Immunoassay Validation - BIOT6002 Lecture 9 PDF

Immunoassay Validation BIOT6002: Lecture 9 Lecturer: Caroline A Browne, Ph.D. Learning Objectives Describe the validation process for immunoassays Understand the importance of the following parameters Precision Sensitivity Accuracy Selectivity and Specificity List comm...

Immunoassay Validation BIOT6002: Lecture 9 Lecturer: Caroline A Browne, Ph.D. Learning Objectives Describe the validation process for immunoassays Understand the importance of the following parameters Precision Sensitivity Accuracy Selectivity and Specificity List common sources of Imprecision Inaccuracy Interferences. Immunoassay Validation Validation is the process of Defining an analytical requirement Confirming that the method has performance capabilities consistent with application needs The method is fit for purpose The validation should follow a plan that includes the scope of the method, the method performance characteristics & acceptance limits. Immunoassay Validation Validation is necessary to verify that the method performance parameters are adequate for a particular analytical problem/application. A method just developed Revised method Review of QC indicates that an established method is changing with time When an established method is used in a different laboratory or with different instrumentation. Immunoassay Validation A Method Validation study should include these parameters Precision Sensitivity Accuracy Recovery Parallelism Linearity Selectivity and Specificity Method Validation - Precision Precision or Reproducibility is a statistical measure of variation between repeated determinations on the same sample. Calculated from mean, standard deviation & % CV at a particular analyte level. Precision profile is the performance (% CV) over full range of analyte concentration. Precision of instruments Error of pipettes. Blue lines are wavelength shifted by 1 nm Precision Profile For competitive assays precision decreases at low & high concentrations For non-competitive assays precision decreases at low concentrations Within run precision (intra-assay) – three concentrations of standard at n=20 Between run precision (inter-assay, inter-day)- three concentrations of standard at n=3 over 20 assays/days. Magnetic bead base immunoassay for Diclofenac - Calibration curve of the optimized assay (orange) and precision profile (purple); n = 6; error bars represent single standard deviation. The arrows indicate lower and upper limits of detection, and thus the measurement range of the assay (400 ng/L–300 µg/L) Sources of imprecision Reagent: Ab capture, Ab conjugate, calibrator, diluent, wash solution, Ab:Ag reaction: Timing, temperature, separation, washing. Enzyme substrate reaction: timing, temperature & quenching. Detection: Plate reader, calibration, filters Interference: non-specific, specific, high dose hook effect Sensitivity Limit of quantitation (LOQ) is the least concentration of analyte that can be determined with an acceptable level of precision. Calculated by assaying 10-20 replicates of zero standard (blank) Mean and standard deviation (SD) are calculated. For non-competitive immunoassays, the mean +3 x SD is interpolated from the standard curve as minimal detectable dose (MDD) value For competitive immunoassays, the mean -3 x SD is interpolated. Accuracy Accuracy is the ability of an assay to measure the true value of an analyte. Calculated as % error of mean. Bias is the measure of the difference between the measured and the true value. Assay bias can be Proportional: higher or lower % than true value Constant: higher or lower concentration than true value. Accuracy - Recovery Recovery studies: The ability of a test to recover/measure an incremental amount of an analyte from a sample matrix. Protocol: Add a known concentration of Analyte (A) to a matrix (B) and measure the concentration (C) in the immunoassay system. % recovery = C –B/A x 100 Accuracy - Parallelism (Relative Recovery) Tested by dilution experiments Sample/standard is diluted with appropriate diluent (e.g. at least three different dilutions per sample) & linearity checked. Good parallelism shows the fundamental principal of immunoassays that unknown Ag gives the same response as standard Ag. Accuracy - Linearity Is an indication that the responses are proportional and that final concentrations calculated from the curve are linear. Good linearity = accurate results Sources of Inaccuracy Poor recovery Cross reactivity Endogenous molecules structurally similar to analyte Metabolites with common cross-reactive epitopes Medications administered with structurally similar analytes Potential cross-reacting substances should be tested (e.g. spiking sample) across a range of concentrations Lack of specificity of Ab Low affinity Ab Method Validation Selectivity: “The ability of the bioanalytical method to measure and differentiate the analytes in the presence of components that may be expected to be present” Specificity: “The degree of interference by other substances also present in the sample while analysing the analyte.” Cross reactivity in immunoassays caused by: Endogeneous molecules structurally similar to analyte Metabolites with common cross-reactive epitopes Medications administered with structurally similar analytes Potential cross-reacting substances should be tested (e.g. By spiking sample) across range of concentrations Interferences Important in accuracy testing to prove the assay is free from non-specific interferences that can increase or decrease result of sample – classed as matrix effects. Matrix effects can be caused by: Alteration of effective analyte concentration by blocking/changing conformation of Ag (Interference with Ab binding) Effecting Ab binding site conformation by changes in ionic strength/pH. (c) Interference with detection system. Enzyme inhibitors Endogenous signal generating system  Method Comparison Comparison with reliable, validated method (reference method) Linear regression analysis performed & correlation coefficient estimated IFN Alpha: Luminex® Assay Vs Quantikine ELISA Kit IFN Alpha: Quantikine ELISA Kit Vs Simple Plex Assay Learning Objectives Describe the validation process for immunoassays Understand the importance of the following parameters Precision Sensitivity Accuracy Selectivity and Specificity List common sources of Imprecision Inaccuracy Interferences.