1887

Chapter 39 : Multiplex Cytokine Assays

MyBook is a cheap paperback edition of the original book and will be sold at uniform, low price.

Preview this chapter:
Zoom in
Zoomout

Multiplex Cytokine Assays, Page 1 of 2

| /docserver/preview/fulltext/10.1128/9781555815905/9781555813642_Chap39-1.gif /docserver/preview/fulltext/10.1128/9781555815905/9781555813642_Chap39-2.gif

Abstract:

This chapter explores both traditional enzyme-linked immunosorbent assay (ELISA) and the newer multiplexed assays. For each of the methods, the author examines the technology, instrumentation, and data analysis. The sequential ELISA still has several pitfalls. First, the sample volume necessary is still greater than that used in most multiplex assays. Second, the amount of time required to measure each one of the cytokines is not reduced in the sequential ELISA, so there is no cost savings with regards to time. Third, if there is potential cross-reactivity of the antibodies, then measurement of the first cytokine will result in an overestimate of the amount of that cytokine present and measurement of the next cytokine in the sequence will result in an underestimate of the amount of that cytokine present. A recent paper described a novel method for rapidly detecting cytokines by capillary electrophoresis. Bead array assays are rapidly becoming the rage in cytokine measurement. There are several commercial vendors marketing the bead array assays The major limitation is obtaining antibody with sufficient specificity in order to prevent cross-reactivity. Finally, the volume of sample required to measure multiple cytokines is extremely small. Multiplex cytokine assays for measuring cytokines are rapidly becoming standard across the world. A clear indicator of the enthusiasm for these assays may be found in the large number of companies which are presently producing and manufacturing multiplex cytokine kits.

Citation: Remick D. 2006. Multiplex Cytokine Assays, p 340-352. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch39

Key Concept Ranking

Tumor Necrosis Factor
0.7327667
Enzyme-Linked Immunosorbent Assay
0.50970876
Flow Cytometric Assay
0.45617905
Immune Response
0.43672046
0.7327667
Highlighted Text: Show | Hide
Loading full text...

Full text loading...

Figures

Image of FIGURE 1
FIGURE 1

Direct ELISA. In the direct ELISA, the analyte is first bound to the bottom of the microtiter well. In this example, the analyte is IL-6. Unbound IL-6 is washed away, and excess protein binding sites are blocked in order to reduce background. In the next step, biotin-labeled antibody (biotin-Ab) directed against IL-6 is added; the small circle represents the biotin moiety directly attached to the antibody. After washing, streptavidin (SA) conjugated to horseradish peroxidase (HRP) is added and a final wash is performed. Following addition of a colorimetric substrate, the color develops. The intensity of the color development is directly proportional to the amount of IL-6 in the first step.

Citation: Remick D. 2006. Multiplex Cytokine Assays, p 340-352. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch39
Permissions and Reprints Request Permissions
Download as Powerpoint
Image of FIGURE 2
FIGURE 2

Indirect or sandwich ELISA. This ELISA shares many features with the assay described in the legend to Fig. 1 . The major difference is in the first step, in which an antibody (Ab) directed against the analyte is bound to the bottom of the microtiter well. This antibody is termed the capture or coating antibody. Following addition of the analyte, the biotin-labeled detection antibody (biotin-Ab) is applied, followed by horseradish peroxidase (HRP)-conjugated streptavidin (SA-HRP). Color development proceeds, and the intensity of the color is directly related to the amount of the analyte. This is termed a sandwich ELISA because the analyte is sandwiched between two antibodies.

Citation: Remick D. 2006. Multiplex Cytokine Assays, p 340-352. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch39
Permissions and Reprints Request Permissions
Download as Powerpoint
Image of FIGURE 3
FIGURE 3

Sequential ELISA. A sample containing a mixture of cytokines is applied to an IL-6 ELISA. IL-6 binds to the anti-IL-6 antibody attached to the bottom of the well. When the sample is removed, cytokines which are not IL-6, such as tumor necrosis factor (TNF), may be used in a subsequent ELISA. Ab, antibody; B, biotin moiety; biotin-Ab, biotin-labeled antibody; HRP, horseradish peroxidase; SA-HRP, horseradish peroxidase-conjugated streptavidin.

