps is necessary. | |||||||||||||||||||||||||||
| Material Required But Not Supplied | |||||||||||||||||||||||||||
| 1.Microplate reader (wavelength: 450nm) 2.37℃ incubator 3.Automated plate washer 4.Precision single and multi-channel pipette and disposable tips 5.Clean tubes and Eppendorf tubes 6.Deionized or distilled water | |||||||||||||||||||||||||||
| Manual Washing | |||||||||||||||||||||||||||
| Discard the solution in the plate without touching the side walls. Clap the plate on absorbent filter papers or other absorbent material. Fill each well completely with 350ul wash buffer and soak for 1 to 2 minutes, then aspirate contents from the plate, and clap the plate on absorbent filter papers or other absorbent material. Repeat this procedure two more times for a total of THREE washes. | |||||||||||||||||||||||||||
| Automated Washing | |||||||||||||||||||||||||||
| Aspirate all wells, then wash plate THREE times with 350ul wash buffer. After the final wash, invert plate, and clap the plate on absorbent filter papers or other absorbent material. It is recommended that the washer be set for a soaking time of 1 minute. | |||||||||||||||||||||||||||
| Assay Procedure | |||||||||||||||||||||||||||
| Remove the kit from the refrigerated environment and let it equilibrate at room temperature for 30 minutes before use 1. Label the sample wells, 3 Negative Controls, 2 Positive Controls and 1 blank wells. 2. Add 100μL Negative Controls and Positive Controls to each wells(except blank well). 3. Add 100μL sample dilution buffer to sample wells and then add 10μL sample serum. Gently tap the plate to ensure thorough mixing. Seal the plate with a cover and incubate at 37℃ for 30 min. 4. Remove the cover, and wash plate 5 times with Wash buffer and let the wash buffer stay in the wells for 0.5-1 minute each time. Wash the board 5 times in a row and pat dry the last time. 5. Add 100 µL HRP- Anti-Rat IgG Antibody to each well, except blank well 6. Seal the plate with a cover and incubate at 37℃ for 30 min. 7. Remove the cover, and wash plate 5 times with Wash buffer and let the wash buffer stay in the wells for 0.5-1 minute each time.Wash the board 5 times in a row and pat dry the last time. 8. Add 50 μl of TMB substrate A and 50 μl of TMB substrate B into each well. Gently tap the plate to ensure thorough mixing. Cover the plate and incubate at 37℃ in dark within 10 min. And the shades of blue can be seen in the Positive Controls. Negative Controls wells show no obvious color. 9. Add 50 μl of Stop solution into each well and mix thoroughly. The color changes into yellow immediately. 10. Read the O.D. absorbance at 450 nm in a microplate reader immediately after adding the stop solution. (Use the blank well to set zero)(Readings are taken within 30 minutes of termination of reaction) | |||||||||||||||||||||||||||
| Data Analysis | |||||||||||||||||||||||||||
| 1. Calculatin of the Cutoff Value Cutoff Value = NCx+0.1 NCx: Mean Absorbance of Negative Control(When the negative mean A value is less than 0.05, it is calculated as 0.05.When the negative mean A value is greater than or equal to 0.05, it is calculated according to the actual value.) 2. Determination of results Sample with absorbance values ＜ Cutoff Value are NON-REACTIVE and are considered NEGATIVE for TOX-IgG. Sample with absorbance values ≥Cutoff Value are considered POSITIVE for TOX-IgG. 3. Quality control The blank well (only adding TMB and Stop solution) should not be greater than 0.08. The positive control (PC) A value was greater than 0.3. The negative mean A value was less than 0.08. | |||||||||||||||||||||||||||
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ADMEbio Team
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ADMEbio Team
