Sortase-Mediated Phage Decoration for Analytical Applications

Abstract

Phage-borne peptides and antibody fragments isolated from phage display libraries have proven to be versatile and valuable reagents for immunoassay development. Due to the lack of convenient and mild-condition methods for the labeling of the phage particles, isolated peptide/protein affinity ligands are commonly removed from the viral particles and conjugated to protein tracers or nanoparticles for analytical use. This abolishes the advantage of isolating ready-to-use affinity binders and creates the risk of affecting the polypeptide activity. To circumvent this problem, we optimized the phage display system to produce phage particles that express the affinity binder on pIII and a polyglycine short peptide fused to pVIII that allows the covalent attachment of tracer molecules employing sortase A. Using a llama heavy chain only variable domain (VHH) against the herbicide 2,4-D on pIII as the model, we showed that the phage can be extensively decorated with a rhodamine-LPETGG peptide conjugate or the protein nanoluciferase (Nluc) equipped with a C-terminal LPETGG peptide. The maximum labeling amounts of rhodamine-LPETGG and Nluc-LPETGG were 1238 ± 63 and 102 ± 16 per phage, respectively. The Nluc-labeled dual display phage was employed to develop a phage bioluminescent immunoassay (P-BLEIA) for the detection of 2,4-D. The limit of detection and 50% inhibition concentration of P-BLEIA were 0.491 and 2.15 ng mL −1 , respectively, which represent 16-fold and 8-fold improvement compared to the phage enzyme-linked immunosorbent assay. In addition, the P-BLEIA showed good accuracy for the detection of 2,4-D in spiked samples.

M13 phage: a versatile building block for a highly specific analysis platform

“…The direct insertion of foreign genes facilitates 100% display of the endogenous peptide on M13 surface, but only within short peptide. With the assistance of transpeptidase, for instance, Sortase A, the size of peptide/protein displayed on M13 surface can be facilely expanded [25,105,106]. However, Sortase A-based displaying strategy faced another issue of reaction reversibility [107].…”

Rapid and sensitive immunoassay for alpha-fetoprotein in serum by fabricating primary antibody–enzyme complexes using protein self-assembly

“…After testing, it was found that each phage can be coupled with up to 102 ± 16 Nluc molecules, and the Nluc-labeled dual display phage was employed to develop a phage bioluminescent immunoassay (P-BLEIA) for the detection of herbicide 2,4-D, which represent a 16-fold improvement compared to the phage enzyme-linked immunosorbent assay (ELISA). 24 However, the traditional multivalent PAEC development strategies are cumbersome and time consuming, which limits its application in immunoassays.…”

“…Ding et al optimized the phage display system to produce M13 phage particles that express the nanobody on pIII and a polyglycine short peptide (LPETGG) fused to pVIII that allows the covalent attachment of nanoluciferase (Nluc) employing sortase A. Aer testing, it was found that each phage can be coupled with up to 102 ± 16 Nluc molecules, and the Nluc-labeled dual display phage was employed to develop a phage bioluminescent immunoassay (P-BLEIA) for the detection of herbicide 2,4-D, which represent a 16-fold improvement compared to the phage enzyme-linked immunosorbent assay (ELISA). 24 However, the traditional multivalent PAEC development strategies are cumbersome and time consuming, which limits its application in immunoassays.…”

Enhancing Oriented Immobilization Efficiency: A One-for-Two Organism-Bispecific Nanobody Scaffold for Highly Sensitive Detection of Foodborne Pathogens

“…10−12 Compared with conventional IgG antibodies, nanobodies, which have determine genetic code, exhibit unique advantages, such as great antigenicity toward cryptic epitope, high thermostability, simple expression system, and ease of genetic engineering, and facilitate the construction of immunoassays. 13−15 At present, nanobody-based immunoassays have found extensive application in diverse analytical domains, including the analysis of mycotoxins, 16−18 pesticide residues, 19 foodborne pathogens, 20,13 environmental pollutants, 21,22 and cancer markers. 23,24 However, their diminutive size poses a challenge as passive adsorption of nanobodies onto physical surfaces in a random manner can significantly compromise their binding function.…”

“…In recent years, nanobodies derived from the heavy chain antibodies of camelids and cartilaginous fish have emerged as a subject of increasing interest in the realm of immunoassays. − Compared with conventional IgG antibodies, nanobodies, which have determine genetic code, exhibit unique advantages, such as great antigenicity toward cryptic epitope, high thermostability, simple expression system, and ease of genetic engineering, and facilitate the construction of immunoassays. − At present, nanobody-based immunoassays have found extensive application in diverse analytical domains, including the analysis of mycotoxins, − pesticide residues, foodborne pathogens, , environmental pollutants, , and cancer markers. , However, their diminutive size poses a challenge as passive adsorption of nanobodies onto physical surfaces in a random manner can significantly compromise their binding function. This arises from the fact that their binding interface constitutes a relatively larger proportion of the total surface area, leading to concealed binding sites and a diminished signal-to-noise ratio, particularly when detecting large molecules like bacteria and proteins .…”