Further Optimizations for Covalent Binding
The previous experiments outlined in this course are a recommended strategy for a first-pass optimization of a stable conjugate. However, further optimization of each of these parameters may yield additional improvements. The reaction buffer formulation and blocking formulations can be varied by salt concentration or by addition of other stabilizing agents. Antibody loading and incubation times may require further tuning up or down. To order more BioReady™ materials to perform additional experiments, visit nanocomposix.com/bioready.
Each antibody and each assay system will require a different optimal storage buffer. The components of the conjugate diluent may be varied or fine-tuned to optimize stability and shelf life of your conjugate. Protein additives such as BSA or casein should be investigated as these components can sometimes eliminate non-specific binding or assist with flow properties.
BioReady™ 40 nm and 80 nm Carboxyl Gold Nanospheres are an alternative to the 150 nm Carboxyl Gold Nanoshells. For lateral flow assays, the smaller diameter particles often require lower OD-loading to achieve their respective maximum sensitivity, reducing the cost per strip. However, using these smaller particle sizes will typically result in a lower maximum possible sensitivity compared to using the gold nanoshells.
A small percentage of antibodies are not suitable for covalent conjugation and will not result in a colloidally stable conjugate. For these cases, passive absorption on “bare” particle surfaces (such as BioReady™ Carbonate or BioReady™ Citrate) should be investigated. The 40 and 80 nm sizes are available with a carbonate surface, which is specifically designed for passive adsorption. For some antibodies passive adsorption works very well, and it is a good idea to try both covalent and passive adsorption during development.
The target sample matrix often requires its own set of optimization steps. Sample matrices such as saliva, serum, whole blood, urine, etc. contain extraneous material that can bind to your conjugate non-specifically, which can result in false-negative or false-positive results. When transitioning to a more complex sample type, it is recommended to rescreen conditions such as conjugate diluent formulation, necessity of a blocking step, and pH.