Immunoassay Data Handling PDF

Immunoassay Data handling BIOT6002: Lecture 13 Lecturer: Dr. Caroline A. Browne Learning Objectives Data handing Serial dilutions Calibration standard concentrations Antibody stock concentrations Data handling for precision profiles Homogenous v’s Heterogenous assays Homogeno...

Immunoassay Data handling BIOT6002: Lecture 13 Lecturer: Dr. Caroline A. Browne Learning Objectives Data handing Serial dilutions Calibration standard concentrations Antibody stock concentrations Data handling for precision profiles Homogenous v’s Heterogenous assays Homogenous immunoassays No separation required before analysis Reagents added at the same time, no wash/blocking steps required Can be competitive or non-competitive Heterogenous immunoassays Separation required prior to analysis Multiple steps of filtration, washing, and/or blocking required. Can be competitive or non-competitive Most common type of assay Direct Immunoassays Serial dilutions In a quantitative ELISA experiment you are asked to perform a serial 1 in 3 dilution from a stock standard (1200ng/ml) on a microtitre plate. 7 standards in total need to be prepared. What is the standard concentration after dilution in standards 3 & 6? Answer - Work out concentrations for each of the standards 1-7 in the plate by dividing by dilution factor (=3) as follows: Standard 1: = 1200ng/ml Standard 2: 1200/3 = 400ng/ml Standard 3: 400/3 = 133.3ng/ml Standard 4: 133.3/3 = 44.4ng/ml Standard 5: 44.4/3 = 14.8ng/ml Standard 6: 14.8/3 = 4.93ng/ml Standard 7: 4.93/3 = 1.65ng/ml Calibration Standard Concentrations Given a 2000ng/ml standard concentration of Antigen in an immunoassay, what is the concentration of Antigen after performing the following dilutions? 1) 1 in 200 2) 1 in 360 3) 1 in 20 Answer: Divide the antigen concentration by each of the dilution factors to give you the final antigen concentration 1) 2000/200 = 10ng/ml 2) 2000/360 = 5.56ng/ml 3) 2000/ 20 = 100ng/ml Indirect Immunoassays Antibody stock concentration The antibody stock concentration you have been given for an immunoassay is 200mg/ml. You are instructed in the protocol to perform a 1 in 2000 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 (i.e. What volume of Ab to what volume of buffer needed to get a 1 in 2000 dilution) in a final volume of 1ml. Answer: 1. 200 / 2000 = 0.1mg/ml = 0.1 x 1000 = 100mg/ml 2. Final volume required 1ml = 1000ml. Divide this volume by dilution factor (2000) 3. 1000/2000 = 0.5ml of Ab required in final volume of 1000ml of buffer Precision The following data for absorbance readings was generated for an Intra-assay precision study for a standard (n=10) in an immunoassay. Standard 3 : 0.645, 0.625, 0.633, 0.602, 0.615, 0.650, 0.640, 0.645, 0.610, 0.600 Calculate the precision for this standard 3 in terms of %CV. Answer: % CV = SD / mean x 100 Calculate the mean, SD for the ten replicates and input into equation to calculate %CV Mean = 0.627 SD = 0.0178 %CV = 2.84% Precision Profiles Sensitivity - What is the dynamic range of the assay? 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