Fenobody: A Ferritin-Displayed Nanobody with High Apparent Affinity and Half-Life Extension

Fan, Kelong; Jiang, Bing; Guan, Zhe; He, Jianxun; Yang, Di; Xie, Ni; Nie, Guohui; Xie, Can; Yan, Xiyun

doi:10.1021/acs.analchem.7b05217

Cited by 69 publications

(65 citation statements)

References 45 publications

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“…To compare the affinity of the fenobody and traditional nanobody, a capture ELISA were designed and performed based on a previous characterisation with some modifications [24,30]. Briefly, after the same amounts of fenobodies and traditional nanobodies were coated into the ELISA plates, NDV particles (1 µg/well), anti-NDV monoclonal antibodies, and HRP labeled goat anti-mouse antibodies were then added to the plates one by one.…”

Section: Affinity and Half-life Extension Test Of Fenobodymentioning

confidence: 99%

“…To analyze the half-life extension, the fenobody and traditional nanobody were both labeled with FITC based on the descriptions by Fan K et al [24] and Fisher et al [31]. The FITC-labeled fenobody and FITC-labeled traditional nanobody were intravenously injected into female BALB/c mice via the tail vein.…”

Section: Affinity and Half-life Extension Test Of Fenobodymentioning

confidence: 99%

“…In this study, NDV specific nanobodies were obtained from a Bactrian camel immunized with inactivated NDV vaccines. Then, fenobodies (ferritin-fused nanobodies) [24] and RANbodies (nanobodies-fused reporter protein) [25,26] against NDV were produced through genetic modification of the anti-NDV nanobodies (Scheme 1a). For the first time, a fenobody was implemented as the capture antibody and RANbody as the detection antibody to develop a sensitive, specific, and easy production sandwich ELISA for detection of NDV in the samples (Scheme 1b).…”

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confidence: 99%

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Fenobody and RANbody-based sandwich enzyme-linked immunosorbent assay to detect Newcastle disease virus

Zhu

et al. 2020

Preprint

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Background: Traditional sandwich enzyme-linked immunosorbent assay (ELISA) using polyclonal and monoclonal antibodies as reagents presents several drawbacks, including limited amounts, difficulty in permanent storage, and required use of a secondary antibody. Nanobodies can be easily expressed with different systems and fused with several tags in their tertiary structure by recombinant technology, thus offering an effective detection method for diagnostic purposes. Recently, the fenobody (ferritin-fused nanobody) and RANbody (nanobody-fused reporter) have been designed and derived from the nanobody for developing the diagnostic immunoassays. However, there was no report about developing the sandwich ELISA using the fenobody and RANbody as pairing reagents.Results: A platform for developing a sandwich ELISA utilizing fenobody as the capture antibody and RANbody as the detection antibody was firstly designed in the study. Newcastle disease virus (NDV) was selected as the antigen, from which 13 NDV-specific nanobodies were screened from an immunized Bactrian camel. Then, 5 nanobodies were selected to produce fenobodies and RANbodies. The best pairing of fenobodies (NDV-fenobody-4, 800 ng/well) and RANbodies (NDV-RANbody-49, 1:10) was determined to develop the sandwich ELISA for detecting NDV. The detection limits of the assay were determined to be 22 of hemagglutination (HA) titers and 10 ng of purified NDV particles. Compared with two commercial assays, the developed assay shows higher sensitivity and specificity. Meanwhile, it exhibits 98.7% agreement with the HA test and can detect the reference NDV strains belonging to Class II but not Class I. Conclusions: In the presented study, the 13 anti-NDV nanobodies binding the NDV particles were first produced. Then, for the first time, the sandwich ELISA to detect the NDV in the different samples has been developed using the fenobody and RANbody as reagents derived from the nanobodies. Considering the rapidly increasing generation of nanobodies, the platform can reduce the cost of production for the sandwich ELISA and be universally used to develop assays for detecting other antigens.

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Section: Affinity and Half-life Extension Test Of Fenobodymentioning

confidence: 99%

Section: Affinity and Half-life Extension Test Of Fenobodymentioning

confidence: 99%

mentioning

confidence: 99%

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Fenobody and RANbody-based sandwich enzyme-linked immunosorbent assay to detect Newcastle disease virus

Zhu

et al. 2020

Preprint

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“…Research shows Balkan endemic nephropathy (BEN) and urinary tract tumors may be introduced by OTA [26]. Hence, to humans, the International Agency for Research on Cancer (IARC) classified OTA as a possible carcinogen [27]. Consequently, it is of supreme importance to establish detecting approaches with high sensitivity and specificity.…”

Section: Introductionmentioning

confidence: 99%

Change of Amino Acid Residues in Idiotypic Nanobodies Enhanced the Sensitivity of Competitive Enzyme Immunoassay for Mycotoxin Ochratoxin A in Cereals

