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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.


(A) Schema of the measuring setup consisting of the flow-through cell with the piezoelectric quartz crystal; opened (B) and assembled (C) views. The sensor is driven by the lever oscillator and the output frequency is recorded using a general-purpose counter and continuously transferred to the computer. Flow is realized using the peristaltic pump.
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f1-sensors-07-00341: (A) Schema of the measuring setup consisting of the flow-through cell with the piezoelectric quartz crystal; opened (B) and assembled (C) views. The sensor is driven by the lever oscillator and the output frequency is recorded using a general-purpose counter and continuously transferred to the computer. Flow is realized using the peristaltic pump.

Mentions: The piezoelectric system in a flow through arrangement was used. The Lever Oscillator (ICM) and counter (Grundig, Fuerth, Germany) worked under the own software LabTools (Fig. 1), sampling time was 1 s and frequency resolution 0.2 Hz. Samples were transported by the peristaltic pump (PCD 21M, Kouřil, Kyjov, Czech Rep.) using silicon tubes into the flow through cell with the piezoelectric biosensor fixed between two silicone rubber o-rings (Fig. 1B and C), internal volume was 10 μl. The cell was oriented vertically in order to facilitate release of occasionally formed bubbles. Flow rate was adjusted to 50 μl/min, this is sufficiently high to avoid mass transfer limitations. PBS was used for dilution of samples and as the carrier buffer. Each measuring cycle consisted of the following steps: 2 min flow of the carrier to stabilize baseline signal, 2 min sample, 2 – 5 min flow of the carrier for signal stabilization. When isotypization was required, 2 min flow of the secondary antibody was applied (either anti IgG or anti IgM, supplied from Serotec, Oxford, UK), followed again with 2 – 5 min of the carrier. Regeneration was realized with a 2 min flow of 50 mM NaOH. Samples and other reagents were diluted with PBS, the dilution ratio for samples was from 1:10 up to 1:300. For statistical purpose, each sample was measured three times.


Rapid Characterization of Monoclonal Antibodies using the Piezoelectric Immunosensor
(A) Schema of the measuring setup consisting of the flow-through cell with the piezoelectric quartz crystal; opened (B) and assembled (C) views. The sensor is driven by the lever oscillator and the output frequency is recorded using a general-purpose counter and continuously transferred to the computer. Flow is realized using the peristaltic pump.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3756724&req=5

f1-sensors-07-00341: (A) Schema of the measuring setup consisting of the flow-through cell with the piezoelectric quartz crystal; opened (B) and assembled (C) views. The sensor is driven by the lever oscillator and the output frequency is recorded using a general-purpose counter and continuously transferred to the computer. Flow is realized using the peristaltic pump.
Mentions: The piezoelectric system in a flow through arrangement was used. The Lever Oscillator (ICM) and counter (Grundig, Fuerth, Germany) worked under the own software LabTools (Fig. 1), sampling time was 1 s and frequency resolution 0.2 Hz. Samples were transported by the peristaltic pump (PCD 21M, Kouřil, Kyjov, Czech Rep.) using silicon tubes into the flow through cell with the piezoelectric biosensor fixed between two silicone rubber o-rings (Fig. 1B and C), internal volume was 10 μl. The cell was oriented vertically in order to facilitate release of occasionally formed bubbles. Flow rate was adjusted to 50 μl/min, this is sufficiently high to avoid mass transfer limitations. PBS was used for dilution of samples and as the carrier buffer. Each measuring cycle consisted of the following steps: 2 min flow of the carrier to stabilize baseline signal, 2 min sample, 2 – 5 min flow of the carrier for signal stabilization. When isotypization was required, 2 min flow of the secondary antibody was applied (either anti IgG or anti IgM, supplied from Serotec, Oxford, UK), followed again with 2 – 5 min of the carrier. Regeneration was realized with a 2 min flow of 50 mM NaOH. Samples and other reagents were diluted with PBS, the dilution ratio for samples was from 1:10 up to 1:300. For statistical purpose, each sample was measured three times.

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.