Product Name:	Human NLRP3 (Nod Like Receptor Pyrins-3) ELISA Kit
Product Code:	HUFI04745
Size:	96 Assays
Alias:	Nod Like Receptor Pyrins-3 ELISA Kit
Detection method:	Sandwich ELISA, Double Antibody
Application:	This immunoassay kit allows for the in vitro quantitative determination of Human NLRP3 (Nod Like Receptor Pyrins-3) concentrations in serum plasma and other biological fluids.
Sensitivity:	< 0.469ng/ml
Range:	0.781-50ng/ml
Storage:	4°C for 6 months
Note:	For Research Use Only

Recovery:

Matrices listed below were spiked with certain level of Human NLRP3 (Nod Like Receptor Pyrins-3) and the recovery rates were calculated by comparing the measured value to the expected amount of Human NLRP3 (Nod Like Receptor Pyrins-3) in samples.

Matrix	Recovery range(%)	Average(%)
serum(n=5)	88-98	93
EDTA plasma(n=5)	87-105	99
heparin plasma(n=5)	86-105	96

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human NLRP3 (Nod Like Receptor Pyrins-3) 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)	87-97%	86-103%	88-101%
EDTA plasma(n=5)	88-100%	82-96%	85-101%
UFH plasma(n=5)	85-98%	80-99%	80-98%

CV(%):

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 Protein Function:	NLRP3: May function as an inducer of apoptosis. Interacts selectively with ASC and this complex may function as an upstream activator of NF-kappa-B signaling. Inhibits TNF-alpha induced activation and nuclear translocation of RELA/NF-KB p65. Also inhibits transcriptional activity of RELA. Activates caspase-1 in response to a number of triggers including bacterial or viral infection which leads to processing and release of IL1B and IL18. Defects in NLRP3 are the cause of familial cold autoinflammatory syndrome type 1 (FCAS1); also known as familial cold urticaria. FCAS are rare autosomal dominant systemic inflammatory diseases characterized by episodes of rash, arthralgia, fever and conjunctivitis after generalized exposure to cold. Defects in NLRP3 are a cause of Muckle-Wells syndrome (MWS); also known as urticaria-deafness-amyloidosis syndrome. MWS is a hereditary periodic fever syndrome characterized by fever, chronic recurrent urticaria, arthralgias, progressive sensorineural deafness, and reactive renal amyloidosis. The disease may be severe if generalized amyloidosis occurs. Defects in NLRP3 are the cause of chronic infantile neurologic cutaneous and articular syndrome (CINCA); also known as neonatal onset multisystem inflammatory disease (NOMID). CINCA is a rare congenital inflammatory disorder characterized by a triad of neonatal onset of cutaneous symptoms, chronic meningitis and joint manifestations with recurrent fever and inflammation. Belongs to the NLRP family. 5 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:	Protein type:Inhibitor Chromosomal Location of Human Ortholog: 1q44 Cellular Component: cytoplasm; cytosol Molecular Function:peptidoglycan binding; protein binding; ATP binding Biological Process: caspase activation; apoptosis; negative regulation of acute inflammatory response; positive regulation of caspase activity; interleukin-1 beta production; defense response; negative regulation of NF-kappaB import into nucleus; positive regulation of interleukin-1 beta secretion; signal transduction; protein oligomerization; activation of NF-kappaB transcription factor; negative regulation of interleukin-1 beta secretion; inhibition of NF-kappaB transcription factor; negative regulation of inflammatory response; interleukin-18 production; innate immune response; interleukin-1 secretion; inflammatory response; detection of biotic stimulus; defense response to virus Disease: Cinca Syndrome; Familial Cold Autoinflammatory Syndrome 1; Muckle-wells Syndrome
NCBI Summary:	This gene encodes a pyrin-like protein containing a pyrin domain, a nucleotide-binding site (NBS) domain, and a leucine-rich repeat (LRR) motif. This protein interacts with the apoptosis-associated speck-like protein PYCARD/ASC, which contains a caspase recruitment domain, and is a member of the NALP3 inflammasome complex. This complex functions as an upstream activator of NF-kappaB signaling, and it plays a role in the regulation of inflammation, the immune response, and apoptosis. Mutations in this gene are associated with familial cold autoinflammatory syndrome (FCAS), Muckle-Wells syndrome (MWS), chronic infantile neurological cutaneous and articular (CINCA) syndrome, and neonatal-onset multisystem inflammatory disease (NOMID). Multiple alternatively spliced transcript variants encoding distinct isoforms have been identified for this gene. Alternative 5' UTR structures are suggested by available data; however, insufficient evidence is available to determine if all of the represented 5' UTR splice patterns are biologically valid. [provided by RefSeq, Oct 2008]
UniProt Code:	Q96P20
NCBI GenInfo Identifier:	262527566
NCBI Gene ID:	114548
NCBI Accession:	Q96P20.3
UniProt Secondary Accession:	Q96P20,O75434, Q17RS2, Q59H68, Q5JQS8, Q5JQS9, Q6TG35 Q8TCW0, B2RC97, B7ZKS9, B7ZKT2, B7ZKT3,
UniProt Related Accession:	Q96P20
Molecular Weight:
NCBI Full Name:	NACHT, LRR and PYD domains-containing protein 3
NCBI Synonym Full Names:	NLR family, pyrin domain containing 3
NCBI Official Symbol:	NLRP3
NCBI Official Synonym Symbols:	AII; AVP; FCU; MWS; FCAS; CIAS1; NALP3; C1orf7; CLR1.1; PYPAF1; AGTAVPRL
NCBI Protein Information:	NACHT, LRR and PYD domains-containing protein 3; cryopyrin; caterpiller protein 1.1; PYRIN-containing APAF1-like protein 1; NACHT, LRR and PYD containing protein 3; cold autoinflammatory syndrome 1 protein; NACHT domain-, leucine-rich repeat-, and PYD-containing protein 3; nucleotide-binding oligomerization domain, leucine rich repeat and pyrin domain containing 3
UniProt Protein Name:	NACHT, LRR and PYD domains-containing protein 3
UniProt Synonym Protein Names:	Angiotensin/vasopressin receptor AII/AVP-like; Caterpiller protein 1.1; CLR1.1; Cold autoinflammatory syndrome 1 protein; Cryopyrin; PYRIN-containing APAF1-like protein 1
Protein Family:	NACHT, LRR and PYD domains-containing protein
UniProt Gene Name:	NLRP3
UniProt Entry Name:	NALP3_HUMAN

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

Before adding to wells, equilibrate the SABC working solution and TMB substrate for at least 30 min at 37 °C. When diluting samples and reagents, they must be mixed completely and evenly. It is recommended to plot a standard curve for each test.

Step	Protocol
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.