ABSTRACT
Contents Introduction ............................................................................................................ 112 List of Protocols ..................................................................................................... 114 Protocol 7.1: Bead Selection .................................................................................. 114
How It Works ................................................................................................ 114 Protocol 7.2: Buffer Exchange ............................................................................... 115
How It Works ................................................................................................ 115 Required Materials ........................................................................................ 115 Step-by-Step Protocol ................................................................................... 115 Protein Desalting Spin Column Preparation ................................................. 115 Sample Loading ............................................................................................ 115
Protocol 7.3: Determination of Antibody IgG Concentration ................................ 116 How It Works ................................................................................................ 116 Required Materials ........................................................................................ 116 Step-by-Step Protocol ................................................................................... 116
Protocol 7.4: Bead Conjugation ............................................................................. 117 How It Works ................................................................................................ 117 Required Materials ........................................................................................ 117 Reagent Preparation ...................................................................................... 117 Step-by-Step Protocol ................................................................................... 118
Protocol 7.5: Biotinylation of Antibodies .............................................................. 119 How It Works ................................................................................................ 119 Required Materials ........................................................................................ 119 Reagent Preparation ...................................................................................... 119 Step-by-Step Protocol ................................................................................... 120
Protocol 7.6: Sandwich Immunoassays ................................................................. 120 How It Works ................................................................................................ 120
Required Materials ........................................................................................ 120 Reagent Preparation ...................................................................................... 121 Protocol 7.6.1: Standard Preparation ............................................................ 121 Protocol 7.6.2: Antibody-Coupled Bead Working
Suspension Preparation ............................................................................. 121 Protocol 7.6.3: Biotinylated Detection Antibody Working
Solution Preparation ................................................................................. 122 Protocol 7.6.4: Streptavidin-PE Working Solution Preparation ................... 122 Protocol 7.6.5: Performing the Bead Immunoassay ..................................... 123 Step-by-Step Protocol ................................................................................... 123
Troubleshooting Guide ..........................................................................................124 Note 1: Selection of Antibody Pairs .............................................................124 Note 2: Antibody Specificity ........................................................................124 Note 3: Recombinant Proteins ...................................................................... 125 Note 4: Assay Diluents ................................................................................. 125 Note 5: Secondary Antibody Diluent ............................................................ 125
Key References ...................................................................................................... 126
Introduction Multiplex, a term once only familiar to moviegoers from the film Multiplicity (1996), has become a commonly used word in the biomedical testing field. After more than a decade of development, multiplexed assay technology has become a regular laboratory tool in many research areas, including immunology, oncology, molecular cell biology, toxicology, neurobiology, etc. A multiplexed assay measures multiple analytes simultaneously in a single reaction. The wealth of information provided by these kinds of high-content assays has revolutionized the way in which drug discovery, drug development, vaccine testing, and diagnostics are conducted. One of the biggest challenges in today’s drug development arena, which includes small molecule, biologics, and vaccine development, is the comprehensive analysis of drug effects. Such effects are both in vitro and in vivo, intercellularly or intracellularly in nature. Analyzing one analyte at a time has become a bottleneck in understanding the mechanism of action, efficacy, and side effects related to these therapeutic candidates.
