The immunoassay handbook / ed. by Wild D. - 3rd ed. - Amsterdam; Boston: Elsevier, 2005. - xxvi, 930 p.: ill. - Ind.: p.909-930. - ISBN: 0-08-044526-8  Место хранения:   040 | Институт биофизики СО РАН | Красноярск | Библиотека

Оглавление / Contents

Contributors ................................................... xx Preface ....................................................... xxi Acknowledgements ............................................. xxiv Foreword ...................................................... xxv PART 1 PRINCIPLES 1. Introduction to Immunoassay Principles Chris Dauies .............................................. 3 Immunoassay for beginners .................................... 3 Kinetics of antibody-antigen interactions .................... 5 Immunoassay design .......................................... 11 Competitive (reagent limited) assays ..................... 11 Single-site immunometric assays .......................... 14 Two-site immunometric assays (reagent excess) ............ 14 Determinants of assay sensitivity ........................ 16 Detection and quantification of antibodies ............... 31 Special considerations for solid-phase immunoassays ...... 34 Comparison of experimental and theoretical immunoassay performance .............................................. 37 2. Non-Competitive Immunoassays for Small Molecules - The Anti-Complex, Selective Antibody and Apposition Systems Colin H. Self, Stephen Thompson and Larry A. Winger ...... 41 Introduction ................................................ 41 The anti-complex assay ...................................... 41 Enhanced specificity ........................................ 43 Multiple binding assay ...................................... 43 Selective antibody immunometric assay ....................... 43 Apposition assay ............................................ 45 Conclusions ................................................. 46 3. Ambient Analyte Assay Roger Ekins .............................................. 48 Introduction ................................................ 48 Basic theoretical principles ................................ 49 Applications of the ambient analyte assay principle ......... 50 Microspot assays ......................................... 51 Free (non-protein bound) hormone immunoassays ............ 58 Other applications of the principle ...................... 61 Summary and conclusion ...................................... 61 4. Free Analyte Immunoassay Nic D. Christofides ...................................... 63 Basic principles governing the free hormone concentration ... 63 Calculation of free analyte concentration ................... 63 Spreadsheet for calculation of free analyte concentration ... 64 Effect of serum proteins on free analyte concentration ...... 67 In vitro measurement of free analyte concentration .......... 68 Direct equilibrium dialysis .............................. 68 Immunoassays for free analytes ........................... 70 Tests of validity (accuracy) ............................. 76 Concluding remarks .......................................... 78 5. The Foundations ot Immunochemistry Robert F. Ritchie ............................................... 79 Introduction ................................................ 79 The immunological reaction (1895-1935) ...................... 80 Summary .................................................. 81 Qualification by diffusion in gel (1861-1977) ............... 82 Summary .................................................. 85 Qualitative analysis - by Immunoelectrophoresis (1953-1978) ................................................. 85 Summary .................................................. 86 Quantification of antigens by in-gel immunochemistry (1963-present) .............................................. 86 Summary .................................................. 88 Quantification of antigens by in-liquid immunochemistry (1935-present) .............................................. 89 Changes in immunization practice ............................ 90 Summary .................................................. 92 Quantification of antigens by particle-enhanced immunochemistry (1972-present) .............................. 93 Summary .................................................. 94 Labeled immunoassay (1959-present) .......................... 94 Summary .................................................. 94 Comparative studies (1907-present) .......................... 95 Summary .................................................. 95 6. Concepts Chris Davies ............................................ 103 Assay concepts ............................................. 103 Assay sensitivity ....................................... 103 Accuracy and bias ....................................... 103 Precision and imprecision ............................... 104 Cross-reactivity ........................................ 108 Interference ............................................ 110 Recovery ................................................ 114 Dilution ................................................ 114 Correlation and method comparison ....................... 115 Assay drift ............................................. 119 Clinical concepts .......................................... 120 Diagnosis ............................................... 120 Reference interval ...................................... 121 Bias .................................................... 121 Clinical sensitivity and specificity .................... 123 Positive and negative predictive values ................. 125 Prevalence and clinical utility ......................... 125 Likelihood ratio ........................................ 126 Continuous measurement, ROC curves ...................... 127 Confidence interval analysis of clinical performance .... 128 Probabilistic interpretation of test results ............ 128 Analytical goals ........................................ 129 Summary .................................................... 133 7. Immunoassay Development in the In Vitro Diagnostic Industry Doug Brandt and Steve Figard ............................ 136 Assay design overview ...................................... 136 The needs of the customer ............................... 136 The capability of the technology ........................ 137 The requirements for consistent manufacturing ........... 137 Experimental design in immunoassay development ............. 137 Preamble ................................................ 137 The process in overview ................................. 138 The problems assuaged by DOE ............................ 138 The particulars ......................................... 138 Four key assay design principles ........................... 141 Plan first .............................................. 141 Think from a theoretical perspective .................... 141 Simplify the approach ................................... 142 Establish specifications before starting ................ 142 Concluding comments ........................................ 143 8. Antibodies Eryl Liddell ............................................ 144 Introduction ............................................... 144 Antibody structure ......................................... 144 The antibody response in vivo .............................. 145 Antibody diversity ......................................... 147 Immunization ............................................... 147 Immunogen ............................................... 147 Polyclonal antisera ........................................ 148 Purification of antisera ................................ 149 Monoclonal antibodies ...................................... 149 Immune lymphocytes ...................................... 150 Myeloma cell lines ......................................... 150 Cell fusion ............................................. 151 Hybrid selection ........................................ 152 Antibody screening tests ................................ 153 Cloning ................................................. 