QUANTITATIVE DETERMINATION NEW PRODUCT OF HUMAN -8

Human Interleukin-8 ELISA

Sensitivity (0.5 pg/ml) Very good analytical characteristics Validated for human serum, plasma (EDTA, citrate, heparin) and saliva samples Preliminary population data

CYTOKINES, AND RELATED MOLECULES , INFECTION AND ONCOLOGY ∙ PERIODONTITIS ∙ SEPSIS HUMAN INTERLEUKIN-8 ELISA

Introduction

Interleukin (IL)-8, a member of the C-X-C gastric, lung, melanoma, mesothelioma, ovarian, prostate, subfamily, is a key mediator of inflammation. It is a product renal, and thyroid) and hematological malignancies (AML, of the CXCL8 mapped to 4, namely CLL, Hodgkin’s lymphoma) [7]. Similarly, a close association 4q.12-q12. It is 3211 bp in length and contains 4 exons. between high serum IL-8 expression and disease progression The is initially produced as a precursor peptide of has been shown in several clinical studies of breast, colon, 99 amino acids which then undergoes cleavage to create ovarian, and prostate cancers, as well as in melanoma [7]. several active IL-8 isoforms, the predominant variants In various cancers, high IL-8 levels have been identified as a consist of 77 and/or 72 amino acids residues [1, 2]. IL-8 is prognostic factor and as a possible treatment target through expressed by several cell types such as activated monocytes inhibition of IL-8 production and/or blockage of IL-8 receptors and , T cells, , NK cells and also [8]. endothelial cells, wide variety of epithelial cells and fibroblasts [3]. Interleukin-8 is a multifunctional mediator associated with inflammation where it plays a key role in recruitment of (IL-8/CXCL8), also known as leukocytes to sites of infection or tissue injury [4]. In addition chemotactic factor, has several main functions: to induce to cancer, elevated serum and plasma interleukin-8 (IL-8) in target cells (primarily neutrophils but also levels were observed in women with severe or milder pre- other granulocytes, causing them to migrate toward the site eclampsia [9, 10] in critically-ill patients within septic shock of infection); to induce phagocytosis (once they have arrived) onset [11], in several chronic inflammatory diseases, such as and to promote [4]. IL-8 mediates its biological , rheumatoid arthritis, as well as pulmonary diseases functions by interacting with two homologous specific [12] or in biliary atresia where IL-8 plays an important role in G-protein-coupled CXC chemokine receptors (CXCR1 and disease inflammation and progression [13]. CXCR2) [5]. Under normal conditions the expression of IL-8 is very low or undetectable. The IL-8 expression is significantly Interleukin 8 (IL-8) has been studied for several years and has increased at early inflammatory phase and remains active for been revealed as a promising putative marker for many clinical a long period of time at the site of inflammation [6]. conditions, and its level is currently being applied by various subspecialties of medicine either for the purpose of rapid An increased IL-8 expression has been detected in numerous diagnosis or as a predictor of prognosis. cancers, including solid tumors (brain, breast, cervical, colon, HUMAN INTERLEUKIN-8 ELISA QUANTITATIVE DETERMINATION OF HUMAN INTERLEUKIN-8

BioVendor Human Interleukin-8 ELISA (RD194558200R)

Intended use Test principle The RD194558200R Human Interleukin-8 ELISA is a sandwich In the BioVendor Human Interleukin-8 ELISA, standards and enzyme immunoassay for the quantitative measurement of samples are incubated in a microtitrate plate wells pre-coated native human Interleukin-8 Protein. with monoclonal anti-human IL-8 antibody. After 60 minutes incubation and a washing, biotin-labelled monoclonal anti- It is intended for research use only human IL-8 antibody is added and incubated with captured The total assay time is less than 4 hours IL-8 for 60 minutes. After another washing, the streptavidin- The kit measures total human IL-8 in serum, HRP conjugate is added. After 60 minutes incubation and the plasma (EDTA, citrate, heparin) and saliva last washing step, the remaining conjugate is allowed to react Assay format is 96 wells with the substrate solution (TMB). The reaction is stopped by addition of acidic solution and absorbance of the resulting Standard is purified E.coli protein yellow product is measured. The absorbance is proportional Components of the kit are provided ready to use, to the concentration IL-8. A standard curve is constructed concentrated or lyophilized by plotting absorbance values against concentrations of IL-8 standards, and concentrations of unknown samples are determined using this standard curve. Clinical application Immune Response, Infection and Inflammation Oncology Periodontitis Sepsis

3.5 HUMAN INTERLEUKIN-8 ELISA CAT. NO.: RD194558200R 3.0 Assay format Sandwich ELISA, Biotin-labelled 2.5 antibody, 96 wells/kit Samples Serum, Plasma (EDTA, citrate, 2.0 heparin) and Saliva 1.5 Standards 1.6 to 100 pg/ml 1.0 Limit of detection 0.5 pg/ml Absorbance at 450 nm 0.5

0.0 0.11 10 100 IL-8 (pg/ml) HUMAN INTERLEUKIN-8 ELISA

Precision Intra-assay (Within-Run) (n=8) Inter-assay (Run-to-Run) (n=7) Sample Mean SD CV Sample Mean SD CV (pg/ml) (pg/ml) (%) (pg/ml) (pg/ml) (%) Serum 1 19.1 1.0 5.2 1 30.7 2.0 6.1 Serum 2 35.1 1.3 3.7 2 37.0 3.0 8.2

