Generation of monoclonal antibodies against native viral proteins using antigen-expressing mammalian cells for mouse immunization

Abstract

BackgroundDue to their rising incidence and progressive geographical spread, infections with mosquito-borne viruses, such as dengue (DENV), chikungunya and zika virus, have developed into major public health challenges. Since all of these viruses may cause similar symptoms and can occur in concurrent epidemics, tools for their differential diagnosis and epidemiological monitoring are of urgent need.ResultsHere we report the application of a novel strategy to rapidly generate monoclonal antibodies (mAbs) against native viral antigens, exemplified for the DENV nonstructural glycoprotein 1 (NS1). The described system is based on the immunization of mice with transfected mammalian cells expressing the target antigens in multiple displays on their cell surface and thereby presenting them efficiently to the host immune system in their native conformation. By applying this cell-based approach to the DENV NS1 protein of serotypes 1 (D1NS1) and 4 (D4NS1), we were able to rapidly generate panels of DENV NS1 serotype cross-reactive, as well as D1NS1- and D4NS1 serotype-specific mAbs. Our data show that the generated mAbs were capable of recognizing the endogenous NS1 protein in DENV-containing biological samples.ConclusionThe use of this novel immunization strategy, allows for a fast and efficient generation of hybridoma cell lines, producing mAbs against native viral antigens. Envisaged applications of the mAbs include the development of test platforms enabling a differentiation of the DENV serotypes and high resolution immunotyping for epidemiological studies.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-016-0314-5) contains supplementary material, which is available to authorized users.

Development of Dengue Virus Serotype–Specific NS1 Capture Assays for the Rapid and Highly Sensitive Identification of the Infecting Serotype in Human Sera

“…Immunization strategy. In a previous study, we have generated panels of serotype-specific mAbs against D1NS1 and D4NS1 (18). This was achieved by applying an efficient mouse immunization strategy using mammalian cells expressing either the D1NS1 or the D4NS1 target Ag on the cell surface, as described earlier (19).…”

“…Briefly, HEK 293 cells were transfected with the expression plasmid pcDNA3.1 (Invitrogen, San Diego, CA) that was modified to supply inserted genes encoding the NS1 protein of the four DENV serotypes with the secretion signal sequence of bee venom melittin (BVM) as well as a transmembrane (TM) domain (pcDNA3.1_BVM_D1NS1_FLAG_TM_HIS, pcDNA3.1_BVM_D2NS1_FLAG_TM_HIS pcDNA3.1_BVM_D3NS1_ FLAG_TM_HIS and pcDNA3.1_BVM_D4NS1_FLAG_TM_HIS) (19), thereby enabling the display of target Ags hooked onto the cell surface. Addition of a FLAG and a hexa-His tag facilitated expression analyses of the target proteins (18). HEK cells (50 ml with a cell density of 10 6 cells/ml) were transfected with 50 mg of plasmid DNA using 150 ml of Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA).…”

“…Characteristics of mAbs generated against D1NS1 and D4NS1 were described previously (18). To analyze the serotype specificity of mAbs generated by immunizing with D2NS1 and D3NS1 HEK 293 transfectants, we performed Western blot analyses with whole protein lysates of the transfected HEK cells.…”

“…One main advantage of this strategy is that it does not require target Ag purification, which bears the danger of perturbing the native protein structure. By immunizing mice using this approach, we have initially generated panels of serotypespecific mAbs against the NS1 protein of DENV-1 (D1NS1) and DENV-4 (D4NS1) (18). In this study, we aimed at the development of highly specific NS1 capture assays for all four DENV serotypes for the rapid screening of dengue patient sera.…”

Establishment of murine hybridoma cells producing antibodies against spike protein of SARS-CoV-2

“…Enzyme-linked immunosorbent assay (ELISA) was performed as described previously [33]. Briefly, 0.5 μg of RBD isolated from E. coli; RBD isolated from or HEK293 cells or 0.5 μg of a control protein isolated from E. coli were immobilized on wells of 96 well EIA plate (Corning).…”