153 Storage and propagation ................................. 153 Human monoclonal antibodies ............................. 154 Purification of monoclonal antibodies ................... 154 Antibody fragments ......................................... 154 Bispecific antibodies ...................................... 155 Chimeric and humanized antibodies .......................... 156 Recombinant antibodies ..................................... 156 Recombinant phage antibody library construction ............ 156 Selection of specific phage antibodies .................. 158 Altering antibody characteristics ....................... 159 Synthetic recombinant antibodies ........................ 160 Bivalent and bispecific recombinant antibodies .......... 160 Antibody conjugates and fusion proteins in diagnostics ..... 161 Polyclonal, monoclonal or recombinant? ..................... 161 9. Standardization and Calibration David Wild .............................................. 167 Standardization ............................................ 167 The role of external quality assessment (proficiency testing) schemes ........................................ 167 International standards ................................. 168 Definitive and reference methods ........................ 169 Other reference materials ............................... 170 Heterogeneity of standard material ...................... 170 Method-related causes of standardization differences .... 170 Special considerations for assay of antibodies .......... 173 Calibration ................................................ 173 Analyte ................................................. 173 International standards ................................. 173 Secondary standards ..................................... 173 Calibrators ............................................. 173 Standard and calibrator matrices ........................ 174 Reduced and stored calibration curves ................... 174 Recovery and dilution ................................... 175 10.Separation Systems David Wild and Wlad Kusnezow ............................ 177 Liquid-phase separations ................................... 177 Electrophoresis ......................................... 177 Gel filtration .......................................... 178 Dextran-coated charcoal ................................. 178 Precipitation by salts, organic solvents and polyethylene glycol (PEG) ............................... 178 Double (second) antibody precipitation .................. 178 PEG-assisted second antibody precipitation .............. 178 Aspiration and decantation methods ...................... 178 Surface-coated solid phases ................................ 179 General principles of protein binding to plastic surfaces ................................................ 179 Covalent attachment of proteins to solid phases ......... 182 Glass and plastic particles ............................. 182 Magnetizable particles .................................. 183 Tubes, wells and microtitration plates .................. 183 Beads ................................................... 184 Common solid phase ...................................... 184 Membrane filtration ..................................... 184 Immunochromatography .................................... 184 Western blot ............................................ 185 Washing .................................................... 185 Competitive assays ...................................... 185 Immunometric assays ..................................... 185 Removal of interfering substances ....................... 186 The mechanics of washing ................................ 186 Microarray Immunoassay Separation .......................... 186 Basic principles of receptor ligand interaction on microarray .............................................. 187 Solid-phase medium ...................................... 188 Protein attachment on microarrays ....................... 188 11.Signal Generation and Detection Systems (Excluding Homogeneous Assays) Larry J. Kricka and David Wild .......................... 192 Radioactive labels ......................................... 192 Radioactivity measurement ............................... 192 Preparation of radioactive tracers ...................... 193 Enzyme labels .............................................. 194 Colorimetry ............................................. 195 Fluorometry ............................................. 196 Chemiluminescence ....................................... 197 Enhanced chemiluminescence .............................. 198 Direct fluorescence ........................................ 199 Long wavelength fluorescence ............................ 201 Time-resolved fluorescence ................................. 201 Direct chemiluminescence ................................... 203 Bioluminescence ............................................ 203 Phosphorescence ............................................ 203 Microparticle labels ....................................... 203 Streptavidin/Avidin-Biotin ................................. 205 Protein A .................................................. 205 Amplification strategies ................................... 205 Multiple analytes and miniaturization ...................... 208 Microarrays ............................................. 208 12.Homogeneous Immunoassays Edwin F. Ullman ......................................... 212 Introduction ............................................... 212 Particle agglutination ..................................... 212 Erythrocytes and latex .................................. 212 Gold sols ............................................... 213 Lysis immunoassays ......................................... 214 Spin immunoassays .......................................... 215 Fluorescent labels ......................................... 215 Fluorescence polarization immunoassay ................... 215 Fluorescence resonance energy transfer .................. 217 Fluorescence protection assays .......................... 218 Fluorescence fluctuation ................................ 220 Enzyme immunoassays ........................................ 220 Enzyme-multiplied immunoassay technique (EMIT®1) ........ 220 Charge induced enzyme activation ........................ 222 Enzyme channeling ....................................... 222 Enzyme effector immunoassays ............................... 224 Substrate linked fluorescence immunoassay (SLFIA) ....... 224 Enzyme cofactor immunoassay ............................. 224 Enzyme inhibitor immunoassay ............................ 225 Enzyme complementation immunoassay ...................... 225 Isotopic labels ............................................ 226 Scintillation proximity assay ........................... 226 Electroactive labels ....................................... 227 Electrochemical detection ............................... 227 Electrochemiluminescence ................................ 227 Oxygen channeling immunoassays ............................. 228 Luminescent oxygen channeling immunoassay ............... 228 Conclusion ................................................. 230 13.Calibration Curve-fitting Barry Nix and David Wild ................................ 233 Introduction ............................................... 233 Dose-response metameter ................................. 233 Response-error relationship ............................. 233 Homo- and heteroscedasticity ............................ 233 Linearizing transformations ............................. 233 Normalizing transformations ............................. 234 Outliers ................................................ 234 Empirical and theoretical curve-fitting methods ......... 234 Least-squares fitting procedure ......................... 234 Weighted least-squares procedure ........................ 235 Precision profiles ...................................... 235 Examples of calibration curve-fitting methods .............. 236 Hand-plots .............................................. 236 Linear interpolation .................................... 