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

View Article: PubMed Central - PubMed

ABSTRACT

Background: Due 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.

Results: Here 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.

Conclusion: The 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 material: The online version of this article (doi:10.1186/s12896-016-0314-5) contains supplementary material, which is available to authorized users.

No MeSH data available.


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ELISA and Western blot analysis of lysates derived from DENV-infected VeroE6 cell lines. a: NS1 relative units were determined in lysates of VeroE6 cells infected with DENV serotypes 1–4 by NS1 capture ELISA (EUROIMMUN, Luzern, Switzerland). NS1 relative units were calculated in accordance with the manufacturer’s instructions and approximate values are shown. Gels for Western blot analysis were loaded with DENV serotype 1–4 infected VeroE6 cell lysates containing 40.000 relative units of NS1, each under non-reducing conditions. The 12 generated mAbs were tested on serotype 1 (b), serotype 2 (c), serotype 3 (d) and serotype 4 (e) virus-infected cell lysates
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Fig6: ELISA and Western blot analysis of lysates derived from DENV-infected VeroE6 cell lines. a: NS1 relative units were determined in lysates of VeroE6 cells infected with DENV serotypes 1–4 by NS1 capture ELISA (EUROIMMUN, Luzern, Switzerland). NS1 relative units were calculated in accordance with the manufacturer’s instructions and approximate values are shown. Gels for Western blot analysis were loaded with DENV serotype 1–4 infected VeroE6 cell lysates containing 40.000 relative units of NS1, each under non-reducing conditions. The 12 generated mAbs were tested on serotype 1 (b), serotype 2 (c), serotype 3 (d) and serotype 4 (e) virus-infected cell lysates

Mentions: Cross-reactivity of the generated mAbs with virus-infected cells was analyzed by Western blot analysis with lysates of VeroE6 cells infected with the four different DENV serotypes (Fig. 6). Results were nearly identical to the ELISA analysis of all four hexa-His tagged NS1 fusion proteins from HEK cell lysates with three exceptions: i) the Western blot cross-reactive mAb NR1.8 did not react with the D2NS1 protein in the ELISA, ii) mAb NR1.2 did recognize D3NS1 in virus material, but not the NS1 protein present in D3NS1-HEK cell lysates and iii) mAb NR1.5 recognized NS1 in D4NS1-HEK cell lysates, but not the one present in the virus-infected cell lysates. Analysis with lysates of the virus-infected cells showed that mAb NR1.8 is cross-reactive with all four DENV serotypes. Five of the mAbs specifically reacted with D1NS1 and four recognized D4NS1 only. One of the mAbs cross-reacted with D1NS1 - D3NS1, but did not recognize D4NS1 and one reacted with D1NS1 and D3NS1 (Fig. 6, Table 1).Fig. 6


Generation of monoclonal antibodies against native viral proteins using antigen-expressing mammalian cells for mouse immunization
ELISA and Western blot analysis of lysates derived from DENV-infected VeroE6 cell lines. a: NS1 relative units were determined in lysates of VeroE6 cells infected with DENV serotypes 1–4 by NS1 capture ELISA (EUROIMMUN, Luzern, Switzerland). NS1 relative units were calculated in accordance with the manufacturer’s instructions and approximate values are shown. Gels for Western blot analysis were loaded with DENV serotype 1–4 infected VeroE6 cell lysates containing 40.000 relative units of NS1, each under non-reducing conditions. The 12 generated mAbs were tested on serotype 1 (b), serotype 2 (c), serotype 3 (d) and serotype 4 (e) virus-infected cell lysates
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Fig6: ELISA and Western blot analysis of lysates derived from DENV-infected VeroE6 cell lines. a: NS1 relative units were determined in lysates of VeroE6 cells infected with DENV serotypes 1–4 by NS1 capture ELISA (EUROIMMUN, Luzern, Switzerland). NS1 relative units were calculated in accordance with the manufacturer’s instructions and approximate values are shown. Gels for Western blot analysis were loaded with DENV serotype 1–4 infected VeroE6 cell lysates containing 40.000 relative units of NS1, each under non-reducing conditions. The 12 generated mAbs were tested on serotype 1 (b), serotype 2 (c), serotype 3 (d) and serotype 4 (e) virus-infected cell lysates
Mentions: Cross-reactivity of the generated mAbs with virus-infected cells was analyzed by Western blot analysis with lysates of VeroE6 cells infected with the four different DENV serotypes (Fig. 6). Results were nearly identical to the ELISA analysis of all four hexa-His tagged NS1 fusion proteins from HEK cell lysates with three exceptions: i) the Western blot cross-reactive mAb NR1.8 did not react with the D2NS1 protein in the ELISA, ii) mAb NR1.2 did recognize D3NS1 in virus material, but not the NS1 protein present in D3NS1-HEK cell lysates and iii) mAb NR1.5 recognized NS1 in D4NS1-HEK cell lysates, but not the one present in the virus-infected cell lysates. Analysis with lysates of the virus-infected cells showed that mAb NR1.8 is cross-reactive with all four DENV serotypes. Five of the mAbs specifically reacted with D1NS1 and four recognized D4NS1 only. One of the mAbs cross-reacted with D1NS1 - D3NS1, but did not recognize D4NS1 and one reacted with D1NS1 and D3NS1 (Fig. 6, Table 1).Fig. 6

View Article: PubMed Central - PubMed

ABSTRACT

Background: Due 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.

Results: Here 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.

Conclusion: The 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 material: The online version of this article (doi:10.1186/s12896-016-0314-5) contains supplementary material, which is available to authorized users.

No MeSH data available.


Related in: MedlinePlus