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1.
FIG. 5.

FIG. 5. From: Flexible Label-Free Quantitative Assay for Antibodies to Influenza Virus Hemagglutinins .

Isotyping the immune response to vaccination. Recombinant H3-BR07 HA (100 μg/ml) was bound to the Octet Red biosensors (HA binding), and after washing to remove excess HA (wash), the biosensors were incubated with human sera (GP3) at a 1:80 dilution, followed by incubation with various anti-human immunoglobulin antibodies (IgG1, IgG2, IgG3, IgG4, IgA, IgE, and IgM), all at 1 μg/ml. Similar results were also obtained with GP3 and TIV1 (results not shown).

Paul J. Carney, et al. Clin Vaccine Immunol. 2010 September;17(9):1407-1416.
2.
FIG. 3.

FIG. 3. From: Flexible Label-Free Quantitative Assay for Antibodies to Influenza Virus Hemagglutinins .

Sensitivity in detection of binding anti-H5-VN04 serum and its ability to inhibit fetuin binding. (A) H5-VN04 HA was bound to the Octet Red biosensors (HA binding), and after washing to remove excess HA (wash), the biosensors were incubated with 2-fold dilutions of anti-H5-VN04 ferret sera (association; 1:80 to 1:5,120), followed by incubation with fetuin (0.5 μg/ml). (B and C) After subtraction of the buffer-only control, results clearly show that serum binding (B) can be detected down to 1:1,280, while fetuin binding (C) can be detected only when the ferret serum is at dilutions of 1:160 or more. This serum has a titer of 160 in HI assays (Table 4).

Paul J. Carney, et al. Clin Vaccine Immunol. 2010 September;17(9):1407-1416.
3.
FIG. 1.

FIG. 1. From: Flexible Label-Free Quantitative Assay for Antibodies to Influenza Virus Hemagglutinins .

Detection of antibody binding to recombinant HA by f-AbBA. (A) HA from Brisbane/10/2007 (H3-BR07) was bound and analyzed, in parallel, to eight Octet Red biosensors (HA binding), and after washing to remove excess HA (wash), the biosensors were incubated with 2-fold dilutions of anti-BR07 ferret sera (association from 1:80 to 1:5,120). Each curve is a separate dilution of serum. (B) After data processing, results clearly show that serum binding can be detected down to 1:1,280 (based on the 10% cutoff criterion described in the text). The instrument detects binding to the biosensor tip, which results in a wavelength shift (measured in nanometers).

Paul J. Carney, et al. Clin Vaccine Immunol. 2010 September;17(9):1407-1416.
4.
FIG. 2.

FIG. 2. From: Flexible Label-Free Quantitative Assay for Antibodies to Influenza Virus Hemagglutinins .

HA antibody binding detection specificity and sensitivity by f-AbBA. Recombinant HAs from H3-BR07 (A), H1-BR07 (B), H1pdm (C), and H5-VN04 (D) were probed with ferret sera raised against homologous and heterologous viruses. Data are presented as percentages of binding compared to those of homologous sera at 1:80 dilution (100%). The highest dilution with binding above 10% (indicated by a horizontal dashed line above the x axis) was the empirical endpoint, and values are presented in tabular form for direct comparison with titers from an HI assay using equivalent viruses (Table 4). Results correspond to the mean and standard error from 3 independent replicate assays.

Paul J. Carney, et al. Clin Vaccine Immunol. 2010 September;17(9):1407-1416.
5.
FIG. 6.

FIG. 6. From: Flexible Label-Free Quantitative Assay for Antibodies to Influenza Virus Hemagglutinins .

Assessment of H5-VN04 stability upon drying/storage during a 2- to 3-month period. H5-VN04-coupled biosensors were dried in different storage buffers, stored for 3 days, and assessed for binding to the homologous polyclonal ferret serum identified formulations containing sucrose as a potential condition to store HA-coupled biosensors in a dry format (A). Ten percent sucrose was selected for experiments to assess longer-term storage of biosensors. At days 12 and 62, biosensors were used to detect binding to a monoclonal antibody (B519M) that can neutralize by HI and thus detects a neutralizing antigenic epitope in the HA (B). Results show only a minimal loss of binding compared to that for biosensors loaded with freshly prepared recombinant HA. The viability of the dried HA after 76 days was also assessed by the biosensor's ability to bind the glycoprotein fetuin with and without preincubation with anti-H5-VN04 ferret serum (which detects a functional receptor binding site) (C).

Paul J. Carney, et al. Clin Vaccine Immunol. 2010 September;17(9):1407-1416.
6.
FIG. 4.

FIG. 4. From: Flexible Label-Free Quantitative Assay for Antibodies to Influenza Virus Hemagglutinins .

Sensitive and specific detection of antibody to HA in human serum. Binding of human sera to a seasonal H3 virus (BR07) (A), a seasonal H1 virus (Sol06) (B), and a pandemic H1 virus (CA409) (C). General population sera randomly collected by the American Red Cross (GP1 to GP10) as well as 12 pairs of pre-and postvaccination sera were RDE treated and analyzed at a 1:80 dilution. TIV1 to TIV6 (highlighted in yellow) received the TIV vaccine, while LAIV1 to LAIV6 (highlighted in green) received the LAIV vaccine. Results, presented as percentage of antibody bound, were calculated for the binding level after 5 min, standardized with ferret sera against the homologous virus (100%). The ferret sera from the H1-SI06 response bound at a reduced level compared to three of the patient sera tested. Values next to the bars represent corresponding pre- and postvaccination titers (single titers for GP1 to GP10 sera) determined independently by HI assay using turkey red blood cells. Results shown are representative of 2 or 3 experiments.

Paul J. Carney, et al. Clin Vaccine Immunol. 2010 September;17(9):1407-1416.

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