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Nanobody-horseradish peroxidase fusion protein as an ultrasensitive probe to detect antibodies against Newcastle disease virus in the immunoassay. 2019

Yamin Sheng, and Kun Wang, and Qizhong Lu, and Pinpin Ji, and Baoyuan Liu, and Jiahong Zhu, and Qingyuan Liu, and Yani Sun, and Jingfei Zhang, and En-Min Zhou, and Qin Zhao
Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling, 712100, Shaanxi, China.

BACKGROUND Sensitive and specific antibodies can be used as essential probes to develop competitive enzyme-linked immunosorbent assay (cELISA). However, traditional antibodies are difficult to produce, only available in limited quantities, and ineffective as enzymatic labels. Nanobodies, which are single-domain antibodies (sdAbs), offer an alternative, more promising tool to circumvent these limitations. In the present work, a cELISA using nanobody-horseradish peroxidase (HRP) fusion protein firstly designed as a probe was developed for detecting anti-Newcastle disease virus (NDV) antibodies in chicken sera. RESULTS In the study, a platform for the rapid and simple production of nanobody-HRP fusion protein was constructed. First, a total of 9 anti-NDV-NP protein nanobodies were screened from a immunised Bactrian camel. Then, the Nb5-HRP fusions were produced with the platform and used for the first time as sensitive reagents for developing cELISA to detect anti-NDV antibodies. The cut-off value of the cELISA was 18%, and the sensitivity and specificity were respectively 100% and 98.6%. The HI test and commercial ELISA kit (IDEXX) separately agreed 97.83% and 98.1% with cELISA when testing clinical chicken sera and both agreed 100% when testing egg yolks. However, for detecting anti-NDV antibodies in the sequential sera from the challenged chickens, cELISA demonstrated to be more sensitive than the HI test and commercial ELISA kit. Moreover, a close correlation (R2 = 0.914) was found between the percent competitive inhibition values of cELISA and HI titers. CONCLUSIONS A platform was successfully designed to easily and rapidly produce the nanobody-HRP fusion protein, which was the first time to be used as reagents for establishing cELISA. Results suggest that the platform supports the development of a cELISA with high sensitivity, simplicity, and rapid detection of anti-NDV antibodies. Overall, we believe that the platform based on nanobody-HRP fusions can be widely used for future investigations and treatment other diseases and viruses.

UI MeSH Term Description Entries
D009522 Newcastle disease virus The most well known avian paramyxovirus in the genus AVULAVIRUS and the cause of a highly infectious pneumoencephalitis in fowl. It is also reported to cause CONJUNCTIVITIS in humans. Transmission is by droplet inhalation or ingestion of contaminated water or food. Avian Paramyxovirus 1,Paramyxovirus 1, Avian
D011994 Recombinant Proteins Proteins prepared by recombinant DNA technology. Biosynthetic Protein,Biosynthetic Proteins,DNA Recombinant Proteins,Recombinant Protein,Proteins, Biosynthetic,Proteins, Recombinant DNA,DNA Proteins, Recombinant,Protein, Biosynthetic,Protein, Recombinant,Proteins, DNA Recombinant,Proteins, Recombinant,Recombinant DNA Proteins,Recombinant Proteins, DNA
D002162 Camelus Two-toed, hoofed mammals with four legs, a big-lipped snout, and a humped back belonging to the family Camelidae. They are native to North Africa, and Western and Central Asia. Camels,Dromedary,Bactrian Camels,Bractrian Camels,Camelus bactrianus,Camelus dromedarius,Bactrian Camel,Bractrian Camel,Camel,Camel, Bactrian,Camel, Bractrian,Camels, Bactrian,Camels, Bractrian,Dromedaries
D002645 Chickens Common name for the species Gallus gallus, the domestic fowl, in the family Phasianidae, order GALLIFORMES. It is descended from the red jungle fowl of SOUTHEAST ASIA. Gallus gallus,Gallus domesticus,Gallus gallus domesticus,Chicken
D004797 Enzyme-Linked Immunosorbent Assay An immunoassay utilizing an antibody labeled with an enzyme marker such as horseradish peroxidase. While either the enzyme or the antibody is bound to an immunosorbent substrate, they both retain their biologic activity; the change in enzyme activity as a result of the enzyme-antibody-antigen reaction is proportional to the concentration of the antigen and can be measured spectrophotometrically or with the naked eye. Many variations of the method have been developed. ELISA,Assay, Enzyme-Linked Immunosorbent,Assays, Enzyme-Linked Immunosorbent,Enzyme Linked Immunosorbent Assay,Enzyme-Linked Immunosorbent Assays,Immunosorbent Assay, Enzyme-Linked,Immunosorbent Assays, Enzyme-Linked
D004926 Escherichia coli A species of gram-negative, facultatively anaerobic, rod-shaped bacteria (GRAM-NEGATIVE FACULTATIVELY ANAEROBIC RODS) commonly found in the lower part of the intestine of warm-blooded animals. It is usually nonpathogenic, but some strains are known to produce DIARRHEA and pyogenic infections. Pathogenic strains (virotypes) are classified by their specific pathogenic mechanisms such as toxins (ENTEROTOXIGENIC ESCHERICHIA COLI), etc. Alkalescens-Dispar Group,Bacillus coli,Bacterium coli,Bacterium coli commune,Diffusely Adherent Escherichia coli,E coli,EAggEC,Enteroaggregative Escherichia coli,Enterococcus coli,Diffusely Adherent E. coli,Enteroaggregative E. coli,Enteroinvasive E. coli,Enteroinvasive Escherichia coli
D006735 Horseradish Peroxidase An enzyme isolated from horseradish which is able to act as an antigen. It is frequently used as a histochemical tracer for light and electron microscopy. Its antigenicity has permitted its use as a combined antigen and marker in experimental immunology. Alpha-Peroxidase,Ferrihorseradish Peroxidase,Horseradish Peroxidase II,Horseradish Peroxidase III,Alpha Peroxidase,II, Horseradish Peroxidase,III, Horseradish Peroxidase,Peroxidase II, Horseradish,Peroxidase III, Horseradish,Peroxidase, Ferrihorseradish,Peroxidase, Horseradish
D006801 Humans Members of the species Homo sapiens. Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man
D000818 Animals Unicellular or multicellular, heterotrophic organisms, that have sensation and the power of voluntary movement. Under the older five kingdom paradigm, Animalia was one of the kingdoms. Under the modern three domain model, Animalia represents one of the many groups in the domain EUKARYOTA. Animal,Metazoa,Animalia
D000914 Antibodies, Viral Immunoglobulins produced in response to VIRAL ANTIGENS. Viral Antibodies

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