HSP70 ELISA 试剂盒

• Anti-Hsp70 Immunoassay Plate
• 5X Hsp70 Extraction Reagent
• Recombinant Hsp70 Standard
• Standard and Sample Diluent
• 10X Wash Buffer Concentrate
• Anti-Hsp70 Biotinylated Antibody Concentrate
• Anti-Hsp70 Biotinylated Antibody Diluent
• Streptavidin: HRP Concentrate
• Streptavidin: HRP Diluent
• TMB Substrate
• Stop Solution
• Pre-treatment Buffer

1. Prepare Standard and samples in Standard and Sample Diluent.
2. Add 100 μL of Standard to appropriate wells.
3. Add 50 μL of Pre-Treatment Buffer to all sample wells.
4. Add 50 μL of sample to appropriate wells.
5. Cover plate with Plate Sealer and incubate at 37 °C for 2 hours.
6. Wash plate four times with 1X Wash Buffer.
7. Add 100 μL of Detection Antibody Working Solution to each well.
8. Cover plate with Plate Sealer and incubate at 37 °C for 2 hours.
9. Wash plate four times with 1X Wash Buffer as described in step
10. 10. Add 100 μL of Streptavidin-HRP Working Solution to each well.
11. Cover plate with Plate Sealer and incubate at room temperature for 30 minutes.
12. Wash plate four times with 1X Wash Buffer as described in step
13. 13. Add 100 μL of TMB Substrate to each well.
14. Develop the plate in the dark at room temperature for 30 minutes.
15. Stop reaction by adding 100 μL of Stop Solution to each well.
16. Measure absorbance on a plate reader at 450 nm.

Sample Handling
All blood components and biological materials should be handled as potentially hazardous. Follow universal precautions when handling and disposing of infectious agents.
100 µl of diluted sample is required per well.
Samples must be assayed in duplicate each time the assay is performed.
Samples should be frozen if not analyzed shortly after harvest. For long-term storage, aliquot and freeze samples. Avoid repeated freeze-thaw cycles when storing samples.
If particulate is present in samples, centrifuge prior to analysis.
If the integrity of the sample is of concern, make a note on the Plate Template and interpret results with caution.

Sample Dilution
Samples must first be diluted prior to testing.
Suggested starting dilutions for samples:
- For cell and tissue lysates, dilute samples 1:4 in Standard and Sample Diluent.
For example, dilute 35 µL of sample in 105 µL Standard and Sample Diluent. Mix well.
Note: If values for samples are not within the range of the standard curve, optimal sample dilutions need to be determined.

Prepare at least 125 µL of sample in Standard and Sample Diluent. Mix samples well prior to analysis.
Note: Plasma samples are not recommended.

1. Prepare Standard and samples in Standard and Sample Diluent.
2. Add 100 μL of Standard to appropriate wells.
3. Add 50 μL of Pre-Treatment Buffer to all sample wells.
4. Add 50 μL of sample to appropriate wells.
5. Cover plate with Plate Sealer and incubate at 37 °C for 2 hours.
6. Wash plate four times with 1X Wash Buffer.
7. Add 100 μL of Detection Antibody Working Solution to each well.
8. Cover plate with Plate Sealer and incubate at 37 °C for 2 hours.
9. Wash plate four times with 1X Wash Buffer as described in step
10. 10. Add 100 μL of Streptavidin-HRP Working Solution to each well.
11. Cover plate with Plate Sealer and incubate at room temperature for 30 minutes.
12. Wash plate four times with 1X Wash Buffer as described in step
13. 13. Add 100 μL of TMB Substrate to each well.
14. Develop the plate in the dark at room temperature for 30 minutes.
15. Stop reaction by adding 100 μL of Stop Solution to each well.
16. Measure absorbance on a plate reader at 450 nm.

Duplicate absorbance values should be within 10% of each other. Care should be taken when interpreting data with differences in absorbance values greater than 10%.

1. Prepare a standard curve to determine the amount of Hsp70 in an unknown sample. Plot the average absorbance obtained for each standard concentration on the vertical (Y) axis versus the corresponding Hsp70 concentration on the horizontal (X) axis using graph paper or curve-fitting software.

2. Calculate the Hsp70 concentration in unknown samples using the prepared standard curve. Determine the amount of Hsp70 in each unknown sample by noting the Hsp70 concentration (X axis) that correlates with the absorbance value (Y axis) obtained for the unknown sample.

3. Multiply the Hsp70 concentration obtained by the dilution factor to determine the amount of Hsp70 in the undiluted sample.