Fig 1.
Binding of mAb F211-11H12 (A) and F211-10A9 (B) to 13 subtypes of influenza. Molecular markers corresponding to HA2 at 30KDa and HA1 at 70KDa are indicated. Full names of the virus strains corresponding to each lane are shown in the table on the right panel.
Table 1.
List of 40 influenza strains and strength of the signal measured when probed with pan-HA mAb cocktail.
Fig 2.
Quantification by dot-blot using the pan-HA cocktail of ten influenza samples (H1N1 A/Puerto Rico/8/34) produced in HEK293 suspension cells.
The calibrating antigen (Ag) was loaded in duplicate in concentrations ranging from 160ng/ml to 20μg/ml, and ten samples were loaded in 4 different dilutions as indicated in a representative blot (A). Using the signal measured with the calibrating antigen, a standard curve was generated with an R squared value of 0.9893 in the linear range (B). Using the standard curve, HA concentrations for the ten non-purified samples (S1 to S10) loaded in A were calculated (C).
Table 2.
Concentrations obtained by dot blot on three different runs for two influenza samples (H1N1 A/Puerto Rico/8/34).
Fig 3.
A standard curve was generated with H5 VLP from 0.25 to 16 μg/ml in a competitive ELISA.
The antibody at a concentration of 800ng/ml was mixed with different concentrations of H5 VLP and added to a pre-coated plate. The standard curve was obtained by plotting the mean absorbance (y axis) against the VLP concentration (x axis). A four parameter logistic regression was performed to fit the curve.
Fig 4.
Comparison of HA concentrations obtained by dot blot and hemagglutination assay.
Thirty-six samples were produced in HEK293 suspension cells and clarified by centrifugation. Samples were quantified by dot blot using the pan-HA cocktail, and by hemagglutination assay with chicken red blood cells. The R-squared value calculated by linear regression is 0.8015.
Table 3.
HA concentrations of three vaccine-like samples measured by SRID and dot blot.
Table 4.
KD values (M) measured by SPR for pan-HA antibodies produced in mouse hybridomas and CHO pools.
Fig 5.
Dot blot results with a pan-HA cocktail obtained from mouse hybridomas (A) and CHO pools (B). Each dot represents a different strain as indicated in the table on the right panel. Recombinant proteins and plant VLPs were loaded at a final concentration of 2.5 μg while the viruses and standard antigens were loaded at a concentration of 5 μg. Legend: rHA = recombinant hemagglutinin, Sd = cell-produced calibrating standards (NIBSC), Virus = viruses produced in-house in HEK293 cells, VLP = Viral like particles produced in plants (Medicago).
Fig 6.
Probability of interaction between the highly-conserved peptide sequence and mAb 11H12 (A) or mAb 10A9 (B). Amino Acids 1 to 14 correspond to the highly-conserved peptide used to generate antibodies (GLFGAIAGFIEGGW). Amino acids 15–17 are the amino acids following the end of the highly conserved sequence.