Enhancement of Biotinidase activity in dried blood spot by Disulfide Reducing Reagent and Comparative Evaluation with Reference Method
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To develop a simple and reliable method for quantification of biotinidase (EC.18.104.22.168) activity in dried blood spot and serum by a modification of existing colorimetric screening test.
The assay involves incubation of DBS, serum sample and controls in 30 μL phosphate buffer containing synthetic substrate B-PABA, EDTA and 10μL of 40mM disulfide reducing reagent Mercaptoethanol at 37ºC for 4 hours. The reaction was further stopped by TCA addition for hemoglobin denaturation. After the coupling reaction was started, the absorbance was measured by a microplate-reader.
A present rapid method using Mercaptoethanol (ME) for biotinidase assay from dried blood spots was developed, validate and compare with Yamaguchi et al method for dried blood spot (n=360). The method performance with DBS was compare with serum by using Wolf et al method. The correlation coefficients obtained by plotting the values obtained by modified method against the values by Wolf et and Yamaguchi et al methods were 0.86 and 0.96 respectively.
Biotinidase activities in DBS (n360) and in serum (n=50) were always enhanced by 2-mercaptoethanol (ME). The modified method has reduced the processing and incubation time up to 4 Hr. and it is significantly correlate with reference method. The present method applicable for newborn screening in DBS as well as test for biotinides deficiency in serum. Modified method is very cost effective due to less run time, precision and accuracy.
Biotinidase enzyme, dried blood spots, Mercaptoethanol, newborn screening.