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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.


ELISAs showing humoral immune responses of mice immunized with D1NS1-HEK and D4NS1-HEK cells. a and b: Mouse antisera of D1NS1-HEK (red, panel a) and D4NS1-HEK (blue, panel b) immunized mouse groups (4 mice each) were analyzed on recombinant D1NS1 and recombinant D4NS1, respectively. c and d: Serotype cross-reactive humoral immune responses of D1NS1-HEK (red, panel c) and D4NS1-HEK (blue, panel d) mouse groups were analyzed on recombinant D4NS1 and recombinant D1NS1, respectively. Blue line (panel c) and red line (panel d) = pool of D4NS1-HEK and D1NS1-HEK mouse antisera, respectively, to enable direct comparison with the level of serotype-specific immune responses. e and f: Analysis of pooled mouse sera (red = D1NS1-HEK (e), blue = D4NS1-HEK (f)) on an unrelated hexa-His tagged protein (MUL_3720) as a control for humoral responses against the hexa-His tag. Black line in all panels = pool of mouse sera before immunization (pre-bleed)
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Fig3: ELISAs showing humoral immune responses of mice immunized with D1NS1-HEK and D4NS1-HEK cells. a and b: Mouse antisera of D1NS1-HEK (red, panel a) and D4NS1-HEK (blue, panel b) immunized mouse groups (4 mice each) were analyzed on recombinant D1NS1 and recombinant D4NS1, respectively. c and d: Serotype cross-reactive humoral immune responses of D1NS1-HEK (red, panel c) and D4NS1-HEK (blue, panel d) mouse groups were analyzed on recombinant D4NS1 and recombinant D1NS1, respectively. Blue line (panel c) and red line (panel d) = pool of D4NS1-HEK and D1NS1-HEK mouse antisera, respectively, to enable direct comparison with the level of serotype-specific immune responses. e and f: Analysis of pooled mouse sera (red = D1NS1-HEK (e), blue = D4NS1-HEK (f)) on an unrelated hexa-His tagged protein (MUL_3720) as a control for humoral responses against the hexa-His tag. Black line in all panels = pool of mouse sera before immunization (pre-bleed)

Mentions: Immunization of mice with either D1NS1-HEK or D4NS1-HEK cells resulted in the development of humoral immune responses specific for the NS1 proteins (Fig. 3). Sera of mice immunized with D1NS1-HEK contained higher titers of antibodies against recD1NS1 (Fig. 3a) than against recD4NS1 (Fig. 3c). In contrast, similar antibody titers to both recD1NS1 and recD4NS1 were detected in mice that were immunized with D4NS1-HEK cells (Fig. 3b and d). Individual mouse antisera of the D1NS1-HEK and D4NS1-HEK mouse groups contained similar levels of antibodies to the NS1 target protein. All mouse antisera showed only very low anti-hexa-His tag antibody levels, as determined by ELISA of the sera on an unrelated hexa-His tagged protein (MUL_3720) (Fig. 3e and f).Fig. 3


Generation of monoclonal antibodies against native viral proteins using antigen-expressing mammalian cells for mouse immunization
ELISAs showing humoral immune responses of mice immunized with D1NS1-HEK and D4NS1-HEK cells. a and b: Mouse antisera of D1NS1-HEK (red, panel a) and D4NS1-HEK (blue, panel b) immunized mouse groups (4 mice each) were analyzed on recombinant D1NS1 and recombinant D4NS1, respectively. c and d: Serotype cross-reactive humoral immune responses of D1NS1-HEK (red, panel c) and D4NS1-HEK (blue, panel d) mouse groups were analyzed on recombinant D4NS1 and recombinant D1NS1, respectively. Blue line (panel c) and red line (panel d) = pool of D4NS1-HEK and D1NS1-HEK mouse antisera, respectively, to enable direct comparison with the level of serotype-specific immune responses. e and f: Analysis of pooled mouse sera (red = D1NS1-HEK (e), blue = D4NS1-HEK (f)) on an unrelated hexa-His tagged protein (MUL_3720) as a control for humoral responses against the hexa-His tag. Black line in all panels = pool of mouse sera before immunization (pre-bleed)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig3: ELISAs showing humoral immune responses of mice immunized with D1NS1-HEK and D4NS1-HEK cells. a and b: Mouse antisera of D1NS1-HEK (red, panel a) and D4NS1-HEK (blue, panel b) immunized mouse groups (4 mice each) were analyzed on recombinant D1NS1 and recombinant D4NS1, respectively. c and d: Serotype cross-reactive humoral immune responses of D1NS1-HEK (red, panel c) and D4NS1-HEK (blue, panel d) mouse groups were analyzed on recombinant D4NS1 and recombinant D1NS1, respectively. Blue line (panel c) and red line (panel d) = pool of D4NS1-HEK and D1NS1-HEK mouse antisera, respectively, to enable direct comparison with the level of serotype-specific immune responses. e and f: Analysis of pooled mouse sera (red = D1NS1-HEK (e), blue = D4NS1-HEK (f)) on an unrelated hexa-His tagged protein (MUL_3720) as a control for humoral responses against the hexa-His tag. Black line in all panels = pool of mouse sera before immunization (pre-bleed)
Mentions: Immunization of mice with either D1NS1-HEK or D4NS1-HEK cells resulted in the development of humoral immune responses specific for the NS1 proteins (Fig. 3). Sera of mice immunized with D1NS1-HEK contained higher titers of antibodies against recD1NS1 (Fig. 3a) than against recD4NS1 (Fig. 3c). In contrast, similar antibody titers to both recD1NS1 and recD4NS1 were detected in mice that were immunized with D4NS1-HEK cells (Fig. 3b and d). Individual mouse antisera of the D1NS1-HEK and D4NS1-HEK mouse groups contained similar levels of antibodies to the NS1 target protein. All mouse antisera showed only very low anti-hexa-His tag antibody levels, as determined by ELISA of the sera on an unrelated hexa-His tagged protein (MUL_3720) (Fig. 3e and f).Fig. 3

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.