Immunochemistry LabFax

Contributors

Abbreviations


1. Plasma Proteins

Plasma and Serum

Anticoagulants

Properties of Commonly Used Anticoagulants (Table 1)

Serum

Preparation of Serum from Plasma (Reversal of Anticoagulants) (Table 2)

Plasma/Serum Proteins

Human Blood Plasma Proteins in Binding and Transport of Ligands (Table 3)

Human Blood Plasma Apolipoproteins (Table 4)

Human Blood Plasma Proteins in Coagulation and Fibrinolysis (Table 5)

Human Blood Plasma Proteinase Inhibitors (Table 6)

Human Immunoglobulins (Table 7)

Human Complement Components (Table 8)

Factors Affecting the Concentration of Plasma Proteins

The Effect of Age

Protein, Albumin, γ-Globulin Plasma Concentrations (Table 9)

Concentration of Immunoglobulin Isotypes in Plasma (Table 10)

Concentration of IgG Subclasses in Plasma (Table 11)

Concentration of C3, C4 and C5 in Plasma (Table 12)

The Acute Phase Response

Acute Phase Proteins (Table 13)

Plasma Proteins in Other Body Fluids

Concentrations of Immunoglobulins and Albumin in Body Fluids (Table 14)

Antibodies Recognizing Plasma/Serum Proteins

Absorption of Unwanted Specificities

Anti-Whole Sera Antibodies

Purified Antibodies

IgG fractions

Affinity Purified Antibodies

Monoclonal Antibodies

Storage of Commercial Antibodies

References


2. Properties of Immunoglobulins

Introduction

Diversity of Immunoglobulins

The General Structure of Immunoglobulins (Figure 1)

General Properties of Human Immunoglobulins (Table 1)

Polypeptide Composition of Human Immunoglobulins (Table 2)

Distribution and Characteristics of Human Immunoglobulins (Table 3)

Biological Properties of Human IgG Subclasses (Table 4)

Reported Antibody Specificities of Human IgG Subclasses (Table 5)

Biological Properties of Human IgA Subclasses (Table 6)

Human Immunoglobulin Allotypes

Specificity and Frequency of Human Immunoglobulin Allotypes (Table 7)

Immunoglobulins from Other Species

Properties of Mouse Immunoglobulins (Table 8)

Properties of Rat Immunoglobulins (Table 9)

Distribution of Light Chain Types Among Various Species (Table 10)

Immunoglobulin Receptors (Fc Receptors)

Molecular Properties of Human Leukocyte Fc Receptors (Table 11)

Distribution and Regulation of Human Leukocyte Fc Receptor Expression (Table 12)

General Characteristics of Other Fc Receptors (Table 13)

References


3. Antibodies Recognizing Immunoglobulins

The Specificity of Antibodies Recognizing Immunoglobulins of Other Species

Anti-Immunoglobulin Whole Molecule Antibodies

Anti-Immunoglobulin Heavy or Light Chain-Specific Antibodies

Antibodies Against Immunoglobulin Subclasses

Cross-Reactions of Anti-Immunoglobulin Reagents

Conjugated and Derivatized Anti-Immunoglobulin Reagents

Commercial Availability of Antibodies Recognizing Immunoglobulins of Different Species

References


4. Producing Antibodies

Polyclonal Antisera

Preparation of Immunogen

Haptenization of Peptides

Adjuvants

Species and Immunization Routes

Immunization Protocols

Bleeding Animals and Preparation and Storage of Sera

Monoclonal Antibodies

'Immune' Lymphocytes

Cell fusion (Hybridoma Production) and Fusion Partners

Cell Culture, Medium and Supplements

Cloning

Production of Monoclonal Antibodies

Production of Human Monoclonal Antibodies by Lymphocyte Transformation

rDNA-Produced Antibodies

Spliced Immunoglobulin Molecules

Recombinant DNA-Derived Immunoglobulin Fragments

References


5. Purification and Fragmentation of Immunoglobulins

General Properties of Antibodies

Stability

Sources of Immunoglobulins

Sources of Immunoglobulins (Table 2)