236 Spline fits ............................................. 237 Polynomial regression ................................... 237 Logit-log and four-parameter log-logistic methods ....... 238 Five-parameter log-logistic ............................. 240 Four-parameter law of mass action ....................... 240 Monitoring the quality of curve-fitting .................... 241 Stored calibration curves, factory master curves, and adjusters .................................................. 241 Master calibration curve ................................ 241 Adjusters ............................................... 241 Modeling calibration curve changes over shelf life ...... 244 Use of electronic data transmission ..................... 244 Summary ................................................. 244 Suitable calibration curve-fit software .................... 244 14.Conjugation Methods Alastair H. Dent ........................................ 246 Categories of conjugates employed in immunoassay ........... 246 Protein-protein coupling ................................... 247 Functional chemistry of proteins ........................ 247 Categories of protein-protein coupling reaction ......... 249 Common protein-protein coupling methods ................. 249 Genetic engineering approaches to protein conjugation ... 254 Protein-small molecule coupling ............................ 255 Introduction ............................................ 255 Common protein-small molecule coupling methods .......... 256 Purification of conjugates ................................. 258 Chromatographic approaches to conjugate purification .... 259 Other approaches to conjugate purification .............. 262 Characterization of conjugates ............................. 262 Conclusion ................................................. 263 15.Immunological Biosensors James K. Gimzewski, Jason Reed, Michael A. Teitell and P. Gordon Malan ..................................... 265 Overview ................................................... 266 Electrochemical sensors .................................... 267 Amperometric sensors .................................... 267 Potentiometric sensors .................................. 268 Nanomechanical sensors ..................................... 268 Piezoelectric mass sensors .............................. 268 Microcantilever sensors ................................. 270 Micromagnetic sensors ................................... 270 Optical sensors ............................................ 270 Fluorescent evanescent wave sensors ..................... 271 Integrated optical sensors .............................. 271 Quantum dots ............................................ 271 Conclusions and future directions .......................... 272 16.Surface Plasmon Resonance in Kinetic, Concentration and Binding Site Analyses Robert Karlsson, Marie Arvola and Gary Franklin ......... 281 Introduction ............................................... 281 Objectives of protein studies in basic research ......... 281 Protein studies and therapeutics ........................ 281 Technology demands ...................................... 281 SPR biosensors ............................................. 282 Technology principles ................................... 282 Sensorgrams and report points ........................... 283 Immobilization .......................................... 283 Surface activity and immobilization levels .............. 284 Binding site analysis - epitope mapping .................... 284 Pair-wise epitope mapping ............................... 284 Kinetic analysis ........................................... 286 SPR in kinetic analysis ................................. 287 Concentration analysis ..................................... 289 Direct binding assays ................................... 289 Inhibition assays ....................................... 290 Summary .................................................... 291 17.Lab-on-a-Chip, Micro-, and Nanoscale Immunoassay Systems Larry J. Kricka and David Wild .......................... 294 Fundamental differences due to miniaturization ............. 296 Viscosity and surface tension ........................... 296 Capillary flow .......................................... 296 Electroosmosis .......................................... 297 The effects of reduced volume on low concentration samples ................................................. 297 Effect of reduced volumes on kinetics ................... 297 Immunoassay design at micro- and nanoscale ................. 298 Assay format ............................................ 298 Antibodies .............................................. 298 Standardization and calibration ......................... 298 Separation .............................................. 298 Signal generation ....................................... 299 Homogeneous immunoassay ................................. 300 On-chip detection methods ............................... 300 Bioelectronic chips and immunoassay ..................... 301 Microarrays and immunoassay ............................. 301 Atomic force microscopy ................................. 304 Manufacturing and business considerations .................. 305 Conclusions ................................................ 305 PART 2 PRODUCT TECHNOLOGY 18.Introduction to Product Technology in Clinical Diagnostic Testing David Wild .............................................. 313 Immunoassay product technologies ........................... 313 Use of fluorescence-labeled antibodies .................. 313 Agglutination assays .................................... 313 Radioimmunoassay and immunoradiometric assay ............ 314 Heterogeneous enzyme immunoassay ........................ 314 Homogeneous enzyme immunoassay .......................... 314 Automated homogeneous batch analyzer .................... 314 Automated heterogeneous batch analyzer .................. 315 Non-radioactive systems with increased sensitivity ...... 315 Semi-automated systems .................................. 315 Automated dual technology batch analyzer ................ 315 Automated, multianalyte batch analyzers ................. 315 Random-access analyzers with bulk reagent packs ......... 315 Unitized random-access systems .......................... 316 Clinical chemistry analyzers ............................ 317 Near-patient tests ...................................... 317 Home-use tests .......................................... 317 Other applications ...................................... 317 Future developments ..................................... 318 19.Market Trends David Huckle and David Wild ............................. 319 Immunoassay market status .................................. 319 Established trends ......................................... 320 Market drivers .......................................... 320 Advances in technology .................................. 321 New analytes ............................................ 324 The future of the immunodiagnostics business ............... 324 Application of marketing theory to immunodiagnostics .... 324 Future market requirements .............................. 326 Changes in the customer base ............................ 328 Potential impact of new technologies .................... 329 New analytes ............................................ 330 Conclusion ................................................. 331 Summary of likely trends in immunodiagnostics and related products ........................................... 331 20.Choosing an Automated Immunoassay System Lori J. Sokoll and Daniel W. Chan ....................... 333 Defining automation goals .................................. 333 Sources of information ..................................... 333 Considerations and criteria ................................ 334 Laboratory environment considerations ................... 334 Test menu considerations ................................ 335 Technical and clinical considerations ................... 335 Financial considerations ................................ 335 Operational considerations .............................. 