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01.12.2018 | Research | Ausgabe 1/2018 Open Access

Virology Journal 1/2018

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

Virology Journal > Ausgabe 1/2018
Violetta Sączyńska, Anna Bierczyńska-Krzysik, Violetta Cecuda-Adamczewska, Piotr Baran, Anna Porębska, Katarzyna Florys, Marcin Zieliński, Grażyna Płucienniczak
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Electronic supplementary material

The online version of this article (https://​doi.​org/​10.​1186/​s12985-017-0886-2) contains supplementary material, which is available to authorized users.



The 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.


We 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.


Our 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.
Additional file 1: Antibodies and antigens used in this work. Table S1. Anti-H5 hemagglutinin antibodies. Table S2. Recombinant H5 hemagglutinin proteins. Figure S1. The MALDI-TOF/TOF mass spectrum of rHA - A/H5N1/Qinghai. Figure S2. The MALDI-TOF/TOF mass spectrum of rHA - A/H5N1/Poland. Figure S3. Antigenicity of rHA - A/H5N1/Qinghai. Figure S4. Antigenicity of rHA - A/H5N1/Poland. Figure S5. Antigenicity of recombinant hemagglutinin proteins. Figure S6. Oligomerization status of recombinant hemagglutinin proteins. Table S3. Hemagglutination activities of recombinant hemagglutinin proteins. Table S4. Avian influenza viruses. (PDF 752 kb)
Additional file 2: Results of hybridoma screening. Table S5. Reactivities of hybridoma culture supernatants with recombinant H5 hemagglutinin proteins. Table S6. ELISA absorbance values from testing of the selected hybridomas. Table S7. Reactivities of culture supernatants from the hybridoma cell lines with H5 hemagglutinin antigens. (PDF 349 kb)
Additional file 3: Raw data from the preliminary immunoreactivity studies. Table S8. The ELISA absorbance values for the selected mAbs tested against recombinant hemagglutinin antigens. Table S9. The ELISA absorbance values for the selected mAbs tested against AIVs of H1-H16 subtypes. Table S10. The ELISA absorbance values for the selected mAbs tested against AIVs of H5 subtype. (PDF 147 kb)
Additional file 4: SDS-PAGE data. (PDF 4249 kb)
Additional file 5: Exemplary MALDI-TOF mass spectra. (PDF 796 kb)
Additional file 6: Data from the advanced immunoreactivity studies. Figure S7. The ELISA titration curves of the mAbs against rHA - A/H5N1/Qinghai from a mammalian expression system. Figure S8. The ELISA titration curves of the mAbs against rHA - A/H5N1/Poland from a baculovirus expression system. Figure S9. The ELISA titration curves of the mAbs against rHA - A/H5N1/Poland from a bacterial expression system. Table S11. The concentration values interpolated from the mAb titration curves. Figure S10. Diversity of the mAb reactivities with recombinant H5 hemagglutinin proteins and H5N3 avian influenza virus. Figure S11. Relative reactivity of the mAbs with recombinant H5 hemagglutinin antigens. Figure S12. Relative reactivity of the mAbs with avian influenza viruses. (PDF 422 kb)
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