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Rat Heat Shock Protein 70 (HSP70) ELISA Kit Instructions for Use
Shanghai Xitang Biotechnology Co., Ltd. 021-55229872, 65333639
Rat Heat Shock Protein 70 (HSP70) ELISA Kit Instructions for Use
principle
This experiment used double antibody sandwich ABC-ELISA. The anti-rat HSP70 mAb was coated on the plate, the standard and the HSP70 in the sample were combined with the mAb, and biotinylated anti-rat HSP70 was added to form an immune complex attached to the plate, horseradish peroxidation. The enzyme-labeled Streptavidin is combined with biotin, and the substrate working solution is blue. Finally, the stop solution sulfuric acid is added, and the OD value is measured at 450 nm. The HSP70 concentration is directly proportional to the OD value, and the HSP70 in the specimen can be obtained by drawing a standard curve. concentration.
Kit composition ( 2-8 ° C preservation)
Coated Wells
96 holes
Enzyme Conjugate
12ml
10× specimen dilution (Sample Buffer)
12ml
20×Wash Buffer
50ml
Standards: 40ng / bottle
2 bottles
Substrate working fluid (TMB Solution)
12ml
Primary antibody working solution (Biotinylated Antibody)
12ml
Stop Solution
12ml
Prepare reagents and collect blood samples
1. Collect specimens: serum, plasma (EDTA), cell culture supernatant, tissue homogenate and other lysate products should be detected as soon as possible, stored at 2-8 ° C for 48 hours; longer time must be frozen (-20 ° C or -70 ° C) to save To avoid repeated freezing and thawing.
2. Standard solution preparation: Add 0.5 ml of distilled water before use and mix well to prepare a solution of 80 ng/ml. Set the standard tube 8 tube, the first tube plus the standard dilution 900ul, the second to the eighth tube to add the sample dilution 500ul. Add 100 ul of the standard solution of 80 ng/ml to the first tube, mix and aspirate 500 ul with the sampler, and transfer to the second tube. Repeat the dilution as described above, and remove 500 ul from the seventh tube and discard it. The eighth tube is a blank control.
3. The 10× specimen dilution was diluted 1:10 with distilled water (example: 1 ml concentrated dilution + 9 ml distilled water).
4. Washing solution: diluted 1:20 with distilled water (example: 1 ml concentrated washing solution added to 19 ml of distilled water)
Specimen activation method
1. Add 450 ul of the sample dilution to a 1.5 ml imported polypropylene tube and add 10 ul of serum or plasma samples.
2. Add 20ul 1 N HCI, cover tightly and mix up and down. Leave at 2-8 ° C for 60 ± 2 minutes.
3. Add 20ul 1 N NaOH, cover tightly, and mix up and down.
4. Use it immediately, or store at -20/-70 °C for 3 days. Multiply the dilution factor by 50 when calculating the result. ( Note: The level of different specimens HSP70 may vary greatly, please flexibly grasp the dilution according to the actual situation)
    5. Cell culture supernatant or tissue homogenate 10-fold dilution (410 ul of sample dilution + 50 ul sample + 20 ul of 1 N HCI + 20 ul of 1 N NaOH).
Test procedure
1. Loading: Add 100 ul of standard or test sample (activated) to each well. Mix the reaction plate thoroughly and let it stand at 37 °C for 120 minutes.
2. Wash the plate: Wash the plate thoroughly with washing solution 4-6 times, and dry it on the filter paper.
3. Add 100 ul of the first antibody working solution to each well. The reaction plate was thoroughly mixed and placed at 37 ° C for 60 minutes.
4. Wash the board: the same as before.
5. Add 100 ul of enzyme-labeled antibody working solution per well. The reaction plate was placed at 37 ° C for 30 minutes.
6. Wash the board: same as before.
7. Add 100 ul of substrate working solution to each well and let it react at 37 ° C for 15 minutes in the dark.
8. Add 100 ul of stop solution to each well and mix.
9. Measure the absorbance at 450 nm using a microplate reader within 30 minutes.
Result calculation and judgment
1. All OD values ​​should be subtracted from the blank value before calculation.
2. Take the standard products 8000, 4000, 2000, 1000, 500, 250, 125, 0 pg/ml as the abscissa and OD as the ordinate. Draw on the coordinate paper and draw the standard curve.
3. According to the OD value of the sample, find the corresponding HSP70 content on the graph, and then multiply the dilution factor.
Kit performance
1. Sensitivity: The minimum HSP70 detection concentration is less than 65pg/ml.
2. Specificity: Recombinant or natural rat HSP70 can be detected simultaneously. Does not cross-react with other cytokines in rats.
3. Repeatability: The coefficient of variation in the plate and plate is less than 12%.
Precautions
1. It is recommended to make double holes for the above standard holes and samples to be tested. The standard curve should be made at the same time for each measurement.
2. The washing process is critical. Insufficient washing will result in an accuracy error and an erroneous rise in the OD value.
3. After the slats are opened, the remaining slats should be sealed again to keep the slats dry .
4. This kit should be stored in a 4oC refrigerator.
5. This kit is for scientific research only and cannot be used for clinical diagnosis!