Citation: Remick D. 2006. Multiplex Cytokine Assays, p 340-352. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch39
Permissions and Reprints Request Permissions
Download as Powerpoint
Image of FIGURE 4
FIGURE 4

Increase in signal intensity with smaller spot size. On the left, each black dot represents the analyte bound to an antibody, which spreads across the entire well. On the right, the same number of antibodies are bound to the analyte, but they now occupy a smaller space, with a resulting increase in signal intensity.

Citation: Remick D. 2006. Multiplex Cytokine Assays, p 340-352. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch39
Permissions and Reprints Request Permissions
Download as Powerpoint
Image of FIGURE 5
FIGURE 5

Example of a microarray. Panel A shows six individual nitrocellulose pads arranged on a glass slide. Each pad has been spotted in an 8-by-12 format. The black area between the pads is the place where a silicon gasket was adhered to the surface to allow each of the wells to function individually, similar to the way in which an individual well in a 96-well plate is independent from its neighbors. The antibodies have been spotted in an identical manner onto each of the nitrocellulose pads. Panel B is an enlarged picture of one of the individual pads and highlights the detail of the spots. The antibodies have been spotted in quadruplicate on the pad in a vertical fashion. Each individual spot has a diameter of 150 μl, and the distance between the spot is 300 μm. The total volume delivered to each spot was 350 to 367 pl. In the far-right column of spots, those in the top eight positions are extremely bright and the lower four have virtually no signal. This line of eight plus four spots may be used for alignment of the protein chip. The intensity of the individual spots may be quantified and used to determine the cytokine concentration in the sample. This image shows excellent reproducibility of the quadruple spots.

Citation: Remick D. 2006. Multiplex Cytokine Assays, p 340-352. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch39
Permissions and Reprints Request Permissions
Download as Powerpoint
Image of FIGURE 6
FIGURE 6

Specificity of the microarray. For this microarray, 18 different cytokines were tested. A cocktail containing recombinant cytokines was prepared, but IL-12 was not added to the cocktail. There is a strong signal from all the other cytokines, but no fluorescence was observed on the array for IL-12. This demonstrates the specificity for the array.

Citation: Remick D. 2006. Multiplex Cytokine Assays, p 340-352. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch39
Permissions and Reprints Request Permissions
Download as Powerpoint
Image of FIGURE 7
FIGURE 7

Mathematical modeling of the standard curve. Data from a microarray standard curve were used to generate a mathematical model for calculations for unknown samples. In panel A, the number of relative light units (RLU) and the concentration of the cytokine (IL-8) were plotted on a linear-linear scale. The correlation coefficient (r) was not very precise. In panel B, the concentration of the IL-8 was plotted on a log scale and the number of RLU was plotted on a linear scale. This resulted in even worse correlation. In panel C, both the number of RLU and the IL-8 concentration were plotted on a log scale and a very good linear regression could be fitted. However, the optimal modeling of the curve was obtained when the number of RLU was plotted on a linear scale, the IL-8 concentration was plotted on a log scale, and a fourth-order polynomial regression line was used. The results for all four panels were obtained using the same data from the IL-8 standard curve, but similar results are observed with most other cytokines.

Citation: Remick D. 2006. Multiplex Cytokine Assays, p 340-352. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch39
Permissions and Reprints Request Permissions
Download as Powerpoint
Image of FIGURE 8
FIGURE 8

Schematic representation of the principle of a flow cytometric bead-based assay. The capture antibody specific for the analyte, in this example IL-6, is attached to a bead. The IL-6 binds to this antibody, which is then followed by a detection antibody which is biotinylated. Streptavidin conjugated to phycoerythrin is then added, and the entire complex is analyzed with the flow cytometer.

Citation: Remick D. 2006. Multiplex Cytokine Assays, p 340-352. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch39
Permissions and Reprints Request Permissions
Download as Powerpoint
Image of FIGURE 9
FIGURE 9

Example of the readout from a bead-based flow cytometry assay. In this example, the individual beads have different concentrations of two fluorescent dyes. The groups of beads are detected on the basis of the fluorescence intensities. The individual dots represent individual beads, and the darker-colored dots are doublets. The clear areas around collections of beads indicate those areas used for analysis. In this example, 22 individual cytokines have been detected. The intensity of the fluorescence of each individual bead is captured in a third fluorescent channel and compared to the standard curve in order to determine the concentration of the cytokine.

Citation: Remick D. 2006. Multiplex Cytokine Assays, p 340-352. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch39
Permissions and Reprints Request Permissions
Download as Powerpoint
Image of FIGURE 10
FIGURE 10

Example of a standard curve generated from the bead-based flow cytometry assay. In this example, a standard curve using recombinant mouse IL-1β is displayed. MFI, mean fluorescence intensity.

