Human IL-8 ELISA Kit (HUFI00184)- High Sensitivity

Description

Description

Human IL-8 ELISA Kit

Interleukin-8 (IL-8) is a chemokine known for its pro-inflammatory properties, actively involved in facilitating the movement of neutrophils across the vascular wall. IL-8 plays a crucial role in the host's defense mechanism by influencing the activation of neutrophils. However, it is important to note that prolonged circulation of IL-8 in response to inflammatory conditions can potentially result in varying levels of tissue damage. The Assay Genie Human IL-8 ELISA Kit is a highly sensitive assay for the quantitative measurement of IL-8 in serum, plasma, cell culture supernatant and tissue samples

Datasheet MSDS

Key Features

	Save Time	Pre-coated 96 well plate
	Quick Start	Kit includes all necessary reagents
	Publication Ready	Reproducible and reliable results

Overview


Product Name:	Human IL-8 ELISA Kit
Product Code:	HUFI00184
Size:	96 Assays
Alias:	IL-8, IL8, CXCL8, GCP-1, GCP1, LECT, LUCT, LYNAP, MDNCF, MONAP, NAF, NAP-1, NAP1, 3-10C, AMCF-I, K60
Detection Method:	Sandwich ELISA, Double Antibody
Application:	This immunoassay kit allows for the in vitro quantitative determination of Human IL-8 concentrations in serum plasma and other biological fluids.
Sensitivity:	18.75pg/ml
Range:	31.25-2000pg/ml
Storage:	4°C for 6 months
Note:	For Research Use Only

Additional Information

Recovery

Matrices listed below were spiked with certain level of Human IL-8 and the recovery rates were calculated by comparing the measured value to the expected amount of Human IL-8 in samples.


Matrix	Recovery Range (%)	Average (%)
serum(n=5)	85-105	98
EDTA plasma(n=5)	88-105	95
UFH plasma(n=5)	86-104	96

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human IL-8 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)	96-101%	88-105%	85-94%
EDTA plasma(n=5)	85-98%	82-99%	83-97%
UFH plasma(n=5)	80-100%	84-95%	82-94%

Kit Components

Component	Quantity	Storage
ELISA Microplate (Dismountable)	8x12 strips	4°C for 6 months
Lyophilized Standard	2	4°C/ -20°C
Sample/Standard Dlution Buffer	20ml	4°C
Biotin-labeled Antibody (Concentrated)	120ul	4°C (Protection 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 (Protection from light)
Stop Solution	10ml	4°C
Wash Buffer (25X)	30ml	4°C
Plate Sealer	5	-

Other materials 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

Protein Information

Heading 1	Heading 2
Uniprot	P10145
NCBI GenInfo Identifier	124359
NCBI Gene ID	3576
Molecular Weight	11,338 Da
NCBI Official Symbol	CXCL8

Protocol

*Note: Protocols are specific to each batch/lot. For the exact 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	Procedure
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.

Sample Type

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.

IL-8 Background

Interleukin-8

IL-8, classified as a pro-inflammatory chemokine, prompts the migration of neutrophils across the vascular wall. Its release is triggered by inflammatory signals from diverse cell types, indicating its multifunctional nature. IL-8 plays a crucial role in the host's defense mechanism by influencing neutrophil activation. However, in the presence of ongoing inflammatory conditions, the continuous presence of IL-8 in the bloodstream may contribute to varying degrees of tissue damage. Similar to other peptide hormones or mediators, IL-8 transmits its signals through specific cell surface receptors.

IL-8 Gene

The IL-8 gene in humans spans a length of 5191 base pairs (bp) and consists of four exons that are separated by three introns. It is located on human chromosome 4q12-q21. The messenger RNA (mRNA) derived from this gene comprises a 101-base 5' untranslated region, a 297-base open reading frame, and a lengthy 3' untranslated region measuring 1.2 kilobases (kb). Within the 5' flanking region of the IL-8 gene, there are potential binding sites for various nuclear factors, such as activated protein-1 (AP-1), activated protein-2 (AP-2), nuclear factor-gene binding (NF-kappa B), nuclear factor-interleukin-6 (NF-IL-6, also known as CAAT/enhancer-binding proteins or C/EBP), IFN regulatory factor-1 (IRF-1), hepatocyte nuclear factor-1 (HNF-1), and others. The expression of the IL-8 gene is initially regulated at the level of gene transcription.

IL-8 Receptor

The IL-8 receptor, which spans the cell membrane and consists of seven helices, is coupled with effector enzyme(s) through the involvement of heterotrimeric GTP-binding regulatory proteins. The IL-8 receptor (IL-8R) is a dimeric glycoprotein composed of a 59 KDa and a 67 KDa subunit, recognized as CDw128. It is expressed in various cell types, even those unresponsive to IL-8. Neutrophils exhibit an estimated receptor density of approximately 20,000/cell, while monocytes and T-lymphocytes have densities of 1,040/cell and 300/cell, respectively. Belonging to the G-protein-coupled receptor family, the IL-8R consists of two distinct types, namely CXCR1 and CXCR2

IL-8 ELISA Kit FAQs

What is the IL-8 ELISA Kit used for?

The Human IL-8 ELISA Kit is specifically designed for the quantitative measurement of Interleukin-8 (IL-8) levels in human samples. IL-8 is a pro-inflammatory chemokine that plays a crucial role in immune responses and inflammatory processes. This kit enables researchers and clinicians to accurately and efficiently assess IL-8 levels, aiding in the investigation of various inflammatory disorders and diseases.

What are the advantages of using the IL-8 ELISA Kit?

The IL-8 ELISA Kit offers several advantages, including high sensitivity, accuracy, and reproducibility. It provides a user-friendly and reliable method to quantify IL-8 levels in biological specimens, allowing for precise measurements and robust data analysis.

What sample types are compatible with the IL-8 ELISA Kit?

The IL-8 ELISA Kit is compatible with various sample types, including serum, plasma, cell lysates, and tissue homogenates. It provides flexibility in sample selection, allowing researchers to analyze IL-8 levels in different biological matrices

What are the storage requirements for the IL-8 ELISA Kit?

The lL-8 ELISA Kit components should be stored according to the instructions provided in the kit manual. Generally, it is recommended to store the kit components at the recommended temperature to ensure their stability and optimal performance.

What should I do if my assay results are not optimal?

If you encounter any issues or have suboptimal assay results, we recommend contacting our dedicated support team for assistance. They will be available to provide troubleshooting guidance, answer your questions, and ensure you achieve the best possible results with the IL-8 ELISA Kit.