Human Treponema pallidum IgG (TP IgG) kit (ELISA) Instructions for Use? This kit is used to quantify the content of Treponema pallidum IgG (TP IgG) in human serum, plasma, tissue, cell supernatant and related liquid samples in vitro. Validity: 6 months? Storage conditions: 2-8 °C experimental principle kit using double antibody one-step sandwich enzyme-linked immunosorbent assay (ELISA). To the coated microwells pre-coated with Treponema pallidum IgG (TP IgG) antibody, the specimen, the standard, and the HRP-labeled detection antibody were sequentially added, and the cells were washed and thoroughly washed. Using the substrate TMB to develop color, TMB is converted to blue under the catalysis of peroxidase and converted to the final yellow color by the action of an acid. The color depth is positively correlated with treponema IgG (TP IgG) in the sample. The absorbance (OD value) was measured at 450 nm using a microplate reader to calculate the sample concentration. Sample processing and requirements 1. Serum: Whole blood samples should be placed at room temperature for 2 hours or 4 °C overnight, centrifuged at 1000g for 20 minutes, the supernatant can be detected, or the specimen can be stored at -20 ° C or -80 ° C, but Repeated freezing and thawing should be avoided. 2. Plasma: EDTA or heparin can be used as an anticoagulant. Centrifuge at 1000g for 2 minutes at 2 - 8 °C for 20 minutes within 30 minutes after collection, or store the specimen at -20 °C or -80 °C, but avoid repeated freezing and thawing. . 3. Cell culture supernatant or other biological specimens: centrifuge at 1000g for 20 minutes, take the supernatant to detect, or store the specimen at -20 °C or -80 °C, but avoid repeated freezing and thawing. Note: Specimen hemolysis will affect the final test results, so hemolysis specimens should not be tested. Reagents and equipment not required 1. Microplate reader (450nm) 2. High-precision sampler and tip: 0.5-10uL, 2-20uL, 20-200uL, 200-1000uL3. 37°C incubator 4. Distilled water Or deionized water kit composition name 96-well configuration 48-well configuration remarks microporous microplates 12 wells × 8 strips 12 wells × 4 no standard 0.5mL * 6 tubes 0.5mL * 6 tubes without sample dilution 6mL 3mL no Detection antibody-HRP 10mL 5mL No 20× Washing buffer 25mL 15mL Diluted substrate A 6mL 3mL without substrate B 6mL 3mL without stop solution 6mL 3mL without sealing film 2 sheets 2 sheets without instructions 1 part 1 without self-sealing One bag has no remarks: 1. The concentration of standard products is: 480, 240, 120, 60, 30, 15 pg/ml2. After a large number of normal specimen tests, the normal concentration values ​​of the specimens are within the detection range provided by the kit. Inside, during the experiment, 50 μL of the sample can be directly loaded. When part of the sample value exceeds the maximum standard concentration, the sample may be diluted with the sample dilution before performing the experiment. Cautions 1. Incubate strictly in accordance with the specified time and temperature to ensure accurate results. All reagents must reach room temperature 20-25 °C before use. Store the reagents refrigerated immediately after use. 2. Incorrect cleaning can result in inaccurate results. Make sure to drain the liquid in the well as much as possible before adding the substrate. Do not let the micropores dry during the incubation. 3. Eliminate residual liquid and fingerprints on the bottom of the board, otherwise it will affect the OD value. 4. The substrate coloring solution should be colorless or very light, and the substrate liquid that has turned blue cannot be used. 5. Avoid cross-contamination of reagents and specimens to avoid erroneous results. 6. Avoid direct exposure to strong light during storage and incubation. 7. After balancing to room temperature, open the sealed bag to prevent the water droplets from condensing on the cold slats. 8. Any reagents should not be exposed to strong gases emitted by bleaching solvents or bleaching solvents. Any bleaching component will destroy the biological activity of the reagents in the kit. 9. Cannot use expired products. 10. If the disease is likely to spread, all samples should be managed and the sample and test device processed in accordance with the prescribed procedures. The reagent preparation kit should be taken out of the refrigerated environment and should be equilibrated at room temperature before use. Dilution of 20× Wash Buffer: Distilled water was diluted 1:20, ie 1 part of 20× wash buffer plus 19 parts of distilled water. Procedure 1. Remove the required slats from the foil pouch after equilibrating for 20 min at room temperature. The remaining slats were sealed back to 4 °C with a ziplock bag. 2. Set standard and sample wells, add 50μL of standard concentration to each standard well; 3. Add 50μL of sample to be tested in the sample well; blank hole is not added. 4. In addition to the blank wells, add 100 μL of horseradish peroxidase (HRP)-labeled detection antibody to each well of the standard wells and sample wells, seal the wells with a sealing membrane, and incubate in a 37 ° C water bath or incubator. 60min. 5. Discard the liquid, pat dry on the absorbent paper, fill each well with the washing solution (350 μL), let stand for 1 min, remove the washing solution, pat dry on the absorbent paper, and repeat the washing 5 times (can also be washed with a washing machine) board). 6. Add 50 μL of substrate A and B to each well and incubate for 15 min at 37 ° C in the dark. 7. Add 50 μL of stop solution to each well, and measure the OD value of each well at a wavelength of 450 nm within 15 min. The experimental results are calculated by taking the OD value of the measured standard as the abscissa and the concentration of the standard as the ordinate. The standard curve is drawn on the coordinate paper or with the relevant software, and the linear regression equation is obtained, and the OD value of the sample is substituted into the equation. Calculate the concentration of the sample. Kit performance 1. Detection range: 15 pg/ml - 480 pg/ml. 2. Sensitivity: The minimum detection concentration is less than 1.0 pg/ml. 3. Specificity: Does not cross-react with other soluble structural analogs. 4. Repeatability: The coefficient of variation between the plates and the plates is less than 15%.
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