Sep 03, 2020
ELISA for quantification of macrophage-colony stimulating factor (M-CSF) in human serum or plasma.
- 1University of the West Indies St. Augustine
- University of the West Indies
- angel.vaillant@sta.uwi.edu
Protocol Citation: Angel A Justiz-Vaillant 2020. ELISA for quantification of macrophage-colony stimulating factor (M-CSF) in human serum or plasma.. protocols.io https://dx.doi.org/10.17504/protocols.io.bks7kwhn
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: In development
We are still developing and optimizing this protocol
Created: September 03, 2020
Last Modified: September 03, 2020
Protocol Integer ID: 41535
Disclaimer
DISCLAIMER – FOR INFORMATIONAL PURPOSES ONLY; USE AT YOUR OWN RISK
The protocol content here is for informational purposes only and does not constitute legal, medical, clinical, or safety advice, or otherwise; content added to protocols.io is not peer reviewed and may not have undergone a formal approval of any kind. Information presented in this protocol should not substitute for independent professional judgment, advice, diagnosis, or treatment. Any action you take or refrain from taking using or relying upon the information presented here is strictly at your own risk. You agree that neither the Company nor any of the authors, contributors, administrators, or anyone else associated with protocols.io, can be held responsible for your use of the information contained in or linked to this protocol or any of our Sites/Apps and Services.
An anti-human macrophage-colony stimulating factor (M-CSF) coating antibody is adsorbed onto the microwells by incubation overnight at 4°C with carbonate-bicarbonate buffer.
Add 50 µl of human serum or plasma. M-CSF present in the serum sample binds to antibodies adsorbed into the microwells.
The microplate is blocked with 3% non-fat milk-PBS buffer and later wash to remove unbound proteins.
Fifty (50) µl of biotin-conjugated anti-M-CSF antibody is added. The optimal dilution must be investigated.
The microplate is rewashed with PBS-Tween 20 buffer, pH 7.4.
One hundred µl of streptavidin-HRP conjugate is added and it binds to the biotin-conjugated anti-M-CSF antibody.
The plate is washed following incubation to remove the unbound Streptavidin-HRP conjugate.
Add 100 µl of 3,3',5,5'- tetramethylbenzidine (TMB; Sigma-Aldrich) into each well.
Incubate the microwells in the dark for 15 min.
A colored product is formed in proportion to the quantity of M-CSF present in the sample or standard.
The reaction is terminated by addition of 100 µl 3M H2SO4 and the absorbance is measured at 450 nm.
A standard curve is made from 7 human M-CSF standard dilutions and the human M-CSF sample concentration is determined.
For better results place the microplate on a microplate shaker in every incubation.