title: An amperometric immunosensor for diagnosis of celiac disease based on covalent immobilization of open conformation tissue transglutaminase for determination of anti-tTG antibodies in human serum.
abstract: A new amperometric immunosensor based on the covalent immobilization of tissue transglutaminase enzyme in its open conformation (open-tTG) was developed and optimized for determination of anti-tissue transglutaminase antibodies (anti-tTG) in human serum. Experimental design allowed us to find the optimal conditions for quantification of both IgA and IgG isotypes of anti-tTG in order to assess suitability of the device for diagnostic purposes. The glassy carbon electrodic substrate was electrochemically functionalized with gold nanoparticles and subsequently derivatized with a self-assembled monolayer of 11-mercaptoundecanoic acid for the covalent anchoring of the enzyme. This step was performed under carefully controlled conditions in order to keep the open conformation of the tTG. The immunosensor showed good analytical performance with limit of detection levels (1.7 AU mL(-1) for IgA and 2.7 AU mL(-1) for IgG) below the diagnostic threshold value (3.0 AU mL(-1)) and inter-sensor reproducibility giving RSD lower than 10%. The developed sensor was validated in serum samples from pediatric patients for clinical applications, using two ELISA kits specific for the determination of anti-tTG IgA and IgG antibodies as reference methods; good recovery rates ranging from 74% to 117% were calculated.
authors.affiliation: Dipartimento di Chimica - Università di Parma, Parco Area delle Scienze 17/A, 43124 Parma, Italy; Centro Interdipartimentale SITEIA.PR - Università di Parma, Parco Area delle Scienze 181/A, 43124 Parma, Italy. Electronic address: marco.giannetto@unipr.it.
authors.forename: Marco
authors.initials: M
authors.lastname: Giannetto
authors.affiliation: Dipartimento di Chimica - Università di Parma, Parco Area delle Scienze 17/A, 43124 Parma, Italy; Centro Interdipartimentale SITEIA.PR - Università di Parma, Parco Area delle Scienze 181/A, 43124 Parma, Italy.
authors.forename: Monica
authors.initials: M
authors.lastname: Mattarozzi
authors.affiliation: Dipartimento di Chimica - Università di Parma, Parco Area delle Scienze 17/A, 43124 Parma, Italy.
authors.forename: Eleonora
authors.initials: E
authors.lastname: Umiltà
authors.affiliation: Dipartimento di Chimica - Università di Parma, Parco Area delle Scienze 17/A, 43124 Parma, Italy.
authors.forename: Anita
authors.initials: A
authors.lastname: Manfredi
authors.affiliation: Institute for Maternal and Child Health - IRCCS "Burlo Garofolo", Via dell'Istria, 65, 34137 Trieste, Italy.
authors.forename: Sara
authors.initials: S
authors.lastname: Quaglia
authors.affiliation: Dipartimento di Chimica - Università di Parma, Parco Area delle Scienze 17/A, 43124 Parma, Italy; Centro Interdipartimentale SITEIA.PR - Università di Parma, Parco Area delle Scienze 181/A, 43124 Parma, Italy.
authors.forename: Maria
authors.initials: M
authors.lastname: Careri
mesh_terms: D001323:Autoantibodies / Q000097:blood*; D015374:Biosensing Techniques / Q000295:instrumentation / Q000379:methods* / Q000706:statistics & numerical data; D002446:Celiac Disease / Q000175:diagnosis* / Q000201:enzymology / Q000276:immunology; D002648:Child; D055664:Electrochemical Techniques; D004797:Enzyme-Linked Immunosorbent Assay; D004800:Enzymes, Immobilized / Q000737:chemistry / Q000276:immunology; D005227:Fatty Acids; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000276:immunology*; D006046:Gold; D006801:Humans; D007070:Immunoglobulin A / Q000097:blood; D007074:Immunoglobulin G / Q000097:blood; D057230:Limit of Detection; D053768:Metal Nanoparticles; D011487:Protein Conformation; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D015203:Reproducibility of Results; D013438:Sulfhydryl Compounds; D011503:Transglutaminases / Q000737:chemistry / Q000276:immunology*
chemical_list: C505222:11-mercaptoundecanoic acid; D001323:Autoantibodies; D004800:Enzymes, Immobilized; D005227:Fatty Acids; D007070:Immunoglobulin A; D007074:Immunoglobulin G; D013438:Sulfhydryl Compounds; D006046:Gold; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D011503:Transglutaminases; D019204:GTP-Binding Proteins
keywords: Amperometric immunosensor; Anti-transglutaminase antibodies; Celiac disease; Diagnosis; Open tissue transglutaminase; Pediatric serum