Citation: Remick D. 2006. Multiplex Cytokine Assays, p 340-352. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch39
Permissions and Reprints Request Permissions
Download as Powerpoint

References

/content/book/10.1128/9781555815905.ch39
1. Akalin, H.,, A. C. Akdis,, R. Mistik,, S. Helvaci, and , K. Kilicturgay. 1994. Cerebrospinal fluid interleukin-1 beta/interleukin-1 receptor antagonist balance and tumor necrosis factor-alpha concentrations in tuberculous, viral and acute bacterial meningitis. Scand. J. Infect. Dis. 26:667674.
2. Atwell, D. M.,, K. P. Grichnik,, M. F. Newman,, J. G. Reves, and , W. T. McBride. 1998. Balance of proinflammatory and antiinflammatory cytokines at thoracic cancer operation. Ann. Thorac. Surg. 66:11451150.
3. Brody, E. N.,, M. C. Willis,, J. D. Smith,, S. Jayasena,, D. Zichi, and , L. Gold. 1999. The use of aptamers in large arrays for molecular diagnostics. Mol. Diagn. 4:381388.
4. Chaouat, G.,, N. Ledee-Bataille,, S. Dubanchet,, S. Zourbas,, O. Sandra, and , J. Martal. 2004. TH1/TH2 paradigm in pregnancy: paradigm lost? Cytokines in pregnancy/early abortion: reexamining the TH1/TH2 paradigm. Int. Arch. Allergy Immunol. 134:93119. (First published 17 May 2004; doi:10.1159/000.74300.)
5. Copeland, S.,, J. Siddiqui, and , D. Remick. 2004. Direct comparison of traditional ELISAs and membrane protein arrays for detection and quantification of human cytokines. J. Immunol. Methods 284:99106.
6. Frengen, J.,, R. Schmid,, B. Kierulf,, K. Nustad,, E. Paus,, A. Berge, and , T. Lindmo. 1993. Homogeneous immunofluorometric assays of alpha-fetoprotein with macroporous, monosized particles and flow cytometry. Clin. Chem. 39:21742181.
7. Herzyk, D. J.,, A. E. Berger,, J. N. Allen, and , M. D. Wewers. 1992. Sandwich ELISA formats designed to detect 17 kDa IL-1 beta significantly underestimate 35 kDa IL-1 beta. J. Immunol. Methods 148:243254.
8. Herzyk, D. J., and , M. D. Wewers. 1993. ELISA detection of IL-1 beta in human sera needs independent confirmation. False positives in hospitalized patients. Am. Rev. Respir. Dis. 147:139142.
9. Huang, R. P. 2001. Detection of multiple proteins in an antibody-based protein microarray system. J. Immunol. Methods 255:113.
10. Huang, R. P. 2003. Protein arrays, an excellent tool in biomedical research. Front. Biosci. 8:d559d576.
11. Huang, R. P. 2001. Simultaneous detection of multiple proteins with an array-based enzyme-linked immunosorbent assay (ELISA) and enhanced chemiluminescence (ECL). Clin. Chem. Lab. Med. 39:209214.
12. Huang, R. P.,, R. Huang,, Y. Fan, and , Y. Lin. 2001. Simultaneous detection of multiple cytokines from conditioned media and patients’ sera by an antibody-based protein array system. Anal. Biochem. 294:5562.
13. Iannone, M. A.,, J. D. Taylor,, J. Chen,, M. S. Li,, P. Rivers,, K. A. Slentz-Kesler, and , M. P. Weiner. 2000. Multiplexed single nucleotide polymorphism genotyping by oligonucleotide ligation and flow cytometry. Cytometry 39:131140.
14. Iannone, M. A.,, J. D. Taylor,, J. Chen,, M. S. Li,, F. Ye, and , M. P. Weiner. 2003. Microsphere-based single nucleotide polymorphism genotyping. Methods Mol. Biol. 226:123134.
15. Kellar, K. L., and , J. P. Douglass. 2003. Multiplexed microsphere-based flow cytometric immunoassays for human cytokines. J. Immunol. Methods 279:277285.
16. [Reference deleted.]
17. Khan, S. S.,, M. S. Smith,, D. Reda,, A. F. Suffredini, and , J. P. McCoy, Jr. 2004. Multiplex bead array assays for detection of soluble cytokines: comparisons of sensitivity and quantitative values among kits from multiple manufacturers. Cytometry 61B:3539.
