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Biotinidase

Continuous Flow |   Microplate Kit

Biotinidase activity is determined colorimetrically by measuring the amount of p-Aminobenzoic Acid (PABA) released from Biotinyl-p-Aminobenzoate (Biotin-PAB).  Samples with biotinidase activity develop a purple color.  Samples without biotinidase activity remain straw-colored.

Astoria-Pacific also supplies Dried Blood Spot Controls, Unassayed (Normal and Deficient) for Biotinidase and GALT testing.  Use with your manual method or with SPOTCHECK  instrumentation.

Biotinidase Continuous Flow System

Positive Attributes of Instrumentation and Method:

  • Semi-quantitative results
  • No operator attendance is required after loading sample plate
  • Modular system for easy maintenance
  • Large capacity autosampler
  • Same day turnaround of results
  • Totally automatic – no transfer of sample or reagents from one microtiter plate to another.
  • Sample throughput is approximately 90 samples per hour after an initial dwell time of approximately 100 minutes
  • Can be run simultaneously with GALT assay
  • Prepared reagents are stable for at least 14 days

Reagent Prep:
The SPOTCHECK Biotinidase 50 hour Reagent Kit contains all necessary reagents required for analysis and will provide approximately 50 hours of analyzer run time.  The approximate number of actual samples analyzed per kit is conservatively 2500.  All reagents are pre-weighed, color coded, and have a 2 year shelf life (with refrigeration).
Sample Prep:

  1. Punch one 3/16” spot or two 1/8″ spots into microtiter filter plate.
  2. Add water for sample elution and shake for 30 minutes.
  3. Filter samples into round bottom microtiter plate.
  4. Place the sample plate on the auto-sampler.

Automated Steps:

  1. Sampler delivers calibrants, controls and samples to the cartridges
  2. Online incubation for 90 minutes
  3. Online dialysis separates the released PABA from other proteins in the sample.
  4. Online addition of 3 color reagents produces an azo dye.
  5. Colorimetric measurement at 550nm

Biotinidase Microplate Kit

Positive Attributes of Instrumentation and Method:

  • Semi-quantitative results
  • Numeric result decreases the number of repeats and/or second screens
  • No subjective visual inspection like the Wolf Method
  • Only 4 hour incubation helps provide same day results
  • Minimal training required and easy to follow procedures
  • Minimal maintenance and troubleshooting
  • Prepared reagents are stable for at least 14 days

Reagent Prep:
The SPOTCHECK Biotinidase Microplate Reagent Kit contains all the necessary reagents required for analysis of approximately 20 96-well microtiter plates.  The approximate number of samples analyzed per kit is conservatively 1760.  All reagents are pre-weighed and color coded.  The shelf life for the kit is 2 years (with refrigeration.)  Filtering of reagents is not required.

Sample Prep:

  1. Punch two 1/8″ spots into microtiter filter plate
  2. Add substrate and incubate/shake for 4 hours.

Operating Procedure:

  1. Add Reagent
  2. Filter samples into microtiter plate
  3. Add 3 color reagents
  4. Read the plate with any commercially available plate reader at wavelengths 550nm and 690nm.  The intensity of the color produced is directly proportional to the amount of biotinidase activity.

Instrumentation:
The flexibility of Astoria-Pacific’s kit allows laboratories to use their own colorimetric plate reader and incubator/shaker or rely on our expertise to recommend and/or supply a complete package.

There are three levels of automation:

  1. Manual; Using an 8 or 12 channel pipette
  2. Semi-automation; A benchtop liquid handling station for pipetting
  3. A fully automated robotic system which does all of the pipetting, incubating, shaking, transferring, filtering, and reading independently.