Amplification of immunoassays using phage-displayed single domain antibodies

Goldman, Ellen R.; Anderson, George P.; Bernstein, Rachael D.; Swain, Marla D.

doi:10.1016/j.jim.2009.10.014

Cited by 23 publications

(12 citation statements)

References 12 publications

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“…For many analytical and diagnostics applications Nbs have been directly used as phage borne antibody fragments. The M13 phage particle, commonly used for phage display, has ~2,700 copies of the pVIII protein providing a large surface for tracer attachment, which translates in higher assay sensitivity that can be up to two orders of magnitude better than that obtained with the soluble Nb ( 91 ). Likewise, the phage DNA can be used for ultrasensitive detection of the phage borne antibody using real-time PCR in different immunoassay formats, which also extends the dynamic range of the assay in several orders of magnitude ( 92 , 93 ).…”

Section: Nbs As Multipurpose Affinity Building Domainsmentioning

confidence: 99%

Single-Domain Antibodies As Versatile Affinity Reagents for Analytical and Diagnostic Applications

González‐Sapienza¹,

Rossotti²,

Rosa³

2017

Front. Immunol.

144

130

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With just three CDRs in their variable domains, the antigen-binding site of camelid heavy-chain-only antibodies (HcAbs) has a more limited structural diversity than that of conventional antibodies. Even so, this does not seem to limit their specificity and high affinity as HcAbs against a broad range of structurally diverse antigens have been reported. The recombinant form of their variable domain [nanobody (Nb)] has outstanding properties that make Nbs, not just an alternative option to conventional antibodies, but in many cases, these properties allow them to reach analytical or diagnostic performances that cannot be accomplished with conventional antibodies. These attributes include comprehensive representation of the immune specificity in display libraries, easy adaptation to high-throughput screening, exceptional stability, minimal size, and versatility as affinity building block. Here, we critically reviewed each of these properties and highlight their relevance with regard to recent developments in different fields of immunosensing applications.

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Section: Nbs As Multipurpose Affinity Building Domainsmentioning

confidence: 99%

Single-Domain Antibodies As Versatile Affinity Reagents for Analytical and Diagnostic Applications

González‐Sapienza¹,

Rossotti²,

Rosa³

2017

Front. Immunol.

144

130

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“…Besides, M13 filamentous phages, which have ∼2700 copies of the 5 kD major pVIII coat protein, can provide great signal amplification when using reporter-conjugated anti-M13 pVIII antibodies. Various studies have shown that M13 phages are valuable reporter elements for use in ELISAs (Goldman, Anderson, Bernstein, & Swain, 2010). In addition, for increasing sensitivity, chemiluminescence has been employed to combine phage-displayed nanobodies, HRP-conjugated anti-M13 pVIII antibodies and chemiluminescent substrates.…”

Section: Assay Validationmentioning

confidence: 99%

Phage-displayed nanobody based double antibody sandwich chemiluminescent immunoassay for the detection of Cry2A toxin in cereals

Qiu

Liu

et al. 2019

Food and Agricultural Immunology

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In this study, we developed a double antibody sandwich chemiluminescent immunoassay (DAS-CLIA) based on phagedisplayed nanobodies for the determination of Cry2A toxin. Nine phage-displayed nanobodies specifically to Cry2A were obtained from a naive nanobody library after four rounds of biopanning. The phage-displayed nanobody P2 with high affinity binding was selected as the detection antibody for the sensitive DAS-CLIA development. The proposed method exhibited high sensitivity with a limit of detection of 0.09 ng/mL, has a wide linear range for Cry2A toxin detection, in the range of 0.1-1000 ng/mL and negligible cross-reactivities to the other Bt toxins. The recoveries of Cry2A toxin spiked in cereal samples (rice, wheat, and corn) ranged from 82.7% to 118%, with a coefficient of variation of less than 10%. This study demonstrated that the DAS-CLIA procedure based on phage-displayed nanobodies may provide an alternative sensitive method for the detection of Bt toxins in agri-products.

