Kristin Cederquist, IMRA America

Kristin Cederquist, Ph.D., Research Scientist, IMRA America, Inc., using Anteo’s Activation Reagent to functionalize bare gold nanoparticles

 

Nanoparticle and bioconjugate stability and activity are crucial parameters for in-vitro diagnostic test development.  Anteo’s Activation Reagent easily circumvents these common problems by:

 

  1. Coating nanoparticles in a thin, yet stabilizing and highly charged layer

  2. Allowing for the gentle conjugation of antibodies and proteins without loss of activity

The functionalization process is simple, and once activated, particles are highly stable over time.  This is an all-encompassing technology that has allowed us to conjugate multiple antibodies to gold particles using the same procedure every time without needing to optimize pH or antibody coupling conditions.

 

 

Materials, Methods and Data

 

IMRA laser-fabricated gold nanoparticles were activated using Anteo’s Activation Reagent for 4 hours as directed, then washed twice by centrifugation to remove excess unbound Reagent before being resuspended in a storage buffer and stored at 4 degrees Celsius in a polypropylene bottle.  

 

The size and charge properties of the particles were monitored over time by dynamic light scattering and zeta potential (Figure 1), and neither property was found to significantly fluctuate during the evaluation period, suggesting that the activated particles were stable over time.

 

To evaluate antibody-binding ability and subsequent bioconjugate activity, we slowly introduced 40 nm gold particles activated by Anteo’s technology to HRP-labeled goat anti-mouse antibody and reacted for 1 hour.  Particles were then blocked with 40 mg/mL (4 % w/v) BSA for 30 minutes and subsequently washed twice by centrifugation and characterized by UV-Vis, DLS, and zeta potential before being resuspended in a storage buffer.  

 

The bioconjugate activity was determined by a functional assay as shown in Figure 2. Briefly, particles were diluted with a BSA-containing buffer to OD 0.1 (read at 520 nm) and added to a microplate (100 uL/well) to which different concentrations of mouse IgG had been adsorbed.  Particle-antibody conjugates were allowed to react for 1 hour at 37 degrees Celsius, after which wells were washed four times with PBS + 0.01% Tween-20.  

 

TMB substrate (100 uL/well) was added and allowed to react for 5 minutes, following which the reaction was quenched with 50 uL of 1M sulfuric acid, and the absorbance was read at 450 nm by a plate reader.  The results (Figure 3) indicated that both the antibody and the HRP enzyme retained activity after the reaction with the Mix&Go-activated surface and also the washing process.   

 

Figure 1: Neither property (hydrodynamic size ± 3 nm and zeta potential ± 5.5 mV) was found to significantly fluctuate during the evaluation period, suggesting that the activated particles were stable over time.

 

 
Figure 2: Bioconjugate activity was determined by a functional assay as depicted in the schematic diagram.  
 

Figure 3: The results indicated that both the antibody and the HRP enzyme retained activity after the reaction with the Mix&Go-activated surface and also the washing process as shown with increasing substrate turnover in a functional assay.   

 

Conclusion

The Anteo protocol process is easy to follow and implement, and it takes the guesswork out of bioconjugation, thereby saving time and biological material.  I like that the system emulates biology and takes advantage of many weak interactions versus one strong, covalent bond. I would definitely use Anteo’s technology again and would like to expand its use to our other lines of nanoparticles that we are developing for IVD.