Human IL-18 ELISA Kit

RACTIVITY	Human
SENSITIVITY	3.25 pg/mL
ASSAY RANGE	31.25 - 2000 pg/mL
REAGENTS PROVIDED	ANTI HUMAN IL-18 COATED MICROTITER PLATE ANTI Human IL-18 BIOTIN CONJUGATE AVIDIN-HRP CONJUGATE HUMAN PD-1 STANDARD - 2000 pg/vial CALIBRATOR DILUENT I CALIBRATOR DILUENT I TMB SUBSTRATE STOP SOLUTION

INTENDED USE

This human IL-18 ELISA kit is to be used for the in vitro quantitative determination of human interleukin-18 (IL-18) concentrations in cell culture supernatant and other biological fluids. This kit is intended for LABORATORY RESEARCH USE ONLY.

 

INTRODUCTION

 IL-18 was first identified and cloned as an interferon-g (IFN-g) inducing factor from a murine liver cell cDNA library after challenging the animal with heat inactivated bacteria and lipopolysaccharide⁸.  It is categorized as pro-inflammatory cytokine based on its structural similarity to the interleukin 1 superfamily and its function in modulating inflammatory immune-response.  IL-18 is constitutively expressed as a precursor in nearly all cells in human and animals.  Sequencing human IL-18 revealed 65% homology with murine IL-18 and an unusual 35 amino acid leader peptide at its N-terminus.  Similar to IL-1β, IL-18 precursor is processed by caspase-1 into the active form.  The primary source of IL-18 is macrophages and dendritic cells and its secretion is increased by IFN-g stimulation.  Circulating IL-18 presents in healthy individuals at substantial levels.  

IL-18 can act with IL-12 synergistically to stimulate T cells and natural killer cells to produce IFN-g and augment T-helper 1 type immune-response¹¹.  It is also be shown that IL-12 and IL-18 induces anti-CD40-activated B cells to produce IFN-g,  inhibits IL-4 dependent IgE and IgG1 production and enhances IgG2a production without inhibiting the B cell proliferative response¹².

IL-18 acts through binding to its receptor IL-18Ra.  Similar to that of IL-1b receptor complex, an accessory protein, known as IL-18 receptor b, can increase the responsiveness of IL-18Ra to IL-18 significantly.  Experiments have shown that IL-12 and IL-1b can increase the expression of IL-18 receptor, and IL-18 receptor is selectively expressed on T-helper 1 cells, but not on T-helper 2 cells.  Resembling that of IL-1, the IL-18 signal transduction involved with the sequential recruitment of MyD88, the four IRAKs and TRAF-6, followed by the degradation of IκB and release of NFκB².  The IL-18 activation of the receptor requires high ligand concentration at 10–20 ng/mL and sometime higher levels, which is a unique feature of this cytokine². 

The function of IL-18 can be neutralized by a constitutively expressed and secreted IL-18 binding protein¹ (IL-18BP). IL-18BP has an immunoglobulin-like structure similar to soluble IL-1 receptor. However, IL-18BP does not have a transmembrane domain.   Through binding to IL-18, IL-18BP suppresses the production of IFN-g, resulting in reduced T-helper type 1 immune responses.

It has been reported that up-regulated IL-18 expression was implicated with autoimmune diseases (Crohn’s disease⁷, Hashimoto’s thyroiditis⁶, Eczema⁵ etc.) and inflammatory conditions (increased risk of atherosclerotic plaque rupture and cardiovascular event in chronic kidney patients⁴, Alzheimer’s disease⁹, age-related macular degeneration³).   IL-18 may play a role in cancer’s immune-evasion through inducing PD-1 expression in mature nature killer cells¹⁰.

The IL-18 ELISA kit is for measurement of IL-18 in cell culture and other biological samples.  The kit is for research only.  The detected value of IL-18 can only be used as reference for scientific study.  The kit should not be used in any diagnostic or therapeutic procedures.

PRINCIPLE OF THE ASSAY

This enzyme-linked immunosorbent assay (ELISA) applies a technique called a quantitative sandwich immunoassay.  The microtiter plate provided in this kit has been pre-coated with a monoclonal antibody specific for human IL-18.  When standards or samples are added to the appropriate microtiter plate wells, human IL-18 in the standards or samples will be immobilized by the pre-coated antibody during incubation.  A biotin-conjugated antibody preparation specific for human IL-18 is added to each well and incubated.  The biotin labelled antibody attaches to the wells by binding to human IL-18.  After plate washing, other proteins, components and unattached biotin labelled antibody is removed.  Then, avidin-horseradish peroxidase (HRP) conjugate is added to each well.  Avidin has a very high affinity for biotin, thus, it links the tracer (HRP) sturdily to the biotin labelled antibody.  The wells are thoroughly washed to remove all unbound avidin-HRP conjugate and a TMB (3,3’,5,5' tetramethyl-benzidine) substrate solution is added to each well.  The enzyme (HRP) and substrate are allowed to react over a short incubation period.  Only wells that contain human IL-18 will exhibit a change in colour.  The extent of colour change is proportional to the quantity of human IL-18 presented in the standards/samples.  The enzyme-substrate reaction is terminated by the addition of a sulphuric acid solution and the colour change is measured spectrophotometrically at a wave length of 450 nm ± 2 nm.

