Mouse PRNP/CD230/Prion ELISA Kit (MOFI00616)

Product Name:	Mouse PRNP/CD230/Prion ELISA Kit
Product Code:	MOFI00616
Size:	96 Assays
Alias:	Prnp, PRNP, PrP33-35C, ASCR, PrP27-30, CJD, GSS, PrP, ASCR, KURU, PRIP, PrPc, CD230, AltPrP, p27-30, PrP33-35C
Detection Method:	Sandwich ELISA
Application:	This immunoassay kit allows for the in vitro quantitative determination of Mouse Prnp concentrations in serum plasma and other biological fluids.
Sensitivity:	0.094ng/ml
Range:	0.156-10ng/ml
Storage:	4°C for 6 months
Note:	For Research Use Only

Recovery:

Matrices listed below were spiked with certain level of Mouse Prnp and the recovery rates were calculated by comparing the measured value to the expected amount of Mouse Prnp in samples.

Matrix	Recovery range(%)	Average(%)
serum(n=5)	94-105	99
EDTA plasma(n=5)	87-105	93
UFH plasma(n=5)	86-97	93

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Mouse Prnp and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.

Sample	1:2	1:4	1:8
serum(n=5)	86-95%	88-104%	92-98%
EDTA plasma(n=5)	82-101%	82-101%	86-94%
UFH plasma(n=5)	81-97%	81-100%	80-100%

Intra Assay:

CV <8%

Inter Assay:

CV <10%

Component	Quantity	Storage
ELISA Microplate (Dismountable)	8-12 strips	4°C for 6 months
Lyophilized Standard	2	4°C/-20°C
Sample/Standard Dilution Buffer	20ml	4°C
Biotin-labeled Antibody(Concentrated)	120ul	4°C (Protect from light)
Antibody Dilution Buffer	10ml	4°C
HRP-Streptavidin Conjugate(SABC)	120ul	4°C (Protect from light)
SABC Dilution Buffer	10ml	4°C
TMB Substrate	10ml	4°C (Protect from light)
Stop Solution	10ml	4°C
Wash Buffer(25X)	30ml	4°C
Plate Sealer	5	-

Other materials and equipment required:

Microplate reader with 450 nm wavelength filter
Multichannel Pipette, Pipette, microcentrifuge tubes and disposable pipette tips
Incubator
Deionized or distilled water
Absorbent paper
Buffer resevoir

