Rat Signaling ELISA Kits 2
Rat GL beta (Galactosidase, Beta) CLIA Kit (RTES00219)
- SKU:
- RTES00219
- Product Type:
- ELISA Kit
- ELISA Type:
- CLIA Kit
- Size:
- 96 Assays
- Sensitivity:
- 18.75pg/mL
- Range:
- 31.25-2000pg/mL
- ELISA Type:
- Sandwich
- Reactivity:
- Rat
- Sample Type:
- Serum, plasma and other biological fluids
Description
| Assay type: | Sandwich |
| Format: | 96T |
| Assay time: | 4.5h |
| Reactivity: | Rat |
| Detection method: | Chemiluminescence |
| Detection range: | 31.25-2000 pg/mL |
| Sensitivity: | 18.75 pg/mL |
| Sample volume: | 100µL |
| Sample type: | Serum, plasma and other biological fluids |
| Repeatability: | CV < 15% |
| Specificity: | This kit recognizes Rat GL beta in samples. No significant cross-reactivity or interference between Rat GL beta and analogues was observed. |
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 Rat GL beta. 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 Rat GL beta 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 Rat GL beta, 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 Rat GL beta. The concentration of Rat GL beta in the samples can be calculated by comparing the RLU of the samples to the standard curve.
| UniProt Protein Function: | GLB1: Cleaves beta-linked terminal galactosyl residues from gangliosides, glycoproteins, and glycosaminoglycans. Defects in GLB1 are the cause of GM1-gangliosidosis type 1 (GM1G1); also known as infantile GM1- gangliosidosis. GM1-gangliosidosis is an autosomal recessive lysosomal storage disease marked by the accumulation of GM1 gangliosides, glycoproteins and keratan sulfate primarily in neurons of the central nervous system. GM1G1 is characterized by onset within the first three months of life, central nervous system degeneration, coarse facial features, hepatosplenomegaly, skeletal dysmorphology reminiscent of Hurler syndrome, and rapidly progressive psychomotor deterioration. Urinary oligosaccharide levels are high. It leads to death usually between the first and second year of life. Defects in GLB1 are the cause of GM1-gangliosidosis type 2 (GM1G2); also known as late infantile/juvenile GM1- gangliosidosis. GM1G2 is characterized by onset between ages 1 and 5. The main symptom is locomotor ataxia, ultimately leading to a state of decerebration with epileptic seizures. Patients do not display the skeletal changes associated with the infantile form, but they nonetheless excrete elevated amounts of beta-linked galactose-terminal oligosaccharides. Inheritance is autosomal recessive. Defects in GLB1 are the cause of GM1-gangliosidosis type 3 (GM1G3); also known as adult or chronic GM1- gangliosidosis. GM1G3 is characterized by a variable phenotype. Patients show mild skeletal abnormalities, dysarthria, gait disturbance, dystonia and visual impairment. Visceromegaly is absent. Intellectual deficit can initially be mild or absent but progresses over time. Inheritance is autosomal recessive. Defects in GLB1 are the cause of mucopolysaccharidosis type 4B (MPS4B); also known as Morquio syndrome B. MPS4B is a form of mucopolysaccharidosis type 4, an autosomal recessive lysosomal storage disease characterized by intracellular accumulation of keratan sulfate and chondroitin-6-sulfate. Key clinical features include short stature, skeletal dysplasia, dental anomalies, and corneal clouding. Intelligence is normal and there is no direct central nervous system involvement, although the skeletal changes may result in neurologic complications. There is variable severity, but patients with the severe phenotype usually do not survive past the second or third decade of life. Belongs to the glycosyl hydrolase 35 family. 3 isoforms of the human protein are produced by alternative splicing. |
