Index. Index 439. Aequorin, 84, 94 Affinity precipitation, 372, AP-1, 100 Asthma, 170, 305

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Index 439 Index A Aequorin, 84, 94 Affinity precipitation, 372, 376 381 AP-1, 100 Asthma, 170, 305 B Bioassay, 185, comparison with ELISA, 318 GM-CSF bioassay, 351 IL-2 bioassay, 185 192, 300 IL-3 IL-6 IL-8 bioassay, 350 IL-15 bioassay, 201 204 interleukins in synovial fluid, 329 330 TNF Blister fluids, 313, collection, 316 318 cytokine concentrations in, 319 passage of molecules into, 315 316 pathological, 313 suction blister fluids, 315 Bradford assay, 404 Breast cancer, 207 Brefeldin A, 44 Bullous dermatoses, 314 315 C C/EBP, 100 Celiac disease, 67 Cerebrospinal fluid, 337, 338, assaying interleukins in, 337 344 Circadian rhythmicity in cytokine levels, 163 168, see also Interleukins Chemiluminescence, 136 137 Chemokines, 125, 129, 138 Chronic obstructive pulmonary disease, 305 Confocal microscopy, 386 390 CRE, 100 CTLL-2 cells, 185 192, effect of cryopreservation, 192 sensitivity over time, 191 Cystic fibrosis, 305 D Depressive illness, 217, cytokine levels, 217 222 E Electrophoresis, 103, 107 Electroporation, 386, 401 ELISA, 3 15, assaying IL-8, 177 183 comparison with bioassay, 318 history, 3, 264 267 mrna ELISAs, 13 overview, 3 5 reagent suppliers, 13 sandwich ELISA, 6 13, 266 267 substrates, 12 ELISPOT, 17 28, 29 39, dual color analysis, 29 39 enumeration of interleukin-secreting cells, 23 24 principals, 17 439

440 Index stardust assay, 32 39 Enzyme-linked immunosorbent assay, see ELISA F FACS, see Flow cytometry Flash-type bioluminescence, 83 97, advantages, 92 93 as an alternative to radioactive methodologies, 94 bioluminescent hybridization assays, 85 93 principals of, 83 84 Flow cytometry, 41 48, assaying intracellular interleukins, 41 48, 288 290 principals, 41 websites, 43 G G-CSF, 129 Gene arrays, see Microarrays Glycoslation analysis, 404 GM-CSF, 164, 346, bioassay, 351 Grave s disease, 251 255, assaying interleukins in, 251 261 Green fluorescent protein, applications in cell signaling, 385 391 H Hyopthalamic-pituitary-adrenal axis, 217 I Interleukins, Interleukin-1 (IL-1), 129, 157, 163, bioassay, 299, membrane bioassay, 299 circadian variation in IL-1, 164 167 in depression, 218 222, 227 230 in seminal plasma, 293 294, 297, 299, 330, 324 single nucleotide polymorphism in IL-1 Interleukin-1 receptor antagonist (IL-1Ra), 129, in seminal plasma, 293, 295 Interleukin-2 (IL-2), 34, 90 92, 163, 185 192, 193, assaying by bioassay, 185 192, 300 assaying by ELISPOT stardust assay, 34 assaying by flash-type bioluminescence, 90 97 in depression, 218, 221, 222, 226 signal transduction, 369 371 Interleukin-2 (IL-2) receptor, 150, 152, 163, circadian variation, 164 Interleukin-3 (IL-3), 253, Interleukin-4 (IL-4), 20, 157, 164, assaying by ELISPOT stardust assay, 34 Interleukin-5 (IL-5), 20, 164,

Index 441 in sputum, 305, 307 use of proteases to improve recovery, 307, 311 Interleukin-6 (IL-6), 111 112, 114 116, 119 123, 163, 164, circadian variation, 164 167 ELISA, 209 216 in breast cancer, 207 216 in depressive illness, 218, 221, 222, 225 in seminal plasma, 293, 295, 297, 299, 329 in tears, 346 mrna detection using Xplore assay, 111 124 Interleukin-8 (IL-8), 129, assaying IL-8, 177 183 bioassay, 350 in sputum, 305 in tears, 346, 350, 351 roles, 177 178 Interleukin-10 (IL-10), 20, 157, 164, in psoriasis, 240 242 in seminal plasma, 293 294 Interleukin-12 (IL-12), 129, 157, in seminal plasma, 293 294 Interleukin-13 (IL-13), 253, activation of phosphoinositide-3- kinase by, 363, 365 Interleukin-15 (IL-15), 193 204, assaying by ELISA, 194, 199 201 assaying by RT-PCR, 197 199 bioassay, 201 204 Interleukin-17 (IL-17), 396 Interleukin-18 (IL-18), 157, Immunoaffinity-receptor affinity chromatography, 145 156, column construction, 145, 149 fluorochrome labeling of samples, 150 principles, 145 Immunoassays, 263 history of, 263 267 interleukins in synovial fluid, 330 331 Immunohistochemistry, 157 162 embedding, 159 fixation, 160 staining of cells in tissue culture, 160, 309 311 tissue processing, 159 Interferon-, 67, 163, detection by quantitative RT-PCR in celiac disease, 67 detection by RNAse protection, 129 circadian variation, 164 167 J J774 cells, 121 L Luminometry, 84 85 Lyophilization, 405 M MCP-1, 328

