Elsevier

Analytica Chimica Acta

Volume 1184, 1 November 2021, 339054
Analytica Chimica Acta

Directional immobilization of antibody onto magnetic nanoparticles by Fc-binding protein-assisted photo-conjugation for high sensitivity detection of antigen

https://doi.org/10.1016/j.aca.2021.339054Get rights and content

Highlights

  • A photoactivable ZBpa-Cys recombinant was genetically engineered by aaRS/tRNA technique.

  • A thiol modified antibody with Fc-specific and covalent conjugation was fabricated by employing ZBpa-Cys.

  • An approach for directional immobilization of antibody onto MNPs was proposed.

  • A MNPs-based ELISA for enhanced antigen detection was developed.

  • The sensitivity was about 100 times more than the traditional microplate ELISA.

Abstract

Immobilized antibodies with site-specific, oriented, and covalent pattern are of great significance to improve the sensitivity of solid-phase immunoassay. Here, we developed a novel antibody conjugation strategy that can immobilize antibodies in a directional and covalent manner. In this study, an IgG-Fc binding protein (Z domain) carrying a site-specific photo-crosslinker, p-benzoyl-L-phenylalanine, and a single C-terminal cysteine (Cys) handle was genetically engineered. Upon UV irradiation, the chimeric protein enables the Cys handle to couple with the native antibody in Fc-specific and covalent conjugation pattern, resulting in a novel thiolated antibody. Thus, an approach for the covalent, directional immobilization of antibodies to maleimide-modified magnetic nanoparticles (MNPs) was developed on the basis of the crosslinking between sulfhydryl and maleimide groups. The antibody-conjugated MNPs were applied in MNP-based enzyme-linked immunosorbent assay (ELISA) for the detection of carcinoembryonic antigen. The MNP-based ELISA presented a quantification linear range of 0.1–100 ng mL−1 and detection limit of 0.02 ng mL−1, which was approximately 100 times more sensitive than the traditional microplate ELISA (2.0 ng mL−1). Thus, the proposed antibody immobilization approach can be used in surface functionalization for the sensitive detection of various biomarkers.

Introduction

Solid-phase immunoassays (SPIs), such as enzyme-linked immunosorbent assays (ELISAs), immunosensors, and antibody chips, etc., have been widely used in disease diagnosis and biological research [1,2]. Among them, the plastic microtiter plate-based ELISA is the most popular format [[3], [4], [5]]. In recent years, magnetic particles (MPs), a substitute of the microplate, have been increasingly used in ELISA to detect various analytes, such as disease-related biomarkers, environmental contaminants, and food toxins, etc. [[6], [7], [8], [9]]. Given their high surface area-to-volume ratio and facile magnetic separation, the analytes would be effectively enriched from a large volume onto the antibody-coated MPs; as a result, the sensitivity of ELISA was significantly improved [[10], [11], [12]]. Despite this advancement, a critical step in SPIs is the immobilization of antibodies to substrates. Antibodies are commonly immobilized onto the surfaces of substrates through physical adsorption of hydrophobic interactions, or covalent conjugation of chemical cross-linkers [13,14]. Covalent immobilization relies on the chemical reaction of amino acid residues (amine/carboxyl groups) within antibody, and antibodies can be stably immobilized onto substrates via chemical cross-linkers [13]. Covalent conjugation strategy is efficient but suffers from non-specificity because all functional groups of antibody can possibly react. Due to uncontrolled and random immobilization, these immobilization methods result in a low density of antigen binding sites, and increasing the amount of antibodies adsorbed on the support cannot effectively increase the antigen binding capacity [15,16].

Several strategies for the oriented immobilization of native antibody have been reported. For example, controlled immobilization can be achieved with excellent steric accessibility of the antigen binding site through conjugation on the carbohydrate moiety located on the Fc region of a glycosylated antibody [17]. Immunoglobulin G (IgG)-binding proteins, such as protein A and G, provide a general strategy to control immobilization due to their selectivity to the Fc region of the antibody [16,18]. These strategies substantially improve the directional immobilization of antibodies and the sensitivity of immunoassay. However, the strategy of linking a carbohydrate moiety for the directional immobilization is well suitable for glycosylated antibody, but not for non-glycosylated antibody [17]. Protein A/G can bind to human and animal IgGs, and the bioaffinity interactions are reversible, which may not be sufficient for certain applications owing to the potential cross-reactivity [19,20]. To date, the development of a profitable approach for controlled orientation of antibodies still remains a challenge.

