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Rapid Characterization of Monoclonal Antibodies using the Piezoelectric Immunosensor

View Article: PubMed Central

ABSTRACT

Monoclonal antibodies with specificity against the Francisella tularensis outer lipopolysaccharide (LPS) membrane were prepared and characterized using the piezoelectric immunosensor with immobilized LPS antigen from F. tularensis. Signals obtained by the immunosensor were compared with ELISA and similar sensitivity was noticed. Signal of negative controls obtained using the biosensor was below 0.5% of the signal obtained for the selected specific antibody clone 4H3B9D3. Testing of cross reactivity based on the sensors with immobilized LPS from Escherichia coli and Bacillus subtilis confirmed selectivity of this antibody. Furthermore, the 4H3B9D3 antibody was successfully isotypized as IgM using the piezoelectric sensors with secondary antibodies. Kinetics parameters of antibody were evaluated in the flow-through arrangement. The kinetic rate constants for the antibody 4H3B9D3 were ka = (2.31 ± 0.20)·105 l mol-1s-1 (association) and kd = (0.0010 ±0.00062) s-1 (dissociation) indicating very good affinity to the LPS antigen.

No MeSH data available.


Isotyping of the mAb 4H3B9D3 using the piezoelectric sensor modified with LPS from F. tularensis. At first, the tested antibody was allowed to bind on the surface and after washing with buffer, either anti-IgG (blue curve) or anti-IgM (red curve) secondary antibody was injected.
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f4-sensors-07-00341: Isotyping of the mAb 4H3B9D3 using the piezoelectric sensor modified with LPS from F. tularensis. At first, the tested antibody was allowed to bind on the surface and after washing with buffer, either anti-IgG (blue curve) or anti-IgM (red curve) secondary antibody was injected.

Mentions: One of the most important antibody characteristics is the known isotype. Here, the piezoelectric immunosensor was able to determine not only the antigen-specific antibody, but also the isotype of the surface-bound antibody can be easily tested during the subsequent injection of secondary antibodies specific against murine either IgM or IgG. Such experiment is demonstrated in Fig. 4. Antibody 4H3B9D3 diluted 100 times provided sufficiently high response (71 ± 5.0 Hz) on the LPS-modified surface; thus formed LPS-antibody complex was allowed to interact with the subsequently added anti - mouse IgM secondary antibody (0.1 mg/ml) which caused change of frequency of 29.0 ± 2.7 Hz. In a similar experiment, anti – mouse IgG provided a negligible signal of 0.9 ± 0.6 Hz. These results clearly confirm the isotype of mAb 4H3B9D3 as IgM. Results correspond also with the Isotyping Kit (Tab. 1).


Rapid Characterization of Monoclonal Antibodies using the Piezoelectric Immunosensor
Isotyping of the mAb 4H3B9D3 using the piezoelectric sensor modified with LPS from F. tularensis. At first, the tested antibody was allowed to bind on the surface and after washing with buffer, either anti-IgG (blue curve) or anti-IgM (red curve) secondary antibody was injected.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3756724&req=5

f4-sensors-07-00341: Isotyping of the mAb 4H3B9D3 using the piezoelectric sensor modified with LPS from F. tularensis. At first, the tested antibody was allowed to bind on the surface and after washing with buffer, either anti-IgG (blue curve) or anti-IgM (red curve) secondary antibody was injected.
Mentions: One of the most important antibody characteristics is the known isotype. Here, the piezoelectric immunosensor was able to determine not only the antigen-specific antibody, but also the isotype of the surface-bound antibody can be easily tested during the subsequent injection of secondary antibodies specific against murine either IgM or IgG. Such experiment is demonstrated in Fig. 4. Antibody 4H3B9D3 diluted 100 times provided sufficiently high response (71 ± 5.0 Hz) on the LPS-modified surface; thus formed LPS-antibody complex was allowed to interact with the subsequently added anti - mouse IgM secondary antibody (0.1 mg/ml) which caused change of frequency of 29.0 ± 2.7 Hz. In a similar experiment, anti – mouse IgG provided a negligible signal of 0.9 ± 0.6 Hz. These results clearly confirm the isotype of mAb 4H3B9D3 as IgM. Results correspond also with the Isotyping Kit (Tab. 1).

View Article: PubMed Central

ABSTRACT

Monoclonal antibodies with specificity against the Francisella tularensis outer lipopolysaccharide (LPS) membrane were prepared and characterized using the piezoelectric immunosensor with immobilized LPS antigen from F. tularensis. Signals obtained by the immunosensor were compared with ELISA and similar sensitivity was noticed. Signal of negative controls obtained using the biosensor was below 0.5% of the signal obtained for the selected specific antibody clone 4H3B9D3. Testing of cross reactivity based on the sensors with immobilized LPS from Escherichia coli and Bacillus subtilis confirmed selectivity of this antibody. Furthermore, the 4H3B9D3 antibody was successfully isotypized as IgM using the piezoelectric sensors with secondary antibodies. Kinetics parameters of antibody were evaluated in the flow-through arrangement. The kinetic rate constants for the antibody 4H3B9D3 were ka = (2.31 ± 0.20)·105 l mol-1s-1 (association) and kd = (0.0010 ±0.00062) s-1 (dissociation) indicating very good affinity to the LPS antigen.

No MeSH data available.