According to the testing system, the provided standard is diluted (2-fold) with the appropriate diluent and assayed at the same time as the samples.  This allows the operator to produce a curve of Optical Density (O.D.) versus human IL-18 concentration (pg/mL) in standards.  The concentration of human IL-18 in the samples is then determined by comparing the O.D. of the samples to the standard curve and multiplying with sample dilution factor.

 

REFERENCES

1. Dinarello C, Novick D, Kim S, and Kaplanski G.  Interleukin-18 and IL-18 binding protein.  Front. Immunology. 2013; 4:289

2. Dinarello C, Giamila F.  Interleukin-18 and Host Defense against Infection.  The Journal of Infectious Diseases.  2003; 187(2)

3. Doyle SL, Ozaki E, Brennan K, Humphries M, Mulfaul K, Keaney J, Kenna P, Maminishkis A, Kiang AS, Saunders SP, Hams E, Lavelle EC, Gardiner C, Fallon PG, Adamson P, Humphries P and Campbell M.   IL-18 attenuates experimental choroidal neovascularization as a potential therapy for wet age-related macular degeneration.  Science Translational Medicine.  2014; 6(230) 230-44

4. Formanowicz D, Wanic-Kossowska M, Pawliczak E, Radom M and Formanowicz P.  Usefulness of serum interleukin-18 in predicting cardiovascular mortality in patients with chronic kidney disease – systems and clinical approach.  Nature, Scientific Reports 2015; Article number: 18332

5. Hu YL, Wang JL, Zhang HY, Xie H,  Song WW, Jiang QJ, Zhao N, and He SH.  Enhanced expression of IL-18 and IL-18BP in plasma of patients with Eczema: Altered expression of IL-18BP and IL-18 receptor on Mast Cells.  Mediators of Inflammation.  2017; Article ID 3090782

6. Liu Z, Wang H, Xiao W, Wang C, Liu G, Hong T.  Thyrocyte interleukin-18 expression is up-regulated by interferon-γ and may contribute to thyroid destruction in Hashimoto's thyroiditis". Int J Exp Pathol. 2010; 91 (5): 420–51

7. Monteleone G1, Trapasso F, Parrello T, Biancone L, Stella A, Iuliano R, Luzza F, Fusco A, Pallone F.  Bioactive IL-18 expression is up-regulated in Crohn's disease.  J Immunol. 1999  ;163(1):143-7.

8. Okamura H, Tsutsi H, Komatsu T, Yutsudo M, Hakura A, Tanimoto T, Torigoe K, Okura T, Nukada Y, Hattori K.  Cloning of a new cytokine that induces IFN-gamma production by T cells. Nature. 1995; 378 (6552): 88–9

10. Ojala J, Alafuzoff I, Herukka SK, van Groen T, Tanila H, Pirttilä T.  Expression of interleukin-18 is increased in the brains of Alzheimer's disease patients.  Neurobiol Aging. 2009; 30(2):198-209.

11. Terme M, Ullrich E, Aymeric L, Meinhardt K, Desbois M, Delahaye N, Viaud S, Ryffel B, Yagita H,  Kaplanski G, Prévost-Blondel A,  Kato M, Schultze J, Tartour E, Kroemer G, Chaput N and Zitvogel L.  IL-18 Induces PD-1–Dependent Immunosuppression in Cancer.  Cancer Research 2011; DOI: 10.1158/0008-5472.CAN-11-0993

12. Tominaga K1, Yoshimoto T, Torigoe K, Kurimoto M, Matsui K, Hada T, Okamura H, Nakanishi K. IL-12 synergizes with IL-18 or IL-1beta for IFN-gamma production from human T cells.  Int Immunol. 2000; 12(2):151-60.

13. Yoshimoto T, Okamura H, Tagawa YI, Iwakura Y, Nakanishi K.  Interleukin 18 together with interleukin 12 inhibits IgE, production by induction of interferon-gamma production from activated B cells.  Proc. Natl. Acad. Sci.  1997; 94:3948–53

14. Wang J, Guo M, Ma R, Wu M, Zhang Y.Tetrandrine alleviates cerebral ischemia/reperfusion injury by suppressing NLRP3 inflammasome activation via Sirt-1. PeerJ 2020; (8):e9042.