Abstract
In general immunoassays, secondary antibodies are covalently linked with enzymes and bind to the Fc region of target-bound primary antibodies to amplify signals of low-abundant target molecules. The antibodies themselves are obtained from large mammals and are further modified with enzymes. In this study, we developed novel recombinant immunoglobulin G (IgG)-binding luciferase-based signal amplifiers (rILSAs) by genetically fusing luciferase (Nluc) with antimouse IgG1 nanobody (MG1Nb) and antibody-binding domain (ABD), individually or together, in a mix-and-match manner. We obtained three different highly pure rILSAs in large quantities using a bacterial overexpression system and one-step purification. Mouse-specific rILSA, MG1Nb-Nluc, and rabbit-specific rILSA, Nluc-ABD, selectively bound to target-molecule-bound mouse IgG1 and rabbit IgG primary antibodies, whereas the bispecific rILSA, MG1Nb-Nluc-ABD, mutually bound to both mouse IgG1 and rabbit IgG primary antibodies. All rILSAs exhibited an outstanding signal-amplifying capability comparable to those of conventional horseradish-peroxidase-conjugated secondary antibodies, regardless of the target molecules, in various immunoassay formats, such as enzyme-linked immunosorbent...Continue Reading
References
Jun 11, 1996·Proceedings of the National Academy of Sciences of the United States of America·A C Braisted, J A Wells
Oct 31, 1997·Journal of Molecular Biology·M TashiroG T Montelione
Jul 1, 1955·The Journal of Experimental Medicine·A H COONSJ M CONNOLLY
Jun 15, 2005·ILAR Journal·Neil S LipmanFrances Weis-Garcia
Jan 13, 2006·British Journal of Cancer·P WentP A Baeuerle
May 9, 2006·Gynecologic Oncology·Gilbert SpizzoAlain G Zeimet
Nov 21, 2007·Analytical Biochemistry·Yongwon JungBong Hyun Chung
Feb 7, 2008·Analytical Biochemistry·Yoshihito YoshidaIwao Waga
Jan 10, 2012·Analytical Chemistry·Vladimir GubalaDavid E Williams
Jun 19, 2012·Analytical Biochemistry·Etvi JuntunenKim Pettersson
Aug 17, 2012·ACS Chemical Biology·Mary P HallKeith V Wood
Nov 2, 2012·Lab on a Chip·Yuriko OyamaShoji Takeuchi
Dec 6, 2012·Current Chemical Genomics·Marjeta Urh, Martin Rosenberg
May 11, 2013·Genetics and Molecular Research : GMR·Jun MaS Q Liao
Dec 18, 2013·Biosensors & Bioelectronics·Jie HuFeng Xu
Oct 25, 2014·Scientific Reports·Rupak DoshiGeoffrey Chang
Apr 14, 2015·Advanced Healthcare Materials·Miju KimJunsang Doh
May 15, 2015·Bioconjugate Chemistry·Christopher G EnglandWeibo Cai
Jun 10, 2015·The Journal of Cell Biology·Jonas HelmaHeinrich Leonhardt
Jun 13, 2015·Chemical Communications : Chem Comm·Jisu LeeSebyung Kang
Aug 13, 2015·Biosensors & Bioelectronics·Gülsen Betül AktasLluis Masip
Mar 1, 2016·Frontiers in Pharmacology·Nicolas BouteMichael Tesar
Apr 12, 2016·Scientific Reports·Junseon MinSebyung Kang
Jun 15, 2016·Biosensors & Bioelectronics·Jia Li, Joanne Macdonald
Nov 3, 2016·Analytical Chemistry·Sukyo JeongHak-Sung Kim
Dec 14, 2016·Analytical Chemistry·Samiksha NayakSamuel K Sia
Jun 3, 2017·Analytical Chemistry·Hong DuanYonghua Xiong
Dec 22, 2017·The Journal of Cell Biology·Tino PleinerDirk Görlich
Feb 9, 2019·Analytical and Bioanalytical Chemistry·J-Pablo SalvadorM-Pilar Marco
Apr 26, 2019·Electrophoresis·Manish MishraAldrin V Gomes
Jul 7, 2019·Biosensors & Bioelectronics·Gaojian YangZiyu He