Human ISG15 ELISA Kit (HUEB1610)- High Sensitivity

Product Name:	Human Ubiquitin-like protein ISG15 (ISG15) ELISA Kit
SKU:	HUEB1610
Size:	96T
Target:	Human Ubiquitin-like protein ISG15 (ISG15)
Synonyms:	Interferon-induced 15 kDa protein, Interferon-induced 17 kDa protein, Ubiquitin cross-reactive protein, IP17, hUCRP, G1P2, UCRP
Assay Type:	Sandwich
Detection Method:	ELISA
Reactivity:	Human
Detection Range:	0.312-20ng/mL
Sensitivity:	0.162 ng/mL

Intra CV:

7.1%

Inter CV:

10.9%

Linearity:

Sample	1:2	1:4	1:8	1:16
Serum(N=5)	82-92%	115-127%	91-102%	101-111%
EDTA Plasma(N=5)	93-103%	81-93%	107-116%	108-117%
Heparin Plasma(N=5)	103-112%	80-90%	97-107%	99-108%

Recovery:

Sample Type	Average(%)	Recovery Range(%)
Serum	103	97-109
Plasma	105	99-111

Function:

Ubiquitin-like protein which plays a key role in the innate immune response to viral infection either via its conjugation to a target protein (ISGylation) or via its action as a free or unconjugated protein. ISGylation involves a cascade of enzymatic reactions involving E1, E2, and E3 enzymes which catalyze the conjugation of ISG15 to a lysine residue in the target protein. Its target proteins include IFIT1, MX1/MxA, PPM1B, UBE2L6, UBA7, CHMP5, CHMP2A, CHMP4B and CHMP6. Can also isgylate: EIF2AK2/PKR which results in its activation, DDX58/RIG-I which inhibits its function in antiviral signaling response, EIF4E2 which enhances its cap structure-binding activity and translation-inhibition activity, UBE2N and UBE2E1 which negatively regulates their activity, IRF3 which inhibits its ubiquitination and degradation and FLNB which prevents its ability to interact with the upstream activators of the JNK cascade therby inhibiting IFNA-induced JNK signaling. Exhibits antiviral activity towards both DNA and RNA viruses, including influenza A, HIV-1 and Ebola virus. Restricts HIV-1 and ebola virus via disruption of viral budding. Inhibits the ubiquitination of HIV-1 Gag and host TSG101 and disrupts their interaction, thereby preventing assembly and release of virions from infected cells. Inhibits Ebola virus budding mediated by the VP40 protein by disrupting ubiquitin ligase activity of NEDD4 and its ability to ubiquitinate VP40. ISGylates influenza A virus NS1 protein which causes a loss of function of the protein and the inhibition of virus replication. The secreted form of ISG15 can: induce natural killer cell proliferation, act as a chemotactic factor for neutrophils and act as a IFN-gamma-inducing cytokine playing an essential role in antimycobacterial immunity.

Uniprot:	P05161
Sample Type:	Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:	Natural and recombinant human Ubiquitin-like protein ISG15
Sub Unit:	(Microbial infection) Interacts with vaccinia virus protein E3 (PubMed:24257616). Interaction with influenza B NS1 protein inhibits its conjugation (PubMed:11157743).
Research Area:	Immunology
Subcellular Location:	Cytoplasm Secreted Exists in three distinct states: free within the cell, released into the extracellular space, or conjugated to target proteins.
Storage:	Please see kit components below for exact storage details
Note:	For research use only

UniProt Protein Function:	ISG15: Ubiquitin-like protein that is conjugated to intracellular target proteins after IFN-alpha or IFN-beta stimulation. Its enzymatic pathway is partially distinct from that of ubiquitin, differing in substrate specificity and interaction with ligating enzymes. ISG15 conjugation pathway uses a dedicated E1 enzyme, but seems to converge with the Ub conjugation pathway at the level of a specific E2 enzyme. Targets include STAT1, SERPINA3G/SPI2A, JAK1, MAPK3/ERK1, PLCG1, EIF2AK2/PKR, MX1/MxA, and RIG-1. Deconjugated by USP18/UBP43. Shows specific chemotactic activity towards neutrophils and activates them to induce release of eosinophil chemotactic factors. May serve as a trans-acting binding factor directing the association of ligated target proteins to intermediate filaments. May also be involved in autocrine, paracrine and endocrine mechanisms, as in cell-to-cell signaling, possibly partly by inducing IFN-gamma secretion by monocytes and macrophages. Seems to display antiviral activity during viral infections. Interacts with, and is conjugated to its targets by the UBE1L (E1 enzyme) and UBE2E2 (E2 enzyme) (Probable). Interaction with influenza B NS1 protein inhibits this conjugation. By type I interferons. Detected in lymphoid cells, striated and smooth muscle, several epithelia and neurons.
UniProt Protein Details:	Protein type:Ubiquitin-like modifier Chromosomal Location of Human Ortholog: 1p36.33 Cellular Component: cytosol; extracellular region; nucleoplasm Molecular Function:protein binding Biological Process: bypass DNA synthesis; defense response to bacterium; defense response to virus; ISG15-protein conjugation; negative regulation of interferon type I production; negative regulation of protein ubiquitination; negative regulation of viral genome replication; positive regulation of bone mineralization; regulation of interferon-gamma production Disease: Immunodeficiency 38
NCBI Summary:	The protein encoded by this gene is a ubiquitin-like protein that is conjugated to intracellular target proteins upon activation by interferon-alpha and interferon-beta. Several functions have been ascribed to the encoded protein, including chemotactic activity towards neutrophils, direction of ligated target proteins to intermediate filaments, cell-to-cell signaling, and antiviral activity during viral infections. While conjugates of this protein have been found to be noncovalently attached to intermediate filaments, this protein is sometimes secreted. [provided by RefSeq, Dec 2012]
UniProt Code:	P05161
NCBI GenInfo Identifier:	52001470
NCBI Gene ID:	9636
NCBI Accession:	P05161.5
UniProt Secondary Accession:	P05161,Q5SVA4, Q7Z2G2, Q96GF0,
UniProt Related Accession:	P05161
Molecular Weight:	17,888 Da
NCBI Full Name:	Ubiquitin-like protein ISG15
NCBI Synonym Full Names:	ISG15 ubiquitin-like modifier
NCBI Official Symbol:	ISG15
NCBI Official Synonym Symbols:	G1P2; IP17; UCRP; IFI15; IMD38; hUCRP
NCBI Protein Information:	ubiquitin-like protein ISG15
UniProt Protein Name:	Ubiquitin-like protein ISG15
UniProt Synonym Protein Names:	Interferon-induced 15 kDa protein; Interferon-induced 17 kDa protein; IP17; Ubiquitin cross-reactive protein; hUCRP
Protein Family:	Ubiquitin-like protein
UniProt Gene Name:	ISG15

