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Decker. R. Oldenburg, S. Covalent Bioconjugation of Antibodies To Carboxyl Terminated Nanoparticles

This document describes a method for covalently binding antibodies to carboxyl-terminated nanoparticles through an amide bond. It involves purifying the antibody using a protein A column and removing any amine-containing buffers or stabilizing proteins. The purified antibody is then conjugated to carboxyl-functionalized nanoparticles by forming an amide bond between the primary amine on the antibody and the carboxyl group on the nanoparticle surface. This allows controlling the amount of antibody attached and improves stability compared to physical adsorption. The method is optimized for 40nm gold nanoparticles but can be adapted to other particle types and sizes.

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70 views1 page

Decker. R. Oldenburg, S. Covalent Bioconjugation of Antibodies To Carboxyl Terminated Nanoparticles

This document describes a method for covalently binding antibodies to carboxyl-terminated nanoparticles through an amide bond. It involves purifying the antibody using a protein A column and removing any amine-containing buffers or stabilizing proteins. The purified antibody is then conjugated to carboxyl-functionalized nanoparticles by forming an amide bond between the primary amine on the antibody and the carboxyl group on the nanoparticle surface. This allows controlling the amount of antibody attached and improves stability compared to physical adsorption. The method is optimized for 40nm gold nanoparticles but can be adapted to other particle types and sizes.

Uploaded by

Stella Aguirre
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Download as PDF, TXT or read online on Scribd
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Optical Imaging 37

Method: Covalent Bioconjugation of


Antibodies to Carboxyl Terminated
Nanoparticles
Rhea Decker, Steven J. Oldenburg*
nanoComposix, Inc
4878 Ronson Ct, San Diego, CA 92111
*Email: [email protected]

Introduction Antibody Purification


Conjugation of antibodies is commonly used as a means of Prior to conjugation, it is critical that the antibody solution does
targeting nanoparticles, either for diagnostics or for delivery not contain additional free amines in the storage buffer as it will
of nanoparticles to a particular location in vitro or in vivo. compete with binding sites on the nanoparticle. Free amines,
In this article, we describe a method for covalently binding including those found in tris buffer or the preservative sodium
antibodies through an amide bond to the nanoparticle surface. azide, must be removed and the antibody must be transferred
There are many different methods for binding antibodies to a to a suitable amine-free buffer. Likewise, any additional
nanoparticle surface. For example, physisorption is a commonly stabilizing proteins in the antibody solution must also be
used technique for gold nanoparticles because a clean surface removed. Protein A or other affinity columns can be employed
on the gold nanoparticle exhibits a high affinity for the various to isolate the antibody from other proteins. It is to be noted that
functional groups present in an antibody. Such binding has been the elution of the antibody from affinity columns often involves
used for decades for the preparation of lateral flow diagnostics. the use of amine containing buffers. A subsequent purification
However, the physisorption method has several drawbacks. of the antibody into an amine-free buffer is then required. Steps
These include: the release of the antibodies off the particles involved in the purification of antibodies are detailed below.
over time, difficulty in controlling the amount of antibody on the
particles, and the need to optimize the exact conditions for the Materials
physisorption with each different antibody. • Millipore Amicon Ultra 0.5 mL 10K filters for protein
purification and concentration (Prod. No. Z677108)
It has been found that the covalent bonding of antibodies
• 2 mL Microcentrifuge tubes (to hold filters) (Prod. No.
through an amide bond from a free amine on the antibody to a
Z628034)
carboxylic acid on the surface of nanoparticles is a simple and
reproducible method for creating conjugates and for controlling • Microcentrifuge
the amount of antibody on the surface. In addition, in the case • Antibody to be purified
of conjugates used in lateral flow diagnostics, substantially less
• Purification buffer (e.g., 10 mM potassium phosphate, or
antibody is required to generate a functional conjugate. The
other amine-free buffer)
example method we provide here has been optimized for 40 nm
• Bicinchoninic acid (BCA) assay kit (Prod. No. QPBCA)
carboxyl functionalized (also known as lipoic acid functionalized)
and plate reader or UV/vis spectrophotometer for protein
gold nanoparticles. Normalization for the total nanoparticle
quantification
surface area in solution allows for extension of this chemistry to
other sizes of nanoparticles with a carboxyl surface. In addition, Purification Protocol
by modifying the centrifugation parameters, the isolation of a 1. Place filter inside microcentrifuge tube.
variety of different particles can be achieved. For example, we 2. Add 450 µL of purification buffer and centrifuge 5 minutes at
have employed a similar method for carboxyl surfaced materials 13,800 RCF to pre-rinse the filter.
including gold nanoshells, gold rods, hollow gold spheres, 3. Dispose of the filtrate at the bottom of the tube.
spherical silver nanoparticles (lipoic acid functionalized), silver 4. Aliquot antibody solution into filter and close cap.
cubes, silica, quantum dots, and latex beads.

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