Human IgA Monoclonal Antibodies That Neutralize Poliovirus, Produced by Hybridomas and Recombinant Expression

Figures

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  Figure 1. M. musculus leader sequences used for recombinant protein expression.

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  Figure 2. Binding of human IgA mAbs to whole Sabin PV by ELISA. Error bars = S.E.M.

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  Figure 3. Binding of type 3 PV to OCMS hybridomas secreting anti-PV IgA mAbs. Hybridomas were incubated overnight with the rabbit mAb specific for human IgA and then with biotinylated PV. Human IgA and PV3 binding to the cells were detected with an Alexa-Fluor 647 anti-human IgA secondary and Alexa-Fluor 488 conjugated streptavidin, respectively. Nuclei were stained with DAPI. Scale bar = 10 µm.

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  Figure 4. Analysis of the human hybridoma IgA mAbs after Jacalin purification. (A) Gel filtration with a Superose 6 HR 10/300 column. Protein peaks for all samples were collected in the two fractions shown (F1, ~27 min; F2, ~33 min). 8C5 IgG and 1G1 IgG are used as monomer controls. (B) Western blot analysis of the F1 and F2 fractions for expression of the Ig J chain. Recombinant 2F7 dIgA1 (Section 3.6) was used as a positive control. Molecular weight markers (in kD) are indicated on the left.

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  Figure 5. Assessment of human κ light chain variants for binding to Protein L. (A) The amino acid sequence of the 1G1 κ light chain (VκII subtype) is shown, as well as the amino acid mutations tested. The framework regions (FR1, FR1, FR3) and complementarity determining regions (CDR1, CDR2, CDR3) are shown above. Also shown are the sequences of 2F7 (VκIV subtype) and 2A2, the VκI light chain used for the reference crystal structure [43]. (B) Western blot of IgGs expressed in supernatants of cells co-transfected with κ LC wt or mutant genes as shown in (A) or the 1G1 IgG heavy-chain gene. Double lanes for some mutants represent independent transfections. (C) Supernatants shown in (B) were run through Protein L columns and retained IgGs were analyzed by SDS-PAGE. IgG, purified 1G1 IgG, expressed with P12S κ mutation.

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  Figure 6. Analysis of the recombinant IgA mAbs after Protein L purification. (A) Gel filtration of 20 µg mAb samples with a Superdex 200 Increase 10/300 GL column. Marker, Bio-Rad Gel Filtration Standards. (B) Analysis of the purified proteins on reducing (left) and non-reducing (right) SDS-PAGE.

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  Figure 7. Expression of recombinant IgA in 293T OCMS cells. 293T OCMS (top row) and 293T (bottom row) cells were transiently transfected with plasmids encoding recombinant 1G1 IgA HC and LC genes. The next day, the cells were incubated overnight with the rabbit anti-human IgA mAb (Linker). Bound Linker was detected with Alexa-Fluor 488 anti-rabbit IgG secondary antibody (green). Captured human IgA was labeled with Alexa-Fluor 647 anti human IgA (magenta). Nuclei were stained with DAPI (blue). Scale bar = 5 µm.