Assay type:	Sandwich
Format:	96T
Assay time:	4.5h
Reactivity:	Mouse
Detection Method:	Colormetric
Detection Range:	0.16-10 mIU/mL
Sensitivity:	0.10 mIU/mL
Sample Volume Required Per Well:	100µL
Sample Type:	Serum, plasma and other biological fluids
Specificity:	This kit recognizes Mouse GLb in samples. No significant cross-reactivity or interference between Mouse GLb and analogues was observed.

This ELISA kit uses Sandwich-ELISA as the method. The micro ELISA plate provided in this kit has been pre-coated with an antibody specific to Mouse GLb. Standards or samples are added to the appropriate micro ELISA plate wells and combined with the specific antibody. Then a biotinylated detection antibody specific for Mouse GLb 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 Mouse GLb, biotinylated detection antibody and Avidin-HRP conjugate will appear blue in color. The enzyme-substrate reaction is terminated by adding Stop Solution and the color turns yellow. The optical density (OD) is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The OD value is proportional to the concentration of Mouse GLb. The concentration of Mouse GLb in samples can be calculated by comparing the OD 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:Carbohydrate Metabolism - galactose; EC 3. 2. 1. 23; Glycan Metabolism - glycosaminoglycan degradation; Glycan Metabolism - glycosphingolipid biosynthesis - ganglio series; Glycan Metabolism - other glycan degradation; Hydrolase; Lipid Metabolism - sphingolipid Chromosomal Location of Human Ortholog: 9 F3\|9 64. 4 cM Cellular Component: cytoplasm; extracellular exosome; extracellular space; Golgi apparatus; intracellular membrane-bound organelle; lysosome; vacuole Molecular Function:beta-galactosidase activity; galactoside binding; hydrolase activity; hydrolase activity, acting on glycosyl bonds; hydrolase activity, hydrolyzing O-glycosyl compounds Biological Process: carbohydrate metabolic process; cellular carbohydrate metabolic process; galactose catabolic process; metabolic process
NCBI Summary:	This gene encodes a preproprotein that is proteolytically cleaved to yield a signal peptide and a proproptein that is subsequently processed to generate the active mature peptide. The encoded protein is a lysosomal enzyme that catalyzes the hydrolysis of terminal beta-D-galactose residues in various substrates like lactose, ganglioside GM1 and other glycoproteins. Mutations in the human gene are associated with GM1-gangliosidosis and Morquio B syndrome. Disruption of the mouse gene mirrors the symptoms of human gangliosidosis. Alternative splicing results in multiple transcript variants. [provided by RefSeq, Dec 2013]
UniProt Code:	P23780
NCBI GenInfo Identifier:	6753190
NCBI Gene ID:	12091
NCBI Accession:	NP_033882. 1
UniProt Related Accession:	P23780
Molecular Weight:
NCBI Full Name:	beta-galactosidase preproprotein
NCBI Synonym Full Names:	galactosidase, beta 1
NCBI Official Symbol:	Glb1
NCBI Official Synonym Symbols:	Bge; Bgl; Bgs; Bgt; Bgl-e; Bgl-s; Bgl-t; AW125515; C130097A14Rik
NCBI Protein Information:	beta-galactosidase
UniProt Protein Name:	Beta-galactosidase
UniProt Synonym Protein Names:	Acid beta-galactosidase; Lactase
Protein Family:	Nitrogen regulatory protein
UniProt Gene Name:	Glb1

As the OD 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 (mIU/mL)	O.D	Average	Corrected
10	2.334 2.352	2.343	2.276
5	1.689 1.693	1.691	1.624
2.5	0.93 0.9	0.915	0.848
1.25	0.459 0.485	0.472	0.405
0.63	0.271 0.241	0.256	0.189
0.32	0.183 0.159	0.171	0.104
0.16	0.115 0.125	0.12	0.053
0	0.061 0.073	0.067	--

Precision

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

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Mouse GLb 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 (mIU/mL)	0.51	1.49	3.63	0.55	1.47	3.79
Standard deviation	0.03	0.07	0.13	0.04	0.06	0.13
C V (%)	5.88	4.70	3.58	7.27	4.08	3.43

Recovery

The recovery of Mouse GLb 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)	86-96	91
EDTA plasma (n=5)	94-109	101
Cell culture media (n=5)	91-104	98

Linearity

Samples were spiked with high concentrations of Mouse GLb 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 (%)	93-105	92-106	88-101
1:2	Average (%)	98	99	95
1:4	Range (%)	92-103	80-91	87-99
1:4	Average (%)	97	86	92
1:8	Range (%)	89-102	82-95	86-99
1:8	Average (%)	96	88	92
1:16	Range (%)	93-108	83-99	87-102
1:16	Average (%)	99	90	93

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 ELISA 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	1 vial, 10 mL	4°C(shading light)
Stop Solution	1 vial, 10 mL	4°C
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 90µL of Substrate Reagent to each well. Cover with a new plate seal and incubate forapproximately 15 min at 37°C. Protect the plate from light. Note: the reaction time can beshortened or extended according to the actual color change, but not by more than 30min.
Add 50 µL of Stop Solution to each well. Note: Adding the stop solution should be done inthe same order as the substrate solution.
Determine the optical density (OD value) of each well immediately with a microplate readerset at 450 nm.