Immunohistochemistry and Immunocytochemistry E-Book

• Herausgeber: John Wiley & Sons
• Kategorie: Wissenschaft und neue Technologien
• Serie: Essential Methods
• Sprache: Englisch

Seitenzahl: 411

Veröffentlichungsjahr: 2017

Beschreibung

Immunohistochemistry and immunocytochemistry are invaluable tools for the visualization of tissue and cellular antigens in diagnostic and biological research environments. The need to obtain accurate, reliable and reproducible results is paramount.

It is with this fundamental aim in mind that we have compiled Immunohistochemistry: Essential Methods. We have achieved this by examining each aspect of immunochemistry in turn, with each chapter including detailed information regarding the subject matter in question. Each chapter is written by an expert in their field and includes protocols that are typically used in their own research. Subjects covered are, amongst others, antibodies and their production; selection of reporter labels; immunochemical staining methods and experimental design (both using single and multiple reporter labels); quality assurance; automated immunochemistry; confocal microscopy and electron microscopy. In addition, benefits and limitations of each approach are discussed within the chapters.

Leseprobe

Table of Contents

Title Page

Copyright

List of Contributors

Preface

Acknowledgements

Chapter 1: Antibodies for Immunochemistry

INTRODUCTION

IMMUNOGENS FOR ANTIBODY PRODUCTION

ANTIBODY PRODUCTION

ANTIBODY PURIFICATION

FRAGMENT ANTIBODY PREPARATIONS

ANTIBODY LABELLING

ANTIBODY STABILITY AND STORAGE

REFERENCES

Chapter 2: The Selection of Reporter Labels

INTRODUCTION

ENZYMATIC LABELS

FLUORESCENCE DETECTION

REFERENCES

Chapter 3: Immunohistochemistry and Immunocytochemistry

SPECIMEN FORMATS FOR IMMUNOCHEMISTRY

FIXATION

PROCESSING TISSUE BLOCKS TO PARAFFIN WAX

MICROTOMY

TISSUE MICROARRAYS

SPECIMEN STORAGE

DECALCIFICATION

ANTIGEN RETRIEVAL

CONTROLS

IMMUNOCHEMICAL STAINING TECHNIQUES (OPTIMIZING A NEW ANTIBODY)

COUNTERSTAINS

MOUNTING

TROUBLESHOOTING

EXAMPLES OF IMMUNOSTAINING PHOTOMICROGRAPHS

ACKNOWLEDGEMENTS

REFERENCES

Chapter 4: Multiple Immunochemical Staining Techniques

INTRODUCTION

METHODS AND APPROACHES

REFERENCES

Chapter 5: Quality Assurance in Immunochemistry

INTRODUCTION

METHODS AND APPROACHES

AUTOMATED IMMUNOCHEMICAL STAINING

TROUBLESHOOTING

REFERENCES

Chapter 6: Automated Immunochemistry

INTRODUCTION

METHODS AND APPROACHES

OTHER FORMS OF AUTOMATION

REFERENCES

Chapter 7: Confocal Microscopy

INTRODUCTION

WHEN SHOULD CONFOCAL BE USED?

APPLICATIONS: FOR EXAMPLE CO-LOCALIZATION, QUANTIFICATION, 3D VISUALIZATION AND KINETICS

HOW TO SET UP A CONFOCAL EXPERIMENT?

REFERENCES

FURTHER READINGS

Chapter 8: Ultrastructural Immunochemistry

INTRODUCTION

METHODS AND APPROACHES

REFERENCES

Index

End User License Agreement

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Guide

Cover

Table of Contents

Preface

Begin Reading

List of Illustrations

Chapter 1: Antibodies for Immunochemistry

Figure 1.1 A Schematic Diagram of an Immunoglobulin MoleculeThe basic antibody molecule is a Y-shaped structure consisting of two heavy and two light polypeptide chains joined by disulfide bonds. The chains are composed of variable (orange) and constant (blue) immunoglobulin domains. The antibody–antigen binding site (paratope) is located at the tip of the Y arms. The Fab and Fc regions of the molecule are tethered together by a hinge region, which provides the antibody molecule with flexibility. Constant domains share the same amino acid sequence for a given antibody isotype. Different immunoglobulin isotypes arise from subtle sequence variations in the constant domains.

