Assay type:	Sandwich
Format:	96T
Assay time:	4.5h
Reactivity:	Human
Detection Method:	Colormetric
Detection Range:	0.31-20 ng/mL
Sensitivity:	0.19 ng/mL
Sample Volume Required Per Well:	100µL
Sample Type:	Serum, plasma and other biological fluids
Specificity:	This kit recognizes Human G6PD in samples. No significant cross-reactivity or interference between Human G6PD 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 Human G6PD. 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 Human G6PD 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 G6PD, 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 Human G6PD. The concentration of Human G6PD in samples can be calculated by comparing the OD of the samples to the standard curve.

UniProt Protein Function:	G6PD: glucose-6-phosphate dehydrogenase. A cytosolic enzyme encoded by a housekeeping X-linked gene whose main function is to produce NADPH, a key electron donor in the defense against oxidizing agents and in reductive biosynthetic reactions. G6PD is remarkable for its genetic diversity. Many variants of G6PD, mostly produced from missense mutations, have been described with wide ranging levels of enzyme activity and associated clinical symptoms. G6PD deficiency may cause neonatal jaundice, acute hemolysis, or severe chronic non-spherocytic hemolytic anemia. Two splice variant isoforms have been described. The long isoform is found in lymphoblasts, granulocytes and sperm.
UniProt Protein Details:	Protein type:EC 1. 1. 1. 49; Carbohydrate Metabolism - pentose phosphate pathway; Cell development/differentiation; Other Amino Acids Metabolism - glutathione; Oxidoreductase Chromosomal Location of Human Ortholog: Xq28 Cellular Component: centrosome; cytoplasm; cytosol; internal side of plasma membrane; intracellular membrane-bound organelle; membrane; microtubule organizing center Molecular Function:glucose binding; glucose-6-phosphate dehydrogenase activity; identical protein binding; NADP binding; protein binding; protein homodimerization activity Biological Process: cholesterol biosynthetic process; erythrocyte maturation; glucose 6-phosphate metabolic process; glutathione metabolic process; lipid metabolic process; NADP metabolic process; NADPH regeneration; pentose biosynthetic process; pentose-phosphate shunt; pentose-phosphate shunt, oxidative branch; ribose phosphate biosynthetic process; substantia nigra development Disease: Anemia, Nonspherocytic Hemolytic, Due To G6pd Deficiency; Favism, Susceptibility To; Malaria, Susceptibility To
NCBI Summary:	This gene encodes glucose-6-phosphate dehydrogenase. This protein is a cytosolic enzyme encoded by a housekeeping X-linked gene whose main function is to produce NADPH, a key electron donor in the defense against oxidizing agents and in reductive biosynthetic reactions. G6PD is remarkable for its genetic diversity. Many variants of G6PD, mostly produced from missense mutations, have been described with wide ranging levels of enzyme activity and associated clinical symptoms. G6PD deficiency may cause neonatal jaundice, acute hemolysis, or severe chronic non-spherocytic hemolytic anemia. Two transcript variants encoding different isoforms have been found for this gene. [provided by RefSeq, Jul 2008]
UniProt Code:	P11413
NCBI GenInfo Identifier:	116242483
NCBI Gene ID:	2539
NCBI Accession:	P11413. 4
UniProt Secondary Accession:	P11413,Q16000, Q16765, Q8IU70, Q8IU88, Q8IUA6, Q96PQ2 D3DWX9,
UniProt Related Accession:	P11413
Molecular Weight:	62,468 Da
NCBI Full Name:	Glucose-6-phosphate 1-dehydrogenase
NCBI Synonym Full Names:	glucose-6-phosphate dehydrogenase
NCBI Official Symbol:	G6PD
NCBI Official Synonym Symbols:	G6PD1
NCBI Protein Information:	glucose-6-phosphate 1-dehydrogenase
UniProt Protein Name:	Glucose-6-phosphate 1-dehydrogenase
UniProt Gene Name:	G6PD
UniProt Entry Name:	G6PD_HUMAN

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 (ng/mL)	O.D	Average	Corrected
20	2.363 2.365	2.364	2.294
10	1.692 1.7	1.696	1.626
5	0.893 0.871	0.882	0.812
2.5	0.463 0.487	0.475	0.405
1.25	0.276 0.262	0.269	0.199
0.63	0.18 0.16	0.17	0.1
0.31	0.114 0.128	0.121	0.051
0	0.067 0.073	0.07	--

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human G6PD were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human G6PD 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 (ng/mL)	1.00	2.07	7.95	0.94	2.18	7.85
Standard deviation	0.05	0.11	0.36	0.05	0.13	0.35
C V (%)	5.00	5.31	4.53	5.32	5.96	4.46

Recovery

The recovery of Human G6PD 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)	92-107	100
EDTA plasma (n=5)	85-99	92
Cell culture media (n=5)	86-99	91

Linearity

Samples were spiked with high concentrations of Human G6PD 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 (%)	84-96	89-102	90-104
1:2	Average (%)	91	96	97
1:4	Range (%)	89-103	85-100	88-102
1:4	Average (%)	94	92	94
1:8	Range (%)	89-103	83-98	83-93
1:8	Average (%)	94	90	88
1:16	Range (%)	89-103	85-98	88-103
1:16	Average (%)	97	90	94

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.