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

Table 2 Kinetic parameters obtained from the optimization of the Peptidomatrix conditions for peptide MXR P2 and the serum from rabbit # 64850 (see Figure 2) (± SD, n = 3).

A : increasing attached peptide at four fixed levels of added antibody
	1 ^stAB Concentration 0.0005 serum/PBS	1 ^stAB Concentration 0.001 serum/PBS	1 ^stAB Concentration 0.005 serum/PBS	1 ^stAB Concentration 0.01 serum/PBS
Kd (μg/ml)	0.49 ± 0.25	0.89 ± 0.56	0.49 ± 0.097	0.24 ± 0.06
Amax (OD units)	0.076 ± 0.01	0.12 ± 0.01	0.22 ± 0.012	0.35 ± 0.024
D (OD units)	0.29 ± 0.008	0.3 ± 0.015	0.29 ± 0.009	0.3 ± 0.02
B: increasing added antibody at three fixed levels of attached peptide
	Attached peptide 0.25 μg/ml	Attached peptide 0.5 μg/ml	Attached peptide 1 μg/ml
Kd (serum dilutions)	0.003 ± 0.0007	0.003 ± 0.001	0.001 ± 0.0002
Amax (OD units)	0.35 ± 0.023	0.56 ± 0.06	0.49 ± 0.02
D (OD units)	0.38 ± 0.01	0.38 ± 0.02	0.38 ± 0.01
C: Competition assay – Kd determined when increasing added free peptide at two fixed levels of attached peptide
	Attached peptide 0.25 μg/ml	Attached peptide 0.5 μg/ml
Kd (μg/ml)	0.14 ± 0.03	0.97 ± 0.15
Amax (OD units)	0.29 ± 0.019	0.37 ± 0.016
D (OD units)	0.39 ± 0.01	0.35 ± 0.01