Figure 1.2 Typical Primary and Secondary Responses to an AntigenUpon first exposure to an antigen, a series of B-cell divisions must occur before enough antibody is present to be effective. This lag phase is significantly reduced upon subsequent exposure to the same antigen. Memory B cells generated from the primary response are available to rapidly divide, thus reducing the time required to produce antibodies and generating larger quantities.

Figure 1.3 An Outline Affinity Purification Chromatogram

Figure 1.4 Fragmentation of IgG Immunoglobulin by Papain and Pepsin

Figure 1.5 Fluorescent Labelling of Antibody with FITC

Chapter 2: The Selection of Reporter Labels

Figure 2.1 Diagram of Fluorochrome Excitation. The Addition of Light of a Certain Wavelength to a Fluorochrome Results in the Excitation of an Electron from the S

0

(Ground State) to the Unstable S

1

′ State. In this State, the Electron only Remains for a Few Nanoseconds before a Conformational Change Results in the Release of Heat and a Drop to the More Stable S

1

State. Once the Electron Moves from the S

1

State Back to S

0

, the Remaining Energy Taken up During the Excitation Process is Emitted as Light of a Longer Wavelength than the One Initially Absorbed

Chapter 3: Immunohistochemistry and Immunocytochemistry

Figure 3.1 Formation of Formaldehyde Polymers.

Figure 3.2 Monomerization of Paraformaldehyde.

Figure 3.3 Formaldehyde Fixation through Cross-Linking of Adjacent Proteins.

Figure 3.4 Chemical Structure of Glutaraldehyde.

Figure 3.5 Glutaraldehyde Polymer Formation.

Figure 3.6 Mechanics of Glutaraldehyde Methylene Bridge Formation.

Figure 3.7 Avidin–Biotin Complex (ABC) Immunochemical Staining Protocol.

Figure 3.8 Label-Conjugated Secondary Antibody Immunochemical Staining Protocol.

Figure 3.9 Compact Polymer Immunochemical Staining Protocol.

Chapter 4: Multiple Immunochemical Staining Techniques

Figure 4.1 Aspects to be Considered When Selecting a Multi-Staining Method. The Choice to Perform Immunofluorescence or an Enzymatic-Based Assay will Depend on the Type of Sample, the Reagents, the Nature of the Target and the Imaging Tools Available

Figure 4.2 Differences between Direct and Indirect Method in Immunochemical Staining. In the Direct Method, the Directly Labelled Primary Antibody Binds the Antigen (a). In the Indirect Method, the Primary Antibody Binds the Antigen, and in the Second Step, Secondary Antibodies (Reporter Labelled) Bind to the Primary Antibody (b), Giving a Degree of Signal Amplification

Figure 4.3 Combinations of Primary and Secondary Antibodies to Be Used to Prevent Cross-Reactivity

Figure 4.4 Double Immunostaining Using Conjugated Fab Fragments

Figure 4.5 Double Immunostaining Using Unconjugated Fab Fragments

Chapter 5: Quality Assurance in Immunochemistry

Figure 5.1 Example of a Microtomy Artefact: An Air Bubble under the Section Has Trapped Antibodies and Chromogen, Leading to the Observed Non-specific Artefact

Figure 5.2 Determination of Optimal Dilution of Antibody: The Poor Reaction in Area 1 Is due to Steric Hindrance of the Primary Antibody Accessing the Antigen as the Antibody Concentration Being Too High. This Is Known as Prozone, but Is a Rare Phenomenon in Immunochemical Staining. The Suboptimal Reaction in Area 2 Is Caused by Insufficient Primary Antibody, That Is the Concentration of Primary Antibody Is Too Low

Figure 5.3 Example of Absorption Control for Immunochemical Staining

Chapter 6: Automated Immunochemistry

Figure 6.1 Leica Biosystems BOND-III, An Example of An Array Stainer.

