1 Non Specific Binding (NSB) in Antigen-Antibody Assays Chem 395 Spring 2007 Instructor : Dr. James Rusling Presenter : Bhaskara V. Chikkaveeraiah
2 OUTLINE Immunoassays Introduction Factors contributing to Non-specific binding (NSB) NSB Blocking agents Minimizing NSB NSB in Immunoassay.
3 Immunoassay Immunoassay A biochemical test-measures levels of a particular molecule in biological samples- e.g. serum uses antibody binding to its antigen (specific binding). Antigen Ab1 Ab 2 Substrate Importance Detecting a disease at very early stage, at lowest antigen concentration. Accuracy of the detection. Non-specific binding (NSB) affects these two factors
4 Non Specific Binding : What is it? Secondary antibody Antigen Primary Antibody PG electrode In addition to binding to receptors of interest, sec. antibody may also bind to other sites. Binding to the receptor of interest is called specific binding, while binding to the other sites is called nonspecific binding (NSB). NSB can be minimized by saturating the unoccupied binding sites with a blocking reagent (NSB agent) without taking active part in specific assay reaction.
5 Immunoassay without NSB blocking agent Enzyme labelled Secondary antibody Antigen Primary Antibody PG electrode I Voltage+H 2 O 2 Signal t
6 Immunoassay with NSB Blocking agent Antigen Enzyme labeled Secondary antibody Primary antibody Blocking agent PG Elecrode I Voltage+H 2 O 2 Signal t
7 Properties of the blocking agent. Inhibit NSB (passive or covalent) of assay components to the surface, Inhibit non-specific protein - protein interaction. Exhibit no cross reactivity with subsequent assay components ( antibodies, protein ) Not disrupt the bonds that immobilize the specific protein or biomolecule to the surface. Exhibit consistent, reproducible performance with every lot.
9 Detergent Blockers Disrupt ionic and hydrophobic biomolecule-surface bonds. Inhibits enzyme-substrate reactions. Inexpensive, stable, can be stored at room temperature ( wash buffers) strips off loosely bound molecules in wash steps % is commonly used.
10 Protein Blockers Blocks the non occupied sites on the surface. Space out and stabilize biomolecules bound to the surface to reduce the steric hindrance. Bovine serum albumin (BSA) Widely used, Inexpensive, blocks non specific protein-surface binding. ~1 3% ic commonly used. Fish Gelatin Mainly blocks protein-protein(ab1-ab2) interactions Whole sera Blocks biomolecule-surface passive, covalent interactions, proteinprotein interactions, and acts as protein stabilizer.
11 Factors affecting NSB Ab1 / Ab2 ratio ( ~ 500:1) Type of blocking agent used. Bovine serum albumin, Casein, etc. Washing steps Consistent and vigorous.
12 PSMA Immunoassay (Prostate Specific Membrane Antigen) HRP labeled anti-psma1 PSMA anti-psma2 Blocking agent SWCNT I PG Electrode t Voltage+H 2 O 2 Signal
13 Effect of blocking agents in PSMA Immunoassay PBS buffer % Tween % BSA PBS buffer % Tween % Casein ng ng ng Control ng Control t / s PSMA ng/ml 10 ng/ml 100 ng/ml Control 0 ng/ml Current na 345 ± ± ± t / s PSMA Current ng/ml na 10 ng/ml 325 ± ng/ml 583 ± 97 Control 246 ± 53 0 ng/ml
14 Reduction of the Nonspecific Binding of a Target Antibody and of Its Enzyme-Labeled Detection Probe Enabling Electrochemical Immunoassay of Antibody through the 7 pg/ml 100 ng/ml (40 fm pm) Range Yongchao Zhang and Adam Heller * Department of Chemical Engineering and Texas Materials Institute University of Texas at Austin. Anal. Chem. 2005, 77,
15 Experimental Procedure I H 2 O 2 H 2 O t Screen Printed Electrode rabbit IgG Biotin-labeled anti-rabbit IgG Avidin - - Os 2+ Os 2+ HRP labeled anti-rabbit IgG PAA / PAMA PAA-PVP-[Os(bpy) 2 Cl] + polymer
16 Results and Discussion Suppression of the nonspecific binding-noise current by polyanions having terminal functions forming covalent bonds with amines and with thiols.
17 Results and Discussion Dependance of H 2 O 2 electroreduction current on the antigen concentration.
18 Summary Selection of appropriate blocking system is essential for the development of a specific and sensitive assay. Empirical testing is required to both choose and optimize the blocking procedure and is influenced by the surface chemistry.
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