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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 NAGase . 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 NAGase 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 NAGase, 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 NAGase. The concentration of Human NAGase in the samples can be calculated by comparing the RLU of the samples to the standard curve.
| Assay type | Sandwich |
| Format | 96T |
| Assay time | 4.5h |
| Reactivity | Human |
| Detection method | Chemiluminescence |
| Detection range | 0.31-20 ng/mL |
| Sensitivity | 0.19 ng/mL |
| Sample volume | 100uL |
| Sample type | Serum, plasma and other biological fluids |
| Repeatability | CV < 15% |
This kit recognizes Human NAGasein samples. No significant cross-reactivity or interference between Human NAGaseand analogues was observed.
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.
| Concentration(ng/mL) | RLU | Average | Corrected |
|---|---|---|---|
| 20 | 3144336521 | 33982 | 33950 |
| 10 | 1133113609 | 12470 | 12438 |
| 5 | 56974803 | 5250 | 5218 |
| 2.5 | 23132735 | 2524 | 2492 |
| 1.25 | 13961370 | 1383 | 1351 |
| 0.63 | 922812 | 867 | 835 |
| 0.31 | 589657 | 623 | 591 |
| 0 | 3133 | 32 | -- |
Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human NAGasewere tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human NAGasewere tested on 3 different plates, 20 replicates in each plate.
| Intra-assay Precision | Inter-assay Precision | |||||
|---|---|---|---|---|---|---|
| Sample | 1 | 2 | 3 | 1 | 2 | 3 |
| n | 20 | 20 | 20 | 20 | 20 | 20 |
| Mean (ng/mL) | 1.02 | 2.28 | 6.93 | 1.07 | 2.18 | 6.30 |
| Standard deviation | 0.09 | 0.18 | 0.61 | 0.13 | 0.19 | 0.42 |
| C V (%) | 8.82 | 7.89 | 8.80 | 12.15 | 8.72 | 6.67 |
The recovery of Human NAGasespiked 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) | 98-116 | 106 |
| EDTA plasma (n=5) | 96-112 | 104 |
| Cell culture media (n=5) | 87-100 | 92 |
Samples were spiked with high concentrations of Human NAGaseand 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) | ||
|---|---|---|---|---|
| 1:2 | Range (%) | 102-115 | 95-109 | 97-113 |
| Average (%) | 107 | 101 | 103 | |
| 1:4 | Range (%) | 103-118 | 95-109 | 85-97 |
| Average (%) | 109 | 101 | 90 | |
| 1:8 | Range (%) | 86-99 | 85-98 | 96-111 |
| Average (%) | 93 | 91 | 102 | |
| 1:16 | Range (%) | 89-103 | 89-107 | 85-98 |
| Average (%) | 95 | 97 | 91 |
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 Standard | 2 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 Diluent | 1 vial, 20 mL | 4"C, 6 months |
| Biotinylated Detection Ab Diluent | 1 vial, 14 mL | |
| HRP Conjugate Diluent | 1 vial, 14 mL | |
| Concentrated Wash Buffer (25X) | 1 vial, 30 mL | |
| Substrate Reagent A | 1 vial, 5 mL | 4"C (shading light) |
| Substrate Reagent B | 1 vial, 5 mL | 4"C (shading light) |
| Plate Sealer | 5 pieces | |
| Product Description | 1 copy | |
| Certificate of Analysis | 1 copy |
| UniProt Protein Function: | OGA: Cleaves GlcNAc but not GalNAc from glycopeptides. Can use p-nitrophenyl-beta-GlcNAc as substrate but not p-nitrophenyl- beta-GalNAc or p-nitrophenyl-alpha-GlcNAc. Possesses hyaluronidase activity. Acetylates "Lys-8" of histone H4 and "Lys-14" of histone H3. 3 isoforms of the human protein are produced by alternative splicing. |
| UniProt Protein Details: | Protein type:Acetyltransferase; Hydrolase; EC 3.2.1.169; EC 3.2.1.52 Chromosomal Location of Human Ortholog: 10q24.1-q24.3 Cellular Component: membrane; cytosol; nucleus Molecular Function:beta-N-acetylglucosaminidase activity; histone acetyltransferase activity; hyalurononglucosaminidase activity Biological Process: positive regulation of DNA metabolic process; positive regulation of protein complex disassembly; dATP metabolic process; positive regulation of proteolysis; protein deglycosylation; N-acetylglucosamine metabolic process; positive regulation of insulin secretion; positive regulation of growth hormone secretion; positive regulation of glucose import; negative regulation of protein amino acid glycosylation; positive regulation of mitochondrial depolarization; response to steroid hormone stimulus; positive regulation of cell killing; glycoprotein catabolic process; histone acetylation; protein targeting to membrane; aging |
| NCBI Summary: | The dynamic modification of cytoplasmic and nuclear proteins by O-linked N-acetylglucosamine (O-GlcNAc) addition and removal on serine and threonine residues is catalyzed by OGT (MIM 300255), which adds O-GlcNAc, and MGEA5, a glycosidase that removes O-GlcNAc modifications (Gao et al., 2001 [PubMed 11148210]).[supplied by OMIM, Mar 2008] |
| UniProt Code: | O60502 |
| NCBI GenInfo Identifier: | 215490056 |
| NCBI Gene ID: | 10724 |
| NCBI Accession: | NP_001135906.1 |
| UniProt Secondary Accession: | O60502,O75166, Q86WV0, Q8IV98, Q9BVA5, Q9HAR0, B7WPB9 D3DR79, E9PGF9, |
| UniProt Related Accession: | O60502 |
| Molecular Weight: | 96,932 Da |
| NCBI Full Name: | bifunctional protein NCOAT isoform b |
| NCBI Synonym Full Names: | meningioma expressed antigen 5 (hyaluronidase) |
| NCBI Official Symbol: | MGEA5 |
| NCBI Official Synonym Symbols: | OGA; MEA5; NCOAT |
| NCBI Protein Information: | bifunctional protein NCOAT; O-GlcNAcase; hyaluronidase in meningioma; meningioma-expressed antigen 5; nuclear cytoplasmic O-GlcNAcase and acetyltransferase |
| UniProt Protein Name: | Bifunctional protein NCOAT |
| UniProt Synonym Protein Names: | Meningioma-expressed antigen 5; Nuclear cytoplasmic O-GlcNAcase and acetyltransferaseIncluding the following 2 domains:Protein O-GlcNAcase (EC:3.2.1.169)Alternative name(s):Glycoside hydrolase O-GlcNAcase; Hexosaminidase C; N-acetyl-beta-D-glucosaminidase; N-acetyl-beta-glucosaminidase; O-GlcNAcase; OGA |
| Protein Family: | Protein O-GlcNAcase |
| UniProt Gene Name: | MGEA5 |
| UniProt Entry Name: | NCOAT_HUMAN |
Reactivity: | Human |
ELISA Type: | CLIA |
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