Figure 6.2 Leica Aperio AT2®, An Example of a High-Throughput Automated Slide Scanner.

Chapter 7: Confocal Microscopy

Figure 7.1 Widefield Epifluorescent Microscopy versus Confocal. (a) A Schematic Diagram of a Widefield Epifluorescent Microscope (Green for Excitation Wavelength of Light, Red for Emitted). (b) Shows the Comparison of a Widefield Epifluorescent with a Confocal Microscopy Image of Gut Epithelia Cells. Nuclei Are Shown in Blue (DAPI), Dividing Cells in Red (EdU) and Cytoskeleton in Green (Cytokeratin)

Figure 7.2 Schematic Diagram Detailing Confocal Set-Up: (a) A Simplified Diagram Showing Confocal Microscope Set-Up; (b) Schematic Diagram Showing Confocal Point-by-Point Scanning, Yellow Dots Indicate the Laser Line Scanning the Sample

Figure 7.7 Comparison of Simultaneous and Sequential Imaging for Confocal Data Acquisition

Figure 7.3 (a) A Typical Objective Lens; (b) Table Explaining the Text Written on the Objective Lens

Figure 7.4 (a) A High-Resolution, Low-Magnification Image; (b) A High-Magnification, Low-Resolution Image

Figure 7.5 Schematic Diagram Showing Confocal Configuration of a Confocal with a Spectral Detector Which Can Detect Emission across the Visible Spectrum

Figure 7.6 Spectral Bleedthrough Can Lead to Misinterpretation of Scientific Data. Confocal Images of Kidney Epithelia Labelled with

N

-Cadherin and CellTracker Orange

Figure 7.8 Best Practice in Alignment of PMTs. Confocal Image of

N

-Cadherin in Kidney Cells. Red, Saturated Pixels; Blue, Under-Exposed Pixels; and Grey, Pixels in Linear Detector Range

Figure 7.9 Scan Averaging and Altering Scan Speed/Pixel Dwell Time Improve Confocal Image Quality: (a) Image of Kidney Epithelia Was Scanned at High-Speed/Short-Pixel Dwell Time; (b) the Image Was Acquired with Longer Pixel Dwell Time/Slow Scan Speed and Displays the Average of Four mages

Figure 7.10 Confocal Microscopy Workflow Summary Diagram

Figure 7.11 Three-Dimensional Confocal Image of Mouse Hair Follicles

Figure 7.12 Resolution Limit in 3D of Microscope: (a) Schematic Diagram Showing How a Point Source of Light Is Distorted by the Optical Elements in a Microscope to Yield a Measureable Point Spread Function. (b) Measuring the Resolution by Looking at the Full Width at Half Maximal Intensity of Image of the Point Source Shows That the Resolution in

x

,

y

Is Half the Size in

z

Figure 7.13 Acquiring

z

Stacks: (a) Diagram of Nyquist Imaging of a Nucleus in 3D with 1-um Optical Sections Using 500 nm Steps (b) Indicated How to Set Up a

z

Stack Where the Bottom and Top of the Object Are Defined and Then Imaged

Figure 7.14 Tiled Image of Mouse Retina Olympus BioScapes 2013

Figure 7.15 Confocal Microscope with Environmental Incubation for Live Imaging

Figure 7.16 Fluorescent Protein Types

Figure 7.17 Neurons in Brainbow Mice http://suzs.tumblr.com/post/4416556844/ryan-sciandra-brainbow-is-a-term-used-to

Figure 7.18 Schematic Diagrams of Different Fast Confocals: (a) Spinning Disk (b) Slit Scanning (c) Light Sheet

Figure 7.19 FRET Theory. (a) Donor Dye Is Too Far to Pass on the Energy for the Laser Light Wave onto the Acceptor. (b) Donor and Acceptor Are Close Enough Together that the Energy from the Donor Can Be Passed to the Acceptor Allowing the Acceptor's Fluorescence to Be Excited by the Energy Emitted by the Donor Dye

