title: Engineering resonance energy transfer for advanced immunoassays: the case of celiac disease.
abstract: Celiac disease (CD) is an immune-mediated disorder affecting genetically predisposed subjects. It is caused by the ingestion of wheat gluten and related prolamins. A final diagnosis for this disease can be obtained by examination of jejunal biopsies. Nevertheless, different analytical approaches have been established to detect the presence of anti-tissue transglutaminase antibodies that represent a serological hallmark of the disease. In this work, we explored a new method for the diagnosis of CD based on the detection of serum anti-transglutaminase antibodies by resonance energy transfer (RET) between donor molecules and acceptor molecules. In particular, we labeled the liver transglutaminase (tTG) enzyme from guinea pig and the rabbit anti-tTG antibodies with a couple of fluorescence probes that are able to make RET if they are located within with Förster distance. We labeled tTG with the fluorescence probe DyLight 594 as donor and the anti-tTG antibodies with the fluorescence probe DyLight 649 as acceptor. However, due to the large size of the formed complex (tTG/anti-tTG), and consequently to the low efficiency energy transfer process between the donor-acceptor molecules, we explored a new experimental approach that allows us to extend the utilizable range of RET between donor:acceptor pairs by using one single molecule as donor and multiple molecules as energy acceptors, instead of using a single acceptor molecule as usually occurs in RET experiments. The obtained results clearly show that the use of one donor and multiacceptor strategy enables for a simple and rapid detection of serum anti-transglutaminase antibodies. In addition, our results point out that it is possible to consider this approach as a new method for a wide variety of analytical assays.
authors.affiliation: Institute of Protein Biochemistry, CNR, Naples, Italy. s.dauria@ibp.cnr.it
authors.forename: Sabato
authors.initials: S
authors.lastname: D'Auria
authors.forename: Elisa
authors.initials: E
authors.lastname: Apicella
authors.forename: Maria
authors.initials: M
authors.lastname: Staiano
authors.forename: Stefano
authors.initials: S
authors.lastname: Di Giovanni
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Ruggiero
authors.forename: Mauro
authors.initials: M
authors.lastname: Rossi
authors.forename: Pabak
authors.initials: P
authors.lastname: Sarkar
authors.forename: Rafal
authors.initials: R
authors.lastname: Luchowski
authors.forename: Ignacy
authors.initials: I
authors.lastname: Gryczynski
authors.forename: Zygmunt
authors.initials: Z
authors.lastname: Gryczynski
mesh_terms: D000818:Animals; D001323:Autoantibodies / Q000097:blood; D002446:Celiac Disease / Q000175:diagnosis* / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D031541:Fluorescence Resonance Energy Transfer; D006168:Guinea Pigs; D011503:Transglutaminases / Q000378:metabolism
chemical_list: D001323:Autoantibodies; D011503:Transglutaminases