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BioReady 150 nm Carboxyl Gold Nanoshells Covalent Conjugation Protocol

nanoComposix BioReady™ 150 nm Carboxyl Gold Nanoshells can be conjugated to proteins through carbodiimide activation chemistry. Covalent coupling of proteins (e.g. antibodies) to a gold nanoparticle surface yields a robust and stable gold particle conjugate. The BioReady™ 150 nm Carboxyl Gold Nanoshells are functionalized with a tightly bound monolayer containing terminal carboxylic acid functional groups which can be activated through EDC/Sulfo-NHS chemistry to generate gold nanoparticle-antibody amide bonds.

Contact us for inquiries regarding custom conjugation, technical support, or determining which nanoparticle is right for your application.

Material Information & Storage

BioReady 150 nm Carboxyl Gold Nanoshells are provided at OD 20 in water. Store at 4 °C. Do not freeze. Thoroughly shake or sonicate contents to disperse particles if settling occurs.

Proper handling and storage of EDC and Sulfo-NHS is critical for successful conjugations. These reagents can be purchased from a number of vendors and the manufacturer guidelines for handling and storage should be followed. EDC and Sulfo-NHS should be stored with desiccant at -20 °C and 4 °C respectively. Ensure that reagents are brought to room temperature before opening to avoid water condensation.


Additional Materials Required

Antibody purification filter columns

DI Water

Antibody Purification Buffer

  • 10 mM potassium phosphate, pH 7.4

Reaction Buffer

  • 5 mM potassium phosphate, 0.5% 20K MW PEG at pH 7.4

EDC

Sulfo-NHS

Quencher

  • 50% (w/v) hydroxylamine

Conjugate Diluent

  • 0.1X PBS, 0.5% BSA, 0.5% casein, 0.5% Tween 20, 0.05% Sodium Azide, pH8

Centrifuge

Centrifuge Tubes

  • 5 mL Labcon SuperSpin® tubes Cat# 3016-870-000
  • 15 mL Labcon SuperClear® conical Cat #3131-335-028
  • 50 mL Labcon SuperClear® conical Cat #3181-345-008

Vortex

Rotator

Important: The reaction buffer used when activating NHS esters and forming covalent bonds with your protein is important. The activation with EDC and Sulfo-NHS is most efficient at pH 5, and the particle solution will self-adjust to this pH after reagent addition. The conjugation of these activated particles with primary amines on the antibody is most efficient at pH 7-7.5. For best results, perform the conjugation step with the suggested reaction buffer or another amine-free, low-salt buffer in the pH 7-7.5 range. Performing the reaction at a higher pH drastically reduces the half-life of the NHS-ester. Once a stable conjugate is formed it can be transferred into the buffer of choice.

Antibody Preparation

The antibody for conjugation should be purified and adjusted to a concentration > 1 mg/mL in a low ionic strength buffer free of additional proteins or free amines, such as 10 mM potassium phosphate. Commercial antibodies may contain protein additives for stabilization (e.g. BSA), preservatives (e.g. sodium azide), or amines in the buffer (e.g. Tris) which all need to be removed before covalent conjugation to nanoparticles. Antibodies can be purified from salt preservatives using spin columns or dialysis tubing with the appropriate molecular weight cutoff, and can be transferred into a non-amine-containing buffer using the same mechanisms. If Tris or another amine-containing buffer is used to elute the antibody from an affinity column during isolation from stabilizing proteins, the antibody will need to be buffer-exchanged to transfer it to a suitable amine-free buffer.

Antibody purification into an amine-free buffer is different from affinity purification of the antibody during development. Even a small concentration of sodium azide (NaN3) will interfere with the efficacy of conjugation.

Antibody Concentration

For covalent conjugation to BioReady™ 150 nm Carboxyl Gold Nanoshells, a typical antibody to gold ratio is 20–30 µg of antibody per 1 mL of gold at OD 20.

Conjugation Protocol

It is important to note that optimal conjugation procedures are antibody-dependent; optimization techniques will differ from antibody to antibody.

This conjugation protocol is for 1 mL of OD 20  BioReady150 nm Carboxyl Gold Nanoshells that will result in 1 mL of antibody-gold conjugate at OD 20. For larger or smaller volumes, scale proportionately. 

  1. Tubes with specialized treatments (e.g. low-bind) or with residual plasticizer from the manufacturing process can cause instability of the particles during activation steps. If using tubes other than those specified in this protocol, thoroughly rinse tubes with isopropyl alcohol and DI water before use.

Important: Steps 2-8 should be completed immediately after solubilizing EDC and Sulfo-NHS to minimize hydrolysis of the Sulfo-NHS ester in water and enhance the efficiency of conjugation.

  1. Prepare EDC and Sulfo-NHS at 10 mg/mL in H2O immediately before conjugation steps.

Hint: Ensure the reagents are at room temperature before opening vials. Weigh out approximately 1-10 mg EDC and Sulfo-NHS in individual microcentrifuge tubes and record mass. Just prior to conjugation, dissolve in the H2O to bring the concentration to 10 mg/mL.

Example:

Mass of EDC = 4.3 mg, add 430 µL H2O

Mass of Sulfo-NHS = 6.1 mg, add 610 µL H2O

  1. Add 80 µg EDC (20 µL freshly prepared EDC at 10 mg/mL) to 1 mL of BioReady™ 150 nm Carboxyl Gold Nanoshells.
  2. Add 160 µg Sulfo-NHS (40 µL of freshly prepared Sulfo-NHS at 10 mg/mL) to the 1 mL BioReady™ 150 nm Carboxyl Gold Nanoshells.
  3. Vortex solution and incubate at room temperature for 30 minutes while rotating.
  4. Centrifuge at 2000 RCF for 5 minutes.
  5. Carefully remove supernatant to remove any excess EDC/Sulfo-NHS and resuspend pelleted nanoparticles with 1 mL reaction buffer. Vortex and/or sonicate (<30 seconds) to fully re-suspend particles.
  6. Repeat step 6-7 to wash particles of any excess EDC/NHS.
  7. Add 30 µg antibody and vortex.
  8. Incubate at room temperature for 1 hour while rotating (shorter or longer incubation times may yield better results).
  9. After incubation, add 10 µL of quencher to deactivate any remaining active NHS-esters. Vortex and incubate at room temperature for 10 minutes while rotating.
  10. Centrifuge at 2000 RCF for 5 minutes. Carefully remove supernatant and resuspend pellet with 1 mL of reaction buffer. Vortex and/or sonicate to fully resuspend conjugate.
  11. Repeat centrifugation and resuspension (Step #12) to remove any excess antibody.
  12. Centrifuge again at 2000 RCF for 5 minutes. Remove supernatant and bring volume of pellet up to 1 mL in conjugate diluent. Vortex and/or sonicate to fully resuspend conjugate.
  13. Store conjugate at 4 °C. Do not freeze.

Frequently Asked Questions

What is the shelf life of BioReady Gold Nanoparticles?

What is the shelf life of the conjugates?

Can I conjugate any type of antibody or protein?

Is there a test to confirm my conjugates are functional?

How do I optimize my conjugate?

What can I do to improve conjugate release and flow on a lateral flow test?