Nanobody-Based Indirect Competitive ELISA for Sensitive Detection of 19-Nortestosterone in Animal Urine

Overview

Journal Biomolecules

Publisher MDPI

Specialties Biochemistry
Molecular Biology

Date 2021 Jan 30

PMID 33513883

Citations 5

Authors

Yuan-Yuan Yang

Yu Wang

Yi-Feng Zhang

Feng Wang

Yi-Fan Liang

Jin-Yi Yang

Zhen-Lin Xu

Yu-Dong Shen

Hong Wang

Affiliations

Soon will be listed here.

Abstract

Nanobody (Nb), a new type of biorecognition element generally from , has the characteristics of small molecular weight, high stability, great solubility and high expression level in . In this study, with 19-nortestosterone (19-NT), an anabolic androgenic steroid as target drug, three specific Nbs against 19-NT were selected from camel immune library by phage display technology. The obtained Nbs showed excellent thermostability and organic solvent tolerance. The nanobody Nb2F7 with the best performance was used to develop a sensitive indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) for 19-NT detection. Under optimized conditions, the standard curve of ic-ELISA was fitted with a half-maximal inhibitory concentration (IC) of 1.03 ng/mL and a detection limit (LOD) of 0.10 ng/mL for 19-NT. Meanwhile, the developed assay had low cross- reactivity with analogs and the recoveries of 19-NT ranged from 82.61% to 99.24% in spiked samples. The correlation coefficient between ic-ELISA and the ultra-performance liquid chromatography/mass spectrometry (UPLC-MS/MS) method was 0.9975, which indicated that the nanobody-based ic-ELISA could be a useful tool for a rapid analysis of 19-NT in animal urine samples.

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References

Goldman E, Liu J, Zabetakis D, Anderson G . Enhancing Stability of Camelid and Shark Single Domain Antibodies: An Overview. Front Immunol. 2017; 8:865. PMC: 5524736. DOI: 10.3389/fimmu.2017.00865. View

Ren W, Li Z, Xu Y, Wan D, Barnych B, Li Y . One-Step Ultrasensitive Bioluminescent Enzyme Immunoassay Based on Nanobody/Nanoluciferase Fusion for Detection of Aflatoxin B in Cereal. J Agric Food Chem. 2019; 67(18):5221-5229. PMC: 7792509. DOI: 10.1021/acs.jafc.9b00688. View

Olichon A, Schweizer D, Muyldermans S, de Marco A . Heating as a rapid purification method for recovering correctly-folded thermotolerant VH and VHH domains. BMC Biotechnol. 2007; 7:7. PMC: 1790891. DOI: 10.1186/1472-6750-7-7. View

Akazawa-Ogawa Y, Takashima M, Lee Y, Ikegami T, Goto Y, Uegaki K . Heat-induced irreversible denaturation of the camelid single domain VHH antibody is governed by chemical modifications. J Biol Chem. 2014; 289(22):15666-79. PMC: 4140921. DOI: 10.1074/jbc.M113.534222. View

Bhasin S, Jasuja R . Selective androgen receptor modulators as function promoting therapies. Curr Opin Clin Nutr Metab Care. 2009; 12(3):232-40. PMC: 2907129. DOI: 10.1097/MCO.0b013e32832a3d79. View

Kaneko R, Kitabatake N . Heat-induced formation of intermolecular disulfide linkages between thaumatin molecules that do not contain cysteine residues. J Agric Food Chem. 1999; 47(12):4950-5. DOI: 10.1021/jf990267l. View

Ebrahimizadeh W, Mousavi Gargari S, Rajabibazl M, Ardekani L, Zare H, Bakherad H . Isolation and characterization of protective anti-LPS nanobody against V. cholerae O1 recognizing Inaba and Ogawa serotypes. Appl Microbiol Biotechnol. 2012; 97(10):4457-66. DOI: 10.1007/s00253-012-4518-x. View

Zabetakis D, Olson M, Anderson G, Legler P, Goldman E . Evaluation of disulfide bond position to enhance the thermal stability of a highly stable single domain antibody. PLoS One. 2014; 9(12):e115405. PMC: 4272287. DOI: 10.1371/journal.pone.0115405. View

Shirpoor A, Heshmatian B, Tofighi A, Eliasabad S, Kheradmand F, Zerehpoosh M . Nandrolone administration with or without strenuous exercise increases cardiac fatal genes overexpression, calcium/calmodulin-dependent protein kinaseiiδ, and monoamine oxidase activities and enhances blood pressure in adult wistar rats. Gene. 2019; 697:131-137. DOI: 10.1016/j.gene.2019.02.053. View

10.

