Dedication
Preface
Contents
Contributors
Chapter 1: A Short History of Hormone Measurement
References
Chapter 2: Immunoassay Techniques
1 Introduction
1.1 Antibodies
1.2 Labeled Reagents (Haptens, Proteins, and Antibodies) or Tracer
2 Materials
2.1 Preparation of Steroid–Albumin Conjugate
2.2 Polyclonal Antibody Production
2.3 Production of Labeled Antigen or Antibody (Tracers)
2.3.1 Iodination Using Chloramine T Oxidation
2.3.2 Iodination Using Lactoperoxidase Oxidation
2.3.3 Iodination of Steroids and Other Haptens
Iodination of Histamine
Formation of a Mixed Anhydride
Conjugation
Separation and Purification of Iodinated Product by TLC
Separation and Purification of Iodinated Product by HPLC
2.3.4 Conjugation of Proteins with Enzymes
2.3.5 Glutaraldehyde Conjugation of Enzyme Label to Protein
2.4 Separation Methods
2.4.1 Coating of Microtiter Plate Wells and the Inside of Polystyrene Tubes for Solid-Phase Assays
2.4.2 Separation of Antibody-Bound and Unbound Antigen by a Second Antibody Method
2.4.3 Antigen Separation of Tritiated Tracers Using Dextran-
Preparation of Dextran-
Separation with Dextran-
3 Methods
3.1 Preparation of Steroid–Albumin Conjugate
3.2 Polyclonal Antibody Production
3.3 Production of Labeled Antigen or Antibody (Tracers)
3.3.1 Iodination Using Chloramine T Oxidation
3.3.2 Iodination Using Lactoperoxidase Oxidation
3.3.3 Iodination of Steroids and Other Haptens
Iodination of Histamine
Formation of a Mixed Anhydride
Conjugation
Separation and Purification of Iodinated Product by TLC
Separation and Purification of Iodinated Steroid by HPLC
3.3.4 Glutaraldehyde Conjugation of Enzyme Labels to Protein
3.4 Separation Methods
3.4.1 Coating of Microtiter Plate Wells and the Inside of Polystyrene Tubes for Solid-Phase Assays
3.4.2 Separation of Antibody-Bound and Unbound Antigen by a Second Antibody Method
3.4.3 Antigen Separation of Tritiated Tracers Using Dextran-Coated Charcoal
Preparation of Dextran-
Separation with Dextran-
4 Notes
References
Chapter 3: Introduction to Gas Chromatography-Mass Spectrometry
1 Introduction
2 Gas Chromatography
2.1 Principle
2.2 Carrier Gas
2.3 Injection Ports
2.4 Columns
3 Interface
4 Mass Spectrometry
4.1 Ionization
4.2 Mass Analyzers
4.2.1 Magnetic Sector Analyzer
4.2.2 Quadrupole Analyzer
4.2.3 Ion Trap Analyzer
4.2.4 Time-of-Flight Analyzer
4.3 Ion Detection and Data Handling
4.3.1 Detector
4.3.2 Scanning Techniques
4.3.3 Quantitative Mass Spectrometry
5 Example of ID-GC-MS Analysis of Hormones: Profiling Plasma Steroids
5.1 Sample Workup
5.2 Method Validation
5.3 Significance and Perspective
References
Chapter 4: Tandem Mass Spectrometry in Hormone Measurement
1 Introduction
2 The LC-MS/MS System
2.1 Liquid Chromatography
2.2 Ion Source
2.3 Ion Separation and Detection
2.4 Data Handling
2.5 Maintenance
2.6 Preparation of Samples for Hormone Assays
2.7 Matrix Effects
2.8 Derivatization
2.9 Quantitation
2.10 Automation and Further Developments
3 Examples of the LC-MS/MS Analysis of Hormones
3.1 Steroid Hormones
3.1.1 Testosterone
3.1.2 Dihydrote-stosterone
3.1.3 17-Hydroxy-progesterone
3.1.4 Cortisol
3.1.5 Estrogens
3.1.6 Aldosterone and Renin
3.2 Thyroid Hormones
3.3 Protein and Peptide Hormones
References
Chapter 5: Methods for the Investigation of Thyroid Function
1 Introduction
1.1 Thyroid-
1.2 Thyroid Hormones
1.3 Thyroid Antibodies
1.4 Sample Collection and Stability
