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Distinction of subtype-specific antibodies against European porcine influenza viruses by indirect ELISA based on recombinant hemagglutinin protein fragment-1.

Zhao N, Lange E, Kubald S, Grund C, Beer M, Harder TC - Virol. J. (2013)

Bottom Line: Serological investigations of swine influenza virus infections and epidemiological conclusions thereof are challenging due to the complex and regionally variable pattern of co-circulating viral subtypes and lineages and varying vaccination regimes.Detection of subtype-specific antibodies currently depends on hemagglutination inhibition (HI) assays which are difficult to standardize and unsuitable for large scale investigations.Proteins were co-translationally mono-biotinylated and refolded in vitro into an antigenically authentic conformation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Diagnostic Virology, Friedrich-Loeffler-Institut, Suedufer 10, Greifswald 17493, Germany.

ABSTRACT

Background: Serological investigations of swine influenza virus infections and epidemiological conclusions thereof are challenging due to the complex and regionally variable pattern of co-circulating viral subtypes and lineages and varying vaccination regimes. Detection of subtype-specific antibodies currently depends on hemagglutination inhibition (HI) assays which are difficult to standardize and unsuitable for large scale investigations.

Methods: The nucleocapsid protein (NP) and HA1 fragments of the hemagglutinin protein (HA) of five different lineages (H1N1av, H1N1pdm, H1pdmN2, H1N2, H3N2) of swine influenza viruses were bacterially expressed and used as diagnostic antigens in indirect ELISA.

Results: Proteins were co-translationally mono-biotinylated and refolded in vitro into an antigenically authentic conformation. Western blotting and indirect ELISA revealed highly subtype-specific antigenic characteristics of the recombinant HA1 proteins although some cross reactivity especially among antigens of the H1 subtype were evident. Discrimination of antibodies directed against four swine influenza virus subtypes co-circulating in Germany was feasible using the indirect ELISA format.

Conclusions: Bacterially expressed recombinant NP and HA1 swine influenza virus proteins served as antigens in indirect ELISAs and provided an alternative to commercial blocking NP ELISA and HI assays concerning generic (NP-specific) and HA subtype-specific sero-diagnostics, respectively, on a herd basis.

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Comparison of HI titres measured against porcine avian-derived H1N1 isolate A/swine/Germany/R248/2010 and S/P ratios obtained by indirect ELISA using recombinant HA1 antigen of avian-derived H1N1 isolate A/swine/Germany/R1738/2011. Black dots – congruent qualitative result; white dots – incongruent qualitative result; HI titre shown as log2 series (starting dilution 1 = 1:10); dotted lines represent cut-off values.
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Figure 5: Comparison of HI titres measured against porcine avian-derived H1N1 isolate A/swine/Germany/R248/2010 and S/P ratios obtained by indirect ELISA using recombinant HA1 antigen of avian-derived H1N1 isolate A/swine/Germany/R1738/2011. Black dots – congruent qualitative result; white dots – incongruent qualitative result; HI titre shown as log2 series (starting dilution 1 = 1:10); dotted lines represent cut-off values.

Mentions: Inter-rater agreement [25] was used to evaluate results of indirect ELISAs using recombinant HA1 compared to HI titres. While excellent and good agreements were seen for the H3 (κR96 = 0,8255 [95% CI 0,6997-0,9513]) and the pandemic H1 antigens (κR26 = 0,7772 [0,6917-0,8627]; κR2035 = 0,661 [0,5643-0,7577]), respectively, only moderate agreement was signaled for the H1av (κR1738 = 0,5722 [0,4401-0,7043]) and H1N2 (κR1207 = 0,5083 [0,3359-0,6807]) antigens. A graphical analysis revealed that correlation between HI titres and S/P ratios was stronger for higher but less tight for sera with lower HI titres (Figure 5, examplified for recombinant H1av HA1 antigen). A good agreement (κHOLDING7 = 0,625 [0,374-0,876]) across all indirect ELISAs was seen when results were compared based on holding level instead of individual sera (Table 5).