Dumoulin M, Conrath K, Van Meirhaeghe A, Meersman F, Heremans K, Frenken L . Single-domain antibody fragments with high conformational stability. Protein Sci. 2002; 11(3):500-15. PMC: 2373476. DOI: 10.1110/ps.34602. View

11.

Zhang J, Wang Y, Dong J, Yang J, Zhang Y, Wang F . Development of a Simple Pretreatment Immunoassay Based on an Organic Solvent-Tolerant Nanobody for the Detection of Carbofuran in Vegetable and Fruit Samples. Biomolecules. 2019; 9(10). PMC: 6843801. DOI: 10.3390/biom9100576. View

12.

Zhao F, Tian Y, Shen Q, Liu R, Shi R, Wang H . A novel nanobody and mimotope based immunoassay for rapid analysis of aflatoxin B1. Talanta. 2019; 195:55-61. DOI: 10.1016/j.talanta.2018.11.013. View

13.

Jiang J, Wang Z, Zhang H, Zhang X, Liu X, Wang S . Monoclonal antibody-based ELISA and colloidal gold immunoassay for detecting 19-nortestosterone residue in animal tissues. J Agric Food Chem. 2011; 59(18):9763-9. DOI: 10.1021/jf2012437. View

14.

Bever C, Dong J, Vasylieva N, Barnych B, Cui Y, Xu Z . VHH antibodies: emerging reagents for the analysis of environmental chemicals. Anal Bioanal Chem. 2016; 408(22):5985-6002. PMC: 4983233. DOI: 10.1007/s00216-016-9585-x. View

15.

Roda A, Manetta A, Portanti O, Mirasoli M, Guardigli M, Pasini P . A rapid and sensitive 384-well microtitre format chemiluminescent enzyme immunoassay for 19-nortestosterone. Luminescence. 2003; 18(2):72-8. DOI: 10.1002/bio.708. View

16.

Seara F, Olivares E, Nascimento J . Anabolic steroid excess and myocardial infarction: From ischemia to reperfusion injury. Steroids. 2020; 161:108660. DOI: 10.1016/j.steroids.2020.108660. View

17.

Pirez-Schirmer M, Rossotti M, Badagian N, Leizagoyen C, Brena B, Gonzalez-Sapienza G . Comparison of Three Antihapten VHH Selection Strategies for the Development of Highly Sensitive Immunoassays for Microcystins. Anal Chem. 2017; 89(12):6800-6806. DOI: 10.1021/acs.analchem.7b01221. View

18.

Akazawa-Ogawa Y, Uegaki K, Hagihara Y . The role of intra-domain disulfide bonds in heat-induced irreversible denaturation of camelid single domain VHH antibodies. J Biochem. 2015; 159(1):111-21. PMC: 4882646. DOI: 10.1093/jb/mvv082. View

19.

Divari S, Berio E, Pregel P, Sereno A, Chiesa L, Pavlovic R . Effects and detection of Nandrosol and ractopamine administration in veal calves. Food Chem. 2016; 221:706-713. DOI: 10.1016/j.foodchem.2016.11.116. View

20.

Ahmadkhaniha R, Kobarfard F, Rastkari N, Khoshayand M, Amini M, Shafiee A . Assessment of endogenous androgen levels in meat, liver and testis of Iranian native cross-breed male sheep and bull by gas chromatography-mass spectrometry. Food Addit Contam Part A Chem Anal Control Expo Risk Assess. 2009; 26(4):453-65. DOI: 10.1080/02652030802627475. View