1.5 Assay Drift
2 Materials
2.1 TSH in Liquid Specimens
2.2 TSH Measurement in Neonatal Dried Blood Spots
2.3 Free T4 by ELISA
2.4 Free T4 by Equilibrium Dialysis
2.5 Total T4
2.6 Free T3
2.7 Thyroxine-
2.8 Antithyroid Peroxidase Antibodies
2.9 Anti-TSH Receptor Antibodies
3 Methods
3.1 TSH
3.2 TSH Measurement in Neonatal Dried Blood Spots
3.3 Free T4
3.4 Free T4 by Equilibrium Dialysis
3.4.1 Dialysis
3.4.2 Radioimmuno-assay
3.5 Total T4
3.6 Free T3
3.7 Thyroxine-
3.8 Antithyroid Peroxidase Antibodies
3.9 Anti-TSH Receptor Antibodies
4 Notes
References
Chapter 6: The Measurement of LH, FSH, and Prolactin
1 Introduction
2 Materials
2.1 Simple Radioimmunoassays for LH, FSH, and Prolactin
2.2 Shortened Radioimmunoassays for LH, FSH, and Prolactin
2.3 Human FSH IRMA (IBL International Gmbh, Hamburg, Germany).
2.4 Human LH ELISA (IBL Immuno-
2.5 Rat EIA for Prolactin (Cayman Chemical Company, MI, USA)
2.6 Use of Blocking Tubes to Remove Heterophilic Antibody Interference
2.7 PEG Precipitation Method for Investigating the Presence of Macroprolactin in Samples
2.8 Column Chromatography Method for Investigating the Presence of Macroprolactin in Samples
3 Methods
3.1 Simple 4-day Radioimmunoassays for LH, FSH, or Prolactin ( See Notes 1 and 2)
3.2 Shortened Radioimmunoassays for LH, FSH, or Prolactin
3.3 Human FSH IRMA
3.4 Human LH ELISA
3.5 Rat EIA for Prolactin
3.6 Use of Blocking Tubes to Remove Heterophilic Antibody Interference
3.7 PEG Precipitation Method for Investigating the Presence of Macroprolactin in Samples
3.8 Column Chromatography Method for Investigating the Presence of Macroprolactin in Samples
4 Notes
References
Chapter 7: Assays for GH, IGF-I, and IGF Binding Protein-3
1 Introduction
2 Materials
2.1 Human GH ELISA
2.2 Human IGF-I ELISA
2.3 Human IGFBP3 ELISA
2.4 General Materials
3 Methods
3.1 Sample Collection, Handling and Storage
3.2 Human GH ELISA
3.3 Human IGF-I ELISA
3.4 Human IGFBP3 ELISA
4 Notes
References
Chapter 8: Measurement of Arginine Vasopressin
1 Introduction
2 Materials
2.1 Extraction
2.2 Radioimmu- noassay
2.3 Separation and Counting
3 Methods
3.1 Plasma AVP (Sample Storage and Preparation)
3.2 Urine AVP (Sample Storage and Preparation)
3.3 Sample Extraction
3.4 Radioimmu- noassay
4 Notes
References
Chapter 9: The Measurement of Anti-Müllerian Hormone (AMH)
1 Introduction
2 Materials
2.1 AMH Gen II ELISA, Beckman Coulter
3 Methods ( See Notes 3 and 4)
3.1 Assay Details
3.2 Calculation of Results
4 Notes
References
Chapter 10: Measurement of Gut Hormones in Plasma
1 Introduction
2 Materials
2.1 General
2.2 Storage of Peptides, Antibodies, and Radiolabelled Peptides
2.3 Production of Radiolabels
2.4 Antibody Production
2.5 Radio
2.6 Separation of RIA Bound and Free Components
2.6.1 Charcoal Separation
2.6.2 Secondary Antibody Separation
3 Methods
3.1 Storage of Peptides, Antibodies, and Radiolabelled Peptides
3.1.1 Storage of Peptides
3.1.2 Storage of Radiolabeled Peptides
3.2 Antibody Production
3.2.1 Glutaraldehyde Conjugation
3.2.2 Carbodiimide Conjugation
3.2.3 Immunization
3.3 Antibody Testing
3.4 Radiolabel Production
3.4.1 Iodogen Method of Iodination
3.4.2 Chloramine T Method of Iodination
3.4.3 Separation of Intact Radioiodinated Peptide
3.4.4 Chemical Assessment of Label
3.4.5 Partial Purification of Iodinated Peptide
3.5 Collection of Blood for Radioimmunoassay of Gut Hormones
