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Table 1 Experimental design for assay development

From: Development and application of assay for determining β-glucosidase activity in human saliva

  Experiment Factors Treatments Outcome
i Validate linearity of the assay signal 7.75 mM p-nitrophenyl-β-O-d-glucosidase + 0.0025 units of almond β-glucosidase, corrected to final volume of 2 mL with 100 mM phosphate buffer (pH 6.5); wavelength 400 nm over 10 min; no α-cyclodextrin Absorbance measured every minute for 10 min after the addition of almond β-glucosidase Linearity of the assay is confirmed
ii Assess interference from saliva See (i) above Absorbance values of buffer compared with and without 25 μL heat-inactivateda human saliva Heat-inactivated saliva does not interfere with the matrix
iii Determine absorbance maxima of p-nitrophenyl See (i) above Assay measured at 400 and 405 nm 400 nm selected
iv Determine substrate saturation concentration Varying substrate concentrations + 0.0025 units of almond β-glucosidase, corrected to final volume of 2 mL with 100 mM phosphate buffer (pH 6.5); wavelength 400 nm over 10 min Substrate concentration varied between 0 and 10 mM 7.75 mM selected
v Determine maximum saliva volume 7.75 mM p-nitrophenyl-β-O-d-glucosidase + 0.0025 units of almond β-glucosidase + varying volumes of heat-inactivated saliva, corrected to final volume of 2 mL with 100 mM phosphate buffer (pH 6.5); wavelength 400 nm over 10 min Different amounts of heat-inactivated saliva evaluated: 25, 50, 100, 150 and 200 μL 200 μL selected
vi Evaluate efficacy of α-cyclodextrin 7.75 mM p-nitrophenyl-β-O-d-glucosidase + 0.0025 units of almond β-glucosidase + 200 μL of heat-inactivated saliva, corrected to final volume of 2 mL with 100 mM phosphate buffer (pH 6.5); wavelength 400 nm over 10 min Assays performed with and without 6 mM α-cyclodextrin Inclusion of 6 mM α-cyclodextrin in assay
  1. aSaliva was heat-inactivated by boiling for 10 min in a water bath, then centrifuged at 8000 rpm for 10 min using a SS-34 rotor (Sorval RC5C Plus centrifuge)