Optimizing a Covalent Conjugation Strategy
Conjugate stability and optimal conditions can vary widely between proteins. The optimization steps outlined in this course will help identify conditions that will produce stable and robust conjugates. We recommend optimizing these initial conjugates by testing against buffered samples that are free of any potential contaminants or non-specific reagents that may exist in a real sample matrix. Another set of optimizations will be necessary after transitioning to the target matrix (e.g. serum, saliva, urine, whole blood etc.).
Common Pitfalls to Avoid
Mis-handling EDC & NHS
The EDC and sulfo-NHS aliquots provided are intended for single use. Be sure to let EDC equilibrate to room temperature (~20 minutes) prior to the opening of the EDC. Use the EDC as soon as possible after resuspension and use a fresh aliquot of EDC for each coupling reaction.
Deviations from protocol:
For conjugation to BioReady™ gold nanoparticles, adding excess activation reagents, protein, or detergents can compromise the performance of the antibody. We recommend following the guidelines in this protocol before trying other conditions.
Inappropriate tubes
Many plastic tubes contain plasticizers or residual mold-release lubricant that can negatively impact the covalent conjugation chemistry used in this protocol. Use the tubes provided for initial conjugation work and then verify that your tube brand is an acceptable substitute before switching to a new type of tube. We recommend LabCon® 1.5 mL, 15 mL and 50 mL volume tubes.
Long periods at room temperature
While covalent conjugates are much more robust than conjugates prepared using passive conjugation methods, avoid long centrifuge times (>30 min) that can inadvertently heat the conjugate.
Unpurified antibody
For covalent conjugation it is critical to purify the antibody or protein of interest away from any primary amines. A purification column is included in the kit to ensure that your antibody is ready to use.
Conjugate Stability and Performance
During the optimization process you will have to assess which conditions yield the best result. The best and most relevant method of determining conjugate performance is with functional testing. Many of our customers use conjugates in lateral flow assay development and this platform is a useful metric for gauging performance. However, initial evaluation of conjugate quality can be performed by simply observing the color of the solution after each step. Gold nanoshells have a distinct visible color that changes when the particles aggregate and simply monitoring the solution color provides a good first assessment of success. If you have access to a UV-Vis spectrophotometer, the "color" of the conjugates can be more precisely tracked.
Visit our module for learning more on how to monitor your conjugate stability with UV-Vis.
Functional Check: Running Lateral Flow Strips to Monitor Conjugate Performance
If you are developing an assay that uses the conjugates as a diagnostic readout, your best test will be to use this functional assay to test your conjugates. Assay results with strong signal intensity and low non-specific background identify optimal conditions. If you are using lateral flow strips to optimize conjugates, we have included Fortis Life Sciences’s standard running buffer formulation that consists of 1× PBS + 1% Tween 20. Using this running buffer by itself or in combination with other protein additives such as BSA and casein is a good starting point for evaluating the quality of your conjugates in lateral flow.
The "best" conjugate depends on the final use. For lateral flow, some typical metrics that mark conjugate quality are:
- Minimal aggregation at nitrocellulose interfaces
- Complete conjugate release from sample pad
- Even flow up the strip
- Low or zero non-specific binding
- The strength of the specific test-line signal
Five Experiments for Optimization
The next sets of modules outline five experiments that will help produce optimal antibodies. The first is the purification of the antibody, followed by 3 conjugation experiments and a final blocking experiment. While this is just the start of the optimization process, in many cases a functional particle can be obtained after following these experiments.
These experiments are discussed in detail in the additional modules:
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