Methods of Purification of IgG

Precipitation with Sodium Sulfate or Ammonium Sulfate

Precipitation with Sodium Sulfate or Ammonium Sulfate Followed by Ion Exchange Chromatography

Precipitation with Caprylic Acid (Octanoic Acid)

Precipitation with Caprylic Acid Followed by Ion Exchange Chromatography

Chromatography on Immobilized Protein A or Protein G

Purification of IgM

Gel Filtration

Purification of Serum IgA

Precipitation with Ammonium Sulfate Followed by Ion Exchange Chromatography

Chromatography on Immobilized Jacalin

Other Techniques for Immunoglobulin or Antibody Purification

Chromatography on Immobilized Anti-Immunoglobulin

Chromatography on Immobilized Antigen

Properties of Bacterial IgG-Binding Proteins: Protein A and protein G

Properties of IgA-Binding Proteins

Purified Immunoglobulins Available Commercially

Preparation of Immunoglobulin Fragments

References


6. Preparation and Use of Radiolabeled Antibodies and Antigens

Labeling with Radioisotopes

Labeling with Iodine-125

Labeling with Other Isotopes

Production of Radiolabeled Antibodies for Radioscintography

Labeling with ß-emitter (Tritium)

Radioimmunoassay

Buffer

Assay Components

Data Analysis

Further Optimization

Immunoradiometric Assay

Assay Components

Data Analysis

Assay Optimization

References


7. Enzyme-Conjugated Antibodies: Preparation and Use

Introduction

Enhanced Enzyme Immunodetection Systems

Enzymes Commonly Used in Immunoconjugates

Horseradish Peroxidase

Alkaline Phosphatase

ß-Galactosidase

Glucose Oxidase

Urease

Methods for the Conjugation of Enzymes to Antibodies

Glutaraldehyde-Mediated Conjugation

Conjugation Mediated by Heterobifunctional Cross-Linking Reagents

Periodate-Mediated Conjugation

Properties of Enzyme Substrates Commonly Used in Immunoassay

Substrates for Horseradish Peroxidase

Substrates for Alkaline Phosphatase

Substrates for ß-Galactosidase

Substrates for Glucose Oxidase

Substrates for Urease

References


8. Derivatization of Antibodies and Antigens: Flourescent Labeling, Biotinylation, Immunogold Derivatives

Fluorescently Labeled Antibodies

Choice of Fluorochrome

Conjugation of Antibodies

Laser-Excitation of Fluorochromes

Biotinylation of Proteins

General

Properties of Biotin

Properties of Avidin

Properties of Streptavidin

Colloidal Gold Derivatives

Properties of Colloidal Gold Conjugates

References


9. Common Immunological Techniques: Elisa, Blotting, Immunohistochemistry and Immunocytochemistry

Elisa

Some Variables to be Considered in Developing Enzyme Immunoassays

Immunoblotting

Alternative Techniques in Immunoblotting

Immunohistochemistry and Immunocytochemistry

Light Microscopy Immunostaining

Immuno-Electron Microscopy

References


10. Complement

Introduction

Nomenclature

General Properties of Complement

Heat Instability

Metal Ion Requirement

Stability on Storage

Concentration Dependence

Proteins of the Complement System

Activation of the Classical Pathway

Activation of the Alternative Pathway

The Cleavage of C5 and Assembly of the Terminal Components

Control of the Complement System

The Fragmentation of C3

Complement Receptors

Detection and Estimation of Complement Components

Hemolytic Assays

Table of Lysis to Sites Values

Antigenic Assays for Complement Components

Functional Assays and Enzyme Assays

Detection of Activation Products

Biological Assays

Complement Measurement in Disease

Genetics of the Complement System

References


11. Safety

Chemical Safety

Risk and Safety Classification Systems

Radiochemical Safety

Definitions

Conversion Between Units

Protection from Radiochemical Hazards

Microbiological Safety

Viral Safety

Introduction

General Requirements

Centrifugation

References

Index