Molecular Biology Space
In addition to enhanced sensitivity, another advantage of isotopic labeling is the incorporation into analytes without altering structure or reactivity. With AMS, the sensitivity of an immunoassay is constrained by the affinity constant of the antibody and not the detection system. In a radioimmunoassay (RIA) using 14C-labeled pesticide atrazine, the detection limit of atrazine was about 10 times lower than the Ka of the capture antibody and the radioactivity used in this assay was 1,000 times lower than in conventional RIA. Knowing a true Ka of an antibody by using AMS would help the understanding of antibody properties and antigen interactions and assist with antibody design and engineering.
AMS could also be applied to elucidation of the role of modified nucleosides in cancer initiation and progression or in viral cycles, for gene-therapy studies, and for the evaluation of novel DNA delivery technologies. In 2005, Vitalea completed an AMS-based study on metabolism of the anti-retroviral compound AZT. This drug is a deoxythymidine analog, which causes termination of elongating DNA chain.
“We were able to quantify the uptake and retention of the orally administered AZT in white blood cells and, specifically, into genetic material of these cells,” explained Le Thuy Vuong, co-founder of Vitalea. “Such data could not have been obtained by any other method and is particularly valuable for a pediatric population.”
“AMS, being the most sensitive analytic technique ever developed, allows us to follow the fate of large biological molecules such as peptides and DNA,” continues Dr. Garner. Radioactive precursors could be supplied directly into cell culture growth media. Addition of 14C deoxyguanosine leads to incorporation of the label into a growing DNA chain. An antibody can be labeled in vivo using radioactive amino acids added to the hybridoma growth media.
Xceleron used AMS to trace CAT-192 (metelimumab) labeled in vivo with 14C. Even though the incorporation of the tracer into the antibody was only 1.4%, AMS was able to detect intact and denatured antibodies, as well as antibody fragments and metabolites. Moreover, the limit of detection was about 1 ng/mL serum, which is ~15 times lower than ELISA, says Dr. Garner.
The success of this study predicts that it may be possible to detect disease-specific biomarkers using ELISA with 14C-labeled antibodies. “We are exploring AMS applications for population monitoring, diagnostics, and pharmacogenomics,” adds Dr. Garner. “And that’s only scratching the surface.”
“We are interested in expanding AMS applications into genomic and proteomic areas,” says Ali Arjomand, Ph.D., president and scientific director at Accium Biosciences (www.acciumbio.com). “A lightly labeled 14C biological, for example, is eventually broken down in the body and its 14C nucleotides or 14C amino acids are recycled. By isolating and quantifying a specific 14C-labeled product, AMS can help to integrate the biochemical, genomic, and proteomic processes involved in a particular disease, eventually leading to discovery of validated biomarkers.”
Even though AMS does not identify the exact metabolite structure but only concentration of the label, it is possible to split HPLC fractions between classical MS and AMS. This way, both the structure and the concentration of the metabolite could be determined. Moreover, AMS could also track interactions between drugs, their metabolites, and cellular machinery such as transporters involved in internalization and excretion of the chemical moieties.