Uniprot

P04925

UniProt Protein Function:	PRNP: May play a role in neuronal development and synaptic plasticity. May be required for neuronal myelin sheath maintenance. May play a role in iron uptake and iron homeostasis. Soluble oligomers are toxic to cultured neuroblastoma cells and induce apoptosis (in vitro). Association with GPC1 (via its heparan sulfate chains) targets PRNP to lipid rafts. Also provides Cu(2+) or ZN(2+) for the ascorbate-mediated GPC1 deaminase degradation of its heparan sulfate side chains. PrP is found in high quantity in the brain of humans and animals infected with neurodegenerative diseases known as transmissible spongiform encephalopathies or prion diseases, like: Creutzfeldt-Jakob disease (CJD), fatal familial insomnia (FFI), Gerstmann-Straussler disease (GSD), Huntington disease-like type 1 (HDL1) and kuru in humans; scrapie in sheep and goat; bovine spongiform encephalopathy (BSE) in cattle; transmissible mink encephalopathy (TME); chronic wasting disease (CWD) of mule deer and elk; feline spongiform encephalopathy (FSE) in cats and exotic ungulate encephalopathy (EUE) in nyala and greater kudu. The prion diseases illustrate three manifestations of CNS degeneration: (1) infectious (2) sporadic and (3) dominantly inherited forms. TME, CWD, BSE, FSE, EUE are all thought to occur after consumption of prion-infected foodstuffs. Defects in PRNP are the cause of Creutzfeldt-Jakob disease (CJD). CJD occurs primarily as a sporadic disorder (1 per million), while 10-15% are familial. Accidental transmission of CJD to humans appears to be iatrogenic (contaminated human growth hormone (HGH), corneal transplantation, electroencephalographic electrode implantation, etc.). Epidemiologic studies have failed to implicate the ingestion of infected annimal meat in the pathogenesis of CJD in human. The triad of microscopic features that characterize the prion diseases consists of (1) spongiform degeneration of neurons, (2) severe astrocytic gliosis that often appears to be out of proportion to the degree of nerve cell loss, and (3) amyloid plaque formation. CJD is characterized by progressive dementia and myoclonic seizures, affecting adults in mid-life. Some patients present sleep disorders, abnormalities of high cortical function, cerebellar and corticospinal disturbances. The disease ends in death after a 3-12 months illness. Defects in PRNP are the cause of fatal familial insomnia (FFI). FFI is an autosomal dominant disorder and is characterized by neuronal degeneration limited to selected thalamic nuclei and progressive insomnia. Defects in PRNP are the cause of Gerstmann-Straussler disease (GSD). GSD is a heterogeneous disorder and was defined as a spinocerebellar ataxia with dementia and plaquelike deposits. GSD incidence is less than 2 per 100 million live births. Defects in PRNP are the cause of Huntington disease-like type 1 (HDL1). HDL1 is an autosomal dominant, early onset neurodegenerative disorder with prominent psychiatric features. Defects in PRNP are the cause of kuru (KURU). Kuru is transmitted during ritualistic cannibalism, among natives of the New Guinea highlands. Patients exhibit various movement disorders like cerebellar abnormalities, rigidity of the limbs, and clonus. Emotional lability is present, and dementia is conspicuously absent. Death usually occurs from 3 to 12 month after onset. Defects in PRNP are the cause of spongiform encephalopathy with neuropsychiatric features (SENF); an autosomal dominant presenile dementia with a rapidly progressive and protracted clinical course. The dementia was characterized clinically by frontotemporal features, including early personality changes. Some patients had memory loss, several showed aggressiveness, hyperorality and verbal stereotypy, others had parkinsonian symptoms. Belongs to the prion family. 2 isoforms of the human protein are produced by alternative initiation.
UniProt Protein Details:	Protein type:Microtubule-binding; Membrane protein, GPI anchor Cellular Component: Golgi apparatus; mitochondrial outer membrane; cell surface; membrane; cell; endoplasmic reticulum; cytoplasm; plasma membrane; lipid raft Molecular Function:tubulin binding; ATP-dependent protein binding; identical protein binding; protein binding; copper ion binding; metal ion binding; chaperone binding; microtubule binding Biological Process: cellular copper ion homeostasis; negative regulation of activated T cell proliferation; negative regulation of transcription factor activity; negative regulation of T cell receptor signaling pathway; negative regulation of interleukin-2 production; learning and/or memory; response to cadmium ion; regulation of protein localization; negative regulation of interleukin-17 production; response to copper ion; negative regulation of protein amino acid phosphorylation; negative regulation of interferon-gamma production; nucleobase, nucleoside, nucleotide and nucleic acid metabolic process; response to oxidative stress; protein homooligomerization; negative regulation of apoptosis
UniProt Code:	P04925
NCBI GenInfo Identifier:	130914
NCBI Gene ID:	19122
NCBI Accession:	P04925.2
UniProt Related Accession:	P04925
Molecular Weight:	27,977 Da
NCBI Full Name:	Major prion protein
NCBI Synonym Full Names:	prion protein
NCBI Official Symbol:	Prnp
NCBI Official Synonym Symbols:	PrP; PrPC; Sinc; CD230; PrPSc; Prn-i; Prn-p; PrP; AA960666; AI325101; prP27-30; prP33-35C
NCBI Protein Information:	major prion protein
UniProt Protein Name:	Major prion protein
UniProt Synonym Protein Names:	PrP27-30; PrP33-35C; CD_antigen: CD230
Protein Family:	Major prion protein
UniProt Gene Name:	Prnp
UniProt Entry Name:	PRIO_MOUSE

*Note: Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.

