Assay type:	Sandwich
Format:	96T
Assay time:	4.5h
Reactivity:	Human
Detection Method:	Colormetric
Detection Range:	0.78-50 ng/mL
Sensitivity:	0.47 ng/mL
Sample Volume Required Per Well:	100µL
Sample Type:	Serum, plasma and other biological fluids
Specificity:	This kit recognizes Human NE/ELA2 in samples. No significant cross-reactivity or interference between Human NE/ELA2 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 NE/ELA2. 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 NE/ELA2 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 NE/ELA2, 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 NE/ELA2. The concentration of Human NE/ELA2 in samples can be calculated by comparing the OD of the samples to the standard curve.

UniProt Protein Function:	ELANE: Modifies the functions of natural killer cells, monocytes and granulocytes. Inhibits C5a-dependent neutrophil enzyme release and chemotaxis. Defects in ELANE are a cause of cyclic haematopoiesis (CH); also known as cyclic neutropenia. CH is an autosomal dominant disease in which blood-cell production from the bone marrow oscillates with 21-day periodicity. Circulating neutrophils vary between almost normal numbers and zero. During intervals of neutropenia, affected individuals are at risk for opportunistic infection. Monocytes, platelets, lymphocytes and reticulocytes also cycle with the same frequency. Defects in ELANE are the cause of neutropenia severe congenital autosomal dominant type 1 (SCN1). SCN1 is a disorder of hematopoiesis characterized by a maturation arrest of granulopoiesis at the level of promyelocytes with peripheral blood absolute neutrophil counts below 0. 5 x 10(9)/l and early onset of severe bacterial infections. Belongs to the peptidase S1 family. Elastase subfamily.
UniProt Protein Details:	Protein type:Motility/polarity/chemotaxis; Cell cycle regulation; Cell surface; Protease; EC 3. 4. 21. 37 Chromosomal Location of Human Ortholog: 19p13. 3 Cellular Component: cell surface; transcriptional repressor complex; cytoplasm; extracellular region; secretory granule Molecular Function:heparin binding; peptidase activity; protein binding; protease binding; cytokine binding; serine-type endopeptidase activity; endopeptidase activity Biological Process: extracellular matrix organization and biogenesis; positive regulation of immune response; positive regulation of smooth muscle cell proliferation; negative regulation of interleukin-8 biosynthetic process; positive regulation of interleukin-8 biosynthetic process; response to lipopolysaccharide; negative regulation of chemotaxis; negative regulation of transcription from RNA polymerase II promoter; proteolysis; phagocytosis; cellular calcium ion homeostasis; positive regulation of MAP kinase activity; extracellular matrix disassembly; collagen catabolic process; negative regulation of inflammatory response; defense response to bacterium; protein catabolic process; response to yeast; leukocyte migration; acute inflammatory response to antigenic stimulus; response to UV; negative regulation of chemokine biosynthetic process Disease: Cyclic Neutropenia; Neutropenia, Severe Congenital, 1, Autosomal Dominant
NCBI Summary:	Elastases form a subfamily of serine proteases that hydrolyze many proteins in addition to elastin. Humans have six elastase genes which encode the structurally similar proteins. The product of this gene hydrolyzes proteins within specialized neutrophil lysosomes, called azurophil granules, as well as proteins of the extracellular matrix following the protein's release from activated neutrophils. The enzyme may play a role in degenerative and inflammatory diseases by its proteolysis of collagen-IV and elastin of the extracellular matrix. This protein degrades the outer membrane protein A (OmpA) of E. coli as well as the virulence factors of such bacteria as Shigella, Salmonella and Yersinia. Mutations in this gene are associated with cyclic neutropenia and severe congenital neutropenia (SCN). This gene is clustered with other serine protease gene family members, azurocidin 1 and proteinase 3 genes, at chromosome 19pter. All 3 genes are expressed coordinately and their protein products are packaged together into azurophil granules during neutrophil differentiation. [provided by RefSeq, May 2009]
UniProt Code:	P08246
NCBI GenInfo Identifier:	119292
NCBI Gene ID:	1991
NCBI Accession:	P08246. 1
UniProt Secondary Accession:	P08246,P09649, Q6B0D9, Q6LDP5,
UniProt Related Accession:	P08246
Molecular Weight:	28,518 Da
NCBI Full Name:	Neutrophil elastase
NCBI Synonym Full Names:	elastase, neutrophil expressed
NCBI Official Symbol:	ELANE
NCBI Official Synonym Symbols:	GE; NE; HLE; HNE; ELA2; SCN1; PMN-E
NCBI Protein Information:	neutrophil elastase; elastase-2; medullasin; PMN elastase; leukocyte elastase; elastase 2, neutrophil; human leukocyte elastase; polymorphonuclear elastase; bone marrow serine protease; granulocyte-derived elastase
UniProt Protein Name:	Neutrophil elastase
UniProt Synonym Protein Names:	Bone marrow serine protease; Elastase-2; Human leukocyte elastase; HLE; Medullasin; PMN elastase
Protein Family:	Elastase
UniProt Gene Name:	ELANE
UniProt Entry Name:	ELNE_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
50	2.471 2.509	2.49	2.432
25	1.596 1.65	1.623	1.565
12.5	0.941 0.907	0.924	0.866
6.25	0.464 0.494	0.479	0.421
3.13	0.231 0.205	0.218	0.16
1.57	0.164 0.152	0.158	0.1
0.78	0.101 0.117	0.109	0.051
0	0.049 0.067	0.058	--

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human NE/ELA2 were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human NE/ELA2 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)	2.58	5.84	23.39	2.53	6.40	25.58
Standard deviation	0.13	0.31	0.91	0.17	0.28	1.21
C V (%)	5.04	5.31	3.89	6.72	4.38	4.73

Recovery

The recovery of Human NE/ELA2 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)	89-103	94
EDTA plasma (n=5)	88-100	94
Cell culture media (n=5)	93-109	100

Linearity

Samples were spiked with high concentrations of Human NE/ELA2 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 (%)	86-98	91-107	95-109
1:2	Average (%)	92	99	101
1:4	Range (%)	91-106	83-95	82-93
1:4	Average (%)	99	88	88
1:8	Range (%)	92-104	82-93	87-102
1:8	Average (%)	98	87	93
1:16	Range (%)	93-106	86-100	89-99
1:16	Average (%)	98	92	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.