The effect of monoclonal antibody (mAb) affinity on the detection limit of enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR), and electrochemiluminescence (ECL) methods was evaluated using a panel of murine mAbs with affinities ranging from 0.057 to 340 nM. M1 and M7 are anti-idiotypic mAbs against a human mAb, ABX10, with dissociation equilibrium constant (KD) values of 0.057 and 7.2 nM, respectively. HP6030 and HP6002 are anti human IgG mAbs with KD values of 30 and 340 nM, respectively. The limit of detection (LOD) for these mAbs was determined using ELISA, SPR, and ECL technologies and was generally correlated with the rank order of their affinities. The LODs for M1, M7, HP6030, and HP6002 by ELISA were 17 +/- 13, 26,000 +/- 9,020, 344,000 +/- 271,000, and 792,000 +/- 1,050,000 ng/ml, respectively. According to an industry-suggested detection limit of 500 ng/ml, the ELISA was not sensitive enough for detecting M7, HP6030, and HP6002, demonstrating its limitation for detection of low- affinity mAbs. The SPR method lowered the LOD for M7 to 3,900 ng/ml, which was above the industry requirement. The ECL method lowered the LOD for all antibodies tested. Importantly, the ECL method lowered the LOD for M7 to 570 +/- 370 ng/ml, which is close to the industry requirement. Since the ECL method had demonstrated a high serum tolerance, its detection capability may be improved by using a higher percentage of serum in the assay matrix. Although a hook effect was observed with ECL methods, the methods could still detect anti-drug antibody (ADA) concentrations greater than 1 mg/ml, which minimizes concerns that high-titer ADA responses could be missed. The results demonstrated the superiority of an ECL method in detecting high- and low- affinity antibodies when compared to the ELISA and SPR methods.