336 Summary .................................................... 336 21.Automated System Features Alain Truchaud, Tanguy Le Neel, Murielle Cazaubiel, Bernard Capolaghi and Jean-Pierre Yvert ................. 338 Introduction ............................................... 338 Specific requirements of immunoassays ...................... 338 Concepts in immunoassay automation: workstations vs. integrated systems ......................................... 339 Reagent features ........................................... 340 Infrequent calibration .................................. 340 Traceability ............................................ 340 Multiselective testing and continuous operation ......... 340 Sample features ............................................ 340 Sample integrity ........................................ 340 Biosafety ............................................... 340 Dilution and reflex testing ............................. 340 Stat testing ............................................ 341 Incubation and washing ..................................... 341 Measurement ................................................ 341 Total quality management/traceability ...................... 341 Maintenance ................................................ 341 Ergonomics ................................................. 341 Integrated automation ...................................... 342 Conclusion ................................................. 342 22.Over-the-Counter Pregnancy Test Kits Michael J. Wheeler ................................. 343 Introduction ............................................... 343 Evaluation of OTC kits ..................................... 344 User preferences ........................................ 345 Factors affecting the measurement of hCG in urine ....... 347 Summary .................................................... 348 23.Fluorescence Microscopy: MicroTrak® Tom Houts ............................................... 349 24.Automated Batch Analyzers: IMx® Kent Ford ............................................... 351 25.Bulk Reagent Random-Access Analyzers: UniCAP® 100 Gareth Evans and Mats Rilven ................................... 358 26.Automated Panel Analyzers PRISMTM Dinesh Shah and Jim Stewart ............................. 362 27.Unitized Reagent Random-Access Analyzers: IMMULITE® and IMMULITE 1000 Arthur L. Babson ........................................ 370 28.Bulk Reagent Random-Access Analyzers: ACS:180® S.E. Elvio Gramignano ................................... 374 29.Bulk Reagent Random-Access Analyzers: AxSYM® Theresa Donahoe ......................................... 379 30.Bulk Reagent Random-Access Analyzers: Elecsys® Immunoassay Systems Mary Beth Myers ......................................... 385 31.Bulk Reagent Random-Access Analyzers: Vitros® ECi David Wild .............................................. 391 32.Bulk Reagent Random-Access Analyzers: IMMULITE® 2000 and IMMULITE 2500 Arthur L. Babson ........................................ 397 33.Bulk Reagent Random-Access Analyzers: ADVIA Centaur® Laura Taylor and Eluio Gramignano ....................... 402 34.Architect® /2000® and /2000fR Analyzers Frank A. Quinn .......................................... 406 35.CEDIA®, a Homogeneous Enzyme Immunoassay System William A. Coty and Rueyming Loor ....................... 412 36.Clinical Chemistry Analyzers: VITROS™® Immuno-Rate and MicroTipTM Assays Susan J. Danielson and David A. Hilborn ................. 418 37.Near-Patient Tests: The TRIAGE® System Kenneth F. Buechler ..................................... 424 38.Near-Patient Tests: Stratus® CS Acute CareTM Diagnostic System W.N. McLellan ........................................... 431 39.Over-the-counter Tests: Clearblue Pregnancy Test™, Clearblue Ovulation TestTM and ClearviewTM Keith May ............................................... 435 40.Over-the-Counter Tests: Persona Keith May ............................................... 438 PART 3 LABORATORY MANAGEMENT 41.Subject Preparation, Sample Collection and Handling Colin Wilde ............................................. 443 Subject state and preparation .............................. 443 Stress .................................................. 443 Exercise ................................................ 443 Food and drink .......................................... 443 Posture ................................................. 444 Medical procedures ...................................... 444 Drugs ................................................... 444 Pregnancy ............................................... 445 Age ..................................................... 445 Race .................................................... 445 Timing ..................................................... 445 Biological rhythms ...................................... 445 Dynamic tests ........................................... 446 Pathological changes .................................... 446 Nonthyroidal illness .................................... 446 Therapeutic drug monitoring ............................. 446 Blood collection by venepuncture ........................... 447 Precautions relating to the patient ..................... 447 Phlebotomy techniques ................................... 447 Blood collection ........................................ 447 Preparation of serum .................................... 448 Preparation of plasma ................................... 448 Whole blood ............................................. 449 Interference by tube and stopper components ................ 449 Interference ............................................ 449 Precautions ............................................. 449 The use of serum separators ................................ 449 Characteristics of separators ........................... 450 Interference ............................................ 450 Other additives ......................................... 450 Hemolysis ............................................... 450 Collection of blood by skin puncture ....................... 450 Skin puncture sites ..................................... 451 Collection into capillary tubes ......................... 451 Urine collection ........................................... 451 Containers .............................................. 451 Preservative ............................................ 451 Types of collection ..................................... 451 Instructions ............................................ 451 Sample handling ......................................... 452 Saliva ..................................................... 452 Procedures for sampling saliva .......................... 452 Special precautions ..................................... 453 Cerebrospinal fluid ........................................ 453 Formation of cerebrospinal fluid ........................ 453 Collection of CSF ....................................... 453 Amniotic fluid ............................................. 453 Collection of amniotic fluid ............................ 453 Amniocentesis ........................................... 453 Special precautions ..................................... 453 Sweat ...................................................... 454 Semen ...................................................... 454 Hair ....................................................... 454 Milk ....................................................... 454 Storage and transportation ................................. 455 Conclusions ................................................ 455 42.Laboratory Quality Assurance Pierre Blockx and Manuella Martin ....................... 456 Staff selection and training ............................... 456 Fixed volume pipettes ................................... 457 Repeating pipettes ...................................... 457 Semi-automated instruments .............................. 457 Manual dilutions ........................................ 457 Equipment maintenance and testing .......................... 458 Laboratory water ........................................... 