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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.


Related in: MedlinePlus

Localization of D1NS1 and D4NS1 on the surface of transfected HEK cells. IFA of methanol fixed D1NS1-HEK, D4NS1-HEK and untransfected HEK cells using mouse anti-hexa-His tag mAbs and Alexa568-labelled anti-mouse IgG antibodies. Nuclei were stained with DAPI
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Fig2: Localization of D1NS1 and D4NS1 on the surface of transfected HEK cells. IFA of methanol fixed D1NS1-HEK, D4NS1-HEK and untransfected HEK cells using mouse anti-hexa-His tag mAbs and Alexa568-labelled anti-mouse IgG antibodies. Nuclei were stained with DAPI

Mentions: After transfection of HEK cells with the NS1 fusion protein-encoding plasmids pcDNA3.1_BVM_D1NS1_FLAG_TM_HIS or pcDNA3.1_BVM_D4NS1_FLAG_TM_HIS, the expression of D1NS1 and D4NS1 by the transfectants was analyzed. No elevated expression levels of a protein corresponding in size to the NS1 protein was visible upon aqua-staining of the SDS-PAGE separated total proteins of D1NS1-HEK and D4NS1-HEK cell lysates when compared to a lysate of untransfected HEK cells (Fig. 1a). However, Western blot analysis with anti-hexa-His tag antibodies (Fig. 1b) confirmed the specific expression of the NS1 fusion proteins (a band at the expected size for the monomer NS1 of 46–55 kDa [20]) by the transfectants. In combination with Western blot analysis with anti-tubulin antibodies serving as a loading control (Fig. 1c), these analyses revealed no significant difference between expression levels of D1NS1 and D4NS1. IFA of D1NS1-HEK, D4NS1-HEK and untransfected HEK cells with anti-hexa-His tag antibodies demonstrated the cell surface localization of the expressed NS1 fusion proteins with similar staining intensities for D1NS1 and D4NS1 (Fig. 2).Fig. 1


Generation of monoclonal antibodies against native viral proteins using antigen-expressing mammalian cells for mouse immunization
Localization of D1NS1 and D4NS1 on the surface of transfected HEK cells. IFA of methanol fixed D1NS1-HEK, D4NS1-HEK and untransfected HEK cells using mouse anti-hexa-His tag mAbs and Alexa568-labelled anti-mouse IgG antibodies. Nuclei were stained with DAPI
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Related In: Results  -  Collection

License 1 - License 2
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getmorefigures.php?uid=PMC5120561&req=5

Fig2: Localization of D1NS1 and D4NS1 on the surface of transfected HEK cells. IFA of methanol fixed D1NS1-HEK, D4NS1-HEK and untransfected HEK cells using mouse anti-hexa-His tag mAbs and Alexa568-labelled anti-mouse IgG antibodies. Nuclei were stained with DAPI
Mentions: After transfection of HEK cells with the NS1 fusion protein-encoding plasmids pcDNA3.1_BVM_D1NS1_FLAG_TM_HIS or pcDNA3.1_BVM_D4NS1_FLAG_TM_HIS, the expression of D1NS1 and D4NS1 by the transfectants was analyzed. No elevated expression levels of a protein corresponding in size to the NS1 protein was visible upon aqua-staining of the SDS-PAGE separated total proteins of D1NS1-HEK and D4NS1-HEK cell lysates when compared to a lysate of untransfected HEK cells (Fig. 1a). However, Western blot analysis with anti-hexa-His tag antibodies (Fig. 1b) confirmed the specific expression of the NS1 fusion proteins (a band at the expected size for the monomer NS1 of 46–55 kDa [20]) by the transfectants. In combination with Western blot analysis with anti-tubulin antibodies serving as a loading control (Fig. 1c), these analyses revealed no significant difference between expression levels of D1NS1 and D4NS1. IFA of D1NS1-HEK, D4NS1-HEK and untransfected HEK cells with anti-hexa-His tag antibodies demonstrated the cell surface localization of the expressed NS1 fusion proteins with similar staining intensities for D1NS1 and D4NS1 (Fig. 2).Fig. 1

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