Immunoassay Data Handling - Part 2 Lecture Notes PDF

Immunoassay Data Handling - Part 2 BIOT6002: Lecture 14 Lecturer: Dr. Caroline A. Browne Learning Objectives Review Competitive Immunoassays Data handing Antibody stock concentrations %CV calculations Data handling for precision profiles Data handling for measures accuracy Reco...

Immunoassay Data Handling - Part 2 BIOT6002: Lecture 14 Lecturer: Dr. Caroline A. Browne Learning Objectives Review Competitive Immunoassays Data handing Antibody stock concentrations %CV calculations Data handling for precision profiles Data handling for measures accuracy Recovery Linearity Competitive Immunoassays Competitive Immunoassays Use either labelled Ag or Ab Both the labelled Ag and the unlabelled specimen Ag (or test sample analyte) compete for a limited amount of antibody. Less label measured in the assay means more of the unlabelled (test sample) Ag is present. Concentration of bound label is plotted against analyte concentration, generating an inverse standard curve Competitive Immunoassays Calibration Curves Antibody stock concentration The labelled antibody stock concentration you have been given for an immunoassay is 100mg/ml. You are instructed in the protocol to perform a 1 in 5000 dilution of the stock for the experiment. 1. What is the final Ab concentration in mg/ml after the dilution is performed? 2. How would you perform this dilution in the lab (What volume of Ab to what volume of buffer needed to get a 1 in 5000 dilution) in a final volume of 1ml. Answer: 1. 100 mg/ml/ 5000 = 0.02mg/ml 2. Part a = 1 ml /5000 = 0.2 ml of Ab required Part b = 1 ml – 0.2 ml = 0.8 ml buffer required Calculating SD on your calculator (casio) 1. Press MENU, then 2(Statistics ), then 1(1-Variable) 2. Input the data into the column.(Press = after inputting each data item) 3. When they are all entered press OPTN 4. Choose 3( 1-Variable Calc) 5. σx is the value for standard deviation Note: If you arrow down the calculator gives the median, Q1, Q3 and more. Precision – SD and % CV Calculate the % CV for the following standard for a competitive immunoassay Standard 3 : 0.555, 0.525, 0.533, 0.522, 0.515, 0.550, 0.540, 0.545, 0.510, 0.546 Answer: % CV = SD / mean x 100 Mean = 0.534 SD = 0.0147 %CV = 2.75 % Precision Profiles For the immunoassay precision profile shown what is the best estimate of the dynamic range of the assay at < 30% CV? For the immunoassay precision profile shown which of the following statements is true? A. Precision improves at low log conc. values B. Precision improves at high log conc. values C. At 30 % CV, a wider dynamic range is shown compared with 20 %CV D. At 30 % CV a narrower dynamic range is shown compared with 20 %CV Sensitivity The sensitivity of an ELISA is a function of the affinity of the Ab to the analyte, the capture efficiency of the plate, signal amplification, buffer and wash conditions, substrate type and detection method and the nature of the sample type being tested. The Lower Limit of Detection (LLD), also known as Minimum Detectable Concentration (MDC), is the lowest measurable value that is statistically different from zero. 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 the MDD or MDC value For competitive immunoassays, the mean -3 x SD is interpolated. Limits of Quantitation - non-competitive immunoassay For the non-competitive ELISA calibration curve shown in the diagram, estimate the Limit of Quantitation (LOQ) given the following: Mean OD450 of zero standard = 0.050 Standard Deviation (SD) of zero standard = 0.002 Answer: Use the following formula: LOQ = mean + (3 x SD) = Interpolate this Absorbance value from the graph to the x- axis. = 20pg/ml (on the x- axis) = Limit of quantitation for the immunoassay. 0.05 + (3 x 0.002) = 0.056 Limits of Quantitation - competitive immunoassay For this competitive ELISA calibration curve, estimate the LOQ given the following: Mean of zero standard (B/Bo) = 0.45 Standard Deviation (SD) of zero standard = 0.048 Calculate LOQ Mean – 3 x SD = 0.31 Interpolate this Absorbance value from the graph to the x-axis.