Creating Successful Covalent Conjugation

In order to consistently fabricate robust and reliable covalent conjugates, it is essential that the correct reagents and consumables are being utilized. 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 conjugation optimization kit that contains pre-validated reagents and consumables that will save time and eliminate issues associated with critical materials.

Purification Columns

The importance of antibody purification from any buffers or blocking agents that contain amines was covered in our NCXU module on Antibody Selection and Purification. At Fortis Life Sciences we typically use the Amicon Ultra-0.5 Centriugal Filter Unit which is one of the components in the optimization kit and comes with 2 column tubes.

Centrifugation Tubes

All plastics will contain residual monomer, catalysts, and additives that support the high temperature molding of the polymer, and also lubricants to release the plastic off of the mold.

Although these are present in relatively small amounts, they can and will leach to some degree into the fluids you put inside them. The amount of leaching is dependent on many factors including time, temperature, the mobility of the additive in the polymer, and the interaction of the container contents with the polymer (solvent effectiveness). The use of the wrong tubes is one of the most common problems we discover when working with clients doing lateral flow covalent conjugations. 

Some of these residuals interfere with EDC and sulfo-NHS chemistry, and will cause the gold to plate out onto the tube or reduce the sensitivity of the assay.

Lo-bind tubes are pre-treated specifically to not bind protein. If you are conjugating a protein you have found to be "sticky" we suggest activating the gold nanoparticles and performing the centrifugation step to remove excess EDC and sulfo-NHS in the recommended tubes, and then transferring the activated nanoparticles to a lo-bind tube prior to adding your protein.

EDC/Sulfo-NHS

The EDC/Sulfo-NHS coupling reagents are critical for obtaining robust conjugates. In our optimization kit we provide 3 single use aliquots of EDC and Sulfo-NHS from CovaChem. 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.

Buffers

There are a number of buffers that are needed for conjugation. The following buffers are included in our optimization kit:

  • Antibody Purification Buffer: 10 mM potassium phosphate buffer
  • Reaction Buffer #1: 50× concentrate of 0.01× PBS with 0.5 PEG 20
  • Reaction Buffer #2: 5 mM potassium phosphate with 0.5% PEG 20, pH 7.4
  • Reaction Buffer #3: 5 mM sodium phosphate with 0.5% PEG 20, pH 7.4
  • Quencher: Hydroxylamine, 50% in water
  • Block Buffer: 4 mM sodium tetraborate, 1% BSA, 0.05% sodium azide, pH 8.2
  • Running Buffer: 1× PBS, 1% Tween 20

Reporter Particles

BioReady 40 nm gold spheres, 80 nm gold spheres, and 150 nm gold nanoshells have carboxyl surfaces that are ready for covalent conjugation. In our conjugation kits, 10 mL of 20 OD solutions of nanoparticles are provided.

Additional Materials Required

In addition to the above materials, you will need:

  • Microcentrifuge
  • Method for determining protein concentration (A280, BCA, Bradford Assay)
  • Single channel pipettes capable of pipetting 5–1000 µL
  • Orbital or end-over-end rotator or mixer for incubation steps
  • UV-Vis spectrophotometer (recommended)
  • Bath sonicator (recommended)
  • Calibrated pH probe (recommended)
  • Lateral flow test strips for functional QC testing of conjugate (recommended)
  • Freshly dispensed deionized water*

* DI water that has been stored for long periods of time can have a low pH. We recommend freshly dispensed DI water for use in dilution buffers.

Covalent Conjugation Optimization Kit

At Fortis Life Sciences, we've found that the best way to maximize rapid success is to make sure that researchers have access to all of the components necessary for successful reactions. We've included all of these elements in an optimization kit that we highly recommend for anyone who wants to fabricate robust and reproducible nanoparticle conjugates.

The following components are included in the kit with their recommended storage conditions.

Item

Amount

Storage

Centrifugal purification column

1 each

2–25°C

Centrifugal column tubes

2 each

2–25°C

Purification buffer, 10 mM potassium phosphate

10 mL

2–25°C

20 OD BioReady™ 150 nm gold nanoshells

10 mL

2–8°C

Potassium phosphate reaction buffer, pH 7.4

50 mL

2–25°C

Sodium phosphate reaction buffer, pH 7.4

50 mL

2–25°C

PBS reaction buffer, 50×

0.5X PBS, 250 mg/mL 20K MW PEG, pH ~7.4

1 mL

2–25°C

60 mL PETG reagent bottle, empty (for dilution of 50× PBS reaction buffer)

1 each

2–25°C

EDC, 10 mg aliquot

3 each

2–8°C

Sulfo-NHS, 10 mg aliquot

3 each

2–8°C

Hydroxylamine quencher

100 µL

2–25°C

NCX block buffer

4 mM sodium tetraborate, 1% BSA, 0.05% sodium azide, pH 8.2

5 mL

2–8°C

NCX conjugate diluent

0.5× PBS, 0.5% BSA, 0.5% casein, 1% Tween 20, 0.05 % azide pH 8

10 mL

2–8°C

1.5 mL microcentrifuge tubes

20 each

2–25°C

NCX lateral flow running buffer (1× PBS, 1% Tween 20

10 mL

2–25°C

Next Steps

Now that you've got all the components you need, the next step is to build and optimize your covalent conjugates.

To save time and ensure the highest possibility of success, we recommend running five experiments that will help you produce high quality conjugates: 1 purification experiment, 3 conjugation experiments, and optional 1 blocking experiment.

These experiments are discussed in detail in the additional modules: