ISG15 Chemi-Luminescent ELISA Kit (Human)

Intra-assay Precision (Precision within an assay) : 3 samples with low, middle and high level Ubiquitin Cross Reactive Protein (UCRP) were tested 20 times on one plate, respectively. Inter-assay Precision (Precision between assays) : 3 samples with low, middle and high level Ubiquitin Cross Reactive Protein (UCRP) were tested on 3 different plates, 8 replicates in each plate. CV (%) = SD/meanX100 Intra-Assay: CV<10% Inter-Assay: CV<12%

G1P2; hUCRP; IFI15; IMD38; interferon, alpha-inducible protein (clone IFI-15K) ; Interferon-induced 15 kDa protein; Interferon-induced 17 kDa protein; interferon-induced 17-kDa/15-kDa protein; interferon-stimulated protein, 15 kDa; IP17; ubiquitin cross-reactive protein; ubiquitin-like protein ISG15; UCRP.

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.

The microplate provided in this kit has been pre-coated with an antibody specific to Ubiquitin Cross Reactive Protein (UCRP) . Standards or samples are then added to the appropriate microplate wells with a biotin-conjugated antibody specific to Ubiquitin Cross Reactive Protein (UCRP) . Next, Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. Then the mixture of substrate A and B is added to generate glow light emission kinetics. Upon plate development, the intensity of the emitted light is proportional to the Ubiquitin Cross Reactive Protein (UCRP) level in the sample or standard.

Reconstitution & Storage Instructions Western Blotting/Immunoblotting (WB/IB) Protocol Immunohistochemistry (IHC) Protocol Immunocytochemistry (ICC) Protocol Enzyme-Linked ImmunoSorbent Assay (ELISA) Protocol Blocking Peptide Competition Protocol (BPCP) Immunoprecipitation (IP) Protocol Antibody Array (AA) Protocol Reconstitution & Storage Instructions Reconstitution & Storage Instructions Western Blotting/Immunoblotting (WB/IB) Protocol Western Blotting/Immunoblotting (WB/IB) Protocol Immunohistochemistry (IHC) Protocol Immunohistochemistry (IHC) Protocol Immunocytochemistry (ICC) Protocol Immunocytochemistry (ICC) Protocol Enzyme-Linked ImmunoSorbent Assay (ELISA) Protocol Enzyme-Linked ImmunoSorbent Assay (ELISA) Protocol Blocking Peptide Competition Protocol (BPCP) Blocking Peptide Competition Protocol (BPCP) Immunoprecipitation (IP) Protocol Immunoprecipitation (IP) Protocol Antibody Array (AA) Protocol Antibody Array (AA) Protocol

Matrix Recovery range (%) Average (%) Serum (n=5) 88-96 93 EDTA Plasma (n=5) 83-101 88 Heparin Plasma (n=5) 91-99 95 Matrix Recovery range (%) Average (%) Matrix Recovery range (%) Average (%) Serum (n=5) 88-96 93 Serum (n=5) 88-96 93 EDTA Plasma (n=5) 83-101 88 EDTA Plasma (n=5) 83-101 88 Heparin Plasma (n=5) 91-99 95 Heparin Plasma (n=5) 91-99 95

Component Amount ISG15 Microplate 96 Wells (12 x 8 Well strips) ISG15 Lyophilized Standard 2 100X ISG15 Biotinylated Detector Anitbody 1 x 120 uL 100X Avidin-HRP Conjugate 1 x 120 uL Standard Diluent 1 x 20 mL Detector Antibody Diluent 1 x 12 mL Conjugate Diluent 1 x 12 mL 30X Wash Buffer 1 x 20 mL 100X Luminol Substrate 1 x 2 mL Substrate Diluent 1 x 20 mL Component Amount Component Amount ISG15 Microplate 96 Wells (12 x 8 Well strips) ISG15 Microplate 96 Wells (12 x 8 Well strips) ISG15 Lyophilized Standard 2 ISG15 Lyophilized Standard 2 100X ISG15 Biotinylated Detector Anitbody 1 x 120 uL 100X ISG15 Biotinylated Detector Anitbody 1 x 120 uL 100X Avidin-HRP Conjugate 1 x 120 uL 100X Avidin-HRP Conjugate 1 x 120 uL Standard Diluent 1 x 20 mL Standard Diluent 1 x 20 mL Detector Antibody Diluent 1 x 12 mL Detector Antibody Diluent 1 x 12 mL Conjugate Diluent 1 x 12 mL Conjugate Diluent 1 x 12 mL 30X Wash Buffer 1 x 20 mL 30X Wash Buffer 1 x 20 mL 100X Luminol Substrate 1 x 2 mL 100X Luminol Substrate 1 x 2 mL Substrate Diluent 1 x 20 mL Substrate Diluent 1 x 20 mL