Figure 7.20 Fluorescent Recovery after Photobleaching. (a) Schematic Diagram of a FRAP Experiment with Slow and Fast Recovery Times. (b) FRAP Quantification (i) Shows Bleach Efficiency and (ii) Indicates the Time for Half of the Fluorescence to Recover. (iii) Once Enough Time Has Passed that the Fluorescence Has Stabilized, the Level of Fluorescence Remaining Divided by the Initial Fluorescence Intensity Shows What Percentage of the Protein of Interest Will Turn Over. This Is Termed the Mobile Fraction

Chapter 8: Ultrastructural Immunochemistry

Figure 8.1 Thin Section of a Rat Pancreatic Acinar Cell. The Section Was Fixed in 3% Formaldehyde, Cryoprotected in 30% Polypropylene Glycol, Dehydrated by Freeze Substitution and Low Temperature Embedded in Lowicryl HM20. Cells Were Immunostained for the Presence of Amylase. Gold Particles Indicate the Rough Endoplasmic Reticulum (Arrows) and Zymogen Granules (Z). Mitochondria (M) Are Unlabelled, Showing That Non-Specific Labelling Is Low. Bar, 200 nm

Figure 8.2 Thin Sections through Single Ommatidia from a Wild-Type or Mutant Drosophila Eye, Immunolabelled for Rhodopsin. The Eyes Were Fixed in 3% Glutaraldehyde, Osmicated and Embedded in Spurr's Resin (a, b; Wild Type) or Fixed in 4% Formaldehyde and Embedded in LR White (c, d; Mutant). Each Ommatidia Contains Seven Rhabdomeres (a). Rhabdomeres 1–6 Express Rhodopsin, while Rhabdomere 7 Does Not (b). In the Mutant Eye, Ommatidia Are Deleted or Altered (c). The Rhabdomeres Are Also Structurally Altered, but Their Staining Pattern for Rhodopsin Remains Unchanged, with No Expression of Rhodopsin in Rhabdomere 7 (d). Bars, 200nm

Figure 8.3 Thin Section through a Rat Pancreatic β-Cell. The Tissue Was Fixed in 4% Glutaraldehyde/1% Osmium Tetroxide, Bulk Stained in Uranyl Acetate and Embedded in Spurr's Resin. The Section Was Treated with Sodium Metaperiodate before Immunostained for Insulin. The Crystalline Cores of the Secretory Granules Are Heavily Labelled with Gold Particles. Bar, 250 nm

Figure 8.4 Thin Section through a Proximal Convoluted Tubule of a Rat Kidney. The Tissue Was Fixed by Immersion in 2% Formaldehyde and Embedded in LR White after Bulk Staining in Uranyl Acetate. The Basal Lamina Is Labelled with Gold Particles after Immunostaining for Laminin. Despite the Weak Fixation, the Outer Mitochondrial Membranes and Cristae of Mitochondria (Arrows) Can Be Clearly Distinguished. Bar, 250 nm

Figure 8.5 Thin Section of a Vero Cell Infected with Human Papilloma Virus. Cells Were Quench Frozen in Melting Propane Cooled in Liquid Nitrogen, Dehydrated by Freeze Substitution against Pure Methanol Containing 0.1% Uranyl Acetate and Low-Temperature Embedded in Lowicryl HM20. Cells Were Immunostained for Glycoprotein D. Gold Particles Indicate the Nuclear Membrane and the Rough Endoplasmic Reticulum (Arrows) and the Membrane Acquired by a Virus Particle (V) That Has Just Budded through the Nuclear Envelope. Bar, 200nm

Figure 8.6 Ultrathin-Thawed Cryosection of Placental Syncytium. Sections Were Fixed in 6% Formaldehyde, Cryoprotected in Sucrose and Polyvinylpyrrolidone and Retrieved from the Microtome on Methylcellulose and Sucrose. Cells Were Immunostained for Copper/Zinc Superoxide Dismutase. Gold Particles Can Be Seen Over Both the Cytoplasm (C) and the Nucleoplasm (N). Bar, 200 nm

