Aug 16, 2023
sCD40L ELISA Assay
- Fang Zhou1,2
- 1Massachusetts General Hospital;
- 2Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network
Protocol Citation: Fang Zhou 2023. sCD40L ELISA Assay. protocols.io https://dx.doi.org/10.17504/protocols.io.5jyl8pp2dg2w/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: August 16, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 86576
Keywords: ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson’s (ASAP)
Grant ID: ASAP-000312
Abstract
This protocol for the sCD40L ELISA kit (Cat#BMS6010, Invitrogen) is used to measure the concentration of sCD40L in serum and CSF samples in mice.
Prepare Wash Buffer.
Wash the microwell strips twice with 300 μL Wash Buffer per well. Allow the Wash buffer to sit for 10-15s before harshly drop it in the strain. Wash again.
Empty wells on the paper towel, use the strip immediately after washing.
Prepare the standard dilution as the protocol. Using Sample Diluent (provided) to dilute samples.
Add 100 μl of Sample Diluent in duplicate to the blank wells.
For serum samples, add 50 μl of Sample Diluent to the sample wells and then add 50 μl of serum samples to the sample well. For CSF samples, add 5 μl of Sample Diluent to the sample wells and then add 95 μl of serum samples to the sample well.
Prepare Bio-Conjugate (fresh) and add 50 μl of Bio-Conjugate to all wells.
Apply an adhesive film to the plate and let it incubate at room temperature (RT) for 2 hrs on a microplate shaker.
Remove adhesive film and empty wells. Wash 4 times using Wash Buffer.
Prepare Streptavidin-HRP and add 100 μl of diluted Streptavidin-HRP to all wells.
Apply an adhesive film to the plate and let it incubate at RT for 1 hr on a microplate shaker.
Remove adhesive film and empty wells. Wash 4 times using Wash Buffer.
Add 100 μl TMB substrate solution to all wells. Incubate the strips at RT until the color changes. Avoid light.
Add 100 μl the stop solution to all wells.
Measure the absorbance of each microwell on a spectro-photometer at a wavelength of 450 nm.