Assay type:	Sandwich
Format:	96T
Assay time:	4.5h
Reactivity:	Human
Detection method:	Chemiluminescence
Detection range:	15.63-1000 pg/mL
Sensitivity:	9.38 pg/mL
Sample volume:	100µL
Sample type:	Serum, plasma and other biological fluids
Repeatability:	CV < 15%
Specificity:	This kit recognizes Human APP in samples. No significant cross-reactivity or interference between Human APP 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 APP. 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 APP 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 APP, 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 APP. The concentration of Human APP in the samples can be calculated by comparing the RLU of the samples to the standard curve.

UniProt Protein Function:	APP: a cell surface receptor that influences neurite growth, neuronal adhesion and axonogenesis. Cleaved by secretases to form a number of peptides, some of which bind to the acetyltransferase complex Fe65/TIP60 to promote transcriptional activation. The Abeta peptide is released from the cell, its extracellular deposition and accumulation form the main components of amyloid plaques in Alzheimer's disease. Mutations in this gene have been implicated in autosomal dominant Alzheimer disease and cerebroarterial amyloidosis. Can promote transcription activation through binding to Fe65-Tip60 and inhibits Notch signaling through interaction with Numb. Couples to apoptosis-inducing pathways such as those mediated by G(O) and JIP. Inhibits G(O) alpha ATPase activity. Acts as a kinesin I membrane receptor, mediating the axonal transport of beta-secretase and presenilin 1. Involved in copper homeostasis/oxidative stress through copper ion reduction. In vitro, copper-metallated APP induces neuronal death directly or is potentiated through Cu(2+)-mediated low-density lipoprotein oxidation. Can regulate neurite outgrowth through binding to components of the extracellular matrix such as heparin and collagen I and IV. Induces a RAGE-dependent pathway that activates p38 MAPK, resulting in internalization of amyloid-beta peptide and leading to mitochondrial dysfunction in cultured cortical neurons. Provides Cu(2+) ions for GPC1 which are required for release of nitric oxide (NO) and subsequent degradation of the heparan sulfate chains on GPC1. Binds, via its C-terminus, to the PID domain of several cytoplasmic proteins, including APBB family members, the APBA family, JIP1, SHC1 and, NUMB and DAB1. Binding to DAB1 inhibits its serine phosphorylation. Associates with microtubules in the presence of ATP and in a kinesin-dependent manner. Amyloid beta-42 binds nAChRA7 in hippocampal neurons. Beta-amyloid associates with HADH2. Soluble APP binds, via its N-terminal head, to FBLN1. Expressed in all fetal tissues examined with highest levels in brain, kidney, heart and spleen. Weak expression in liver. In adult brain, highest expression found in the frontal lobe of the cortex and in the anterior perisylvian cortex- opercular gyri. Moderate expression in the cerebellar cortex, the posterior perisylvian cortex-opercular gyri and the temporal associated cortex. Weak expression found in the striate, extra- striate and motor cortices. Expressed in cerebrospinal fluid, and plasma. 10 isoforms of the human protein are produced by alternative splicing. Isoform APP695 is the predominant form in neuronal tissue, isoform APP751 and isoform APP770 are widely expressed in non- neuronal cells. Isoform APP751 is the most abundant form in T-lymphocytes. Appican is expressed in astrocytes. The splice isoforms that contain the BPTI domain possess protease inhibitor activity. Belongs to the APP family.
UniProt Protein Details:	Protein type:Receptor, misc. ; Membrane protein, integral; Cell surface; Transcription factor; Apoptosis Chromosomal Location of Human Ortholog: 21q21. 