459 Water-purification methods .............................. 459 Water purity measurement ................................ 459 Recommended water quality ............................... 460 Sample control ............................................. 460 Sample collection ....................................... 460 Centrifugation .......................................... 460 Storage ................................................. 460 Reagent and control preparation ............................ 460 Assay quality control ...................................... 461 Within-assay precision .................................. 461 Between-assay precision ................................. 462 Internal QC programs .................................... 462 QC software on automated analyzers ...................... 465 New generation QC software programs ..................... 465 QC parameters other than controls ....................... 467 QC in infectious disease tests .......................... 468 Point-of-care tests ........................................ 470 43.Point-of-Care Testing James H. Nichols ........................................ 472 Delivery options ........................................... 472 Terminology ............................................. 472 Sites ................................................... 472 Operators ............................................... 473 Menu .................................................... 473 Economics ............................................... 473 Clinical outcome ........................................ 475 Quality assurance .......................................... 476 Components of good laboratory practice .................. 476 Regulations ............................................. 477 Data management ......................................... 478 Practical management ....................................... 479 Technical validation .................................... 479 Quality control ......................................... 480 Administration .......................................... 480 Summary .................................................... 481 44.Immunoassay Troubleshooting Guide David Wild .............................................. 483 Instructions ............................................... 483 Troubleshooting guide ...................................... 484 Control bias - consistent change in values from one period of time to another (or from one reagent lot to another) ............................................. 484 Gradual change in control values (not consistent change at one time or with change in reagent lot) ....... 485 Commercial controls consistently biased from target mean (not change in values across same lot of controls) ............................................... 486 Bias in proficiency testing (external quality assessment) scheme from other users of the same assay ... 487 Bias in proficiency testing (external quality assessment) scheme of one method from all-laboratory mean or reference method ................................ 489 Poor within-assay precision ............................. 489 Poor between-assay precision or individual control failure ................................................. 491 Assay drift ............................................. 494 Low signal level ........................................ 496 Low binding in a competitive immunoassay ................ 497 Low binding in an immunometric assay .................... 499 Increase in binding in a competitive immunoassay ........ 501 Reduction in ED50 (estimated dose at 50% of binding at zero concentration) - competitive immunoassays only .................................................... 502 Increase in ED50 (estimated dose at 50% of binding at zero concentration) competitive immunoassays only .... 503 High non-specific binding ............................... 505 Poor linearity of dilution .............................. 506 Unexpected or inconsistent clinical classification ...... 507 Apparent shift in reference interval (normal range) ..... 510 Negative patient sample concentrations .................. 512 Poor recovery ........................................... 514 Poor assay sensitivity .................................. 516 Poor correlation between two immunoassays ............... 518 PART 4 APPLICATIONS 45.Thyroid Rhys John, Carole A. Spencer, Nic Christofides and David Wild .......................................... 523 Normal thyroid function .................................... 523 Clinical disorders ......................................... 523 Hypothyroidism (thyroid hormone deficiency) ............. 523 Hyperthyroidism ......................................... 524 Nontoxic goiter ......................................... 525 Nonthyroidal illness/sick euthyroid syndrome ............ 525 L-thyroxine replacement therapy ......................... 526 Endogenous antibodies to T3 and T4 ...................... 526 Familial dysalbuminemic hyperthyroxinemia ............... 526 Analytes ................................................... 526 Thyrotropin ................................................ 526 Thyroxine ............................................... 528 T3 or T uptake and calculation of free thyroxine index ................................................... 529 Free T4 ................................................. 531 Triiodothyronine ........................................ 534 Free T3 ................................................. 535 Thyroxine-binding globulin .............................. 537 Thyrotropin receptor antibodies ......................... 538 Thyroid peroxidase antibodies ........................... 539 Thyroglobulin antibodies ................................ 540 Thyroglobulin ........................................... 540 Thyroid testing strategies ................................. 541 46.The Adrenal Cortex Sami Medbak ............................................. 544 Normal adrenocortical function ............................. 544 Clinical disorders ......................................... 545 Hypercortisolemia ....................................... 545 Hypocortisolemia ........................................ 545 Analytes ................................................... 546 Cortisol ................................................ 546 Adrenocorticotropic hormone ............................. 548 Management of patients with Cushing's syndrome ............. 550 Pre-admission ........................................... 550 Admission to endocrine ward (e.g. on Monday) ............ 550 47.Bone Metabolism Kay W. Colston and John C. Stevenson .................... 552 Normal calcium metabolism .................................. 552 Clinical disorders ......................................... 552 Hypercalcemia ........................................... 552 Parathyroid disorders ................................... 553 Hypocalcemia ............................................ 554 Vitamin D disorders ..................................... 554 Medullary carcinoma of the thyroid ...................... 555 Paget's disease ......................................... 555 Osteoporosis ............................................ 555 Analytes ................................................... 555 Vitamin D metabolites: 25-hydroxyvitamin D,l,25- dihydroxyvitamin D ...................................... 555 Parathyroid hormone ..................................... 558 Parathyroid hormone-related protein ..................... 559 Calcitonin .............................................. 559 Biochemical markers of bone turnover ....................... 560 Markers of bone formation ............................... 560 Markers of bone resorption .............................. 561 Serum tartrate resistant acid phosphatase (TRAP) ........ 563 Conditions associated with changes in bone markers ......... 563 48.Infertility Michael J. Wheeler ...................................... 565 Clinical disorders ......................................... 566 Primary hypogonadism in the female ...................... 566 Secondary hypogonadism in the female .................... 567 Infertility and normal menstrual function ............... 