Spiking recovery Linearity Serum samples were spiked with different amounts of human Serum, plasma and saliva samples were serially diluted with IL-8 and assayed. Dilution Buffer and assayed. Sample Observed Expected Recovery O/E Sample Dilution Observed Expected Recovery O/E (pg/ml) (pg/ml) (%) (pg/ml) (pg/ml) (%) 41.7 - - 71.6 - - 61.4 60.3 101.8 2x 37.3 35.8 104.3 Serum 1 Serum 1 75.6 79.5 95.2 4x 17.6 17.9 98.5 108.2 116.7 92.7 8x 8.6 8.6 95.8 42.9 - - - 68.0 - - 59.2 61.5 96.2 2x 34.0 34.0 100.1 Serum 2 Serum 2 72.0 80.7 89.2 4x 17.1 17.0 100.3 109.3 117.9 92.7 8x 8.7 8.5 102.7

Sample Dilution Observed Expected Recovery O/E (pg/ml) (pg/ml) (%) 1110.8 - - 2x 526.6 555.4 94.8 Saliva 1 4x 260.3 277.7 93.7 8x 133.0 138.9 95.8 - 451.1 - - 2x 225.4 225.6 99.9 Saliva 2 4x 111.4 112.8 98.8 8x 54.1 56.4 95.9

Effect of sample matrix 40 ¢ Serum ¢ EDTA Plasma ¢ Citrate Plasma ¢ Heparin Plasma EDTA, citrate and heparin plasma samples were compared 35 to respective serum samples from the same 11 individuals. Results are shown below: 30

25

20

15

10 Concentration of human IL-8 (pg/ml)

5

0 132 45678910 11 Volunteers QUANTITATIVE DETERMINATION OF HUMAN INTERLEUKIN-8

Preliminary population data

The following results were obtained when serum samples from 155 unselected donors (89 men + 66 women) 20–65 years old were assayed with the BioVendor Human IL-8 ELISA in our laboratory. Age and Sex Dependent Distribution of Interleukin-8

Sex Age n Mean Median SD Min. Max. (years) IL-8 (pg/ml) IL-8 (pg/ml) IL-8 (pg/ml) IL-8 (pg/ml) IL-8 (pg/ml) 20-29 17 18.8 28.7 12.9 2.3 60.4 Male 30-39 25 19.3 14.0 15.9 5.0 84.2 40-49 31 17.9 17.1 12.7 2.0 70.1 50-65 16 13.4 8.9 11.3 4.1 50.7 20-29 12 15.8 15.5 9.9 0.0 32.1 Female 30-39 26 17.3 13.6 21.0 0.0 107.9 40-49 20 21.1 12.6 32.2 0.0 150.2 50-61 8 13.4 9.6 8.4 5.3 29.0

160 Men Women 140

120

100

80

60

40

20 Concentration of human IL-8 (pg/ml ) 0 0310 20 04050760 0 Age (years)

Summary of protocol • Reconstitute Master Standard and prepare set of Standards • Add 100 μl Streptavidin – HRP Conjugate • Dilute samples (serum 6x, plasma 3x, saliva 20x) • Incubate at RT for 1 hour/300 rpm • Add 100 µl Standards and samples • Wash plate 3 times • Incubate at RT for 1 hour/300 rpm • Add 100 μl Substrate Solution • Wash plate 3 times • Incubate at RT for 10 min • Add 100 μl Biotin Labelled Antibody solution • Add 100 μl Stop Solution • Incubate at RT for 1 hour/300 rpm • Read absorbance and calculate results • Wash plate 3 times References

1. Baggiolini, M. and I. Clark-Lewis, Interleukin-8, a chemotactic and inflammatory . FEBS Lett, 1992. 307(1): p. 97-101. 2. Matsushima, K. and J.J. Oppenheim, Interleukin 8 and MCAF: novel inflammatory cytokines inducible by IL 1 and TNF. Cytokine, 1989. 1(1): p. 2-13. 3. Mukaida, N., et al., Properties of pro-inflammatory cell type-specific leukocyte chemotactic cytokines, interleukin 8 (IL-8) and monocyte chemotactic and activating factor (MCAF). Microbiol Immunol, 1992. 36(8): p. 773-89. 4. David, J.M., et al., The IL-8/IL-8R Axis: A Double Agent in Tumor Immune Resistance. Vaccines (Basel), 2016. 4(3). 5. Holmes, W.E., et al., Structure and functional expression of a human interleukin-8 receptor. Science, 1991. 253(5025): p. 1278-80. 6. Brat, D.J., A.C. Bellail, and E.G. Van Meir, The role of interleukin-8 and its receptors in gliomagenesis and tumoral angiogenesis. Neuro Oncol, 2005. 7(2): p. 122-33. 7. Xie, K., Interleukin-8 and human cancer biology. Cytokine Rev, 2001. 12(4): p. 375-91. 8. Zarogoulidis, P., et al., Interleukin-8 and interleukin-17 for cancer. Cancer Invest, 2014. 32(5): p. 197-205. 9. Sharma, A., A. Satyam, and J.B. Sharma, Leptin, IL-10 and inflammatory markers (TNF-alpha, IL-6 and IL-8) in pre-eclamptic, normotensive pregnant and healthy non-pregnant women. Am J Reprod Immunol, 2007. 58(1): p. 21-30. 10. Kauma, S., et al., Increased endothelial monocyte chemoattractant protein-1 and interleukin-8 in preeclampsia. Obstet Gynecol, 2002. 100(4): p. 706-14. 11. Livaditi, O., et al., Neutrophil CD64 expression and serum IL-8: sensitive early markers of severity and outcome in sepsis. Cytokine, 2006. 36(5-6): p. 283-90. 12. Palomino, D.C. and L.C. Marti, Chemokines and immunity. Einstein (Sao Paulo), 2015. 13(3): p. 469-73. 13. Dong, R. and S. Zheng, Interleukin-8: A critical chemokine in biliary atresia. J Gastroenterol Hepatol, 2015. 30(6): p. 970-6.

Contact Information

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Date of issue November 2016 about BioVendor products.