List of Tables

Chapter 1: Antibodies for Immunochemistry

Table 1.1 A Comparison of Immunoglobulin ClassesIgG has the longest half-life of all the antibody classes and is produced during the secondary immune response. IgG, IgD and IgE are monomeric structures consisting of a single antibody unit. IgA can occur as a monomer or dimer (two units). IgM exists as a pentameric molecule, with five basic immunoglobulin units joined by an additional polypeptide chain (J chain), making it the largest antibody class with a molecular weight of 970 kDa

Table 1.2 Binding Affinity of Antibodies for Proteins L, A and G

Chapter 2: The Selection of Reporter Labels

Table 2.1 Overview of HRP Chromogens

Table 2.2 Overview of AP Chromogens

Table 2.3 Absorption and Emission Spectra for Common Fluorochromes

Chapter 3: Immunohistochemistry and Immunocytochemistry

Table 3.1 Classification of Fixatives

Table 3.2 Example of Antibody Optimization Experiment

Table 3.3 Detection Systems

Table 3.4 Common Fluorescent Nuclear and Cell Membrane Counterstains

Chapter 4: Multiple Immunochemical Staining Techniques

Table 4.1 Controls Required for a Multiple Immunochemical Staining Experiment, Using Two Primary Antibodies (Ab1 and Ab2), and Their Corresponding Secondary Antibodies (S1 and S2), Respectively

Chapter 5: Quality Assurance in Immunochemistry

Table 5.1 Troubleshooting Immunochemical Staining Procedures

Chapter 6: Automated Immunochemistry

Table 6.1 Common Automated Immunochemical Staining Platforms Available at the Time of Publication

Table 6.2 Common Antigen Retrieval Systems Available at the Time of Publication

Table 6.3 Higher Profile Digital Pathology Solutions Available at the Time of Publication

Chapter 7: Confocal Microscopy

Table 7.1 Relationship between Maximum Resolution and Magnification, Numeric Aperture, Immersion Medium and Emission Wavelength

Table 7.2 Guide to Standard Laser and Emission Filter Configurations on Confocal Microscopes

Table 7.3 Types of Fast Scanning Confocal Microscopes

Table 7.4 Image Processing Packages for Confocal Microscopy

Immunohistochemistry and Immunocytochemistry

Essential Methods

EDITED BY

SIMON RENSHAW

Second Edition

This edition first published 2017 © 2017 by John Wiley & Sons, Ltd.

First edition published 2005 by Scion Publishing Ltd.

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Cover image: © Abcam plc

List of Contributors

Mark Cooper

Abcam plc, Cambridge, UK

Michael Gandy

The Doctors Laboratory Ltd, London, UK

Peter Jackson

Department of Histopathology, Leeds General Infirmary, Leeds, UK (retired)

Sofia Koch

Abcam plc, Cambridge, UK

Judith Langenick

AbD Serotec, Oxford, UK

Sheriden Lummas

Abcam plc, Cambridge, UK

Janet Powell

Cambridge Advanced Imaging Centre, Department of Anatomy, University of Cambridge, Cambridge, UK

Simon Renshaw

Abcam plc, Cambridge, UK

Emanuel Schenck

Medimmune LLC, Gaithersburg, MD, USA

Jeremy Skepper

Cambridge Advanced Imaging Centre, Department of Anatomy, University of Cambridge, Cambridge, UK

Ann Wheeler

Institute of Genetics and Molecular Medicine Advanced Imaging Resource, University of Edinburgh, Edinburgh, UK

Preface

IMMUNOCHEMISTRY IS AN INVALUABLE TOOL for the visualization of cellular antigens in diagnostic and biological research environments. The need to obtain accurate, reliable and reproducible results is of paramount importance.

It is with this fundamental aim in mind that we have compiled Immunohistochemistry and Immunocytochemistry: Essential Methods. We have achieved this by examining each aspect of immunochemistry in turn, with each chapter including detailed information regarding the subject matter in question. Each chapter is written by an expert in their field and includes protocols that are typically used in their own research. In addition, benefits and limitations of each approach are discussed within the chapters.

