Aug 24, 2020
Copy of ELISA for quantification of human C5 in serum or plasma.
- Angel A Justiz-Vaillant1,
- Belkis Ferrer-Cosme2
- 1University of the West Indies St. Augustine;
- 2"Saturnino Lora Torres' Provincial Teaching Clinical Surgical Hospital. Cuba
Protocol Citation: Angel A Justiz-Vaillant, Belkis Ferrer-Cosme 2020. Copy of ELISA for quantification of human C5 in serum or plasma.. protocols.io https://dx.doi.org/10.17504/protocols.io.bj76krre
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: August 24, 2020
Last Modified: August 24, 2020
Protocol Integer ID: 40926
An anti-human C5 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. Human C5 present in the serum or plasma 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-C5 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-C5 antibody. The optimal dilution of this conjugate must be investigated.
The plate is washed following incubation to remove the unbound Streptavidin-HRP.
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 C5 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 C5 standard dilutions and the human C5 sample concentration is determined.