Human IL8 ELISA Kit (HUES01335)

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HUES01335
€499

Description

ELISA Kit Technical ManualMSDS

Human IL8 (Interleukin 8) 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 IL8 . 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 IL8 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 IL8, 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 IL8. The concentration of Human IL8 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 Range31.25-2000 pg/mL
Sensitivity18.75 pg/mL
Sample Volume Required Per Well100uL
Sample TypeSerum, plasma and other biological fluids

Specificity

This kit recognizes Human IL8 ELISA Kit in samples. No significant cross-reactivity or interference between Human IL8 ELISA Kit 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
20002.477
2.491
2.4842.397
10001.588
1.634
1.6111.524
5000.979
0.943
0.9610.874
2500.47
0.482
0.4760.389
1250.264
0.264
0.2640.177
62.50.201
0.177
0.1890.102
31.250.135
0.143
0.1390.052
00.086
0.088
0.087--

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human IL8 ELISA Kit were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human IL8 ELISA Kit were tested on 3 different plates, 20 replicates in each plate.

Intra-assay Precision Inter-assay Precision
Sample123123
n202020202020
Mean (pg/mL)111.10220.20878.40108.90237.70865.90
Standard deviation7.1012.8040.407.4012.4046.80
C V (%)6.395.814.606.805.225.40

Recovery

The recovery of Human IL8 ELISA Kit 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)89-10697
EDTA plasma (n=5)94-106101
Cell culture media (n=5)90-10497

Linearity

Samples were spiked with high concentrations of Human IL8 ELISA Kit 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 (%)91-10496-10992-106
Average (%)9710298
1:4Range (%)100-11181-9398-114
Average (%)10687104
1:8Range (%)96-10784-9593-107
Average (%)1029098
1:16Range (%)95-11083-9391-105
Average (%)1038899

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 IL8 ELISA Kit (HUES01335) 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 IL8 ELISA Kit (HUES01335) Protein Information

UniProt Protein Function:IL8: IL-8 is a chemotactic factor that attracts neutrophils, basophils, and T-cells, but not monocytes. It is also involved in neutrophil activation. It is released from several cell types in response to an inflammatory stimulus. IL-8(6-77) has a 5-10-fold higher activity on neutrophil activation, IL-8(5-77) has increased activity on neutrophil activation and IL-8(7-77) has a higher affinity to receptors CXCR1 and CXCR2 as compared to IL-8(1-77), respectively. Belongs to the intercrine alpha (chemokine CxC) family. 2 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:

Protein type:Secreted, signal peptide; Cytokine; Secreted; Motility/polarity/chemotaxis

Chromosomal Location of Human Ortholog: 4q13-q21

Cellular Component: extracellular space; extracellular region

Molecular Function:protein binding; chemokine activity; interleukin-8 receptor binding

Biological Process: regulation of cell adhesion; neutrophil chemotaxis; neutrophil activation; negative regulation of G-protein coupled receptor protein signaling pathway; unfolded protein response; calcium-mediated signaling; chemotaxis; signal transduction; regulation of retroviral genome replication; induction of positive chemotaxis; G-protein coupled receptor protein signaling pathway; negative regulation of cell proliferation; cellular protein metabolic process; unfolded protein response, activation of signaling protein activity; response to molecule of bacterial origin; receptor internalization; immune response; angiogenesis; cell cycle arrest; cell motility; inflammatory response; embryonic gut development

NCBI Summary:The protein encoded by this gene is a member of the CXC chemokine family. This chemokine is one of the major mediators of the inflammatory response. This chemokine is secreted by several cell types. It functions as a chemoattractant, and is also a potent angiogenic factor. This gene is believed to play a role in the pathogenesis of bronchiolitis, a common respiratory tract disease caused by viral infection. This gene and other ten members of the CXC chemokine gene family form a chemokine gene cluster in a region mapped to chromosome 4q. [provided by RefSeq, Jul 2008]
UniProt Code:P10145
NCBI GenInfo Identifier:124359
NCBI Gene ID:3576
NCBI Accession:P10145.1
UniProt Secondary Accession:P10145,Q6FGF6, Q6LAE6, Q96RG6, Q9C077, Q9UCE1, Q9UCR8 Q9UCR9, Q9UCS0, B2R4L8,
UniProt Related Accession:P10145
Molecular Weight:11,338 Da
NCBI Full Name:Interleukin-8
NCBI Synonym Full Names:chemokine (C-X-C motif) ligand 8
NCBI Official Symbol:CXCL8  
NCBI Official Synonym Symbols:IL8; NAF; GCP1; LECT; LUCT; NAP1; GCP-1; LYNAP; MDNCF; MONAP; NAP-1  
NCBI Protein Information:interleukin-8; emoctakin; interleukin 8; T-cell chemotactic factor; neutrophil-activating peptide 1; beta-thromboglobulin-like protein; granulocyte chemotactic protein 1; tumor necrosis factor-induced gene 1; alveolar macrophage chemotactic factor I; monocyte-derived neutrophil chemotactic factor; monocyte-derived neutrophil-activating peptide; small inducible cytokine subfamily B, member 8; lymphocyte derived neutrophil activating peptide; lung giant cell carcinoma-derived chemotactic protein; beta endothelial cell-derived neutrophil activating peptide
UniProt Protein Name:Interleukin-8
UniProt Synonym Protein Names:C-X-C motif chemokine 8; Chemokine (C-X-C motif) ligand 8; Emoctakin; Granulocyte chemotactic protein 1; GCP-1; Monocyte-derived neutrophil chemotactic factor; MDNCF; Monocyte-derived neutrophil-activating peptide; MONAP; Neutrophil-activating protein 1; NAP-1; Protein 3-10C; T-cell chemotactic factorCleaved into the following 7 chains:MDNCF-aAlternative name(s):GCP/IL-8 protein IV; IL8/NAP1 form I
UniProt Gene Name:CXCL8  
UniProt Entry Name:IL8_HUMAN
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Additional Information

Reactivity:
Human
ELISA Type:
Sandwich
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