Assay type:	Sandwich
Format:	96T
Assay time:	4.5h
Reactivity:	Human
Detection method:	Chemiluminescence
Detection range:	78.13-5000 pg/mL
Sensitivity:	46.88 pg/mL
Sample volume:	100µL
Sample type:	Serum, plasma and other biological fluids
Repeatability:	CV < 15%
Specificity:	This kit recognizes Human HTRA1 in samples. No significant cross-reactivity or interference between Human HTRA1 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 HTRA1. 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 HTRA1 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 HTRA1, 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 HTRA1. The concentration of Human HTRA1 in the samples can be calculated by comparing the RLU of the samples to the standard curve.

UniProt Protein Function:	HTRA1: Serine protease with a variety of targets, including extracellular matrix proteins such as fibronectin. HTRA1-generated fibronectin fragments further induce synovial cells to up-regulate MMP1 and MMP3 production. May also degrade proteoglycans, such as aggrecan, decorin and fibromodulin. Through cleavage of proteoglycans, may release soluble FGF-glycosaminoglycan complexes that promote the range and intensity of FGF signals in the extracellular space. Regulates the availability of insulin-like growth factors (IGFs) by cleaving IGF-binding proteins. Inhibits signaling mediated by TGF-beta family members. This activity requires the integrity of the catalytic site, although it is unclear whether TGF-beta proteins are themselves degraded. By acting on TGF-beta signaling, may regulate many physiological processes, including retinal angiogenesis and neuronal survival and maturation during development. Intracellularly, degrades TSC2, leading to the activation of TSC2 downstream targets. Variations in the promoter region of HTRA1 are the cause of susceptibility to age-related macular degeneration type 7 (ARMD7). ARMD is the leading cause of vision loss and blindness among older individuals in the developed word. It is classified as either dry (nonneovascular) or wet (neovascular). ARMD7 is a wet form, in which new blood vessels form and break beneath the retina. This leakage causes permanent damage to surrounding retinal tissue, distorting and destroying central vision. Wet ARMD is more prevalent among Asians than Caucasians. Defects in HTRA1 are the cause of cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL). CARASIL is characterized by nonhypertensive cerebral small-vessel arteriopathy with subcortical infarcts, alopecia, and spondylosis, with an onset in early adulthood. On neuropathological examination, atherosclerosis associated with intimal thickening and dense collagen fibers, loss of vascular smooth-muscle cells, and hyaline degeneration of the tunica media has been observed in cerebral small arteries. Belongs to the peptidase S1B family.
UniProt Protein Details:	Protein type:EC 3. 4. 21. -; Secreted, signal peptide; Secreted; Protease Chromosomal Location of Human Ortholog: 10q26. 3 Cellular Component: extracellular matrix; extracellular space; cytosol Molecular Function:insulin-like growth factor binding; serine-type peptidase activity; serine-type endopeptidase activity Biological Process: negative regulation of defense response to virus; regulation of cell growth; negative regulation of transforming growth factor beta receptor signaling pathway; proteolysis; positive regulation of epithelial cell proliferation; negative regulation of BMP signaling pathway Disease: Macular Degeneration, Age-related, 7; Cerebral Autosomal Recessive Arteriopathy With Subcortical Infarcts And Leukoencephalopathy
NCBI Summary:	This gene encodes a member of the trypsin family of serine proteases. This protein is a secreted enzyme that is proposed to regulate the availability of insulin-like growth factors (IGFs) by cleaving IGF-binding proteins. It has also been suggested to be a regulator of cell growth. Variations in the promoter region of this gene are the cause of susceptibility to age-related macular degeneration type 7. [provided by RefSeq, Jul 2008]
UniProt Code:	Q92743
NCBI GenInfo Identifier:	18202620
NCBI Gene ID:	5654
NCBI Accession:	Q92743. 1
UniProt Secondary Accession:	Q92743,Q9UNS5, D3DRE4,
UniProt Related Accession:	Q92743
Molecular Weight:
NCBI Full Name:	Serine protease HTRA1
NCBI Synonym Full Names:	HtrA serine peptidase 1
NCBI Official Symbol:	HTRA1
NCBI Official Synonym Symbols:	L56; HtrA; ARMD7; ORF480; PRSS11; CARASIL
NCBI Protein Information:	serine protease HTRA1; IGFBP5-protease; protease, serine, 11 (IGF binding); high-temperature requirement A serine peptidase 1
UniProt Protein Name:	Serine protease HTRA1
UniProt Synonym Protein Names:	High-temperature requirement A serine peptidase 1; L56; Serine protease 11
Protein Family:	Serine protease
UniProt Gene Name:	HTRA1
UniProt Entry Name:	HTRA1_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
5000	56050 66462	61256	61233
2500	31448 37260	34354	34331
1250	19775 16563	18169	18146
625	8836 9948	9392	9369
312.5	5037 4629	4833	4810
156.25	2678 2344	2511	2488
78.13	1317 1361	1339	1316
0	22 24	23	--

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human HTRA1 were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human HTRA1 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)	250.96	619.38	1810.37	244.59	672.66	1936.43
Standard deviation	25.37	57.91	141.57	28.79	63.63	145.43
C V (%)	10.11	9.35	7.82	11.77	9.46	7.51

Recovery

The recovery of Human HTRA1 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)	94-108	101
EDTA plasma (n=5)	97-113	104
Cell culture media (n=5)	95-107	101

Linearity

Samples were spiked with high concentrations of Human HTRA1 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 (%)	83-97	95-105	102-120
1:2	Average (%)	90	100	110
1:4	Range (%)	85-97	87-101	96-111
1:4	Average (%)	92	93	102
1:8	Range (%)	96-109	95-108	87-98
1:8	Average (%)	102	101	93
1:16	Range (%)	103-117	98-116	87-100
1:16	Average (%)	109	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.