3 Cellular Component: Golgi apparatus; extracellular space; cell surface; integral to plasma membrane; integral to membrane; coated pit; intercellular junction; cytosol; ER to Golgi transport vesicle; lipid raft; ciliary rootlet; perinuclear region of cytoplasm; nuclear envelope lumen; cytoplasm; synapse; dendritic shaft; neuromuscular junction; endosome; receptor complex; intracellular membrane-bound organelle; extracellular region; dendritic spine; axon; apical part of cell; plasma membrane; spindle midzone Molecular Function:serine-type endopeptidase inhibitor activity; heparin binding; identical protein binding; protein binding; protease activator activity; enzyme binding; DNA binding; transition metal ion binding; PTB domain binding; acetylcholine receptor binding; receptor binding Biological Process: extracellular matrix organization and biogenesis; adult locomotory behavior; mRNA polyadenylation; locomotory behavior; positive regulation of mitotic cell cycle; protein amino acid phosphorylation; regulation of translation; platelet degranulation; synaptic growth at neuromuscular junction; forebrain development; dendrite development; visual learning; collateral sprouting in the absence of injury; neuromuscular process controlling balance; cell adhesion; neurite development; cholesterol metabolic process; platelet activation; Notch signaling pathway; cellular copper ion homeostasis; regulation of epidermal growth factor receptor activity; axon cargo transport; mating behavior; regulation of multicellular organism growth; endocytosis; axon midline choice point recognition; smooth endoplasmic reticulum calcium ion homeostasis; negative regulation of neuron differentiation; neuron apoptosis; axonogenesis; suckling behavior; ionotropic glutamate receptor signaling pathway; regulation of synapse structure and activity; regulation of protein binding; innate immune response; positive regulation of transcription from RNA polymerase II promoter; response to oxidative stress; blood coagulation; neuron remodeling Disease: Alzheimer Disease
NCBI Summary:	This gene encodes a cell surface receptor and transmembrane precursor protein that is cleaved by secretases to form a number of peptides. Some of these peptides are secreted and can bind to the acetyltransferase complex APBB1/TIP60 to promote transcriptional activation, while others form the protein basis of the amyloid plaques found in the brains of patients with Alzheimer disease. In addition, two of the peptides are antimicrobial peptides, having been shown to have bacteriocidal and antifungal activities. Mutations in this gene have been implicated in autosomal dominant Alzheimer disease and cerebroarterial amyloidosis (cerebral amyloid angiopathy). Multiple transcript variants encoding several different isoforms have been found for this gene. [provided by RefSeq, Aug 2014]
UniProt Code:	P05067
NCBI GenInfo Identifier:	112927
NCBI Gene ID:	351
NCBI Accession:	P05067. 3
UniProt Secondary Accession:	P05067,P09000, P78438, Q13764, Q13778, Q13793, Q16011 B2R5V1, B4DII8, D3DSD1, D3DSD2, D3DSD3,
UniProt Related Accession:	P05067
Molecular Weight:	770
NCBI Full Name:	Amyloid beta A4 protein
NCBI Synonym Full Names:	amyloid beta (A4) precursor protein
NCBI Official Symbol:	APP
NCBI Official Synonym Symbols:	AAA; AD1; PN2; ABPP; APPI; CVAP; ABETA; PN-II; CTFgamma
NCBI Protein Information:	amyloid beta A4 protein; preA4; protease nexin-II; peptidase nexin-II; beta-amyloid peptide; beta-amyloid peptide(1-40); beta-amyloid peptide(1-42); alzheimer disease amyloid protein; cerebral vascular amyloid peptide
UniProt Protein Name:	Amyloid beta A4 protein
UniProt Synonym Protein Names:	ABPP; APPI; APP; Alzheimer disease amyloid protein; Cerebral vascular amyloid peptide; CVAP; PreA4; Protease nexin-II; PN-II
Protein Family:	Amyloid beta A4 protein
UniProt Gene Name:	APP
UniProt Entry Name:	A4_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
1000	48997 58353	53675	53650
500	20827 24151	22489	22464
250	11012 9410	10211	10186
125	4825 4977	4901	4876
62.5	2545 2361	2453	2428
31.25	1334 1228	1281	1256
15.63	662 754	708	683
0	25 25	25	--

Precision

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

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human APP 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)	48.42	135.33	437.24	47.86	129.70	438.18
Standard deviation	5.78	11.26	42.41	5.07	11.18	40.40
C V (%)	11.94	8.32	9.70	10.59	8.62	9.22

Recovery

The recovery of Human APP 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	97
EDTA plasma (n=5)	88-102	93
Cell culture media (n=5)	84-96	90

Linearity

Samples were spiked with high concentrations of Human APP 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 (%)	94-108	92-106	93-108
1:2	Average (%)	100	98	100
1:4	Range (%)	87-98	89-103	87-99
1:4	Average (%)	93	95	94
1:8	Range (%)	92-108	97-113	87-98
1:8	Average (%)	100	104	92
1:16	Range (%)	93-105	89-103	86-100
1:16	Average (%)	99	94	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 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.