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). You’ve already learned about passive conjugation in the previous module. Here we will cover protein coupling to reporter particles via covalent conjugation.
Carboxyl-functionalized nanoparticles are recommended for covalent binding. Antibodies are permanently attached onto 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 its 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™ 150 nm Gold Nanoshells are designed to combine the advantages of both covalent conjugation and nanoshell technology for up to 20× increased sensitivity:
Other BioReady™ products are optimized for use in conjugations and lateral flow diagnostic assays, available in different surface chemistry, material type, and size options:
Covalent Conjugation – 40 nm and 80 nm Gold Nanospheres
Passive Conjugation – 40 nm and 80 nm Gold Nanospheres
Steps to Covalent Conjugation
In order to consistently fabricate robust and reliable covalent conjugates, it is essential that the correct reagents and consumables are used. Key components are listed below. We highly recommend that if you are just starting out with covalent conjugation that you take advantage of our Covalent Conjugate Optimization Kit which contains pre-validated reagents and consumables that will save time and eliminate risks associated with using potentially incompatible critical materials.
Recommended reagents and consumables:
Purification column – to prepare antibodies free of amines and other proteins and preservatives that can interfere with conjugation process. At nanoComposix we typically use the Amicon Ultra-0.5 Centrifugal Filter Unit which is one of the components in the Optimization Kit and comes with 2 column tubes.
EDC & Sulfo-NHS – the critical reagents for the covalent coupling process. EDC is used to activate the carboxylic acid group on nanoparticles. Sulfo-NHS is added with EDC to increase conjugation efficiency and create a more stable amine-reactive intermediate for binding with antibodies.
Buffers – buffering salts and pH are important components that ensure the appropriate reaction matrix during the conjugation process of antibodies to gold nanoparticles.
Reporter Particles – nanoparticles with the correct surface functionalization (usually a carboxyl group) for covalently coupling to antibodies. See our BioReady™ products.
After procuring all necessary materials and reagents for covalent conjugation, make sure the antibodies are in a buffer free of amines (Tris), stabilizing proteins (BSA), or any preservatives (sodium azide). Dialysis tubing, spin columns, or Amicon concentrators are all suitable for use in purifying and exchanging antibodies. Read more on Antibody Purification here.
Prepare EDC and Sulfo-NHS at 10 mg/mL in diH2O. Make sure both reagents are desiccated and at room temperature prior to resuspending in water. Use within 5 minutes of preparation.
Add 8uL of freshly-prepared EDC to 1 mL of BioReady™ 150 nm Carboxyl Gold Nanoshells.
Add 16 uL of sulfo-NHS to 1 mL of BioReady™ 150 nm Carboxyl Gold Nanoshells. Note: sulfo-NHS must be added immediately after EDC in order to ensure high conjugation efficiency.
Vortex solution and incubate for 30 minutes at room temperature (RT).
Centrifuge at 2000 RCF for 5 minutes and remove supernatant containing excess EDC/NHS.
Resuspend pellet with 1 mL reaction buffer. Vortex or sonicate to break up pellet.
Add 25 ug of purified antibody to the activated nanoshells and incubate for 1 hour at RT on rotator or rocker.
After incubation, add 5 uL of quencher (50% w/v hydroxylamine) or 1/10 volume of 10% BSA for blocking. Incubate at RT for 10 minutes on rocker/rotator.
Centrifuge at 2000 RCF for 5 minutes and remove supernatant. Resuspend with 1 mL of reaction buffer and vortex/sonicate to mix.
Repeat step 10 one more time to remove any excess antibody.
Centrifuge at 2000 RCF for 5 minutes and remove supernatant. Resuspend pellet in 1 mL of conjugate diluent. Vortex/sonicate to break up pellet and store at 4°C until use. Do not freeze.
During the conjugation procedure, make note of any staining on the side of the tube or change in color as these may be signs of flocculation and unstable gold conjugates. Taking a UV-Vis scan is a simple and reliable method to check for changes in the baseline level or any drop in optical density should aggregation occur.
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 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 nanoshells conjugated antibodies. The crescent speck on 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).
Optimization of Covalent Antibody Conjugation
Many steps of this conjugation protocol 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 un-reacted/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.
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.
Unpurified antibody: For covalent conjugation it is critical to purify the antibody or protein of interest away from any primary amines. While many commercial antibodies are listed as “purified”, this does not mean the antibody is free of amines and some may still be present in the form of Tris or sodium azide. A purification column is included in the Optimization Kit to ensure your antibody is ready to use.