Aug 23, 2020

Public workspace ELISA for quantification of human C4 in serum or plasma.

DOI
dx.doi.org/10.17504/protocols.io.bj75krq6
  • 1University of the West Indies St. Augustine;
  • 2"Saturnino Lora Torres' Provincial Teaching Clinical Surgical Hospital. Cuba
  • University of the West Indies
  • angel.vaillant@sta.uwi.edu
Angel A Justiz-Vaillant
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DOI: dx.doi.org/10.17504/protocols.io.bj75krq6
Protocol Citation: Angel A Justiz-Vaillant, Belkis Ferrer-Cosme 2020. ELISA for quantification of human C4 in serum or plasma.. protocols.io https://dx.doi.org/10.17504/protocols.io.bj75krq6
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 23, 2020
Last Modified: August 23, 2020
Protocol Integer ID: 40925
An anti-human C4 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 C4 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-C4 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-C4 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 C4 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 C4 standard dilutions and the human C4 sample concentration is determined.