Human PAH (Phenylalanine Hydroxylase) CLIA Kit (HUES00845)

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ELISA Kit Technical ManualMSDS

Human PAH (Phenylalanine Hydroxylase) CLIA Kit

This kit uses Sandwich-CLIA as the method. The micro CLIA plate provided in this kit has been pre-coated with an antibody specific to Human PAH . Standards or samples are added to the appropriate micro CLIA plate wells and combined with the specific antibody. Then a biotinylated detection antibody specific for Human PAH and Avidin-Horseradish Peroxidase (HRP) conjugate are added to each micro plate well successively and incubated. Free components are washed away. The substrate solution is added to each well. Only those wells that contain Human PAH, biotinylated detection antibody and Avidin-HRP conjugate will appear fluorescence. The Relative light unit (RLU) value is measured spectrophotometrically by the Chemiluminescence immunoassay analyzer. The RLU value is positively associated with the concentration of Human PAH. The concentration of Human PAH in the samples can be calculated by comparing the RLU of the samples to the standard curve.

Assay typeSandwich
Assay time4.5h
Detection methodChemiluminescence
Detection range15.63-1000 pg/mL
Sensitivity9.38 pg/mL
Sample volume100uL
Sample typeSerum, plasma and other biological fluids
RepeatabilityCV < 15%


This kit recognizes Human PAH in samples. No significant cross-reactivity or interference between Human PAH and analogues was observed.

Typical Data

As the RLU values of the standard curve may vary according to the conditions of the actual assay performance (e.g. operator, pipetting technique, washing technique or temperature effects), the operator should establish a standard curve for each test. Typical standard curve and data is provided below for reference only.

RLU Average Corrected


Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human PAH were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human PAH were tested on 3 different plates, 20 replicates in each plate.

  Intra-assay Precision Inter-assay Precision
Mean (pg/mL)49.95150.40492.6653.50160.45534.63
Standard deviation4.9013.5137.004.4212.8448.54
C V (%)9.818.987.518.268.009.08


The recovery of Human PAH spiked at three different levels in samples throughout the range of the assay was evaluated in various matrices.

Sample Type Range (%) Average Recovery (%)
Serum (n=5)100-115106
EDTA plasma (n=5)88-10394
Cell culture media (n=5)93-10599


Samples were spiked with high concentrations of Human PAH and diluted with Reference Standard & Sample Diluent to produce samples with values within the range of the assay.

    Serum (n=5) EDTA plasma (n=5) Cell culture media (n=5)
1:2Range (%)90-104102-11593-108
Average (%)96108101
1:4Range (%)100-11394-10995-108
Average (%)106100102
1:8Range (%)87-10298-113100-116
Average (%)93106108
1:16Range (%)91-10294-10897-112
Average (%)96101104

Kit Components & Storage

An unopened kit can be stored at 4'C for 1 month. If the kit is not used within 1 month, store the items separately according to the following conditions once the kit is received.

Item Specifications Storage
Micro CLIA Plate(Dismountable)8 wells X12 strips-20'C, 6 months
Reference Standard2 vials
Concentrated Biotinylated Detection Ab (100X)1 vial, 120 uL
Concentrated HRP Conjugate (100X)1 vial, 120 uL-20'C(shading light), 6 months
Reference Standard & Sample Diluent1 vial, 20 mL4'C, 6 months
Biotinylated Detection Ab Diluent1 vial, 14 mL
HRP Conjugate Diluent1 vial, 14 mL
Concentrated Wash Buffer (25X)1 vial, 30 mL
Substrate Reagent A1 vial, 5 mL4'C (shading light)
Substrate Reagent B1 vial, 5 mL4'C (shading light)
Plate Sealer5 pieces
Product Description1 copy
Certificate of Analysis1 copy

Human PAH (Phenylalanine Hydroxylase) CLIA Kit (HUES00845) Assay procedure

    1. Set standard, test sample and control (zero) wells on the pre-coated plate and record theirpositions. It is recommended to measure each standard and sample in duplicate. Note: addall solutions to the bottom of the plate wells while avoiding contact with the well walls. Ensuresolutions do not foam when adding to the wells.
    2. Aliquot 100µl of standard solutions into the standard wells.
    3. Add 100µl of Sample / Standard dilution buffer into the control (zero) well.
    4. Add 100µl of properly diluted sample (serum, plasma, tissue homogenates and otherbiological fluids.) into test sample wells.
    5. Cover the plate with the sealer provided in the kit and incubate for 90 min at 37°C.
    6. Aspirate the liquid from each well, do not wash. Immediately add 100µL of BiotinylatedDetection Ab working solution to each well. Cover the plate with a plate seal and gently mix.Incubate for 1 hour at 37°C.
    7. Aspirate or decant the solution from the plate and add 350µL of wash buffer to each welland incubate for 1-2 minutes at room temperature. Aspirate the solution from each well andclap the plate on absorbent filter paper to dry. Repeat this process 3 times. Note: a microplatewasher can be used in this step and other wash steps.
    8. Add 100µL of HRP Conjugate working solution to each well. Cover with a plate seal andincubate for 30 min at 37°C.
    9. Aspirate or decant the solution from each well. Repeat the wash process for five times asconducted in step 7.
    10. Add 100µL of Substrate mixture solution to each well. Cover with a new plate seal andincubate for no more than 5 min at 37°C. Protect the plate from light.
    11. Determine the RLU value of each well immediately.

Human PAH (Phenylalanine Hydroxylase) CLIA Kit (HUES00845) Protein Information

UniProt Protein Function:PAH: phenylalanine hydroxylase that is the rate-limiting step in phenylalanine catabolism. Belongs to the biopterin-dependent aromatic amino acid hydroxylase family. Deficiency of this enzyme activity results in the autosomal recessive disorder phenylketonuria.
UniProt Protein Details:

Protein type:EC; Amino Acid Metabolism - phenylalanine, tyrosine and tryptophan biosynthesis; Oxidoreductase

Chromosomal Location of Human Ortholog: 12q22-q24.2

Cellular Component: cytosol

Molecular Function:amino acid binding; iron ion binding; phenylalanine 4-monooxygenase activity

Biological Process: L-phenylalanine catabolic process; catecholamine biosynthetic process; amino acid biosynthetic process; neurotransmitter biosynthetic process

Disease: Phenylketonuria

NCBI Summary:PAH encodes the enzyme phenylalanine hydroxylase that is the rate-limiting step in phenylalanine catabolism. Deficiency of this enzyme activity results in the autosomal recessive disorder phenylketonuria. [provided by RefSeq, Jul 2008]
UniProt Code:P00439
NCBI GenInfo Identifier:129973
NCBI Gene ID:5053
NCBI Accession:P00439.1
UniProt Secondary Accession:P00439,Q16717, Q8TC14,
UniProt Related Accession:P00439
Molecular Weight:452
NCBI Full Name:Phenylalanine-4-hydroxylase
NCBI Synonym Full Names:phenylalanine hydroxylase
NCBI Official Symbol:PAH  
NCBI Official Synonym Symbols:PH; PKU; PKU1  
NCBI Protein Information:phenylalanine-4-hydroxylase; phe-4-monooxygenase; phenylalanine 4-monooxygenase
UniProt Protein Name:Phenylalanine-4-hydroxylase
UniProt Synonym Protein Names:Phe-4-monooxygenase
UniProt Gene Name:PAH  
UniProt Entry Name:PH4H_HUMAN
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