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“…Diagnostic and therapeutic [143] HER2 Breast cancer Diagnostic [144,145] HPV-16 L1 protein Cervical cancer Diagnostic and therapeutic [146,147] HSP-60 Brucellosis (livestock) Diagnostic and vaccine [23,148] VCAM1 Atherosclerotic lesions Molecular imaging [149][150][151] VEGFR2 Angiogenesis Therapeutic [152] TNT Explosive Diagnostic [133,153] SEB Toxin Sensor and diagnostic [134] Ricin Toxin Sensor and diagnostic [134] BoNT/A Toxin Sensor and diagnostic [90,154,155] Scorpion AahII Toxin Neutralizing and therapeutic [135] EGFR binding reagents, derived from conventional polyclonal sera, monoclonal antibodies, small fragments (Fab and scFv) and peptides [145]. Foley and co-workers demonstrated the heat stability of purified recombinant V NAR was superior to that of conventional mAbs by targeting immobilized P. falciparum AMA1 in various formats at 45°C, and the refolding property of V NAR was retained when the temperature increased to 80°C.…”

Section: Vegf-a 165 Neoangiogenesismentioning

confidence: 99%

“…As in the case with camelids nanobodies, highly diversified shark V NAR libraries have also been used to detect different kind of toxins, including staphylococcal enterotoxin B (SEB), ricin and botulinum toxin A (BoNT/A) complex toxoid [153] and cholera toxin (CT) [113]. In addition, V NAR sdAbs have been reported to recognize immunosilent targets in human, for example, the 70 kDa translocase of outer membrane (Tom70) [154]. Owing to the findings of negligible cross-reactivity with other antigens and superior heat stability, shark V NAR domains may be a potent source of sdAbs with thermal stability over conventional antibodies in diagnostic and biotherapeutic applications [155,156].…”

Section: Vegf-a 165 Neoangiogenesismentioning

confidence: 99%

The Development of Single Domain Antibodies for Diagnostic and Therapeutic Applications

Leow¹,

Cheng²,

Fischer³

et al. 2018

Antibody Engineering

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Monoclonal antibodies have become increasingly accepted as diagnostics and therapeutics for various human diseases due to their high affinity and specificity. However, several practical drawbacks are apparent for the reagents based on conventional IgG antibodies. With the emergence of antibody engineering, many problems were overcome when the recombinant antibody fragments such as Fabs, scFvs, diabodies and single domain antibodies (sdAbs), are developed. These fragments not only retain the specificity of the whole monoclonal antibodies, but are also easy to express and produce in prokaryotic expression systems. Rather unexpectedly, the natural sdAbs namely V HH s, V NAR s and variable lymphocyte receptors (VLRs) that comprise excellent biological activities were recently discovered in camelids, cartilaginous fish and lampreys, respectively. Due to their unique characteristics, including small size, high thermostability, stable folding in the nucleus and cytosol and long CDR3 regions which have access to cavities or clefts on the surface of proteins, these new binders are now investigated extensively as a substitute for conventional antibodies. This review describes the potential of sdAbs selected using in vitro display systems and their use in multiple applications.(polyclonal Abs) are heterogeneous antibody mixtures that are derived from multiple plasma cell lines. Because polyclonal antibodies comprise a mixture of different antibodies carrying numerous paratopes, they have excellent properties for recognizing antigens [2]. A monoclonal antibody (mAb) is a homogeneous antibody generated from a single B lymphocyte clone. Antibodies produced in mAb format have an extremely high specificity against a single epitope on antigens [3]. Recombinant antibodies (rAbs) are antibodies generated using molecular biology techniques. They are aimed to improve the sensitivity, selectivity, stability and immobilization properties in diagnostic applications, for example, in biosensors [4]. In making decision to use or generate polyclonal, monoclonal or recombinant antibodies, several factors should be considered, including commercial availability, possibility to raise animals, types of applications, time length of a project and costs [1]. Although a vast number of rAbs has been proposed [5][6][7][8], the natural sdAb fragments that were recently discovered from camelids (V HH s), sharks (V NAR s) and lampreys (VLRs) have shown to possess extraordinary features that are not found in conventional antibodies, such as a small dimension, an elevated stability and the capability of recognizing cavities and clefts on the surface of proteins that cannot be reached by conventional recombinant antibodies [9][10][11]. This chapter will discuss the availability of new binders derived from vertebrates and give an overview of their applications in a biomedical platform by recognizing specified targets from various diseases. Monoclonal antibodies and their limitationsThe first description of monoclonal antibody (mAbs) production was published ...

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Amplification of immunoassays using phage-displayed single domain antibodies

Cited by 23 publications

References 12 publications

Single-Domain Antibodies As Versatile Affinity Reagents for Analytical and Diagnostic Applications

Single-Domain Antibodies As Versatile Affinity Reagents for Analytical and Diagnostic Applications

Phage-displayed nanobody based double antibody sandwich chemiluminescent immunoassay for the detection of Cry2A toxin in cereals

The Development of Single Domain Antibodies for Diagnostic and Therapeutic Applications

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