Normal mammalian antibodies are Y-shaped compounds made up of two different protein chains: one heavy and one light. The heavy chain typically has about two and a half times as many amino acids as the light one. At each short-end of the antibody are the sites of the complementarity determining regions (or CDRs), where they bind antigens, or invading substances. Although these doubled proteins can identify and fight many foreign proteins within the mammalian immune system, their use in the emerging field of biosensors has been limited by their instability at high temperatures of about 60 to 70 degrees Celsius. "That's the Achilles' heel of these antibodies--when you start heating them up, those domains come apart and they catastrophically aggregate and they refold and they can't reassemble," says Andrew Hayhurst, a virologist and immunologist at the Southwest Foundation for Biomedical Research, who coauthored the current study, published in November in the journal Analytical Chemistry.
Blood from llamas (as well as camels and sharks), however, contains antibodies that are only single-domain, or chain. In 1999 Dutch scientists found that these molecules, which do not contain the light chain, could remain functional even when exposed to temperatures as high as 90 degrees C. "If they do unfold, they can actually completely refold on cooling and they can cycle over and over again," Hayhurst explains. "For field use, where you don't have very much refrigeration--in the developing world, for example--you could have an infinitely stable diagnostic assay for infectious disease." So, Hayhurst, along with Ellen Goldman of the U.S. Naval Research Laboratory, decided to try these antibodies as biosensors.
When the team tried to clone the heavy chain-only antibodies from three llamas, they could not get a set that would bind well to the several biothreats they desired to test--among them ricin, cholera toxin and vaccinia (a surrogate of smallpox virus). Normally, within the llama, these antibodies would adapt to a new antigen (via a process called somatic hypermutation), but the researchers could not inject the animals with cholera and wait for them to create the correct antibodies. So, instead, they genetically manipulated the genes that controlled the CDRs of the heavy chain-only antibodies, chopping them up and randomly mutating them to generate a varied library of binding sites for their biosensor. These semisynthetic antibodies have, according to Hayhurst, "far more diversity, so there's far more chance of actually pulling out a binder to any target you want." Once they had their better-binding antibodies, the group tested their thermal stability by exposing them to a temperature of 95 degrees C. Compared with antibodies found in sheep, rabbits and mice, the natural ones typically broke down completely within five minutes, whereas the semisynthetic variety retained at least some of their functionality for as long as 80 minutes.
Hayhurst believes his and Goldman's genetically manipulated antibodies will be able to withstand field use in hot, dry areas. "We're looking at diagnostic assays for Ebola and Marburg viruses that we can actually take out into the field in Africa--dreadfully resource-poor environments, incredibly hot and where we need diagnostic assays," Hayhurst says, referring to the Southwest Foundation's intentions for this technology. (Goldman could not be reached for comment on what applications the Navy is planning for the new antibodies.) Clement Furlong, a researcher in medical and genomic sciences at the University of Washington notes that the "huge advantage" of these heavy-chain-only antibodies "is that you can make them by fermentation in microbial systems and make a lot of antibodies inexpensively." Then, he adds, "you have extremely inexpensive reagents, so you can then develop technologies for Third World countries as well as First World countries."
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