Step	Procedure
1.	Set standard, test sample and control (zero) wells on the pre-coated plate respectively, and then, record their positions. It is recommended to measure each standard and sample in duplicate. Wash plate 2 times before adding standard, sample and control (zero) wells!
2.	Aliquot 0.1ml standard solutions into the standard wells.
3.	Add 0.1 ml of Sample / Standard dilution buffer into the control (zero) well.
4.	Add 0.1 ml of properly diluted sample (Human serum, plasma, tissue homogenates and other biological fluids.) into test sample wells.
5.	Seal the plate with a cover and incubate at 37 °C for 90 min.
6.	Remove the cover and discard the plate content, clap the plate on the absorbent filter papers or other absorbent material. Do NOT let the wells completely dry at any time. Wash plate X2.
7.	Add 0.1 ml of Biotin- detection antibody working solution into the above wells (standard, test sample & zero wells). Add the solution at the bottom of each well without touching the side wall.
8.	Seal the plate with a cover and incubate at 37°C for 60 min.
9.	Remove the cover, and wash plate 3 times with Wash buffer. Let wash buffer rest in wells for 1 min between each wash.
10.	Add 0.1 ml of SABC working solution into each well, cover the plate and incubate at 37°C for 30 min.
11.	Remove the cover and wash plate 5 times with Wash buffer, and each time let the wash buffer stay in the wells for 1-2 min.
12.	Add 90 µL of TMB substrate into each well, cover the plate and incubate at 37°C in dark within 10-20 min. (Note: This incubation time is for reference use only, the optimal time should be determined by end user.) And the shades of blue can be seen in the first 3-4 wells (with most concentrated standard solutions), the other wells show no obvious color.
13.	Add 50 µL of Stop solution into each well and mix thoroughly. The color changes into yellow immediately.
14.	Read the O.D. absorbance at 450 nm in a microplate reader immediately after adding the stop solution.

When carrying out an ELISA assay it is important to prepare your samples in order to achieve the best possible results. Below we have a list of procedures for the preparation of samples for different sample types.

Sample Type	Protocol
Serum:	If using serum separator tubes, allow samples to clot for 30 minutes at room temperature. Centrifuge for 10 minutes at 1,000x g. Collect the serum fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. If serum separator tubes are not being used, allow samples to clot overnight at 2-8°C. Centrifuge for 10 minutes at 1,000x g. Remove serum and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles.
Plasma:	Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples at 4°C for 15 mins at 1000 - g within 30 mins of collection. Collect the plasma fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. Note: Over haemolysed samples are not suitable for use with this kit.
Urine & Cerebrospinal Fluid:	Collect the urine (mid-stream) in a sterile container, centrifuge for 20 mins at 2000-3000 rpm. Remove supernatant and assay immediately. If any precipitation is detected, repeat the centrifugation step. A similar protocol can be used for cerebrospinal fluid.
Cell culture supernatant:	Collect the cell culture media by pipette, followed by centrifugation at 4°C for 20 mins at 1500 rpm. Collect the clear supernatant and assay immediately.
Cell lysates:	Solubilize cells in lysis buffer and allow to sit on ice for 30 minutes. Centrifuge tubes at 14,000 x g for 5 minutes to remove insoluble material. Aliquot the supernatant into a new tube and discard the remaining whole cell extract. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20°C.
Tissue homogenates:	The preparation of tissue homogenates will vary depending upon tissue type. Rinse tissue with 1X PBS to remove excess blood & homogenize in 20ml of 1X PBS (including protease inhibitors) and store overnight at ≤ -20°C. Two freeze-thaw cycles are required to break the cell membranes. To further disrupt the cell membranes you can sonicate the samples. Centrifuge homogenates for 5 mins at 5000xg. Remove the supernatant and assay immediately or aliquot and store at -20°C or -80°C.
Tissue lysates:	Rinse tissue with PBS, cut into 1-2 mm pieces, and homogenize with a tissue homogenizer in PBS. Add an equal volume of RIPA buffer containing protease inhibitors and lyse tissues at room temperature for 30 minutes with gentle agitation. Centrifuge to remove debris. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.
Breast Milk:	Collect milk samples and centrifuge at 10,000 x g for 60 min at 4°C. Aliquot the supernatant and assay. For long term use, store samples at -80°C. Minimize freeze/thaw cycles.

Mouse PRNP/CD230/Prion ELISA Kit (MOFI00616)

Description