567 Primary hypogonadism in the male ........................ 567 Secondary hypogonadism in the male ...................... 568 Impaired sperm transport and sperm viability ............ 568 Analytes ................................................... 568 Luteinizing hormone (lutropin) .......................... 568 Follicle stimulating hormone (follitropin) .............. 570 Prolactin ............................................... 571 Inhibin ................................................. 572 Estradiol ............................................... 572 Progesterone ............................................ 574 Testosterone ............................................ 574 Dihydrotestosterone ..................................... 577 Test strategy for infertility in women ..................... 578 49.In Vitro Fertilization and Embryo Transfer (IVF-ET) August C. Olivar ........................................ 580 Stage I: induction of ovulation ......................... 581 Stage II: oocyte retrieval .............................. 581 Stage III: oocyte insemination and incubation ........... 581 Stage IV: embryo transfer ............................... 581 Other assisted reproductive technology procedures .......... 581 Gamete intra-Fallopian tube transfer .................... 581 Zygote intra-Fallopian transfer ......................... 582 Micromanipulation of gametes ............................... 582 Assisted hatching ....................................... 582 Intracytoplasmic sperm injection ........................ 582 Analytes ................................................... 582 Estradiol ............................................... 582 50.Hirsutism and Virilization in the Female Michael J. Wheeler ...................................... 586 Clinical disorders ......................................... 586 Polycystic ovarian syndrome ............................. 586 Androgen-secreting tumors of the ovary .................. 587 Congenital adrenal hyperplasia .......................... 588 Cushing's syndrome ...................................... 589 Analytes ................................................... 589 Luteinizing hormone and follicle-stimulating hormone .... 589 Testosterone ............................................ 589 Sex hormone-binding globulin ............................ 590 Free testosterone ....................................... 591 Androstenedione ......................................... 592 Dehydroepiandrosterone sulfate .......................... 593 17a-Hydroxyprogesterone ................................. 594 Dihydrotestosterone ..................................... 595 Androstanediols and their glucuronides .................. 596 51.Pregnancy Tim Chard ........................................ 598 Clinical disorders ......................................... 599 Detection of early pregnancy ............................ 599 Threatened abortion ..................................... 599 Ectopic pregnancy ....................................... 599 Chromosome defects of the fetus ......................... 599 Neural tube defects of the fetus ........................ 600 Premature labor ......................................... 600 Placental insufficiency ................................. 600 Pre-eclampsia ........................................... 601 Miscellaneous disorders ................................. 601 Analytes ................................................... 601 Alphafetoprotein ........................................ 601 Human chorionic gonadotropin ............................ 604 Estriol ................................................. 606 Pregnancy-associated plasma protein-A ................... 607 Other analytes .......................................... 608 Screening for Down's syndrome ........................... 609 52.Growth and Growth Hormone Deficiency Michael Preece and Jane Pringle ......................... 611 Normal childhood growth .................................... 611 Clinical disorders ......................................... 612 Growth hormone deficiency or insufficiency .............. 612 Growth hormone resistance ............................... 612 Excessive growth hormone secretion ...................... 612 Analytes ................................................... 612 Growth hormone .......................................... 612 Growth hormone binding protein .......................... 614 Insulin-like growth factors: IGF-I (somatomedin C) and IGF-II .............................................. 614 Insulin-like growth factor binding proteins ............. 615 General strategy ........................................... 616 53.Diabetes Mellitus Penny Clark ............................................. 617 Analytes ................................................... 618 Insulin, proinsulin(s) and C-peptide .................... 618 Glycohemoglobin (GHb) ................................... 621 Other glycated proteins ................................. 624 Microalbumin ............................................ 625 Autoimmune and other assays ............................. 626 54.Hematology Derek Dawson, Harry Waters and John Ardern .............. 629 Normal blood function ...................................... 629 Clinical disorders ......................................... 630 Anemia .................................................. 630 Polycythemia ............................................ 630 Iron overload and hemochromatosis ....................... 630 Hemostatic disorders .................................... 631 Leukemia and lymphoma ................................... 631 Analytes ................................................... 631 Anemia .................................................. 631 Intrinsic factor antibody ............................... 636 Ferritin ................................................ 636 Transferrin ............................................. 638 Transferrin receptors ................................... 638 Erythropoietin .......................................... 639 Thrombosis and hemostasis .................................. 640 Thrombophilia .............................................. 640 Proteins С and S ........................................ 640 Antithrombin ............................................ 641 Coagulation ................................................ 641 Coagulation factors ..................................... 641 Fibrinogen .............................................. 642 Evidence of disseminated intravascular coagulation ......... 642 Fibrinogen/Fibrin degradation products .................. 642 D-Dimer test ............................................ 643 Infrequently used and research assays ...................... 643 P-Thromboglobulin ....................................... 643 Platelet factor 4 ....................................... 644 Heparin cofactor II ..................................... 644 Plasminogen ............................................. 644 Tissue plasminogen activator ............................ 644 Immunodetection methods .................................... 645 Leukemia and lymphoma analytes .......................... 645 Malarial parasites ...................................... 646 Hemoglobinopathies ...................................... 646 55.Cardiac Markers Alan H.B. Wu ............................................ 649 Normal heart function ...................................... 649 Clinical disorders ......................................... 649 Coronary artery disease ................................. 649 Heart failure ........................................... 651 Hypertension ............................................ 651 Analytes ................................................... 651 Creatine kinase and the MB isoenzyme .................... 651 Myoglobin ............................................... 652 Cardiac troponin (T and I) .............................. 653 Free fatty acid binding proteins and carbonic anhydrase III isoenzyme ................................. 655 Apolipoproteins AI, AII, and В .......................... 655 Lipoprotein (a) ......................................... 