Oriented antibody immobilization by site-specific UV photocrosslinking of biotin at the conserved nucleotide binding site for enhanced antigen detection

Alves, Nathan J.; Mustafaoğlu, Nur; Bilgiçer, Başar

doi:10.1016/j.bios.2013.05.052

Cited by 39 publications

(54 citation statements)

References 38 publications

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“…33–38 In multiple publications, we established that the UV-NBS method can be used to functionalize antibodies without unfavorably impacting their antigen binding activity. Furthermore, we also demonstrated many utilities that the NBS can provide in a variety of applications beyond covalent linking such as oriented immobilization, 35,36 enhanced antigen detection, 35,37,38 as well as inhibition of allergic reactions 39–41 .…”

Section: Introductionmentioning

confidence: 90%

Site-specific conjugation of an antibody on a gold nanoparticle surface for one-step diagnosis of prostate specific antigen with dynamic light scattering

2017

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Small dimensions of gold nanoparticles (AuNPs) necessitate antibodies to be immobilized in an oriented fashion in order to conserve their antigen binding activity for proper function. In this study, we used the previously described UV-NBS method to site-specifically incorporate thioctic acid (TA) functionality into antibodies at the conserved nucleotide-binding site (NBS). Modified antibodies were immobilized on AuNP surface in an oriented manner utilizing the newly incorporated TA functionality while maintaining antibody structure and activity. The resulting antibody functionalized AuNPs via the UV-NBS method demonstrated significantly enhanced antigen detection capabilities and improved antigen detection sensitivity with high level of selectivity when compared to other commonly used AuNP functionalization methods. Our results demonstrate that the limit of detection (LOD) for AuNPs functionalized via the UV-NBS method was 55 pM PSA, which is 40, 851, and 5873-fold improved over the other immobilization methods: EDC-NHS, thiol reduction, and ionic interaction, respectively. Consequently, the UV-NBS method provides a universal, site-specific functionalization method that generates highly sensitive and more stable antibody functionalized AuNPs that is amenable to any available detection and treatment assay with potential significant implications.

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Section: Introductionmentioning

confidence: 90%

Site-specific conjugation of an antibody on a gold nanoparticle surface for one-step diagnosis of prostate specific antigen with dynamic light scattering

2017

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“…1 Various immobilization strategies, such as adsorption, 2,3 covalent coupling, 4−6 entrapment, 7−9 crosslinking, 10,11 and affinity, 12,13 have been developed to construct biosensors. Adsorption technique presented drawbacks, such as desorption of the immobilized biomolecules, nonspecific adsorption of other substances, and poor operational and storage stability.…”

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confidence: 99%

Biomimetic Superoxide Dismutase Stabilized by Photopolymerization for Superoxide Anions Biosensing and Cell Monitoring

Yuan

Liu

et al. 2014

Anal. Chem.

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Photopolymerization strategy, as one of the immobilization methods, has attracted considerable interest because of some advantages, such as easy operation, harmlessness to the biomolecules, and long storage stability. (E)-4-(4-Formylstyryl) pyridine (formylstyrylpyridine) was prepared through Heck reaction and used as a photopolymer material to immobilize biomimetic superoxide dismutase under ultraviolet irradiation (UV) irradiation in a short time. The styrylpyridinium moiety of Formylstyrylpyridine was photoreactive and formed a dimer under UV irradiation. Mn2P2O7 multilayer sheet, a novel superoxide dismutase mimic, was synthesized. The formed photopolymer can immobilize Mn2P2O7 firmly under UV irradiation. On the basis of high catalytic activity of Mn2P2O7 biomimetic enzyme and long-term stability of Mn2P2O7-formylstyrylpyridine film, after introducing multiwalled carbon nanotubes (MWCNTs), a novel electrochemical biosensing platform called MWCNTs/Mn2P2O7-formylstyrylpyridine for superoxide anion (O2(•-)) detection was constructed. The biosensor displayed good performance for O2(•-) detection and provided a reliable platform to adhere living cells directly on the modified electrode surface. Therefore, the biosensor was successfully applied to vitro determination of O2(•-) released from living cells, which had a promising prospect for living cells monitoring and diagnosis of reactive oxygen species-related diseases.

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“…They also used the same photo-cross-linking strategy to prepare biotin labeled antibodies and coated such conjugates onto a streptavidin plate for enhanced antigen detection. [96,97] Although NBS is present in the variable region of an antibody but it is still distant from the antigen binding site ( Figure 6 ),[93,94] and doesn't affect antigen binding capacity of the antibodies. In addition to NBS, other unconventional binding sites on antibodies, which are specific for small molecules, have been identified and may offer alternative conjugation/immobilization strategies.…”

Section: Antibody Immobilization Chemistriesmentioning

confidence: 99%

Site-selective orientated immobilization of antibodies and conjugates for immunodiagnostics development

Shen

Rusling

Dixit

2017

Methods

162

109

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Immobilized antibody systems are the key to develop efficient diagnostics and separations tools. In the last decade, developments in the field of biomolecular engineering and crosslinker chemistry have greatly influenced the development of this field. With all these new approaches at our disposal, several new immobilization methods have been created to address the main challenges associated with immobilized antibodies. Few of these challenges that we have discussed in this review are mainly associated to the site-specific immobilization, appropriate orientation, and activity retention. We have discussed the effect of antibody immobilization approaches on the parameters on the performance of an immunoassay.

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Oriented antibody immobilization by site-specific UV photocrosslinking of biotin at the conserved nucleotide binding site for enhanced antigen detection

Cited by 39 publications

References 38 publications

Site-specific conjugation of an antibody on a gold nanoparticle surface for one-step diagnosis of prostate specific antigen with dynamic light scattering

Site-specific conjugation of an antibody on a gold nanoparticle surface for one-step diagnosis of prostate specific antigen with dynamic light scattering

Biomimetic Superoxide Dismutase Stabilized by Photopolymerization for Superoxide Anions Biosensing and Cell Monitoring

Site-selective orientated immobilization of antibodies and conjugates for immunodiagnostics development

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