Hi, my name is Jean.
I'm a research scientist here at NanoComposix.
Today we're going to go over the simple process of covalently conjugating antibodies to our
BioReady Carboxyl gold nanoshells.
Covalent conjugation of proteins or antibodies to nanoparticle probes is a very useful tool
while developing lateral flow assays, and for other applications.
It yields reproducible and robust conjugates with just a few simple steps.
When screening antibodies for lateral flow using passive conjugation methods, it can
take days to weeks to optimize each antibody conjugate.
Using covalent conjugation methods, it's possible for a scientist to screen 10 x 10 antibody
matrices in one day or even in as little as one afternoon.
Using covalent conjugation methods substantially reduces the time and effort required to perform
antibody screens for lateral flow assay development.
First, remove the EDC & Sulfo-NHS from cold storage.
Allow the bottles to sit at room temperature for 15-20 minutes before opening.
This is important because moisture from the atmosphere can condense on the cold powders,
causing them to hydrolyze and degrade.
Before you start, you'll need an Eppendorf tube labeled EDC, another labeled Sulfo-NHS,
an aliquot of BioReady gold, and your purified antibody.
Place the tube labeled EDC on the balance and tare.
Remove the EDC from the storage container.
Aliquot between 2 and 10 mg into the Eppendorf tube and record the mass.
When putting the reagents back for storage, wrap them with parafilm to lessen their exposure to air.
Store them in a sealed package with desiccant.
Repeat the process for the Sulfo-NHS, taring the vial on the balance, removing the bottle
from storage, and weighing out between 2 and 10 mg.
Bring your aliquot of EDC to 10 mg/mL with water.
For example, if you have 8.3 mg of EDC, add 830 microliters of water.
Vortex to resuspend.
For 1 mL of BioReady Gold Nanoshells, you'll need to add 8 uL of your 10 mg/mL EDC solution.
Repeat the process with the Sulfo-NHS.
Vortex to resuspend.
For the same aliquot of Gold Nanoshells, you'll need to add 16 uL of Sulfo-NHS.
Vortex to mix and place on a rotating incubator for 30 minutes.
After 30 minutes have passed, centrifuge your nanoparticle solution at 3600 RCF for 5 minutes.
After 5 minutes carefully remove your Eppendorf tube from the centrifuge, being careful not
to disturb the pellet.
Decant the supernatant and discard.
Resuspend the pellet with reaction buffer at pH 7.4.
Vortex to resuspend the pellet or bath sonicate it if needed.
Your particles are now activated, and you can add your purified antibody.
For 1 mL of Gold Nanoshells, we recommend starting at around 20 micrograms of purified
antibody per mL.
Vortex to mix, and incubate the antibody with the gold colloid for 1 hour.
After the antibody incubation is complete, quench any remaining active groups with an
excess of primary amine.
We use 10 uL of hydroxylamine.
Incubate for 10 minutes and repeat the centrifugation step to remove any excess hydroxylamine or
antibody.
You now have antibody conjugated to your gold nanoparticles.
At this point, you can continue blocking to increase stability and decrease potential
for non-specific binding, or transfer directly to your conjugate diluent.
Thanks for watching!
Let us know if you have any questions in the comments below.