Assay type:	Sandwich
Format:	96T
Assay time:	4.5h
Reactivity:	Human
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 Human AZU1 in samples. No significant cross-reactivity or interference between Human AZU1 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 Human AZU1. 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 AZU1 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 AZU1, 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 AZU1. The concentration of Human AZU1 in the samples can be calculated by comparing the RLU of the samples to the standard curve.

UniProt Protein Function:	AZU1: This is a neutrophil granule-derived antibacterial and monocyte- and fibroblast-specific chemotactic glycoprotein. Binds heparin. The cytotoxic action is limited to many species of Gram- negative bacteria; this specificity may be explained by a strong affinity of the very basic N-terminal half for the negatively charged lipopolysaccharides that are unique to the Gram-negative bacterial outer envelope. It may play a role in mediating recruitment of monocytes in the second wave of inflammation. Has antibacterial activity against the Gram-nagative bacterium P. aeruginosa, this activity is inhibited by LPS from P. aeruginosa. Acting alone, it does not have antimicrobial activity against the Gram-negative bacteria A. actinomycetemcomitans ATCC 29532, A. actinomycetemcomitans NCTC 9709, A. actinomycetemcomitans FDC-Y4, H. aphrophilus ATCC 13252, E. corrodens ATCC 23834, C. sputigena ATCC 33123, Capnocytophaga sp ATCC 33124, Capnocytophaga sp ATCC 27872 or E. coli ML-35. Has antibacterial activity against C. sputigena ATCC 33123 when acting synergistically with either elastase or cathepsin G. Belongs to the peptidase S1 family. Elastase subfamily.
UniProt Protein Details:	Chromosomal Location of Human Ortholog: 19p13. 3 Cellular Component: azurophil granule; extracellular region Molecular Function:heparin binding; toxin binding; serine-type endopeptidase activity Biological Process: positive regulation of tumor necrosis factor biosynthetic process; positive regulation of cell adhesion; microglial cell activation; glial cell migration; proteolysis; defense response to Gram-negative bacterium; macrophage chemotaxis; induction of positive chemotaxis; cellular extravasation; monocyte activation; positive regulation of phagocytosis; positive regulation of MHC class II biosynthetic process; positive regulation of fractalkine biosynthetic process; protein kinase C activation; positive regulation of interleukin-1 beta biosynthetic process; protein processing; immune response; regulation of vascular permeability; inflammatory response; negative regulation of apoptosis
NCBI Summary:	Azurophil granules, specialized lysosomes of the neutrophil, contain at least 10 proteins implicated in the killing of microorganisms. The protein encoded by this gene is an azurophil granule antibiotic protein, with monocyte chemotactic and antimicrobial activity. It is also an important multifunctional inflammatory mediator. This encoded protein is a member of the serine protease gene family but it is not a serine proteinase, because the active site serine and histidine residues are replaced. The genes encoding this protein, neutrophil elastase 2, and proteinase 3 are in a cluster located 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, Dec 2014]
UniProt Code:	P20160
NCBI GenInfo Identifier:	416746
NCBI Gene ID:	566
NCBI Accession:	P20160. 3
UniProt Secondary Accession:	P20160,P80014, Q52LG4, Q9UCM1, Q9UCT5,
UniProt Related Accession:	P20160
Molecular Weight:	251
NCBI Full Name:	Azurocidin
NCBI Synonym Full Names:	azurocidin 1
NCBI Official Symbol:	AZU1
NCBI Official Synonym Symbols:	AZU; HBP; NAZC; AZAMP; CAP37; HUMAZUR
NCBI Protein Information:	azurocidin; neutrophil azurocidin; heparin-binding protein; cationic antimicrobial protein 37; cationic antimicrobial protein CAP37
UniProt Protein Name:	Azurocidin
UniProt Synonym Protein Names:	Cationic antimicrobial protein CAP37; Heparin-binding protein; HBP
Protein Family:	Azurocidin
UniProt Gene Name:	AZU1
UniProt Entry Name:	CAP7_HUMAN

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	50244 61198	55721	55693
1000	23033 24915	23974	23946
500	11674 10390	11032	11004
250	4947 5641	5294	5266
125	2813 2403	2608	2580
62.5	1313 1309	1311	1283
31.25	643 705	674	646
0	27 29	28	--

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human AZU1 were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human AZU1 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)	98.61	283.65	896.67	89.68	275.15	946.34
Standard deviation	11.19	20.79	62.95	11.51	21.02	87.82
C V (%)	11.35	7.33	7.02	12.83	7.64	9.28

Recovery

The recovery of Human AZU1 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)	100-114	107
EDTA plasma (n=5)	94-107	100
Cell culture media (n=5)	91-105	98

Linearity

Samples were spiked with high concentrations of Human AZU1 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 (%)	89-102	100-116	92-109
1:2	Average (%)	96	107	99
1:4	Range (%)	91-105	86-100	95-108
1:4	Average (%)	98	92	101
1:8	Range (%)	104-120	91-103	101-117
1:8	Average (%)	110	98	107
1:16	Range (%)	96-110	98-116	86-97
1:16	Average (%)	102	106	92

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.