18. Lewkowich, I. P.,, J. D. Campbell, and , K. T. HayGlass. 2001. Comparison of chemiluminescent assays and colorimetric ELISAs for quantification of murine IL-12, human IL-4 and murine IL-4: chemiluminescent substrates provide markedly enhanced sensitivity. J. Immunol. Methods 247:111118.
19. Lin, Y.,, R. Huang,, N. Santanam,, Y. G. Liu,, S. Parthasarathy, and , R. P. Huang. 2002. Profiling of human cytokines in healthy individuals with vitamin E supplementation by antibody array. Cancer Lett. 187:1724.
20. Martinon, F., and , J. Tschopp. 2004. Inflammatory caspases: linking an intracellular innate immune system to autoinflammatory diseases. Cell 117:561574.
21. McHugh, T. M. 1994. Flow microsphere immunoassay for the quantitative and simultaneous detection of multiple soluble analytes. Methods Cell Biol. 42:575595.
22. Mendoza, L. G.,, P. McQuary,, A. Mongan,, R. Gangadharan,, S. Brignac, and , M. Eggers. 1999. High-throughput microarray-based enzyme-linked immunosorbent assay (ELISA). BioTechniques 27:778780, 782786, 788.
23. Moody, M. D.,, S. W. Van Arsdell,, K. P. Murphy,, S. F. Orencole, and , C. Burns. 2001. Array-based ELISAs for high-throughput analysis of human cytokines. BioTechniques 31:186190, 192194.
24. Morgan, E.,, R. Varro,, H. Sepulveda,, J. A. Ember,, J. Apgar,, J. Wilson,, L. Lowe,, R. Chen,, L. Shivraj,, A. Agadir,, R. Campos,, D. Ernst, and , A. Gaur. 2004. Cytometric bead array: a multiplexed assay platform with applications in various areas of biology. Clin. Immunol. 110:252266.
25. Nemzek, J. A.,, J. Siddiqui, and , D. G. Remick. 2001. Development and optimization of cytokine ELISAs using commercial antibody pairs. J. Immunol. Methods 255:149157.
26. Nielsen, U. B., and , B. H. Geierstanger. 2004. Multiplexed sandwich assays in microarray format. J. Immunol. Methods 290:107120.
27. Obenauer-Kutner, L. J.,, S. J. Jacobs,, K. Kolz,, L. M. Tobias, and , R. W. Bordens. 1997. A highly sensitive electrochemiluminescence immunoassay for interferon alfa-2b in human serum. J. Immunol. Methods 206:2533.
28. O’Connor, K. A.,, A. Holguin,, M. K. Hansen,, S. F. Maier, and , L. R. Watkins. 2004. A method for measuring multiple cytokines from small samples. Brain Behav. Immun. 18:274280.
29. Olson, J. A., Jr. 2004. Application of microarray profiling to clinical trials in cancer. Surgery 136:519523.
30. Pei, R.,, J. Lee,, T. Chen,, S. Rojo, and , P. I. Terasaki. 1999. Flow cytometric detection of HLA antibodies using a spectrum of microbeads. Hum. Immunol. 60:12931302.
31. Petrovas, C.,, S. M. Daskas, and , E. S. Lianidou. 1999. Determination of tumor necrosis factor-alpha (TNF-alpha) in serum by a highly sensitive enzyme amplified lanthanide luminescence immunoassay. Clin. Biochem. 32:241247.
32. Phillips, T. M. 2004. Rapid analysis of inflammatory cytokines in cerebrospinal fluid using chip-based immunoaffinity electrophoresis. Electrophoresis 25:16521659.
33. Prabhakar, U.,, E. Eirikis, and , H. M. Davis. 2002. Simultaneous quantification of proinflammatory cytokines in human plasma using the LabMAP assay. J. Immunol. Methods 260:207218.
34. Prabhakar, U.,, E. Eirikis,, M. Reddy,, E. Silvestro,, S. Spitz,, C. Pendley II,, H. M. Davis, and , B. E. Miller. 2004. Validation and comparative analysis of a multiplexed assay for the simultaneous quantitative measurement of Th1/Th2 cytokines in human serum and human peripheral blood mononuclear cell culture supernatants. J. Immunol. Methods 291:2738.
35. Remick, D. G. 2003. Cytokine therapeutics for the treatment of sepsis: why has nothing worked? Curr. Pharm. Des. 9:7582.
36. Remick, D. G., and , J. S. Friedland (ed.). 1997. Cytokines in Health and Disease, 2nd ed., revised and expanded. Marcel Dekker, Inc., New York, N.Y.
