Table 4 Validated assay summary

 Screening cut point

Cut point factor = 1.11, based on a nonparametric estimate, and representing a 4.6% false-positive error rate

 Precision (%CV) intra-batch by operator

3.6 to 4.7% LPC (40.0 ng/mL)

2.5 to 4.5% MPC

4.1 to 7.6% HPC

1.3 to17.2% NC

 Precision (%CV) inter-batch overall (n = 12 assay by 2 operators)

10.5% LPC (3.45 ng/mL)^a

8.3% LPC (40.0 ng/mL)

8.5% MPC

7.2% HPC

 Hook effect (313 ng/mL to 40,000 ng/mL)

ECL values increase with increasing concentration and/or plateau at the highest level. No hook effect detected

 Selectivity of spiked monoclonal antibody

 10 Matrix lots of normal human serum

 10 Matrix lots of cancer human serum

80% of spikes prepared in normal samples recovered within 80-120%

70% of spikes prepared in cancer samples recovered within 80-120%

 Hemolyzed serum (approximately 1000 mg/dL)

HPC and LPC (40 ng/mL) spiked into hemolyzed serum samples correctly

score positive. Unspiked score negative

 Lipemic serum (visually lipemic)

Spike samples score positive. Unspiked score negative

 Sensitivity

The calculated sensitivity using mouse monoclonal antibody is 0.328 ng/mL and with the rabbit polyclonal antibody, 2.83 ng/mL. Both were determined without spiking-in drugs

 Drug tolerance

The screening assay is tolerant to 250 μg/mL of drug when 500 ng/mL of antibody is present

 Room temperature stability in serum: 23 h 55 min

%CV ≤ 20% for the replicates of PC samples and remain positive

 Refrigerated stability in serum: 24 h

%CV ≤ 20% for the replicates of PC samples and remain positive

 Freeze/thaw stability in serum: 1 to 5 cycles (−80 °C/RT)

%CV ≤ 20% for the replicates of PC samples and remain positive

 Robustness of screening assay

 Block (25 to 35 min)

 Master mix (170 to 190 min)

 Sample (50 to 70 min)

% Difference for the robustness timing is ≤ 20% compared with the recommended incubation times

 Confirmatory cut point

Cut point = 40.7% inhibition, based on a nonparametric estimate, representing a 1.0% false-positive rate

 Precision (%CV) of ECL values with drug intra-batch

HPC: 3.1%

MPC: 2.4%

LPC: 4.3% (40 ng/mL)

 Precision (%CV) of ECL values with drug inter-batch

HPC: 9.8%

MPC: 9.1%

LPC: 8.9% (40 ng/mL)

 Robustness of confirmatory assay

 Block (25 to 35 min)

 Master mix (170 to 190 min)

 Drug (57 to 65 min)

 Streptavidin plate (55 to 65 min)

% Difference for the robustness timing is ≤ 20% compared with

 Titer cut point

Calculated to represent a 0.1% false-positive rate, used to determine the end point titer of a sample titered in the method

 Precision of titer determination

Endpoint titers between analysts are within one dilution step for 3 of 4 samples and within 2 dilutions steps for the remaining sample.