Monoclonal antibodies can be great tools for detecting toxins in the body, in diagnostic modalities such as immunohistochemistry, and, in some cases, for treatment of cancer. One problem with developing devices that actually take advantage of the antibodies’ natural ability to detect the presence of pathogens is the fragility of these complex molecules. Outside of a regulated environment, antibodies are not very stable, hence they are not well suited for point-of-care diagnostic devices. Now researchers at Argonne National Laboratory have developed a method to stabilize antibody proteins systematically, allowing them to survive in a more “rugged” environment.
Antibodies are made up of four polypeptides—two light chains and two heavy chains. These chains are made up of modules known as constant and variable domains. The light and heavy chain each have a variable domain, which come together to form the antigen binding site. Because of the great diversity of amino acids in the variable domains, different antibodies are capable of interacting with an effectively unlimited number of targets.
Sometimes this variability comes at a price; the amyloid-forming light chains were less stable than their normal counterparts. However, even amyloid-forming light chains have amino acid substitutions that improve stability. When seven of these amino acid changes were introduced into an amyloid-forming variable domain, a billion-fold improvement in thermodynamic stability was obtained reflecting a much higher ratio of native protein folds to unfolded proteins—a major determinate of antibody shelf life.
"Our work at this detailed level had taught us that antibody stabilization is possible, but we needed to find out if antibodies could be stabilized without compromising their function and do so with moderate experimental investment," Stevens said. Recent work suggests these goals are potentially achievable. To proactively improve the stability of a different antibody variable domain, Argonne researchers drew up a short list of 11 candidate amino acid changes. Four of the amino acid changes improved antibody stability and when combined together in the original domain, they provided a 2,000-fold improvement in stability.
A follow up experiment using a functional antibody fragment was able to improve antibody stability comparably, with no loss of antibody functionality. Both experiments required approximately one month to accomplish instead of the potentially open-ended time required for most protein stabilization projects.
There is a correlation between thermodynamic stability and thermal stability; the billion-fold improvement in thermodynamic stability increased the thermal resistance of the protein to heating, resulting in a “melting temperature” of about 160 degrees Fahrenheit. "However, still unanswered is whether it is possible to be confident about improving the stability of any antibody generated against a particular target," Stevens said. "Our research indicates that stabilization of antibodies is possible. We project that it could be possible to generate the data to guide stabilization of every future antibody in the near future."
Image: Protein stability arises from networks of inter-atomic interaction. In this protein, a network is formed when Q37, a surface amino acid residue, forms a hydrogen bond with amino acid residue Y86 and interacts with amino acid residue D82 through a bridging water molecule.
Press release: Argonne researchers develop method that aims to stabilize antibodies…