656 Homocysteine ............................................ 656 C-reactive protein ...................................... 657 Brain natriuretic peptide and NT-PRO-BNP ................ 659 Plasma renin ............................................ 660 Plasminogen activators and inhibitors ................... 661 56.Cancer Markers Mavanur R. Suresh ....................................... 664 Introduction ............................................... 664 History and classification ................................. 667 Nomenclature and idiosyncrasies of glycoprotein tumor antigens ................................................ 668 New developments ........................................ 669 Analytes ................................................... 669 Carcino-embryonic antigen ............................... 669 α-fetoprotein ........................................... 671 Carbohydrate antigen 19-9 (sialyl Lewis3) ............... 672 Cancer antigen 125 (Muc-16) ............................. 674 Cancer antigen 15-3 ..................................... 675 Estrogen receptor and progesterone receptor ............. 676 Fecal occult blood ...................................... 677 Prostate-specific antigen ............................... 678 Prostatic acid phosphatase .............................. 680 β2-Microglobulin ........................................ 680 Neuron-specific enolase ................................. 681 Carbohydrate antigen 50 ................................. 682 Squamous cell carcinoma antigen ......................... 683 Tissue polypeptide antigen, tissue polypeptide specific antigen and Cyfra 21-1 ......................... 684 Placental alkaline phosphatase .......................... 684 Human chorionic gonadotropin ............................ 685 Cathepsin D ............................................. 686 Interleukin-2 receptor .................................. 686 C-Erb B-2 (Her-2/Neu) oncoprotein ....................... 686 P53 ..................................................... 687 Bladder tumor antigen ................................... 688 Immunochromatography assays for tumor markers ........... 688 Free light chain (FLC) assays ........................... 689 Novel experimental and other minor markers ................. 690 Cancer antigen 195 (CA 195) ............................. 690 Tumor-associated glycoprotein 72.4 (TAG 72.4) ........... 690 S-100 Antigen ........................................... 690 Sialyl Lewisx (SLX-I) ................................... 690 Mucin-like carcinoma-associated antigen (MCA) ........... 691 Cancer-associated antigen 549 (CA 549) .................. 691 Bone alkaline phosphatase ............................... 691 B/T gene rearrangement test ............................. 691 BRCA 1 and BRCA 2 ....................................... 691 P21 or RAS .............................................. 691 DR-70 ................................................... 691 90K/MAC-2BP ............................................. 691 Bcl-2 ................................................... 691 NMP-22 .................................................. 691 MMP assays .............................................. 692 TAG-12 .................................................. 692 CA 1-18 ................................................. 692 Angiogenic factors ...................................... 692 Chromogranin A .......................................... 692 Telomerase .............................................. 692 Urinary bladder cancer (UBC) antigen .................... 692 HHV-8 antibody .......................................... 692 Nor-/Metanephrine RIA ................................... 693 HER2/neu ................................................ 693 PML protein ............................................. 693 57.Allergy Lars Yman ............................................... 695 Allergic disease ........................................... 695 Diagnosis and therapy ................................... 695 Allergens ............................................... 695 Analytes ................................................... 696 Total serum IgE ......................................... 696 Allergen-specific IgE antibody .......................... 698 IgG antibody assays ..................................... 704 IgA antibody assay ...................................... 704 Markers of cell activation .............................. 705 Standardization and evaluation ............................. 705 Quantitative measurement of allergen-specific IgE antibodies .............................................. 705 Reference values ........................................ 706 Quality control ......................................... 706 Clinical applications ...................................... 707 Diagnosis of atopic disease and identification of offending allergen ...................................... 707 Prediction and monitoring of the development of allergic disease ........................................ 707 58.Autoimmune Disease David F. Keren .......................................... 712 Analytes ................................................... 713 Anti-nuclear antibodies ................................. 713 Anti-double-stranded (ds) DNA ........................... 716 Anti-Sm ................................................. 716 Anti-RNP ................................................ 717 Anti-SSA/Ro ............................................. 717 Anti-SSB/La ............................................. 718 Anti-histone ............................................ 719 Anti-DNP ................................................ 719 Anti-centromere ......................................... 720 Anti-Scl-70 ............................................. 720 Rheumatoid factor ....................................... 720 Anti-cyclic citrullinated (anti-CCP) peptide ............ 721 Anti-neutrophil cytoplasmic antibodies (C-ANCA, P-ANCA) ................................................. 721 Anti-glomerular basement membrane (GBM) ................. 722 Anti-Jo-1 ............................................... 723 Anti-microsomal (thyroid peroxidase - TPO) .............. 723 Islet cell autoantibodies (ICA) ......................... 724 Anti-adrenal cortical antibodies ........................ 724 Anti-parietal cell antibodies (PCA) ..................... 725 Anti-mitochondrial antibodies (AMA, or M2) .............. 725 Anti-smooth muscle antibodies (SMA) ..................... 726 Anti-liver-kidney microsomal (LKM) ...................... 726 IgA anti-endomysium ..................................... 726 IgA anti-tissue transglutaminase (anti-tTG) ............. 727 IgG and IgA anti-gliadin ................................ 727 Anti-acetylcholine receptor (ACHR) ...................... 728 Striational antibodies .................................. 728 Calcium channel antibodies .............................. 729 Anti-cardiolipin antibodies ............................. 729 Anti-myelin oligodendrocyte glycoprotein (MOG) and anti-myelin basic protein (MBP) antibodies .............. 730 59.Sexually Transmitted Diseases Bruce J. Dille, Alan S. Armstrong and Isa K. Mushahwar ............................................ 733 Neisseria gonorrhoeae ...................................... 733 Chlamydia trachomatis ...................................... 735 Trichomonas vaginalis ...................................... 738 Candida albicans - vaginitis ............................... 739 Gardnerella vaginalis ...................................... 741 Herpes simplex virus type 2 ................................ 742 Syphilis (Treponema pallidum) .............................. 744 60.Congenital Diseases of Microbiological Origin Bruce J. Dille, John W. Sajford Jr. and Isa K. Mushahwar ............................................... 746 Cytomegalovirus ............................................ 746 Rubella virus .............................................. 748 Toxoplasma gondii .......................................... 750 IgG avidity tests .......................................... 