3.6 Procedure for Radioimmunoassay (See Note 5)
3.7 Separation of Radioimmunoassay
3.7.1 Charcoal–Dextran Separation (See Fig. 6 and Note 8)
3.7.2 Secondary Antibody Separation
4 Notes
References
Chapter 11: Measurement of Melatonin and 6-Sulphatoxymelatonin
1 Introduction
2 Materials
2.1 Plasma 3 H-Melatonin Assay (Assay Has Been GCMS Validated)
2.2 Saliva and Plasma 125 I-Melatonin Assay (Assay Has Been GCMS Validated)
2.3 Saliva and Plasma 125 I-Melatonin Assay: Bühlmann Test Kit
2.4 Plasma 125 I-Melatonin Assay: IBL Test Kit
2.5 Plasma Melatonin ELISA Assay: IBL Test Kit
2.6 Direct Saliva Melatonin ELISA: Bühlmann and IBL Test Kits
2.7 Urine 125 I-6-
2.8 Urine 6-Sulpha-toxymelatonin ELISA: Bühlmann and IBL Test Kits
3 Methods
3.1 Plasma 3 H Melatonin Assay
3.2 Saliva and Plasma 125 I-Melatonin Assay
3.3 Plasma 125 I-Melatonin Assay: Bühlmann Test Kit
3.4 Serum, Plasma, or Saliva 125 I-Melatonin: IBL Test Kit
3.4.1 Plasma and Serum
3.4.2 Saliva
3.4.3 Plasma, Serum, and Saliva
3.5 Direct Saliva 125 I-Melatonin Assay: Bühlmann Test Kit
3.6 Plasma or Serum Melatonin ELISA: IBL Test Kit
3.7 Direct Saliva Melatonin ELISA: IBL Test Kit
3.8 Direct Saliva Melatonin ELISA: Bühlmann Test Kit
3.9 Urine 125 I-6-
3.10 Urine 6-Sulpha-toxymelatonin ELISA: Bühlmann Test Kit
3.11 Urine 6-Sulpha-toxymelatonin ELISA: IBL Test Kit
4 Notes
References
Chapter 12: Measurement of Glucocorticoids in Biological Fluids
1 Introduction
1.1 Glucocorticoids in Plasma and Serum
1.2 Glucocorticoids in Saliva
1.3 Glucocorticoids in Urine
2 Materials
2.1 Cortisol and 11-DOC in Serum or Plasma
2.2 Cortisol and Cortisone in Saliva
2.3 Cortisol in Urine
3 Methods
3.1 Measurement of Cortisol and 11-DOC in Serum or Plasma [ 3 ]
3.2 Measurement of Cortisol and Cortisone in Saliva [ 4 ]
3.3 Measurement of Cortisol in Urine [ 5 ]
3.4 Calculation of Results
4 Notes
References
Chapter 13: The Measurement of Androgens
1 Introduction
2 Materials
2.1 Materials for Testosterone Extraction Assay
2.2 Measurement of Testosterone by LC-MSMS Following Protein Precipitation
2.3 Solvent Extraction of Testosterone Before LC-MSMS
2.4 LC-MSMS
2.5 Measurement of Testosterone in Saliva
2.6 Measurement of Testosterone in Hair
2.7 Measurement of Free Testosterone
2.8 Measurement of Dehydroepian
2.9 Measurement of Dihydrotestosterone
3 Methods
3.1 Extraction of Testosterone for Immunoassay
3.2 Radio
3.3 Protein Precipitation of Samples Before TMS
3.4 Solvent Extraction of Samples Before TMS
3.5 LC-MSMS
3.6 Measurement of Testosterone in Saliva
3.7 Measurement of Testosterone in Hair
3.8 Measurement of Free Testosterone in Serum
3.9 Measurement of Dehydroepi
3.10 Measurement of DHT
4 Notes
References
Chapter 14: Measurement of Aldosterone in Blood
1 Introduction
2 Materials
2.1 Solvent Extraction
2.2 Radio-immunoassay
2.3 Charcoal Separation
3 Methods
3.1 Solvent Extraction
3.2 Radio-immunoassay
3.3 Charcoal Separation and Results Calculation
4 Notes
References
Chapter 15: Measurement of Plasma Renin Activity
1 Introduction
2 Materials
2.1 Generation of Angiotensin I
2.2 Radio-immunoassay
2.3 Charcoal Separation
3 Methods
3.1 Generation of Angiotensin I
3.2 Radio-immunoassay of Angiotensin I
3.3 Charcoal Separation and Results Calculation
4 Notes
References
Chapter 16: Measurement of Vitamin D
1 Introduction
2 Materials
2.1 Serum and Plasma LC-MS/MS Analysis
2.2 Dried Blood Spots
3 Methods
