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Building Better Reagents

Facing problems of inconsistent, time-consuming, and costly antibody production, some researchers are turning to alternatives to target specific proteins of interest, in the lab and in the clinic.

Feb 1, 2016
Jane McLeod, Paul Ko Ferrigno

© STEVE GRAEPELAntibodies are large proteins, weighing in at about 150 kDa. Four polypeptides—two heavy chains and two light chains—are linked by disulfide bonds to form a Y-shape molecule. The amino acid sequences at tips of the short ends of the Y vary greatly between antibodies produced by different B cells, while the rest of the molecule is relatively consistent. The variable portion of the antibody binds in a specific region (epitope) on a foreign protein (antigen) and signals the immune system to the presence of an invader.

 

 

 

 

 

 

 

 

To produce antibodies, researchers immunize lab animal with protein of interest. The animal’s B cells then generate antibodies that bind to different regions, or epitopes, on the protein. The diverse antibodies that bind to the target protein can then be isolated and purified for use. Because these bind numerous epitopes, they are called polyclonal antibodies.

© STEVE GRAEPEL

 

 

Alternatively, the immunized animals’ B cells can be isolated from the spleen or lymph
nodes and fused with a tumor cell to generate immortal hybridoma lines. Those cell lines
that produce the desired antibody against a specific epitope of the target protein can
then be grown in large bioreactors to scale up production of the antibody.

© STEVE GRAEPEL

 

POLYCLONAL ANTIBODIES MONOCLONAL ANTIBODIES
• Size: Large (about 150 kDa)
• Binds specific epitope?: Typically no. Diverse antibodies against different epitopes, making them less sensitive to antigen changes than monoclonal antibodies. Antibodies will also vary in affinity and specificity for a given target.
• Production: 2–4 months; entirely in animal models
• Lot-to-lot heterogeneity: High
• Shelf life: Limited
• Size: Large (about 150 kDa)
• Binds specific epitope?: Yes. As they offer specific recognition of a single epitope on the target protein, monoclonal antibodies are sensitive to molecular changes of that epitope and offer precise molecular recognition of a group of structurally similar molecules.
• Production: Six months; requires animal models and the use of expensive cell cultures of
higher eukaryotes for growth in bioreactors of up to 2,000 L
• Lot-to-lot heterogeneity: Low, though downstream production processes and drift in the cell line’s antibody expression can introduce variation
• Shelf life: Limited

© STEVE GRAEPEL© STEVE GRAEPEL

NUCLEIC ACID APTAMERS PROTEIN SCAFFOLDS
• Size: Small (<25 kDa), opening up new targets that were previously
inaccessible to antibodies
• Binds specific epitope?: Yes
• Production: Weeks; chemically synthesized
• Lot-to-lot heterogeneity: Very low
• Shelf life: Stable at room temperature for months
• Size: Small (~15 kDa), opening up new targets that were previously
inaccessible to antibodies
• Binds specific epitope?: Yes
• Production: Weeks; entirely in vitro with lower organisms such as
bacteria
• Lot-to-lot heterogeneity: Very low
• Shelf life: Stable at room temperature for months

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