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The detection range of this kit is from 96T12μg/ml to 400μg/ml. It is specifically designed for the quantitative determination of immunoglobulin G (IgG) in rat serum, plasma, and related liquid samples. The assay is based on the double-antibody sandwich ELISA method. A microplate is pre-coated with purified anti-rat IgG antibodies to serve as a solid-phase capture antibody. After adding the sample containing IgG, the antigen binds to the immobilized antibody. Then, HRP-conjugated anti-IgG antibody is introduced, forming a complex of antibody-antigen-enzyme-labeled antibody. Following thorough washing, the TMB substrate is added, which turns blue under the action of HRP and then changes to yellow when an acidic stop solution is added. The intensity of the color is directly proportional to the concentration of IgG in the sample. The optical density (OD) at 450 nm is measured using a microplate reader, and the IgG concentration is calculated by comparing the OD values with a standard curve.
The kit includes the following components:
1. 30× Washing Solution (20ml × 1 bottle)
2. Enzyme Standard Reagent (6ml × 1 bottle)
3. Enzyme-Labeled Coating Plate (12 wells × 8)
4. Sample Diluent (6ml × 1 bottle)
5. Color Reagent A (6ml × 1 bottle)
6. Color Reagent B (6ml × 1 bottle)
7. Stop Solution (6ml × 1 bottle)
8. Standard (800μg/ml) (0.5ml × 1 bottle)
9. Standard Dilutions (1.5ml × 1 bottle)
10. Instruction Manual (1 part)
11. Sealing Film (2 sheets)
12. Seal Bag
Sample requirements: Specimens should be processed as soon as possible after collection, following the relevant literature. If testing cannot be performed immediately, store the samples at -20°C, avoiding repeated freeze-thaw cycles. Samples containing NaN3 are not suitable for testing, as it may inhibit HRP activity.
Procedure:
1. Prepare standard dilutions according to the provided chart.
2. Add 50 μl of each standard, 40 μl of sample diluent, and 10 μl of sample into designated wells (final dilution factor: 5×).
3. Seal the plate and incubate at 37°C for 30 minutes.
4. Prepare the 30× washing solution by diluting with distilled water.
5. Wash the plate 5 times with the washing solution.
6. Add 50 μl of enzyme reagent to each well except the blank.
7. Incubate again at 37°C for 30 minutes.
8. Wash the plate again.
9. Add 50 μl of TMB A and 50 μl of TMB B, incubate at 37°C for 15 minutes away from light.
10. Add 50 μl of stop solution to each well to terminate the reaction.
11. Measure the absorbance at 450 nm within 15 minutes after stopping the reaction.
This detailed procedure ensures accurate and reliable quantification of rat IgG levels in various biological samples.
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