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BINDING ASSAY

Key Reagents 1. Antibodies 3. Matrices . Labeled analytes (tracer) 2.

Bioanalytical Method Development and Validation Including: 1. Selectivity 2. Calibration curve 4. Sensitivity 5. precision. Accuracy. Stability of analyte . recovery 3. Reproducibility 6.

Interference from substances physicochemically similar to analyte 2. Selectivity There are 2 selectivity issues should be considered: 1.1. Matrix effects .

Precision.2. Accuracy. Recovery Accuracy: • Determined by replicate analysis of samples containing known amounts of the analytes. • Measured using minimum 5 determination per concentration and minimum 3 concentrations in the range of expected concentration • The mean value should be within 20% of the actual value (except LLOQ not more than 25%) .

Precision: • Measured using minimum 5 determination per concentration and minimum 3 concentrations in the range of expected concentration • The precision determined at each concentration level should not exceed 20% of CV (except LLOQ not more than 25% of CV) • Precision is divided into: a. equipment. Within-run precision / intrabatch precision Assessment of the precision during a single analytical run b. reagents and laboratories . Between-run precision / interbatch precision Assessment of precision with time and may involve different analysts.

Recovery: • Recovery is the measured concentration relative to the known amount added to the matrix • For assay that employ extraction • Performed at three concentration .

Calibration Curve • Most are nonlinear and more concentration points may be recommended • Should consist of a minimum of 6 non-zero calibrator concentrations covering the entire range (including LLOQ.3. medium and high) analysed duplicate in each run • Should be prepared in the same matrix as the study sample . low.

. including quality controls and possibly incurred samples.4. Reproducibility Reproducibility of the method is assessed by replicate measurements using the assay. Sensitivity Sensitivity is defined as the lowest analyte concentration that can be measured with acceptable accuracy and precision 5.

including conditions at clinical site.6. shipment and at all other secondary sites • Stability samples should be compared to freshly made calibrators and or freshly made QCs • At least 3 replicates at each of the low and high concentrations should be assessed • Assessment of analyte stability should be conducted in the same matrix as that of the study samples and prepared from a freshly made stock solution . Stability • Pre-study stability evaluation should cover the expected sample handling and storage condition during the study.

e. c.• Conditions used in stability experiments should reflect situations likely to be encountered during actual sample handling and analysis. b. • Stability experiments including: a. Long-term Stability Bench-top Stability Stock Solution Stability Freeze and Thaw Stability Processed Sample • Stability sample results should be within 15% of nominal concentrations . d.