Experiment #3 - Antibody Loading Sweep

Make sure to check out experiment #2 - reaction buffer screen before completing this step.

The antibody-to-gold ratio is important to optimize in order to prevent non-specific binding and increase assay sensitivity. For the BioReady carboxyl gold 150 nm nanoshells and for the 80 nm carboxyl gold, we recommend beginning with an antibody loading of 20 µg antibody for 1 mL of particles at 20 OD. For the 40 nm carboxyl gold we recommend starting at 50 µg antibody for 1 mL (adjust swept values in the protocol below appropriately based on this starting value).

In this experiment we will evaluate antibody loading above and below this standard ratio.

Scientist Extracting a Rack Tube With Urine Samples. Closeup of a Scientist Working With Urine Samples in Lab.

Technical Notes

  • We begin screening antibody increments of ± 5 µg, however smaller or larger increments may be suitable. Excess antibody often negatively impacts the conjugation quality.
  • For competitive format assays where it is desirable to limit the number of antibodies per particle, we recommend screening
  • 1, 5, and 10 µg of antibody per mL of 20 OD gold nanoshells and reducing the antibody incubation time to 5–30 minutes.

Materials

  • DI water
  • 3 × 1.5 mL LabCon® test tubes
  • 3 mL BioReady™ 150 nm Carboxyl Gold Nanoshells, 20 OD
    • 1 mL each aliquoted into 3 × 1.5 mL LabCon® tubes (provided)
  • Reaction buffer selected in Conjugate Optimization #1
  • EDC: 10 mg aliquot
  • Sulfo-NHS: 10 mg aliquot
  • Hydroxylamine solution
  • Conjugate diluent
  • Microcentrifuge
  • Rotator or end-over-end mixer for antibody incubation

Antibody Loading Sweep Protocol

  1. Remove 1 × 10 mg aliquot of EDC and 1 × 10 mg aliquot of sulfo-NHS from cold storage and allow it to come to room temperature prior to use (~20 minutes).
  2. Thoroughly shake the BioReady™ 150 nm Carboxyl Gold Nanoshells to disperse particles and aliquot 1 mL of particles into three separate tubes. Label tubes “A”, “B”, and “C”.

    IMPORTANT: Steps 3–6 should be completed within 5 minutes of solubilizing EDC and sulfo-NHS to minimize hydrolysis of the sulfo-NHS ester in water and enhance the efficiency of conjugation.

  3. Pipette 1 mL of fresh DI water into the 10 mg aliquot of EDC for a final concentration of 10mg/mL. Vortex for 10 seconds.
  4. Add 8 µL of 10 mg/mL EDC to each of the three 1 mL aliquots of BioReady™ 150 nm Carboxyl Gold Nanoshells.
  5. Pipette 1 mL of fresh DI water into the 10 mg aliquot of sulfo-NHS for a final concentration of 10mg/mL. Vortex for 10 seconds.
  6. Add 16 µL of 10 mg/mL sulfo-NHS to each of the three 1 mL aliquots of BioReady™ 150 nm Carboxyl Gold Nanoshells.
  7. Vortex each solution and incubate at room temperature for 30 minutes while mixing.
  8. After the 30-minute incubation, balance tubes in a microcentrifuge and spin at 2.0k RCF for 5 minutes.
  9. Carefully remove supernatant (~950 µL) to remove any excess EDC/sulfo-NHS and resuspend each pelleted nanoparticle with 1 mL of reaction buffer selected in Conjugate Optimization Step #1. Vortex and/or sonicate (< 30 seconds) to fully resuspend particles.
  10. Add the following amount of antibody to each corresponding labeled 1 mL tube of activated gold nanoshells:
  11. 15 µg antibody
  12. 20 µg antibody
  13. 25 µg antibody

    Note: For competitive format assays where it is desirable to limit the number of antibodies per particle, we recommend adding 1, 5, and 10 µg of antibody per mL of 20 OD gold nanoshells and reducing the antibody incubation time to 30 minutes.

  14. Vortex each solution and incubate at room temperature for 60 minutes with adequate mixing.
  15. After incubation, add 10 µL of hydroxylamine quencher to deactivate any remaining active NHS-esters. Vortex and incubate at room temperature for 10 minutes while rotating on a mixer.

    Note: Observe the tube and the color of the conjugates after quenching and compare to the “parent” unconjugated material. A lighter blue or the presence of black “specks” is a sign of colloidal instability.

  16. Centrifuge aliquots at 2.0k RCF for 5 minutes and carefully remove supernatant. Resuspend pellet with 1 mL of reaction buffer. Vortex and/or sonicate to fully resuspend conjugate.
  17. Repeat centrifugation and resuspension step #13 to remove any excess antibody.
  18. Centrifuge one final time at 2.0k 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.
  19. Store conjugate at 4°C. Do not freeze.

Next Steps

Proceed to experiment #4 - Optimize Antibody Incubation Time