This book offers a wealth of knowledge to the novice immunochemist, who, from the outset, wishes to fully understand the theory and practice of immunochemical staining techniques and obtain reliable and reproducible data time and time again. For the experienced immunochemist, this book is a comprehensive reference guide to the theory and practice of immunochemical staining techniques, allowing further optimization of existing immunochemical staining protocols.

Simon Renshaw January 2017

Acknowledgements

THANK YOU TO ALL of my friends, family and colleagues for your continued support throughout this project.

A special thank you goes to the contributing authors, without whom this book would have taken considerably longer to write!

Thank you to Elsevier Ltd, Abcam plc and Leica Biosystems for kindly agreeing to reproduction of copyrighted materials.

Finally, a very special dedication goes to Chris van der Loos, who had very kindly agreed to be the author of the ‘Multiple Immunochemical Staining Techniques’ chapter, but sadly passed away before beginning the work. He was incredibly gifted in his field and delivered a most informative and entertaining lecture. He will be missed by many.

Chapter 1Antibodies for Immunochemistry

Mark Cooper and Sheriden Lummas

Abcam plc, Cambridge, UK

INTRODUCTION

Unlike innate immunity, the adaptive immune response recognizes, reacts to and remembers foreign substances invading an organism. Antibodies play a central role in the function of adaptive immunity. Their roles are to detect, specifically bind and facilitate the removal of foreign substances from the body. Memory B cells create an immunological memory that allows the immune system to respond quicker upon subsequent exposure to the same foreign substance.

A substance not recognized by the immune system as being native to the host and therefore stimulates an immune response is known as an antigen (antibody generator). Binding of an antigen to an antibody is specific. Biochemical research utilizes the ability of antibodies to distinguish between antigens and to detect biological molecules (commonly proteins) in cells and tissues using immunochemical staining techniques. Immunochemistry is the focus of this text, and its practice is discussed in detail throughout later chapters (see p 35).

Typical Antibody Structure

Antibodies are immunoglobulin (Ig) proteins produced by B cells in the presence of an antigen. Immunoglobulins exist as five main classes or isotypes: IgA, IgD, IgE, IgG and IgM. Each isotype performs a different function in the immune system. IgG has a long half-life in serum (Table 1.1), which means its clearance from the circulatory system is slow. The abundance and retention of IgG in circulation compared to the other classes make it the most common antibody isotype reagent used in biochemical research.

Table 1.1 A Comparison of Immunoglobulin ClassesIgG has the longest half-life of all the antibody classes and is produced during the secondary immune response. IgG, IgD and IgE are monomeric structures consisting of a single antibody unit. IgA can occur as a monomer or dimer (two units). IgM exists as a pentameric molecule, with five basic immunoglobulin units joined by an additional polypeptide chain (J chain), making it the largest antibody class with a molecular weight of 970 kDa

Immunoglobulin

IgG1

IgG2

IgG3

IgG4

IgM

IgA1

IgA2

IgD

IgE

Molecular weight (kDa)

146

146

165

146

970

160

160

184

188

Serum level (mean adult mg ml

−1

)

9

3

1

0.5

1.5

3

0.5

0.03

5 × 10

−5

Half-life in serum (days)

21

20

7

21

10

6

6

3

2

Location

Bloodstream. Can pass through blood vessel walls readily and cross into the placenta.

Bloodstream

Body secretions: tears, sweat, saliva; breast milk

B-cell surface

Bound to mast cells

Function

Activates complement pathway

Produced during primary immune response and activates complement system

Form a defence on the surface of body cells. Immune protection to newborn.

Unknown

Stimulates allergy response

The basic antibody unit is shared across all five isotypes. Two identical heavy (H) and light (L) polypeptide chains connected by a disulfide bond form the commonly illustrated Y-shaped antibody structure (Fig. 1.1). The arms of the Y structure form the Fab (fragment antigen-binding) region while the base is the Fc (fragment crystallizable) region.