| UniProt Protein Details: | Protein type:Hydrolase; EC 3. 2. 1. 23; Glycan Metabolism - glycosphingolipid biosynthesis - ganglio series; Glycan Metabolism - other glycan degradation; Carbohydrate Metabolism - galactose; Glycan Metabolism - glycosaminoglycan degradation; Lipid Metabolism - sphingolipid Cellular Component: Golgi apparatus; lysosome; cytoplasm Molecular Function:galactoside binding; beta-galactosidase activity Biological Process: galactose catabolic process; cellular carbohydrate metabolic process |
| NCBI Summary: | mutation of the human homolog causes GM1 gangliosidosis and Morquio B disease [RGD, Feb 2006] |
| UniProt Code: | D3ZUM4 |
| NCBI GenInfo Identifier: | 157824103 |
| NCBI Gene ID: | 316033 |
| NCBI Accession: | NP_001101662. 1 |
| Molecular Weight: | 27. 3kDa |
| NCBI Full Name: | beta-galactosidase |
| NCBI Synonym Full Names: | galactosidase, beta 1 |
| NCBI Official Symbol: | Glb1 |
| NCBI Protein Information: | beta-galactosidase |
| UniProt Protein Name: | Beta-galactosidase |
| Protein Family: | Nitrogen regulatory protein |
| UniProt Gene Name: | Glb1 |
| UniProt Entry Name: | D3ZUM4_RAT |
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 (pg/mL) | RLU | Average | Corrected |
| 2000 | 54246 57196 | 55721 | 55693 |
| 1000 | 22507 25441 | 23974 | 23946 |
| 500 | 11410 10654 | 11032 | 11004 |
| 250 | 5078 5510 | 5294 | 5266 |
| 125 | 2620 2596 | 2608 | 2580 |
| 62.5 | 1360 1262 | 1311 | 1283 |
| 31.25 | 649 699 | 674 | 646 |
| 0 | 28 28 | 28 | -- |
Precision
Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Rat GL beta were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Rat GL beta were 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 (pg/mL) | 93.84 | 254.47 | 811.24 | 89.37 | 256.68 | 789.77 |
| Standard deviation | 10.05 | 29.93 | 70.90 | 11.15 | 27.23 | 89.17 |
| C V (%) | 10.71 | 11.76 | 8.74 | 12.48 | 10.61 | 11.29 |
Recovery
The recovery of Rat GL beta 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) | 92-109 | 99 |
| EDTA plasma (n=5) | 88-99 | 93 |
| Cell culture media (n=5) | 92-108 | 100 |
Linearity
Samples were spiked with high concentrations of Rat GL beta 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) | ||
| 1:2 | Range (%) | 101-117 | 86-98 | 90-101 |
| Average (%) | 107 | 92 | 96 | |
| 1:4 | Range (%) | 95-111 | 96-109 | 84-96 |
| Average (%) | 102 | 101 | 90 | |
| 1:8 | Range (%) | 89-103 | 99-116 | 95-108 |
| Average (%) | 95 | 106 | 101 | |
| 1:16 | Range (%) | 99-112 | 97-112 | 99-111 |
| Average (%) | 105 | 104 | 105 |
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 ×12 strips | -20°C, 6 months |
| Reference Standard | 2 vials | |
| Concentrated Biotinylated Detection Ab (100×) | 1 vial, 120 µL | |
| Concentrated HRP Conjugate (100×) | 1 vial, 120 µL | -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 (25×) | 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 |
- 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.
- Aliquot 100µl of standard solutions into the standard wells.
- Add 100µl of Sample / Standard dilution buffer into the control (zero) well.
- Add 100µl of properly diluted sample (serum, plasma, tissue homogenates and otherbiological fluids. ) into test sample wells.
- Cover the plate with the sealer provided in the kit and incubate for 90 min at 37°C.
- 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.
- 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.
- Add 100µL of HRP Conjugate working solution to each well. Cover with a plate seal andincubate for 30 min at 37°C.
- Aspirate or decant the solution from each well. Repeat the wash process for five times asconducted in step 7.
- 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.
- Determine the RLU value of each well immediately.
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