Immunoassay Optimisation Lecture 8 PDF

Immunoassay Optimisation BIOT6002: Lecture 8 Lecturer: Caroline A Browne, Ph.D. Learning Objectives List the stages of assay development Understand what the 10 key immunoassay parameters for success are. Describe the optimisation parameters Reagents Coating Buff...

Immunoassay Optimisation BIOT6002: Lecture 8 Lecturer: Caroline A Browne, Ph.D. Learning Objectives List the stages of assay development Understand what the 10 key immunoassay parameters for success are. Describe the optimisation parameters Reagents Coating Buffers Wash solutions Stop solutions Specific antibodies Instrumentation Basic Steps for Developing and Running an Immunoassay 1. Establish assay critical success factors (i.e. sensitivity required). 2. Ensure appropriate antibody and antigen reagents are available. 3. Adsorb antigen or capture antibody to a solid surface. 4. Wash off unbound reagents. 5. Block nonspecific binding sites to reduce background. 6. Incubate the secondary antibody with the sample. 7. Wash off unbound reagents. 8. Incubate secondary antibody-conjugate with sample. 9. Wash off unbound reagents. 10. Incubate substrate to generate signal. 11. Calibration curve fitting, data analysis and quantitation by non-linear regression. Stages of Assay Development 1. Critical Success Factors: sensitivity, range, reagents etc. 2. Acquire reagents: Ab, analyte standards, sample matrices, labels, substrates. 3. Instrument Testing: Calibration, performance testing. 4. Proof of concept experiments: establish preliminary assay parameters, reagent suitability etc. 5. Assay Optimisation: Select format to be optimised 6. Assay Validation: robustness, %recovery analysis, reproducibility. 7. Method documentation (SOP): implementation. Immunoassay Optimisation It is important to establish a set of critical success factors before beginning the development, optimisation & validation of an immunoassay. Critical Success Factors to be considered: 1. Analyte to be measured 2. Sample matrices (e.g. Serum, culture media etc.) 3. Sources of Ab, analyte standards & detection reagents (labelled Ab, enzyme substrates) 4. Detection mode (colorimetric, fluorescence) & plate readers Immunoassay Optimisation 5. Type of immunoassay to develop - sandwich, competitive 6. Expected analyte concentration ranges to be measured: pg/ml, ng/ml or mg/ml. 7. Data analysis models & format for reporting results. 8. Recovery, accuracy & precision expected at the limits of quantitation & the measurable range. 9. Sample throughput, frequency of use, automation 10. Control samples to be used for optimisation, validation & Quality Control. Optimisation Parameters - Reagents 1. Quality of standards and antibodies. 2. Quantity of standards and antibodies. 3. Purity of standards and antibodies (when possible, antibodies are affinity purified). 4. Selectivity and specificity of antibodies. Optimisation Parameter - Coating Buffers 50 mM sodium bicarbonate, pH 9.6 0.2 M sodium bicarbonate, pH 9.4 PBS - 50 mM Phosphate, pH 8.0, 0.15 M NaCl Carbonate-bicarbonate Phosphate Buffer: 1.7 mM NaH2PO4, 98 mM Na2HPO4·7H2O, 0.1% NaN3, pH 8.5 TBS - 50 mM TRIS, pH 8.0, 0.15 M NaCl Optimisation Parameter: - Blocking Buffers 1% BSA or 10% host serum in TBS, or TBS with 0.05% Tween-20 Phosphate Buffer: 73 mM Sucrose, 1.7 mM NaH2PO4, 98 mM Na2HPO4·7H2O, 0.1% NaN3, pH 8.5 1% HSA in PBS Casein Buffer: Pierce Blocker cat# 37528 Protein Free Block: Pierce cat# 37573 Pierce has many blocking buffers that are available in their catalog. Heterophilic Blocking Reagent (HBR): Scantibodies Laboratory, Inc., cat# 3KC533 Coating and blocking Optimisation Parameter: Wash Buffers PBST, 0.05% Tween-20 TBST, 0.05% Tween-20 Enzymes & Substrates Horseradish peroxidase (HRP) substrates: TMB: 3, 3’, 5,5'-tetramethyl benzidine (colorimetric) OPD: o-phenylene diamine (colorimetric) ABTS: 2, 2'-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (colorimetric) Pierce Supersignal (chemiluminescent) Pierce QuantaBlu (chemiluminescent) Pierce QuantaRed (chemiluminescent) Pierce has other substrates that provide strong signal and sensitivity with HRP enzyme conjugates that are available in their catalogue. Alkaline phosphatase substrate: pNpp (p-Nitrophenyl Phosphate) Optimisation Parameter - Stop solutions HRP/TMB: 2M H2SO4 solution (at a 1:1 volume with the HRP/TMB substrate/enzyme solution) OPD: 3M H2SO4 solution, (at a 1:1 volume with the OPD substrate/enzyme solution) ABTS: 1% Sodium dodecyl sulfate (SDS) Optimisation Parameter - Specific Antibodies Sandwich immunoassay: matched pairs of Abs, one for analyte capture on a solid surface and one for detection that binds to the Ag/hapten/analyte. Competitive immunoassay: a single antibody specific for the hapten/analyte. Optimisation Parameter - Standards Enough standard should be obtained for use in the development phase, validation phase & the continued support of the method to avoid changing lots. Standard quality: Can vary from vendor to vendor & lot to lot. Standard stability: Information on the stability of a standard can be obtained from the vendor and their recommendations should be followed in storing the standards. Optimisation Parameter - Control samples Spiked Controls are created by adding a known concentration of the standard analyte into the matrix (e.g. Tissue culture, serum) Control samples are real samples where the Ag level has been determined by another validated method. Samples are aliquoted, frozen & used as control samples in each experiment to track assay performance. Optimisation Parameter - Instrumentation Instrument should be tested for both linearity & performance. Regular calibration required according to manufacturer's specifications Plate readers employ UV-Vis absorbance, fluorescence or chemiluminescence signals as the measured response. Linearity should be checked at the appropriate wavelength. Learning Objectives List the stages of assay development Understand what the 10 key immunoassay parameters for success are. Describe the optimisation parameters Reagents Coating Buffers Wash solutions Stop solutions Specific antibodies Instrumentation