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The GSP Neonatal Biotinidase kit is intended for the quantitative in vitro determination of human biotinidase activity in blood specimens dried on filter paper as an aid in screening newborns for biotinidase deficiency using the GSP instrument.

The GSP Neonatal Biotinidase kit contains sufficient reagents to perform 1152 assays. The GSP Neonatal Biotinidase test system measures biotinidase activity, combining an enzyme reaction with a solid phase time-resolved immunofluorescence assay. The GSP Neonatal Biotinidase assay is based on the ability of the biotinidase enzyme to cleave the amide bond in Eu-labeled biotin. The enzyme reaction is stopped by addition of streptavidin which has high affinity for biotin (either Eu-labeled or free biotin). The streptavidin-biotin complexes are captured by the solid phase monoclonal antibody directed against streptavidin. DELFIA Inducer dissociates the molecules into the solution where the europium fluorescence is measured. The measured fluorescence is inversely proportional to the biotinidase activity of the sample.

The Neonatal Biotinidase kit is intended for the semiquantitative determination of biotinidase activity in blood specimens dried on filter paper as an aid in screening newborns for biotinidase deficiency.

Biotinidase is found in the blood sample itself. Filter paper disks from newborn dried blood spot samples, calibrators and controls are punched into the wells of a microplate. When biotin substrate reagent containing biotin 6-aminoquinoline (6-AQ) is added to a well containing a punched dried blood spot, the reagent extracts and reconstitutes the proteins and enzymes in the spot. The biotinidase enzyme in the sample cleaves the substrate to biotin and fluorescent 6-AQ The addition of the ethanol stops the reaction and precipitates the proteins to cover the bottom of the well and the extracted spot. The fluorescent product (6-AQ) formed during the reaction is measured with a fluorometer. The biotinidase activity is defined against a calibration curve. The biotinidase activity of the sample is determined by comparing the fluorescence intensity of the sample to a calibration curve.

This method is for the semi-quantitative determination of biotinidase, EC 3.5.1.12, activity in dried whole blood spots using a spectrophotometer. Measurement of biotinidase activity is primarily for the diagnosis and treatment of biotinidase deficiency in newborns. This method is intended for in vitro diagnostic use in screening for decreased levels of biotinidase activity and not for monitoring purposes.

Biotinidase activity is determined by measuring the color that develops from p-Aminobenzoic Acid (PABA) after PABA is released from Biotinyl-p-Aminobenzoate (Biotin-PAB). Samples with biotinidase activity develop a purple color. Samples without biotinidase activity remain straw-colored. Patient samples of whole blood collected on standardized filter paper are eluted in a standard 96 well microplate. The plate is incubated with Biotin-PAB in a buffer at 37℃ for 240 minutes on a combination incubator/shaker. Following incubation, TCA is added to the sample mixture and the resulting precipitate is removed through filtration. The PABA in the filtrate is subsequently diazotized and coupled to a napthol derivative to form an azo dye by the successive addition of sodium nitrite, acidic ammonium sulfamate and finally, N-1-naphthylethylenediamine dihydrochloride (NED). The azo dye is measured colorimetrically at 550 nm on a commercial microplate absorbance reader with a reference measurement at 690 mm.