If the reversible bio-interaction could be transformed into irreversible covalent conjugation, protein A/G will play a more important role in controlled immobilization of antibody. The bioaffinity molecules between IgG-binding proteins and IgGs could be crosslinked via chemical crosslinkers such as dimethyl pimelimidate (DMP) and bis-sulfosuccinimidyl suberate [21,22]; however, these crosslinkers may also conjugate the amine groups at the antigen binding sites, leading to decreased antigen binding efficiency. Photo-affinity labeling technique is another attractive strategy for forming the irreversible conjugation between antibody-binding protein and antibody [23]. For example, photoactivatable probes, such as p-benzoyl-L-phenylalanine (Bpa), an unnatural amino acid, can be introduced in the IgG-binding protein domain by employing solid phase peptide synthesis, post-translational modification of protein, or aminoacyl-tRNA synthetase/suppressor tRNA technique (aaRS/tRNA) [[24], [25], [26]]. By employing the inherent Fc-binding capability of IgG-binding protein, Bpa modified IgG-binding protein first forms into bio-affinity complex with IgG. Then, upon 365 nm irradiation, Bpa is activated and can form a covalent bond to an amino acid situated in close proximity, thereby forming the irreversible pair between IgG and IgG-binding protein [24,27]. Those functional groups or molecules such as alkyne, azide, and biotin, can be covalently conjugated to IgGs through the engineered photoreactive IgG-binding proteins without interfering with the binding site of antibodies [26,28].

Herein, we describe a general and effective approach for site-specific and covalent immobilization of native antibodies onto MP support via photoactivatable Fc-binding protein-assisted immobilization strategy. In this study, the Z domain, a well-known Fc-binding molecule, was incorporated in a photoactivatable probe (Bpa) and then tagged with Cys handle. Then, aaRS/tRNA technology was used to obtain the photoactivatable ZBpa–Cys recombinant. Subsequently, by employing the bioaffinity with Fc region and covalent bond-forming ability of the ZBpa moiety, the unique functional Cys handle was covalently conjugated to native antibody, resulting in the Fc-specific thiolated antibody. On the basis of the reaction between sulfhydryl and maleimide groups, the thiolated antibody was covalently immobilized onto the maleimide-modified magnetic nanoparticles (MNPs). Thus, a general platform for directional immobilization of antibody with a covalent and homogeneous pattern was developed. A schematic of this proposed approach is shown in Fig. 1. On the basis of MNP-based ELISA, the performance of the proposed antibody immobilization platform for sensitive immunoassay was discussed by detecting carcinoembryonic antigen (CEA) as a model study.

Section snippets

Materials

pTXB1 plasmid was purchased from New England Biolabs (UK), and pEVOL-pBpF plasmid was purchased from Addgene (UK). Bpa was purchased from Codow (China). CEA and horseradish peroxidase (HRP) conjugated goat anti-mouse IgG (HRP-goat IgG) were from Boster (China). Rabbit anti-CEA IgG and mouse anti-CEA IgG (IgG2b) were from ZSGB-Bio (China) and OriGene (USA), respectively. F(ab')2 fragment (anti-goat IgG) was from Jackson ImmunoResearch (USA). Amine-modified MNPs (Affimag FEO, Fe3O4 core

Production of photoactivatable ZBpa–Cys

The sulfhydryl group is a useful reactive group for protein conjugation. For instance, by employing the specific reaction of sulfhydryl and maleimide (Mal) group, a stable thioether linkage is formed between the –SH of protein and Mal reagent [17,30]. Typically, only free –SH groups are available for thiol-reactive conjugation. The native antibody does not contain free –SH, and the reactive –SH can be obtained by reducing antibody; however, it is usually accompanied by a decrease in antigen

Conclusions

In this study, a Cys handle was successfully conjugated to the Fc region of native antibody by employing Fc-binding protein-assisted photo-conjugation. Thus, a specific and selective site for further antibody conjugation was provided. Moreover, a general platform was developed for covalent, oriented immobilization of antibodies onto maleimide group-modified supports. The feasibility and effectiveness of the proposed antibody immobilization platform for enhancing the sensitivity of detection

CRediT authorship contribution statement

Jin-Bao Tang: Methodology, experiment and writing. Hong-Ming Yang: Data curation. Xiao-Yi Gao: Data curation. Xian-Zhong Zeng: Software. Feng-Shan Wang: Writing – review & editing.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

This work was supported by the National Natural Scientific Foundation of China (81971998), and the Natural Science Foundation of Shandong Province (ZR2018MC009).

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