Human IFN-gamma (Interferon Gamma) ELISA Kit (HUES01394)

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HUES01394
€499

Description

ELISA Kit Technical ManualMSDS

Human IFN-gamma (Interferon Gamma) ELISA Kit

This ELISA kit uses Sandwich-ELISA as the method. The micro ELISA plate provided in this kit has been pre-coated with an antibody specific to Human IFN-gamma . Standards or samples are added to the appropriate micro ELISA plate wells and combined with the specific antibody. Then a biotinylated detection antibody specific for Human IFN-gamma 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 IFN-gamma, biotinylated detection antibody and Avidin-HRP conjugate will appear blue in color. The enzyme-substrate reaction is terminated by adding Stop Solution and the color turns yellow. The optical density (OD) is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The OD value is proportional to the concentration of Human IFN-gamma. The concentration of Human IFN-gamma in samples can be calculated by comparing the OD of the samples to the standard curve.

Assay typeSandwich
Format96T
Assay time4.5h
ReactivityHuman
Detection MethodColormetric
Detection Range15.63-1000 pg/mL
Sensitivity9.38 pg/mL
Sample Volume Required Per Well100uL
Sample TypeSerum, plasma and other biological fluids

Specificity

This kit recognizes Human IFN-gamma in samples. No significant cross-reactivity or interference between Human IFN-gamma and analogues was observed.

Typical Data

As the OD 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)
O.D Average Corrected
10002.475
2.517
2.4962.438
5001.552
1.602
1.5771.519
2500.963
0.953
0.9580.9
1250.463
0.487
0.4750.417
62.50.251
0.241
0.2460.188
31.250.171
0.145
0.1580.1
15.630.107
0.113
0.110.052
00.048
0.068
0.058--

Precision

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

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human IFN-gamma were tested on 3 different plates, 20 replicates in each plate.

Intra-assay Precision Inter-assay Precision
Sample123123
n202020202020
Mean (pg/mL)51.9098.00447.2055.80100.10476.70
Standard deviation3.204.8016.102.805.4024.30
C V (%)6.174.903.605.025.395.10

Recovery

The recovery of Human IFN-gamma 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)96-107101
EDTA plasma (n=5)89-10496
Cell culture media (n=5)86-9791

Linearity

Samples were spiked with high concentrations of Human IFN-gamma 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)
1:2Range (%)99-11492-10589-105
Average (%)1059896
1:4Range (%)98-11086-9991-105
Average (%)1039297
1:8Range (%)96-11082-9898-114
Average (%)10289104
1:16Range (%)97-11185-9995-109
Average (%)10590102

Kit Components & Storage

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 ELISA Plate(Dismountable)8 wells X12 strips-20'C, 6 months
Reference Standard2 vials
Concentrated Biotinylated Detection Ab (100X)1 vial, 120 uL
Concentrated HRP Conjugate (100X)1 vial, 120 uL-20'C(shading light), 6 months
Reference Standard & Sample Diluent1 vial, 20 mL4'C, 6 months
Biotinylated Detection Ab Diluent1 vial, 14 mL
HRP Conjugate Diluent1 vial, 14 mL
Concentrated Wash Buffer (25X)1 vial, 30 mL
Substrate Reagent1 vial, 10 mL4'C(shading light)
Stop Solution1 vial, 10 mL4'C
Plate Sealer5 pieces
Product Description1 copy
Certificate of Analysis1 copy

Human IFN-gamma (Interferon Gamma) ELISA Kit (HUES01394) Assay procedure

    1. 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.
    2. Aliquot 100µl of standard solutions into the standard wells.
    3. Add 100µl of Sample / Standard dilution buffer into the control (zero) well.
    4. Add 100µl of properly diluted sample (serum, plasma, tissue homogenates and otherbiological fluids) into test sample wells.
    5. Cover the plate with the sealer provided in the kit and incubate for 90 min at 37°C.
    6. 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.
    7. 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.
    8. Add 100µL of HRP Conjugate working solution to each well. Cover with a plate seal andincubate for 30 min at 37°C.
    9. Aspirate or decant the solution from each well. Repeat the wash process for five times asconducted in step 7.
    10. Add 90µL of Substrate Reagent to each well. Cover with a new plate seal and incubate forapproximately 15 min at 37°C. Protect the plate from light. Note: the reaction time can beshortened or extended according to the actual color change, but not by more than 30min.
    11. Add 50 µL of Stop Solution to each well. Note: Adding the stop solution should be done inthe same order as the substrate solution.
    12. Determine the optical density (OD value) of each well immediately with a microplate readerset at 450 nm.