Distinction of subtype-specific antibodies against European porcine influenza viruses by indirect ELISA based on recombinant hemagglutinin protein fragment-1.

Zhao N, Lange E, Kubald S, Grund C, Beer M, Harder TC - Virol. J. (2013)

Comparison of HI titres measured against porcine avian-derived H1N1 isolate A/swine/Germany/R248/2010 and S/P ratios obtained by indirect ELISA using recombinant HA1 antigen of avian-derived H1N1 isolate A/swine/Germany/R1738/2011. Black dots – congruent qualitative result; white dots – incongruent qualitative result; HI titre shown as log2 series (starting dilution 1 = 1:10); dotted lines represent cut-off values.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3733666&req=5

Figure 5: Comparison of HI titres measured against porcine avian-derived H1N1 isolate A/swine/Germany/R248/2010 and S/P ratios obtained by indirect ELISA using recombinant HA1 antigen of avian-derived H1N1 isolate A/swine/Germany/R1738/2011. Black dots – congruent qualitative result; white dots – incongruent qualitative result; HI titre shown as log2 series (starting dilution 1 = 1:10); dotted lines represent cut-off values.
Mentions: Inter-rater agreement [25] was used to evaluate results of indirect ELISAs using recombinant HA1 compared to HI titres. While excellent and good agreements were seen for the H3 (κR96 = 0,8255 [95% CI 0,6997-0,9513]) and the pandemic H1 antigens (κR26 = 0,7772 [0,6917-0,8627]; κR2035 = 0,661 [0,5643-0,7577]), respectively, only moderate agreement was signaled for the H1av (κR1738 = 0,5722 [0,4401-0,7043]) and H1N2 (κR1207 = 0,5083 [0,3359-0,6807]) antigens. A graphical analysis revealed that correlation between HI titres and S/P ratios was stronger for higher but less tight for sera with lower HI titres (Figure 5, examplified for recombinant H1av HA1 antigen). A good agreement (κHOLDING7 = 0,625 [0,374-0,876]) across all indirect ELISAs was seen when results were compared based on holding level instead of individual sera (Table 5).

Bottom Line: Serological investigations of swine influenza virus infections and epidemiological conclusions thereof are challenging due to the complex and regionally variable pattern of co-circulating viral subtypes and lineages and varying vaccination regimes.Detection of subtype-specific antibodies currently depends on hemagglutination inhibition (HI) assays which are difficult to standardize and unsuitable for large scale investigations.Proteins were co-translationally mono-biotinylated and refolded in vitro into an antigenically authentic conformation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Diagnostic Virology, Friedrich-Loeffler-Institut, Suedufer 10, Greifswald 17493, Germany.

ABSTRACT

Background: Serological investigations of swine influenza virus infections and epidemiological conclusions thereof are challenging due to the complex and regionally variable pattern of co-circulating viral subtypes and lineages and varying vaccination regimes. Detection of subtype-specific antibodies currently depends on hemagglutination inhibition (HI) assays which are difficult to standardize and unsuitable for large scale investigations.

Methods: The nucleocapsid protein (NP) and HA1 fragments of the hemagglutinin protein (HA) of five different lineages (H1N1av, H1N1pdm, H1pdmN2, H1N2, H3N2) of swine influenza viruses were bacterially expressed and used as diagnostic antigens in indirect ELISA.

Results: Proteins were co-translationally mono-biotinylated and refolded in vitro into an antigenically authentic conformation. Western blotting and indirect ELISA revealed highly subtype-specific antigenic characteristics of the recombinant HA1 proteins although some cross reactivity especially among antigens of the H1 subtype were evident. Discrimination of antibodies directed against four swine influenza virus subtypes co-circulating in Germany was feasible using the indirect ELISA format.

Conclusions: Bacterially expressed recombinant NP and HA1 swine influenza virus proteins served as antigens in indirect ELISAs and provided an alternative to commercial blocking NP ELISA and HI assays concerning generic (NP-specific) and HA subtype-specific sero-diagnostics, respectively, on a herd basis.

Show MeSH
Related in: MedlinePlus