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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.


Specificity of the piezoelectric sensor with immobilized LPS from F. tularensis (left, blue bars). The specific sample (100 μl) was the monoclonal antibody 4H3B9D3 diluted 1:10 (final protein concentration 0.67 mg/ml). As negatives controls, commercials antibodies anti-IgM (Ab1) and anti-IgG (Ab2) (both adjusted to the total protein 0.67 mg/ml) were used as well as BSA at 1 mg/ml. At right (red bars), specificity of the monoclonal antibody 4H3B9D3 (100 μl sample, protein concentration 0.67 mg/ml) was tested using piezoelectric immunosensors with immobilized LPS obtained from either F. tularensis, E. coli or B. subtilis, and also immobilized albumin (BSA) was used. The error bars indicate estimated standard deviations (n = 3).
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f3-sensors-07-00341: Specificity of the piezoelectric sensor with immobilized LPS from F. tularensis (left, blue bars). The specific sample (100 μl) was the monoclonal antibody 4H3B9D3 diluted 1:10 (final protein concentration 0.67 mg/ml). As negatives controls, commercials antibodies anti-IgM (Ab1) and anti-IgG (Ab2) (both adjusted to the total protein 0.67 mg/ml) were used as well as BSA at 1 mg/ml. At right (red bars), specificity of the monoclonal antibody 4H3B9D3 (100 μl sample, protein concentration 0.67 mg/ml) was tested using piezoelectric immunosensors with immobilized LPS obtained from either F. tularensis, E. coli or B. subtilis, and also immobilized albumin (BSA) was used. The error bars indicate estimated standard deviations (n = 3).

Mentions: For evaluation of the sensing selectivity, the signal of mAb 4H3B9D3 diluted 1:10 was compared with negative controls - commercial antibodies without any specificity against Francisella; anti-IgM and anti-IgG antibodies were used; in all cases the total protein was adjusted to 0.67 mg/ml. Commonly used albumin was included, too (1 mg/ml solution). The binding signals obtained using the sensor with immobilized Francisella LPS proved good selectivity of the immunosensor (Fig. 3, left panel). The signal of mAb 4H3B9D3 (264 ± 11 Hz) was approximately two hundred times higher than the negative controls providing 0.7, 1.0 and 1.0 Hz for anti-IgM, anti-IgG and BSA, respectively. Such a high difference is quite sufficient for distinguishing monoclonal antibodies against the target antigen from other immunoglobulins.


Rapid Characterization of Monoclonal Antibodies using the Piezoelectric Immunosensor
Specificity of the piezoelectric sensor with immobilized LPS from F. tularensis (left, blue bars). The specific sample (100 μl) was the monoclonal antibody 4H3B9D3 diluted 1:10 (final protein concentration 0.67 mg/ml). As negatives controls, commercials antibodies anti-IgM (Ab1) and anti-IgG (Ab2) (both adjusted to the total protein 0.67 mg/ml) were used as well as BSA at 1 mg/ml. At right (red bars), specificity of the monoclonal antibody 4H3B9D3 (100 μl sample, protein concentration 0.67 mg/ml) was tested using piezoelectric immunosensors with immobilized LPS obtained from either F. tularensis, E. coli or B. subtilis, and also immobilized albumin (BSA) was used. The error bars indicate estimated standard deviations (n = 3).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3756724&req=5

f3-sensors-07-00341: Specificity of the piezoelectric sensor with immobilized LPS from F. tularensis (left, blue bars). The specific sample (100 μl) was the monoclonal antibody 4H3B9D3 diluted 1:10 (final protein concentration 0.67 mg/ml). As negatives controls, commercials antibodies anti-IgM (Ab1) and anti-IgG (Ab2) (both adjusted to the total protein 0.67 mg/ml) were used as well as BSA at 1 mg/ml. At right (red bars), specificity of the monoclonal antibody 4H3B9D3 (100 μl sample, protein concentration 0.67 mg/ml) was tested using piezoelectric immunosensors with immobilized LPS obtained from either F. tularensis, E. coli or B. subtilis, and also immobilized albumin (BSA) was used. The error bars indicate estimated standard deviations (n = 3).
Mentions: For evaluation of the sensing selectivity, the signal of mAb 4H3B9D3 diluted 1:10 was compared with negative controls - commercial antibodies without any specificity against Francisella; anti-IgM and anti-IgG antibodies were used; in all cases the total protein was adjusted to 0.67 mg/ml. Commonly used albumin was included, too (1 mg/ml solution). The binding signals obtained using the sensor with immobilized Francisella LPS proved good selectivity of the immunosensor (Fig. 3, left panel). The signal of mAb 4H3B9D3 (264 ± 11 Hz) was approximately two hundred times higher than the negative controls providing 0.7, 1.0 and 1.0 Hz for anti-IgM, anti-IgG and BSA, respectively. Such a high difference is quite sufficient for distinguishing monoclonal antibodies against the target antigen from other immunoglobulins.

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.