Human IFN-gamma (Interferon Gamma) ELISA Kit (HUES01394) Protein Information

UniProt Protein Function:IFNG: Produced by lymphocytes activated by specific antigens or mitogens. IFN-gamma, in addition to having antiviral activity, has important immunoregulatory functions. It is a potent activator of macrophages, it has antiproliferative effects on transformed cells and it can potentiate the antiviral and antitumor effects of the type I interferons. Homodimer. Released primarily from activated T lymphocytes. Belongs to the type II (or gamma) interferon family.
UniProt Protein Details:

Protein type:Membrane protein, integral; Secreted; Secreted, signal peptide; Cytokine

Chromosomal Location of Human Ortholog: 12q14

Cellular Component: extracellular space; extracellular region; external side of plasma membrane

Molecular Function:interferon-gamma receptor binding; cytokine activity

Biological Process: positive regulation of isotype switching to IgG isotypes; positive regulation of nitric oxide biosynthetic process; apoptosis; positive regulation of interleukin-23 production; negative regulation of smooth muscle cell proliferation; positive regulation of interleukin-12 production; positive regulation of osteoclast differentiation; positive regulation of interleukin-6 biosynthetic process; negative regulation of epithelial cell differentiation; positive regulation of interleukin-1 beta secretion; positive regulation of killing of cells of another organism; negative regulation of transcription from RNA polymerase II promoter; sensory perception of mechanical stimulus; positive regulation of membrane protein ectodomain proteolysis; cell surface receptor linked signal transduction; positive regulation of MHC class II biosynthetic process; positive regulation of cell proliferation; positive regulation of mesenchymal cell proliferation; positive regulation of T cell proliferation; cell cycle arrest; defense response to virus; regulation of the force of heart contraction; positive regulation of peptidyl-serine phosphorylation of STAT protein; response to drug; positive regulation of synaptic transmission, cholinergic; neutrophil chemotaxis; adaptive immune response; CD8-positive, alpha-beta T cell differentiation during immune response; negative regulation of myelination; unfolded protein response; response to virus; cytokine and chemokine mediated signaling pathway; defense response to protozoan; positive regulation of tumor necrosis factor production; humoral immune response; antigen processing and presentation; positive regulation of tyrosine phosphorylation of Stat1 protein; positive regulation of chemokine biosynthetic process; negative regulation of interleukin-17 production; positive regulation of interleukin-12 biosynthetic process; protein import into nucleus, translocation; defense response to bacterium; neutrophil apoptosis; positive regulation of transcription from RNA polymerase II promoter; cell motility; positive regulation of neuron differentiation; regulation of insulin secretion

Disease: Hepatitis C Virus, Susceptibility To; Aplastic Anemia; Tuberous Sclerosis 2; Mycobacterium Tuberculosis, Susceptibility To; Human Immunodeficiency Virus Type 1, Susceptibility To

NCBI Summary:This gene encodes a member of the type II interferon family. The protein encoded is a soluble cytokine with antiviral, immunoregulatory and anti-tumor properties and is a potent activator of macrophages. Mutations in this gene are associated with aplastic anemia.[provided by RefSeq, Nov 2009]
UniProt Code:P01579
NCBI GenInfo Identifier:124479
NCBI Gene ID:3458
NCBI Accession:P01579.1
UniProt Secondary Accession:P01579,Q53ZV4, B5BU88,
UniProt Related Accession:P01579
Molecular Weight:
NCBI Full Name:Interferon gamma
NCBI Synonym Full Names:interferon, gamma
NCBI Official Symbol:IFNG  
NCBI Official Synonym Symbols:IFG; IFI  
NCBI Protein Information:interferon gamma; IFN-gamma; immune interferon
UniProt Protein Name:Interferon gamma
UniProt Synonym Protein Names:Immune interferon
Protein Family:Interferon
UniProt Gene Name:IFNG  
UniProt Entry Name:IFNG_HUMAN
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Additional Information

Reactivity:
Human
ELISA Type:
Sandwich
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