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

Abstract

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.

Anti-SARS-CoV-1 and −2 nanobody engineering towards avidity-inspired therapeutics

“…In this case, protein cages such as ferritin (24-mer) [123] , dihydrolipoyl acetyltransferase [124] , and Lumazine synthase (60-mer) [125] could be exploited. Notably, these scaffolds have previously been used to display several proteins [126] , [127] , [128] , [129] .…”

Screening and identification of nucleocapsid protein-nanobodies that inhibited Newcastle disease virus replication in DF-1 cells

“…After the cells had been transfected for 72 h, the supernatant containing the recombinant lentivirus was collected at 1,500× g for 10 min and used to infect the DF-1 cells. Subsequently, the recombinant DF-1 cells stably expressing Nbs were screened using a limited dilution method, according to a method described previously ( Ji et al, 2020 ). The cells in each well were observed using fluorescence microscopy (Leica AF6000).…”

Review detection of Newcastle disease virus

“…The specificity results showed that the OD450nm values of other poultry viruses, H9N2, H5N1, H7N9, IBV, IBDV, FADV, and ALV-J were all less than 0.058. In addition, this method can detect different class II strains (LaSota, F48E9, and sx10), but cannot detect class I strains (QH-1), which have good specificity ( 59 ).…”

“…The specificity results showed that the OD450nm values of other poultry viruses, H9N2, H5N1, H7N9, IBV, IBDV, FADV, and ALV-J were all less than 0.058. In addition, this method can detect different class II strains (LaSota, F48E9, and sx10), but cannot detect class I strains (QH-1), which have good specificity (59). To detect NDV antibodies in chicken serum more accurately and specifically, Sun established a competitive ELISA (cELISA) detection method using nanobody-horseradish peroxidase (HRP) fusion protein as a probe.…”