37. Remick, D. G.,, G. R. Bolgos,, J. Siddiqui,, J. Shin, and , J. A. Nemzek. 2002. Six at six: interleukin-6 measured 6 h after the initiation of sepsis predicts mortality over 3 days. Shock 17:463467.
38. Roda, A.,, M. Guardigli,, C. Russo,, P. Pasini, and , M. Baraldini. 2000. Protein microdeposition using a conventional ink-jet printer. BioTechniques 28:492496.
39. Rongen, H. A.,, H. M. van der Horst,, A. J. van Oosterhout,, A. Bult, and , W. P. van Bennekom. 1996. Application of xanthine oxidase-catalyzed luminol chemiluminescence in a mouse interleukin-5 immunoassay. J. Immunol. Methods 197:161169.
40. Schweitzer, B.,, S. Roberts,, B. Grimwade,, W. Shao,, M. Wang,, Q. Fu,, Q. Shu,, I. Laroche,, Z. Zhou,, V. T. Tchernev,, J. Christiansen,, M. Velleca, and , S. F. Kingsmore. 2002. Multiplexed protein profiling on microarrays by rollingcircle amplification. Nat. Biotechnol. 20:359365.
41. Siddiqui, J., and , D. G. Remick. 2003. Improved sensitivity of colorimetric compared to chemiluminescence ELISAs for cytokine assays. J. Immunoassay Immunochem. 24:273283.
42. Spieles, G.,, E. Grossi,, A. Kishbaugh,, K. Johnson,, R. Calamunci,, G. Sigal,, S. Leytner, and , J. N. Wohlstadter. 2004, posting date. Multiplex Measurements of Cytokines in High Density Formats Using Multi-Array Technology. [Online.] http://www.meso-scale.com/CatalogSystemWeb/WebRoot/literature/applications/pdf/MultiplexCyt_2003.pdf.
43. Sreekumar, A.,, M. K. Nyati,, S. Varambally,, T. R. Barrette,, D. Ghosh,, T. S. Lawrence, and , A. M. Chinnaiyan. 2001. Profiling of cancer cells using protein microarrays: discovery of novel radiation-regulated proteins. Cancer Res. 61:75857593.
44. Swartzman, E. E.,, S. J. Miraglia,, J. Mellentin-Michelotti,, L. Evangelista, and , P. M. Yuan. 1999. A homogeneous and multiplexed immunoassay for high-throughput screening using fluorometric microvolume assay technology. Anal. Biochem. 271:143151.
45. Tonkinson, J. L., and , B. A. Stillman. 2002. Nitrocellulose: a tried and true polymer finds utility as a postgenomic substrate. Front. Biosci. 7:c1c12.
46. Turnbull, I. R.,, P. Javadi,, T. G. Buchman,, R. S. Hotchkiss,, I. E. Karl, and , C. M. Coopersmith. 2004. Antibiotics improve survival in sepsis independent of injury severity but do not change mortality in mice with markedly elevated interleukin 6 levels. Shock 21:121125.
47. Vignali, D. A. 2000. Multiplexed particle-based flow cytometric assays. J. Immunol. Methods 243:243255.
48. Wiese, R.,, Y. Belosludtsev,, T. Powdrill,, P. Thompson, and , M. Hogan. 2001. Simultaneous multianalyte ELISA performed on a microarray platform. Clin. Chem. 47:14511457.
49. Yalow, R. S., and , S. A. Berson. 1959. Assay of plasma insulin in human subjects by immunological methods. Nature 184:16481649.
50. Zhu, H., and , M. Snyder. 2003. Protein chip technology. Curr. Opin. Chem. Biol. 7:5563.

Tables

Generic image for table
TABLE 1

Cost analysis for cytokine measurements

Citation: Remick D. 2006. Multiplex Cytokine Assays, p 340-352. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch39
Generic image for table
TABLE 2

Sliding-scale cost of measuring cytokines

Citation: Remick D. 2006. Multiplex Cytokine Assays, p 340-352. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch39
Generic image for table
TABLE 3

Sample volumes required for performing assays

Citation: Remick D. 2006. Multiplex Cytokine Assays, p 340-352. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch39

This is a required field
Please enter a valid email address
Please check the format of the address you have entered.
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error