Component	Quantity (96 Assays)	Storage
ELISA Microplate (Dismountable)	8×12 strips	-20°C
Lyophilized Standard	2	-20°C
Sample Diluent	20ml	-20°C
Assay Diluent A	10mL	-20°C
Assay Diluent B	10mL	-20°C
Detection Reagent A	120µL	-20°C
Detection Reagent B	120µL	-20°C
Wash Buffer	30mL	4°C
Substrate	10mL	4°C
Stop Solution	10mL	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

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

Allow all reagents to reach room temperature (Please do not dissolve the reagents at 37°C directly). All the reagents should be mixed thoroughly by gently swirling before pipetting. Avoid foaming. Keep appropriate numbers of strips for 1 experiment and remove extra strips from microtiter plate. Removed strips should be resealed and stored at -20°C until the kits expiry date. Prepare all reagents, working standards and samples as directed in the previous sections. Please predict the concentration before assaying. If values for these are not within the range of the standard curve, users must determine the optimal sample dilutions for their experiments. We recommend running all samples in duplicate.

Step
1.	Add Sample: Add 100µL of Standard, Blank, or Sample per well. The blank well is added with Sample diluent. Solutions are added to the bottom of micro ELISA plate well, avoid inside wall touching and foaming as possible. Mix it gently. Cover the plate with sealer we provided. Incubate for 120 minutes at 37°C.
2.	Remove the liquid from each well, don't wash. Add 100µL of Detection Reagent A working solution to each well. Cover with the Plate sealer. Gently tap the plate to ensure thorough mixing. Incubate for 1 hour at 37°C. Note: if Detection Reagent A appears cloudy warm to room temperature until solution is uniform.
3.	Aspirate each well and wash, repeating the process three times. Wash by filling each well with Wash Buffer (approximately 400µL) (a squirt bottle, multi-channel pipette,manifold dispenser or automated washer are needed). Complete removal of liquid at each step is essential. After the last wash, completely remove remaining Wash Buffer by aspirating or decanting. Invert the plate and pat it against thick clean absorbent paper.
4.	Add 100µL of Detection Reagent B working solution to each well. Cover with the Plate sealer. Incubate for 60 minutes at 37°C.
5.	Repeat the wash process for five times as conducted in step 3.
6.	Add 90µL of Substrate Solution to each well. Cover with a new Plate sealer and incubate for 10-20 minutes at 37°C. Protect the plate from light. The reaction time can be shortened or extended according to the actual color change, but this should not exceed more than 30 minutes. When apparent gradient appears in standard wells, user should terminatethe reaction.
7.	Add 50µL of Stop Solution to each well. If color change does not appear uniform, gently tap the plate to ensure thorough mixing.
8.	Determine the optical density (OD value) of each well at once, using a micro-plate reader set to 450 nm. User should open the micro-plate reader in advance, preheat the instrument, and set the testing parameters.
9.	After experiment, store all reagents according to the specified storage temperature respectively until their expiry.

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.

ELISA	Antibodies
Human ISG15 ELISA Kit	Anti-ISG15 Antibody (CAB1182)
Human UCRP (Ubiquitin Cross Reactive Protein) CLIA Kit (HUES01281)	Anti-ISG15 Antibody (CAB2416)
Human ISG15 (Interferon Stimulated Gene 15) ELISA Kit (HUES01468)	ISCU Antibody (PACO09996)
	fliC Antibody, Biotin conjugated (PACO32735)
	ISG15 Antibody (PACO32736)