One of the key components to developing successful lateral flow assays is a sensitive and stable conjugate, typically achieved via passive or covalent conjugation to the reporter particle (colloidal gold, latex, gold nanoshells, etc.). Here we will cover protein coupling to reporter particles via covalent conjugation.
Previous: Reporter Nanoparticle Selection for Lateral Flow Immunoassays
Ready to optimize a covalent conjugation?
Use our BioReady Conjugation Kit with compatible carboxyl-functionalized nanoparticles to streamline antibody and protein conjugation.
How Covalent Nanoparticle Conjugation Works
Carboxyl-functionalized nanoparticles are recommended for covalent binding. Antibodies are permanently attached to the carboxyl-modified surface of colloidal gold, latex, or nanoshells via carbodiimide activation chemistry (EDC/sulfo-NHS). For this surface, EDC and sulfo-NHS reactive groups are used for amide bond formation, linking the carboxylic acid on the nanoparticle to primary amines in the lysine residues of the antibody or protein. A typical IgG antibody will have 80–100 lysine residues, of which 30–40 will be accessible for EDC/NHS binding.
In the EDC/NHS activation chemistry used for covalent conjugation, EDC is used to activate the carboxyl group on the surface of nanoparticles to create a crosslinker. The resulting intermediate can bind to primary amines on the antibody but is unstable and susceptible to hydrolysis. Sulfo-NHS is added with EDC to create a more stable amine-reactive intermediate, which will bind to the primary amines on the antibody.
Source: Thermo Fisher.
Benefits of Covalent Conjugation
Covalent conjugation of antibodies to gold nanoparticles continues to become more widely used in commercial assays because of the following benefits:
- Less antibody is needed to maximize sensitivity, reducing the overall cost of an assay.
- Covalent conjugates offer increased stability in difficult sample matrices and harsh buffering conditions (high salt or detergent concentrations).
- Stable and reproducible conjugates provide reliable quantitation of analytes.
- Conjugates are easily and consistently prepared, which can save time when performing antibody screening experiments.
- The antibody-to-particle ratio can be precisely controlled, which is important for adjusting the dynamic range in competitive assays and optimizing sensitivity when using antibodies with varying binding kinetics.
BioReady Products for Covalent Conjugation
BioReady™ gold nanoparticles are available with carboxyl-functionalized surfaces for covalent attachment of antibodies and other proteins using EDC/sulfo-NHS chemistry. Choose the particle size and format based on the sensitivity and performance requirements of your lateral flow assay.
- Gold Nanospheres: 40 nm and 80 nm carboxyl gold nanospheres for EDC/sulfo-NHS conjugation.
- Gold Nanoshells: 150 nm carboxyl gold nanoshells for EDC/sulfo-NHS conjugation, or NHS-activated gold nanoshells for direct reaction with primary amines.
Explore the full range of BioReady nanoparticles and conjugates for diagnostic applications.
Covalent Conjugation Protocols
For step-by-step covalent conjugation procedures, including antibody preparation, particle activation, coupling, blocking, and cleanup, visit our conjugation protocols library.
During conjugation, make note of any staining on the side of the tube or change in color, as these may be signs of flocculation or an unstable gold conjugate. Taking a UV-Vis scan is a simple and reliable way to evaluate changes in optical density or spectral shape that may indicate aggregation.
Conjugate Functionality Test
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. A functionality test can also be performed for the conjugated antibodies to ascertain conjugation efficiency. The two most common methods to validate functionality of the conjugate are anti-species testing and full functionality testing on lateral flow strips.
Below: A quick test with anti-species antibodies spot-dried on membrane and run with nanoshell-conjugated antibodies. The crescent speck on the membrane is evidence that antibodies were conjugated to nanoparticles and recognized by anti-species antibodies (n = 2).
Below: Conjugate run with negative, 0.5 mIU, and 50 mIU samples of analyte. The signal intensity of the test line is proportional to the concentration of analyte spiked in buffer, indicating that the conjugated antibody is working (sandwich format assay, n = 2).
Optimizing Covalent Antibody Conjugation
Many steps of the conjugation process can be adjusted and optimized depending on the specific antibodies and assay application. When starting out with any new conjugation or assay, we recommend examining the following parameters to improve the performance of your conjugate. Click the title of the study for more details and a protocol for each experiment.
- Reaction Buffer Screen – because each antibody and each assay are unique, the buffering condition may be specific to the antibodies used. Screen a few different buffer salts at around pH 7.4–7.5.
- Antibody Loading – the ratio of antibody conjugated onto nanoparticles will need to be titrated to boost performance and conserve reagents if necessary. To start out, conjugate nanoparticles with 3 different amounts of antibody (low, medium, high). Then choose the best loading and further titrate the ratio around that concentration to select the optimal condition. Repeat as necessary.
- Antibody Incubation Time – normally, antibodies require at least 30 minutes to 2 hours of incubation time at room temperature for coupling with nanoparticles. Longer and shorter incubation times at different temperatures can be evaluated for better conjugate stability and performance.
- Conjugate Blocking – for covalent conjugation, the quenching and blocking steps can sometimes be combined into one. The quenching/blocking step stops the coupling reaction and saturates unreacted/open sites on the surface of nanoparticles with proteins (casein or BSA). Incubation time and temperature for the blocking step can be evaluated if necessary.
Common Covalent Conjugation Pitfalls
It is important to understand that each antibody is unique and may exhibit different performance when conjugated under different conditions compared to other proteins. There is no one-size-fits-all conjugation method, and each antibody or assay may need to be optimized individually for best results. However, there are several areas that should be controlled across different conjugation experiments in order to ensure reproducible and robust conjugate performance:
- Mis-handling EDC & NHS: Make sure all reagents and materials are handled and stored at the appropriate temperature. Critical reagents like EDC and NHS, for example, are extremely hygroscopic and tend to hydrolyze and lose activity quickly upon contact with water. Be sure to let EDC equilibrate to room temperature (~20 minutes) prior to opening 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 and resulting conjugate. We recommend following the guidelines in the provided protocols 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 the provided protocols. Use the tubes provided for initial conjugation work and then verify that your tube brand is an acceptable substitute before switching to a different 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.
- Incompatible antibody buffer or additives: For EDC/sulfo-NHS conjugation, the antibody should be transferred into a compatible, primary-amine-free buffer before coupling. Primary amines in buffers such as Tris or glycine can compete with the antibody for NHS-reactive groups and reduce conjugation efficiency. Other proteins and additives may also interfere with the conjugation or downstream assay performance. Use a purification or buffer-exchange step as needed before conjugation. A purification column is included in the Optimization Kit to help prepare the antibody for coupling.
For additional assay-development and troubleshooting guidance, see our Lateral Flow Frequently Asked Questions.
Next: Prepare for covalent conjugation