442 Index M-CSF, 328, assaying in cerebrospinal fluid, 337 344 Messenger RNA, see mrna Mononuclear cells, 21 23, mucosal mononuclear cells: isolation, 22 23 separation from PBMCs, 21, 426 427 Microarrays, 419 438, commercially available arrays, 423 424 comparison of gene array techniques, 422 cdna arrays, 419 438, principal of assay, 419 420 hybridization, 430 431 overview of cdna array for cytokine detection, 421 quantification, 434 types of DNA arrays, 420 Migraine, 268, assaying interleukins in, 268 272 recruitment of patients, 268 269 mrna, 51 52, measurement by nonradioactive Northern blotting, 99 109 measurement by RNAse protection assay, 125 143 preparation of digoxigenin-labeled probes, 102, 106 quantitation by flash-type bioluminescence, 83 98 standards for quantification of cytokines, 65 total RNA extraction, 51, 63 64, 68 69, design of probes, 116 from biopsy material, 63 from cartilage, 102, 105 from cells in culture, 101, 104 from skin, 243, 246 from synovial membranes, 101, 104 105 mechanism, 113 preparation of standards, 114 Xplore assay, 111 124 Myocardial infarction, acute, 273 role of interleukins, 273 275 testing strategy for interleukins, 275 N NFAT, 100 NF- B, 100 NK cells, 170 Northern blotting, 99 109, hybridization and detection, 108 nonradioactive detection method, 99 109 P Peritoneal fluids, 285 286, assaying cytokines in, 285 291 sample collection, 286 Phosphoinositide-3-kinase, 357, assays, 359 368 classes, 357 358 Phosphotyrosine analysis, 373 Phosphospecific antibodies, 374 375 Preanalytical factors in interleukin assays, 276 Psoriasis, 239, assaying interleukins in, 239 250 cytokine concentration in sera, 319 Q Quantitative RT-PCR, 56, 59 76, standards used, 66 principals, 60 61 R Ras, affinity precipitation of, 376 377 Recombinant cytokine protein production, 393 407, use of Pichia pastoris, 393 407, cytokines produced, 394

Index 443 Reverse transcriptase polymerase chain reaction, see RT-PCR RNAse protection assay, 125 143, advantages of multiprobe method, 125 analysis of results, 137 139 cdna template sets, 129 detection by chemiluminescence, 136 137 comparison with direct methods, 126 overview, 127 psoralen-biotin labeling of RNA, 132 133 RT-PCR, 49 58, advantages, 50 PCR reaction, 52 53 primer design, 73 74 principals, 49 quantitative RT-PCR, see Quantitative RT-PCR single cell RT-PCR, 77 81 S Seminal plasma, 293 295, 297 Shaw-Kamen instability sequences, 101 Single nucleotide polymorphisms, 409, detection by allelic discrimination, 410 417 definition, 409 in IL-1 gene, 411 417, probes for analysis, 412 413 SNPs, see Single nucleotide polymorphisms Sputum, 305, assaying interleukins in, 305 312, processing, 305 use of proteases to improve recovery, 307 STATs, affinity precipitation of, 373, 378 381 Synovial fluid, 327 334, assaying interleukins in, 327 334 bioassays for interleukins, 329 330 cytokine levels in arthritis, 328 immunoassays for interleukins, 330, 331 Taqman analysis, 413 417 Tears, 345, assaying interleukins in, 345 353 collection of, 347 348 Th1 cells, 164, 166, 285, 288 Th2 cells, 164, 166, 285, 288 Th3 cells, 286 TNF, 47, 111 124, 163, assaying intracellularly, 47 assaying mrna using Xplore assay, 111 124 circadian variation in p75 receptor, 164 in depressive illness, 218 222, 228 in seminal plasma, 293 294, 296 300, 330, 333, 334 Tumor necrosis factor, see TNF TGF-, 20, 163, Transforming growth factor-, see TGF- W Western blotting, 372 376 Whole blood assay for cytokines, 163 174 X Xplore assays, 111 124, design of probes, 116 mechanism, 113 preparation of standards, 114

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