Computational protocol: Competition between bound and free peptides in an ELISA-based procedure that assays peptides derived from protein digests

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Protocol publication

[…] Polyclonal antibodies were custom made by Affinity Bioreagents (ABR, Golden, CO)Peptides: Synthetic peptides were obtained by a number of sources, ABR, Biosight Ltd (Karmiel, Israel) and the service department of the Hebrew University in Jerusalem [...] The data from the ELISA plate reader are fed into a data analysis template in the program Sigmaplot (SPSS, Chicago, IL). A plot of the OD450 vs. the concentration of the antibody or the concentration of the free peptide is drawn. The plot is then fitted, using a regression program, to a hyperbola fitting an ascending (Equation. 1) or a descending (Equation. 2) hyperbolic 3-parameter equation, as described in the text:Equation 1 Equation 2 where Amax is the calculated maximal amplitude of the curve, D the predicted minimum of the ELISA readings, corresponding essentially to the background signal, S the concentration of the antibody or the attached peptide (in equation 1) or the free peptide (in equation 2) and Kd is the concentration that gives one-half of the shift between maximum and minimum readings. […]

Pipeline specifications

Software tools BioSight, SigmaPlot, SPSS
Applications Drug design, Miscellaneous