Zhang

Tang

et al. 2020

Toxins

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Anti-idiotypic nanobodies, usually expressed by gene engineering protocol, has been shown as a nontoxic coating antigen for toxic compound immunoassays. We here focused on how to increase immunoassay sensitivity by changing the nanobody's primary sequence. In the experiments, two anti-idiotype nanobodies against monoclonal antibody 1H2, which is specific to ochratoxin A, were obtained and named as nontoxic coating antigen 1 (NCA1) and nontoxic coating antigen 2 (NCA2). Three differences between the nanobodies were discovered. First, there are six amino acid residues (AAR) of changes in the complementarity determining region (CDR), which compose the antigen-binding site. One of them locates in CDR1 (I-L), two of them in CDR2 (G-D, E-K), and three of them in CDR3 (Y-H, Y-W). Second, the affinity constant of NCA1 was tested as 1.20 × 10 8 L mol −1 , which is about 4 times lower than that of NCA2 (5.36 × 10 8 L mol −1 ). Third, the sensitivity (50% inhibition concentration) of NCA1 for OTA was shown as 0.052 ng mL −1 , which was 3.5 times lower than that of nontoxic coating antigen 2 (0.015 ng mL −1 ). The results indicate that the AAR changes in CDR of the anti-idiotypic nanobodies, from nonpolar to polar, increasing the affinity constant may enhance the immunoassay sensitivity. In addition, by using the nontoxic coating antigen 2 to substitute the routine synthetic toxic antigen, we established an eco-friendly and green enzyme-linked immunosorbent assay (ELISA) method for rapid detection of ochratoxin A in cereals. The half-maximal inhibitory concentration (IC 50 ) of optimized ELISA was 0.017 ng mL −1 with a limit of detection (LOD) of 0.003 ng mL −1 . The optimized immunoassay showed that the average recoveries of spiked corn, rice, and wheat were between 80% and 114.8%, with the relative standard deviation (RSD) ranging from 3.1-12.3%. Therefore, we provided not only basic knowledge on how to improve the structure of anti-idiotype nanobody for increasing assay sensitivity, but also an available eco-friendly ELISA for ochratoxin A in cereals.Key Contribution: Two anti-idiotype nanobodies were developed and used as non-toxic antigens against monoclonal antibody 1H2, which is specific to ochratoxin A. A basic knowledge was discovered Toxins 2020, 12, 273 2 of 15 that the change of amino acid residues in idiotypic nanobodies enhanced the sensitivity by comparing the amino acid sequences, structure, affinity constants, and sensitivities between both nanobodies.

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“…Nanobody as the reagents for developing the sandwich ELISA can overcome the drawback of the commericial ELISA kit. In a previous study, the fenobody (ferritin-fused nanobodies) can apparently enhance the affinity of univalent nanobody against H5N1 avian influenza virus (AIV) [24].…”

mentioning

confidence: 99%

Fenobody and RANbody-based sandwich enzyme-linked immunosorbent assay to detect Newcastle disease virus

Zhu

et al. 2020

J Nanobiotechnol

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Background: Traditional sandwich enzyme-linked immunosorbent assay (ELISA) using polyclonal and monoclonal antibodies as reagents presents several drawbacks, including limited amounts, difficulty in permanent storage, and required use of a secondary antibody. Nanobodies can be easily expressed with different systems and fused with several tags in their tertiary structure by recombinant technology, thus offering an effective detection method for diagnostic purposes. Recently, the fenobody (ferritin-fused nanobody) and RANbody (nanobody-fused reporter) have been designed and derived from the nanobody for developing the diagnostic immunoassays. However, there was no report about developing the sandwich ELISA using the fenobody and RANbody as pairing reagents. Results: A platform for developing a sandwich ELISA utilizing fenobody as the capture antibody and RANbody as the detection antibody was firstly designed in the study. Newcastle disease virus (NDV) was selected as the antigen, from which 13 NDV-specific nanobodies were screened from an immunized Bactrian camel. Then, 5 nanobodies were selected to produce fenobodies and RANbodies. The best pairing of fenobodies (NDV-fenobody-4, 800 ng/well) and RANbodies (NDV-RANbody-49, 1:10) was determined to develop the sandwich ELISA for detecting NDV. The detection limits of the assay were determined to be 2 2 of hemagglutination (HA) titers and 10 ng of purified NDV particles. Compared with two commercial assays, the developed assay shows higher sensitivity and specificity. Meanwhile, it exhibits 98.7% agreement with the HA test and can detect the reference NDV strains belonging to Class II but not Class I. Conclusions: In the presented study, the 13 anti-NDV nanobodies binding the NDV particles were first produced. Then, for the first time, the sandwich ELISA to detect the NDV in the different samples has been developed using the fenobody and RANbody as reagents derived from the nanobodies. Considering the rapidly increasing generation of nanobodies, the platform can reduce the cost of production for the sandwich ELISA and be universally used to develop assays for detecting other antigens.

show abstract

Fenobody: A Ferritin-Displayed Nanobody with High Apparent Affinity and Half-Life Extension

Cited by 69 publications

References 45 publications

Fenobody and RANbody-based sandwich enzyme-linked immunosorbent assay to detect Newcastle disease virus

Fenobody and RANbody-based sandwich enzyme-linked immunosorbent assay to detect Newcastle disease virus

Change of Amino Acid Residues in Idiotypic Nanobodies Enhanced the Sensitivity of Competitive Enzyme Immunoassay for Mycotoxin Ochratoxin A in Cereals

Fenobody and RANbody-based sandwich enzyme-linked immunosorbent assay to detect Newcastle disease virus

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