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. 2008 Oct 28;8(10):6727-6746.
doi: 10.3390/s8106727.

Immunoglobulin G Determination in Human Serum and Milk Using an Immunosensor of New Conception Fitted with an Enzyme Probe as Transducer

Luigi Campanella  1 , Dalina Lelo  2 , Elisabetta Martini  2 , Mauro Tomassetti  3
Affiliations

Immunoglobulin G Determination in Human Serum and Milk Using an Immunosensor of New Conception Fitted with an Enzyme Probe as Transducer

Luigi Campanella et al. Sensors (Basel). .

Abstract

To completely overcome the problem of the presence of urea in the serum, which can be the cause (especially at low immunoglobulin G concentrations) of a small but non negligible interference in the enzyme reaction of the enzymatic marker, when the measurement was performed by a potentiometric immunosensor that we constructed and characterized in previous work, and which used urease as marker, we have now constructed an entirely different and highly innovative immunosensor. This new device uses the enzyme alkaline phosphatase as marker, sodium phenylphosphate as substrate but above all, a tyrosinase biosensor obtained by coupling a Clark type gas diffusion amperometric electrode and the tyrosinase enzyme, immobilized in a cellulose triacetate membrane, as transducer. After optimizing the 'competitive' measurement procedures, the new immunosensor was used to determine both HIgG and the anti-HIgG, with a limit of detection (LOD) of the order of 3x10-11 M. Clearly this highly innovative construction geometry makes the immunosensor extremely selective. This makes it possible to determine immunoglobulin G both in human serum and milk without the slightest interference by any urea present in these biological matrixes.

Keywords: Immunoglobulin G detection; Immunosensor; enzymatic transducer; human milk; human serum; urea interference.

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Figures

Figure 1.
Figure 1.
New immunosensor assembly.
Figure 2.
Figure 2.
Determination of antibody (Anti-HIgG) by new immunosensor using tyrosinase enzyme electrode as transducer. Test geometry: competition for HIgG immobilized in membrane, between Anti-HIgG alkaline phosphatase conjugated and non-conjugated, both free in solution.
Figure 3.
Figure 3.
Determination of antigen (HIgG) by new immunosensor, using tyrosinase enzyme electrode as transducer. Test geometry: competition for anti-HIgG urease conjugated, between HIgG immobilized on membrane and HIgG free in solution.
Figure 4.
Figure 4.
Optimization (slope and linear range during lifetime) of the membrane binding tyrosinase enzyme using three different immobilization methods, to be used for the immunoglobulin G measurement by the new immunosensor.
Figure 5.
Figure 5.
(a) Behaviour of the new immunosensor response as a function of growing anti-HIgG concentration, using Pall-Biodyne or Immobilon membrane; (b) corresponding calibration curve and confidence interval for the anti-HIgG determination, obtained using a semilogarithmic scale.
Figure 6.
Figure 6.
(a) Behaviour of the new immunosensor response as a function of growing HIgG concentration, using Pall-Biodyne or Immobilon membrane; (b) corresponding calibration curve and confidence interval for the HIgG determination, obtained using a semilogarithmic scale.
Figure 6.
Figure 6.
(a) Behaviour of the new immunosensor response as a function of growing HIgG concentration, using Pall-Biodyne or Immobilon membrane; (b) corresponding calibration curve and confidence interval for the HIgG determination, obtained using a semilogarithmic scale.
Figure 7.
Figure 7.
Reutilization of the immunosensor for anti-HIgG analysis after washing with glycine solution 0.1 M, at pH = 2 and MgCl2 2.5 M, using (a) Pall-Biodyne membrane, (b) Immobilon membrane.
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