3.1 25-Hydroxyvi- tamin D 2 and D 3 in Serum/Plasma
3.1.1 Liquid/Liquid Extraction
3.1.2 Detection
3.2 25-Hydroxyvi- tamin D 2 and D 3 in Dried Blood Spots
3.2.1 Elution
3.2.2 Derivatization
3.2.3 Detection
4 Notes
References
Chapter 17: Urinary Steroid Profiling
1 Introduction
2 Materials
2.1 Solvents
2.2 Solvents Requiring Preparation
2.3 Derivatizing Reagents
2.4 Chemicals
2.5 Gases
2.6 Other Materials and Equipment
2.7 Internal Standards
2.8 Calibration Standards
2.9 Clean Working Practices
3 Methods
3.1 Urine Extraction
3.2 Hydrolysis of Conjugates ( See Note 5)
3.3 Addition of Internal Standards and Purification
3.4 Derivatization
3.5 Gas Chromatography/Gas Chromatography-Mass Spectrometry
3.6 Variant Method for Infants Under 12 Weeks
3.6.1 Conjugate Separation ( See Note 15)
3.6.2 Derivatization
3.7 Evaluation of GC and GC-MS Data
3.8 Summary of Profiling Findings in Steroid-Related Disorders (Affected Gene Given in Parentheses)
4 Notes
References
Chapter 18: Internal Quality Control
1 Introduction
1.1 Quantitative Assays
1.2 Qualitative Assays
2 IQC Material
2.1 Commercial IQC
2.1.1 Availability
2.1.2 Cost
2.1.3 Convenience
2.1.4 Stability
2.1.5 Safety
2.1.6 Storage
2.1.7 Assayed Versus Unassayed Material
2.1.8 Matrix
2.1.9 Concentration Range
2.2 Patient Material for IQC
2.2.1 Availability
2.2.2 Cost
2.2.3 Stability
2.2.4 Safety
2.2.5 Storage
2.2.6 Assayed Versus Unassayed Material
2.2.7 Matrix
2.2.8 Concentration Range
3 Deriving IQC Ranges
3.1 Random Access Assays
3.2 Batch Assays
3.3 Data Collection
3.4 IQC Data Calculation
3.5 Recording IQC Data
3.5.1 Automatic Plotting
3.5.2 Manual Plotting
3.6 Interpretating IQC Data
3.6.1 Multirule IQC
3.7 Acting on IQC Failures
3.8 Common Causes of IQC Failure
4 Notes
References
Additional Reading
Useful Websites
Chapter 19: External Quality Assessment Schemes for Immunoassays
1 Introduction
2 Requirements for Effective EQA Provision
2.1 Scheme Infrastructure
2.1.1 Organization and Quality Management System
2.1.2 Personnel
2.1.3 Premises and Environment
2.1.4 Equipment, Information Systems, and Materials
2.2 Scheme Design
2.2.1 Frequency of Sample Distribution
2.2.2 Return of EQA Reports to Participants
2.2.3 Accurate Recording of Methods Used and Units for Reporting
2.2.4 Treatment of EQA Specimens as Clinical Specimens
2.3 Sample Material Distributed
2.3.1 Essential Requirements
2.3.2 Commutable EQA Samples
2.3.3 EQA Samples of Appropriate Concentration
2.3.4 Homogeneity and Stability of EQA Specimens
2.3.5 Minimizing the Risk of Infectious Hazards
2.4 Definition of Target Values
2.4.1 Reference Measurement Target Values
2.4.2 Consensus Target Values
3 Assessment of Analytical Performance
3.1 Overall Performance
3.2 Method Performance
3.2.1 Between-
3.2.2 Accuracy
3.2.3 Sensitivity
3.2.4 Specificity
3.2.5 Interferences from Other Substances
3.2.6 High Dose Hook Effects
3.2.7 Individual Laboratory Performance
Statistical Calculations
Criteria for Acceptable Performance
3.2.8 Provision of Informative EQA Reports
3.2.9 Provision of Customized Reports for Networks of Laboratories
3.2.10 Method-Related Reports
3.2.11 Assessment of Other Aspects of Laboratory Performance
Interpretation of Laboratory Results
Questionnaire Data
4 Other Aspects of EQA Provision
4.1 Literature Surveys
4.2 Communication with Participants
4.3 Evaluation and Improvement Processes
References
Index