US AIR FORCE, STAFF SGT ARACELI ALARCON
The polyclonal antibodies used to detect the “exercise hormone” irisin in commercial ELISA kits are nonspecific and give false positive results, thereby negating much of the research into the hormone’s function over the last three years, according to a study published this week (March 9) in Scientific Reports. (See “Validating Antibodies: An Urgent Need,” The Scientist, December 2014.)
First announced in a 2012 Nature paper, irisin was identified as the cleaved glycosylated tail of the membrane-bound muscle protein FNDC5, which is thought to travel through the blood and convert white fat cells into calorie-burning brown fat. More than 80 studies that followed up on this finding and its implications for diabetes and obesity used commercial ELISA kits based on three antibodies to identify the protein. When researchers from the Leibniz Institute for Farm Animal Biology in Germany, the University of Oslo, Duke University, and the University of Berne tested the antibodies used in these commercially available ELISA kits, they found that the antibodies did indeed detect lab-synthesized forms of irisin. However, the antibodies were unable to recognize protein of the correct size in blood samples from humans, exercising horses, or a variety of other animals. Instead, the antibodies cross-reacted with a wide range of other unidentified proteins. Because ELISA assaysdo not distinguish proteins by their size, this suggested that many of the previously published results were false positives. For the present study, the Leibniz Institute-led team used a Western blot approach.
Moreover, in this latest analysis, the scientists found that human FNDC5 carried a mutation at the beginning of the gene that results in extremely low levels of the protein, approximately 1 percent of the amount that would be expected from a non-mutated protein. It is thus unlikely that ELISA could have detected the reported levels of irisin, the authors wrote.
“From the start, the study of irisin has been complicated by unvalidated reagents and contradictory data,” study coauthor Harold Erickson of Duke University said in a statement. “Hopefully, our findings will finally convince other researchers to stop chasing a myth.”