Figure 1.1 A Schematic Diagram of an Immunoglobulin MoleculeThe basic antibody molecule is a Y-shaped structure consisting of two heavy and two light polypeptide chains joined by disulfide bonds. The chains are composed of variable (orange) and constant (blue) immunoglobulin domains. The antibody–antigen binding site (paratope) is located at the tip of the Y arms. The Fab and Fc regions of the molecule are tethered together by a hinge region, which provides the antibody molecule with flexibility. Constant domains share the same amino acid sequence for a given antibody isotype. Different immunoglobulin isotypes arise from subtle sequence variations in the constant domains.

Both H and L chains consist of variable (V) and constant (C) domains, named according to the conservation of their amino acid sequence. One variable domain is present for each H chain and L chain and is situated at the amino terminus. VL and VH domains are paired together to create the antigen-binding site (paratope). Specificity of antigen binding is determined by the variation in amino acid sequence in this region. This enables antibodies to recognize a diverse range of antigens, even though only a single amino acid difference between antigens exists. The remainder of the H and L chains are composed of constant domains: one domain in the L chain and three domains in the H chains denoted as CH1, CH2 and CH3. For a given isotype, the entire CH amino acid sequence is conserved. Subtle differences in the sequence occur and give rise to isotype subclasses (as provided in Table 1.1). IgG sub-classes are present across species, with IgG1, IgG2, IgG2a and IgG3 subclasses existing within mice. The subclasses have different binding affinities for the purification of protein resins, which are discussed in the ‘Antibody Labelling’ Section. It is therefore important for the sub-class to be accurately determined in order to efficiently purify antibodies from sera. Furthermore, primary antibody subclass determination is one of the critical parameters (among others) that enables the end user to select an appropriate secondary antibody.

Differences in CL sequence equate to the type of L chain, out of which two types are found in antibodies. L chains exist as lambda (λ) or kappa (k) and are identically present in one form or the other in a single antibody. At the centre of the Y-shaped structure is the hinge region that acts as a tether, linking the Fab and Fc regions of the molecule. The two Y arms of the Fab region are able to move independently, providing the molecule with flexibility when the antibody binds two identical antigens, particularly when the antigens are distances apart [1]. This is a property that contributes to the use of antibodies in immunoassays. Being glycoproteins, antibodies contain a sugar side chain (carbohydrate moiety). This is bound to the CH2 region and contributes to antibody destination within tissues and the type of immune response initiated depending on antibody class [1].

Antibody Structure Is Optimized for Its Function

In order to understand how antibodies are engineered for their function, an overview of protein structure organization has been presented.

Proteins are composed of polypeptide chains consisting of basic units called amino acids. The consecutive sequence of amino acids is the primary structure. Hydrogen bonds within the polypeptide chain generate alpha helices and beta sheets to create the secondary protein structure. As the chains are pulled into close proximity of each other, additional bonds and interactions form between amino acid side chains, and hydrophobic bonds and van der Waals interactions form between non-polar amino acids. Further reinforcement of the conformational structure is achieved by the formation of disulfide bonds between cysteine sulfhydryl groups. The result of these bonds is the generation of polypeptide subunits, that is, the tertiary protein structure. The arrangement into multi-subunit structures creates the final quaternary protein structure [2]. Two main types of quaternary protein structure exist: globular and fibrous.

Immunoglobulins are a superfamily of globular proteins with roles associated with the immune system. Examples include cell surface receptors (Fc) and antibodies. Members of this superfamily exhibit a common structural motif, that is, the immunoglobulin domain [2]. The immunoglobulin domain is approximately 110 amino acids in length. Two immunoglobulin domains are present in the light chain, whereas the heavy chain has four domains, numbered from the amino (N-) terminus to carboxyl (C-) terminus. Each domain is a sandwich-like structure formed from anti-parallel beta-pleated sheets of the polypeptide chain bound together by disulfide bonds. This structure is known as the immunoglobulin fold. Loops are created at the ends of the immunoglobulin folds where the beta-pleated sheets change the direction. These loops are 5–10 amino acids residues in length and reside within the variable regions, protruding from the surface. They are designated hypervariable (HV) loops because the amino acid sequence variation within this region is considerable. HV loops are also referred to as complementarity-determining regions (CDRs). The three HV loops present in the variable region are denoted HV1, HV2 and HV3, with HV3 containing the greatest sequence variation. The remainder of the variable region is composed of framework regions FR1, FR2, FR3 and FR4, respectively. These regions lie between the HV loops, have less sequence variation and provide structural integrity to the immunoglobulin molecule. Pairing of the HV loops from the heavy and light chains in the antibody molecule creates a single antibody–antigen binding site at the tip of the Y arms. The amino acid sequence determines the tertiary structure of this site, described as the paratope. The surface of the paratope is complementary to a specific amino acid sequence on the antigen's surface (the epitope), and thus dictates antibody specificity. Pairing different combinations of VL and VH regions generates the diverse repertoire of antibodies [1].

