Limits...
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

IFA of selected generated anti-NS1 mAbs on HEK cells. IFA of methanol fixed D1NS1-HEK, D4NS1-HEK and untransfected HEK cells after staining with selected generated anti-NS1 mAbs and Alexa568-labelled anti-mouse IgG antibodies. a: D1NS1 and D4NS1 serotype cross-reactive mAb NR1.8. b: D1NS1 serotype-specific mAb NR1.1. c: D4NS1 serotype-specific mAb NR4.4. Nuclei were stained with DAPI
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig4: IFA of selected generated anti-NS1 mAbs on HEK cells. IFA of methanol fixed D1NS1-HEK, D4NS1-HEK and untransfected HEK cells after staining with selected generated anti-NS1 mAbs and Alexa568-labelled anti-mouse IgG antibodies. a: D1NS1 and D4NS1 serotype cross-reactive mAb NR1.8. b: D1NS1 serotype-specific mAb NR1.1. c: D4NS1 serotype-specific mAb NR4.4. Nuclei were stained with DAPI

Mentions: Fusion of spleen cells obtained from mice immunized with D1NS1-HEK or D4NS1-HEK cells with PAI myeloma cells enabled the generation of anti-NS1 antibody-producing hybridoma cell lines. In order to identify cell lines that produce IgG specific for NS1, hybridoma supernatants were screened by ELISA on recD1NS1 or recD4NS1, respectively. Supernatants from ELISA-positive wells were subsequently tested by IFA on D1NS1-HEK and D4NS1-HEK cells. IFA on HEK cells transfected with an unrelated hexa-His tagged protein served as control to identify those hybridoma cell lines that generated NS1-specific antibodies. Screening of D1NS1-HEK and D4NS1-HEK mouse spleen cell fusions yielded eight and four anti-DENV NS1 IgG-producing hybridoma cell lines, respectively. These cell lines were cloned by limiting dilution to obtain hybridoma clones producing the mAbs NR1.1 - NR1.8 and NR4.1 - NR4.4, respectively. Five mAbs were of the IgG1(κ), six of the IgG2a(κ) and one of the IgG2b(κ) IgG subclass (Table 1). IFA with these mAbs on the D1NS1 and D4NS1 transfectants revealed that five specifically recognized D1NS1-HEK but not D4NS1-HEK cells, three only reacted with D4NS1-HEK cells and the remaining four were cross-reactive with both transfectants (Table 1, Fig. 4).Table 1


Generation of monoclonal antibodies against native viral proteins using antigen-expressing mammalian cells for mouse immunization
IFA of selected generated anti-NS1 mAbs on HEK cells. IFA of methanol fixed D1NS1-HEK, D4NS1-HEK and untransfected HEK cells after staining with selected generated anti-NS1 mAbs and Alexa568-labelled anti-mouse IgG antibodies. a: D1NS1 and D4NS1 serotype cross-reactive mAb NR1.8. b: D1NS1 serotype-specific mAb NR1.1. c: D4NS1 serotype-specific mAb NR4.4. Nuclei were stained with DAPI
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig4: IFA of selected generated anti-NS1 mAbs on HEK cells. IFA of methanol fixed D1NS1-HEK, D4NS1-HEK and untransfected HEK cells after staining with selected generated anti-NS1 mAbs and Alexa568-labelled anti-mouse IgG antibodies. a: D1NS1 and D4NS1 serotype cross-reactive mAb NR1.8. b: D1NS1 serotype-specific mAb NR1.1. c: D4NS1 serotype-specific mAb NR4.4. Nuclei were stained with DAPI
Mentions: Fusion of spleen cells obtained from mice immunized with D1NS1-HEK or D4NS1-HEK cells with PAI myeloma cells enabled the generation of anti-NS1 antibody-producing hybridoma cell lines. In order to identify cell lines that produce IgG specific for NS1, hybridoma supernatants were screened by ELISA on recD1NS1 or recD4NS1, respectively. Supernatants from ELISA-positive wells were subsequently tested by IFA on D1NS1-HEK and D4NS1-HEK cells. IFA on HEK cells transfected with an unrelated hexa-His tagged protein served as control to identify those hybridoma cell lines that generated NS1-specific antibodies. Screening of D1NS1-HEK and D4NS1-HEK mouse spleen cell fusions yielded eight and four anti-DENV NS1 IgG-producing hybridoma cell lines, respectively. These cell lines were cloned by limiting dilution to obtain hybridoma clones producing the mAbs NR1.1 - NR1.8 and NR4.1 - NR4.4, respectively. Five mAbs were of the IgG1(κ), six of the IgG2a(κ) and one of the IgG2b(κ) IgG subclass (Table 1). IFA with these mAbs on the D1NS1 and D4NS1 transfectants revealed that five specifically recognized D1NS1-HEK but not D4NS1-HEK cells, three only reacted with D4NS1-HEK cells and the remaining four were cross-reactive with both transfectants (Table 1, Fig. 4).Table 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