751 Parvovirus B19 ............................................. 752 Group В streptococci ....................................... 754 61.Hepatitis Isa K. Mushahwar ........................................ 756 Hepatitis A virus, anti-HAV IgM, anti-HAV IgG .............. 756 Hepatitis В virus, HBsAg, HBcAg, HBeAg, anti-HBs, anti-HBc, anti-HBc IgM, anti-HBe ........................... 758 Hepatitis С virus .......................................... 764 Hepatitis D virus .......................................... 765 Hepatitis E virus .......................................... 766 GB viruses ................................................. 768 GB virus С .............................................. 769 62.Human Retroviruses George J. Dawson and Isa K. Mushahwar ................... 771 Etiologic agents ........................................... 771 HTLV-I/-II ................................................. 771 HIV-1/-2 ................................................... 774 63.Dengue Virus Infections Bruce J. Dille and Isa K. Mushahwar ..................... 779 64.Miscellaneous Diseases of Microbiological Origin Bruce J. Dille and Isa K. Mushahwar ..................... 784 Herpes simplex virus type 1 ................................ 784 Epstein -Barr virus ........................................ 786 Chagas' disease ............................................ 788 Candida albicans - invasive candidiasis .................... 789 Borrelia burgdorferi ....................................... 790 Helicobacter pylori ........................................ 792 Group A streptococcus ...................................... 793 Human herpesvirus 6 ........................................ 794 Human herpesvirus 8 (Kaposi's sarcoma herpesvirus) ......... 796 65.Therapeutic Drug Monitoring (TDM) Philip A. Routledge and Alun D. Hutchings ............... 798 Introduction ............................................... 798 Assay technology ........................................ 798 Measurement of free drug concentration .................. 799 Practical aspects of TDM ................................ 800 Antiarrhythmic drugs ....................................... 800 Acecainide (N-acetylprocainamide) ....................... 800 Disopyramide ............................................ 801 Lidocaine ............................................... 801 Procainamide ............................................ 802 Propranolol ............................................. 802 Quinidine ............................................... 803 Amiodarone .............................................. 803 Flecainide .............................................. 804 Antibiotics ................................................ 804 Amikacin ................................................ 804 Kanamycin ............................................... 805 Gentamicin .............................................. 806 Tobramycin .............................................. 806 Netilmicin .............................................. 807 Chloramphenicol ......................................... 807 Vancomycin .............................................. 808 Anticonvulsants ............................................ 808 Phenytoin ............................................... 808 Phenobarbital (phenobarbitone) .......................... 809 Primidone ............................................... 809 Carbamazepine ........................................... 810 Valproic acid ........................................... 810 Ethosuximide ............................................ 811 Chemotherapeutic agents .................................... 811 Methotrexate ............................................ 811 Miscellaneous .............................................. 812 Acetaminophen (paracetamol) ............................. 812 Tricyclic antidepressants ............................... 812 Cyclosporin ............................................. 812 Digoxin ................................................. 813 Digitoxin ............................................... 814 Theophylline ............................................ 814 66.Drugs of Abuse Brian Widdop ............................................ 816 Applications ............................................... 816 Drug dependence treatment centers ....................... 816 Psychiatric clinics ..................................... 817 Medical-legal applications .............................. 817 Drug abuse in the workplace ............................. 817 Immunoassays for drugs of abuse ............................ 817 Amphetamine ............................................. 818 Methamphetamine ......................................... 819 Methylenedioxyamphetamine ............................... 819 Methylenedioxy-methamphetamine .......................... 819 Barbiturates ............................................ 824 Benzodiazepines ......................................... 825 Cannabis ................................................ 828 Cocaine ................................................. 832 Fentanyl ................................................ 835 Lysergic acid diethylamide .............................. 836 Methadone ............................................... 838 Opiates ................................................. 841 Phencyclidine ........................................... 844 Propoxyphene ............................................ 847 Legal addictive substances ................................. 848 Alcohol (ethyl alcohol) and the use of carbohydrate- deficient transferrin ................................... 848 Nicotine and the use of cotinine ........................ 848 Steroid abuse in sport ..................................... 849 Anabolic androgenic steroids ............................ 849 Rapid immunoassay tests for drugs of abuse ................. 850 67.Assays for Drug-screening Applications and Research Jeffrey K. Horton, Stephen J. Capper, Molly J. Price Jones and Kelvin T. Hughes .............................. 854 Assay technology ........................................... 856 Tritium radioimmunoassays ............................... 856 Iodine-125 radioimmunoassays ............................ 857 Scintillation proximity assay ........................... 857 The application of SPA technology to the measurement of protein: protein interactions using an antibody capture format .......................................... 859 Imaging technologies and instrumentation for ultra high-throughput drug-screening .......................... 861 Enzyme immunoassays ..................................... 862 Analytes ................................................... 863 Prostaglandins .......................................... 863 Leukotrienes ............................................ 865 Rat hormone assays ...................................... 866 Cytokines ............................................... 868 Signal transduction assays .............................. 873 Cell proliferation immunoassay .......................... 877 Matrix metalloproteinases (MMPs) ........................ 878 Cardiovascular peptides ................................. 881 Neurodegeneration assays ................................ 883 68.Immunoassay Applications in Veterinary Diagnostics Erwin Workman ........................................... 885 Infectious disease diagnosis/assessment of immune status ... 885 Feline .................................................. 885 Canine .................................................. 888 Porcine ................................................. 891 Equine .................................................. 893 Bovine .................................................. 894 Avian ................................................... 899 Murine .................................................. 903 Assessment of reproductive/metabolic status ................ 904 Feline/Canine ........................................... 904 Equine .................................................. 905 Bovine .................................................. 905 Conclusion ................................................. 906