Human Alk-Smase (Alkaline Sphingomyelinase) CLIA Kit (HUES00225)

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ELISA Kit Technical ManualMSDS

Human Alk-Smase (Alkaline Sphingomyelinase) 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 Alk-Smase . 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 Alk-Smase 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 Alk-Smase, 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 Alk-Smase. The concentration of Human Alk-Smase 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 range78.13-5000 pg/mL
Sensitivity46.88 pg/mL
Sample volume100uL
Sample typeTissue homogenates
RepeatabilityCV < 15%


This kit recognizes Human Alk-Smase in samples. No significant cross-reactivity or interference between Human Alk-Smase 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 Alk-Smase were tested 20 times on one plate, respectively.

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

  Intra-assay Precision Inter-assay Precision
Mean (pg/mL)237.78485.982153.04259.83511.811965.23
Standard deviation20.9544.37168.1527.0545.86136.78
C V (%)8.819.137.8110.418.966.96


The recovery of Human Alk-Smase 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)93-107100
EDTA plasma (n=5)91-10297
Cell culture media (n=5)100-115106


Samples were spiked with high concentrations of Human Alk-Smase 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 (%)89-104104-11688-104
Average (%)9511095
1:4Range (%)99-11591-10787-99
Average (%)1089992
1:8Range (%)86-9995-10999-113
Average (%)91101107
1:16Range (%)86-10094-10791-106
Average (%)9110097

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 Alk-Smase (Alkaline Sphingomyelinase) CLIA Kit (HUES00225) 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 Alk-Smase (Alkaline Sphingomyelinase) CLIA Kit (HUES00225) Protein Information

UniProt Protein Function:ENPP7: Converts sphingomyelin to ceramide. Also has phospholipase C activity toward palmitoyl lyso-phosphocholine. Does not appear to have nucleotide pyrophosphatase activity. Belongs to the nucleotide pyrophosphatase/phosphodiesterase family.
UniProt Protein Details:

Protein type:Phosphodiesterase; Lipid Metabolism - sphingolipid; Cell cycle regulation; DNA replication; Motility/polarity/chemotaxis; Membrane protein, integral; EC

Chromosomal Location of Human Ortholog: 17q25.3

Cellular Component: Golgi apparatus; microvillus; membrane; integral to membrane; plasma membrane

Molecular Function:sphingomyelin phosphodiesterase activity

Biological Process: negative regulation of cell proliferation; sphingomyelin metabolic process; sphingolipid metabolic process; negative regulation of DNA replication; glycosphingolipid metabolic process; sphingomyelin catabolic process

UniProt Code:Q6UWV6
NCBI GenInfo Identifier:134047772
NCBI Gene ID:339221
NCBI Accession:Q6UWV6.3
UniProt Secondary Accession:Q6UWV6,Q6ZTS5, Q8IUS8,
UniProt Related Accession:Q6UWV6
Molecular Weight:458
NCBI Full Name:Ectonucleotide pyrophosphatase/phosphodiesterase family member 7
NCBI Synonym Full Names:ectonucleotide pyrophosphatase/phosphodiesterase 7
NCBI Official Symbol:ENPP7  
NCBI Official Synonym Symbols:NPP7; NPP-7; E-NPP 7; ALK-SMase  
NCBI Protein Information:ectonucleotide pyrophosphatase/phosphodiesterase family member 7; ENPP7; alkaline sphingomyelinase; intestinal alkaline sphingomyelinase; alkaline sphingomyelin phosphodiesterase
UniProt Protein Name:Ectonucleotide pyrophosphatase/phosphodiesterase family member 7
UniProt Synonym Protein Names:Alkaline sphingomyelin phosphodiesterase; Intestinal alkaline sphingomyelinase; Alk-SMase
Protein Family:Ectonucleotide pyrophosphatase/phosphodiesterase family
UniProt Gene Name:ENPP7  
UniProt Entry Name:ENPP7_HUMAN
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