Production of highly and broad-range specific monoclonal antibodies against hemagglutinin of H5-subtype avian influenza viruses and their differentiation by mass spectrometry

Abstract

BackgroundThe highly pathogenic avian influenza viruses of the H5 subtype, such as the H5N1 viral strains or the novel H5N8 and H5N2 reassortants, are of both veterinary and public health concern worldwide. To combat these viruses, monoclonal antibodies (mAbs) against H5 hemagglutinin (HA) play a significant role. These mAbs are effective diagnostic and therapeutic agents and powerful tools in vaccine development and basic scientific research. The aim of this study was to obtain diagnostically valuable mAbs with broad strain specificity against H5-subtype AIVs.ResultsWe applied the hybridoma method to produce anti-HA mAbs. The cloning and screening procedures resulted in the selection of 7 mouse hybridoma cell lines and their respective antibody clones. Preliminary immunoreactivity studies showed that these newly established mAbs, all of the IgG1 isotype, had high specificity and broad-range activities against the H5 HAs. However, these studies did not allow for a clear distinction among the selected antibodies and mAb-secreting hybridoma clones. To differentiate the analyzed mAbs and determine the exact number of hybridoma clones, peptide mapping of the Fc and Fab fragments was performed using a Matrix-Assisted Laser Desorption Ionization Time of Flight (MALDI-TOF/TOF) mass spectrometer. Detailed analyses of the acquired MS and MS/MS spectra confirmed that the Fc fragments constituted highly conserved species- and isotype-immunoglobulin components, whereas the Fab fragments exhibited considerable variation in the sequences that determine antibody specificity. This approach enabled unambiguous characterization of the selected mAbs according to their peptide composition. As a result, 6 different clones were distinguished.ConclusionsOur work provided a unique panel of anti-H5 HA mAbs, which meets the demand for novel, high-specificity analytical tools for use in serologic surveillance. Applications of these mAbs in areas other than diagnostics are also possible. Moreover, we demonstrated for the first time that peptide mapping of antibody fragments with mass spectrometry is an efficient method for the differentiation of antibody clones and relevant antibody-producing cell lines. The method may be successfully used to characterize mAbs at the protein level.Electronic supplementary materialThe online version of this article (10.1186/s12985-017-0886-2) contains supplementary material, which is available to authorized users.

Immobilization/Stabilization of Ficin Extract on Glutaraldehyde-Activated Agarose Beads. Variables That Control the Final Stability and Activity in Protein Hydrolyses

“…It has also been used for bioactive peptides production [13,14] and meat tenderization [15,16]. Ficin is considered to give a more reproducible hydrolysis map and it has been employed in the generation of antibodies via specific hydrolysis of some peptide bonds, [17][18][19]. Four isoforms of ficin have been described (A, B, C and D) and crystallized, resolving their structure [20,21].…”

A Novel Epitope-Blocking ELISA for Specific and Sensitive Detection of Antibodies Against H5-Subtype Influenza Virus Hemagglutinin

“…MAbs against H5 HA were generated according to the procedure described previously [9]. Brie y, female BALB/c mice were immunized with puri ed recombinant H5 HA protein produced in a mammalian expression system (Immune Technology Corp., New York, NY, USA).…”

“…The nally selected, a nity-puri ed mAbs, denoted G-1-31-22, G-2-14-10, G-5-32-5, G-6-42-42, G-6-42-71, G-7-24-17 and G-7-27-18, were characterized by isotyping, immunoreactivity studies and peptide mapping, as described previously [9]. Isotyping was performed using a commercial kit: 'Mouse Monoclonal Antibody Isotyping Reagents' (ISO-2; Sigma-Aldrich).…”

“…MAbs against H5 HA were produced with hybridoma technology as described previously [9]. For mouse immunization, the puri ed, ectodomain-based H5 HA protein (aa 17-530, ΔRRRKKR, 6x His) from a mammalian expression system was used (Additional le 1: Table S1).…”

“…In particular, the development of diagnostically valuable cELISA or bELISA tests requires the use of mAbs recognizing intrasubtype-conserved epitopes in the HA antigen. This requirement is met by the highly and broad-range speci c mAbs against H5 HAs, which were produced and differentiated by us according to the procedures presented previously, together with the results from immunoreactivity studies [9]. In this work, the diagnostic value of the newly generated mAbs was positively veri ed by using one of the 6 antibody clones, the G-7-27-18 mAb, in epitope-blocking (EB) ELISA for detecting H5 subtype-speci c antibodies in chicken sera.…”