Antibodies must be able to perform their biological function under broad and changing conditions. Intermolecular disulfide bonds occur between the heavy and light chains along the entire length of the antibody and contribute to the stability of the molecule. Out of academic interest, by the use of reducing agents such as dithiothreitol (DTT) and 2-mercaptoethanol, the disulfide bonds can be removed to denature the antibody into its heavy and light chain fragments, with molecular weights of 50 and 25 kDa, respectively. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) using reducing conditions is routinely employed to resolve the antibody fragments.

IMMUNOGENS FOR ANTIBODY PRODUCTION

Protein Structure Considerations for Antibody Generation

The large globular structure of proteins means binding to an antibody is achieved through continuous or discontinuous epitopes. Antibodies raised using a full-length protein recognize a combination of amino acids brought together in the protein's three-dimensional conformation (tertiary structure). The amino acids are discontinuous, and this type of epitope is termed ‘conformational’. Antibodies also recognize linear epitopes by the consecutive sequence of amino acids (primary structure) [3]. In vivo digestion of a foreign substance by macrophages yields small segments of its sequence. Commercially available antibodies raised against peptide immunogens mimic this biological process.

The likely success of an antibody recognizing a continuous versus conformational epitope generally cannot be predicted. Antibodies that recognize the native protein structure are more likely to perform well in immunochemistry, due to the protein being in a more in vivo state when compared to other immunoassay techniques such as western blotting, when performed under reducing conditions. However, fixation, tissue preparation and antigen retrieval methods can potentially have a detrimental effect on the tertiary protein structure by chemically modifying amino acids [4]. It is therefore recommended that researchers evaluate and optimize their antibody in a given application (see p 59). Antibodies purchased from commercial suppliers should be supported by characterization data. Often, the protocol employed to obtain the desired staining pattern will be provided or made available upon request.

Types of Immunogens for Antibody Production

The range of antigens (immunogens) able to facilitate an immune response is diverse and may originate from within an organism or from the external environment. Antigens employed to produce antibodies to specific protein targets in industry and academia include peptides, whole cells, nucleotides and recombinant proteins. However, peptides are the most commonly used antigen, which is represented by the vast number of custom peptide suppliers available commercially. Peptides offer two main advantages over other antigens. Firstly, they are simple and quick to synthesize. Secondly, cross-reactivity with related proteins can be minimized through considered selection. Furthermore, antibodies can be raised to specific post-translational modifications such as methylation, acetylation and phosphorylation, which are important for epigenetic advances. The main limitation of peptide immunogens is that antibodies generated are less likely to recognize the protein's native structure. If candidates are poorly selected, the likelihood of epitopes lying within a region that is not accessible (due to the tertiary protein structure) is greater. This may be seen in immunochemical assays as poor or no positive staining [4]. The following sections aim to alleviate this by presenting tools and guidelines for the determination of protein structure and the subsequent selection of an antigenic and immunogenic peptide immunogen. It should be noted that this information will only be of any real use to people who are designing peptide immunogens for antibody production. Most end users will simply purchase an antibody from a commercial supplier, where this will have already have been done. However, the information will help to give the end user a wider appreciation of how antibodies are designed and produced, and how immunogen design can either negatively or positively affect the performance of an antibody in any given immunoassay.

Epitope Prediction Tools

Traditional methods for predicting a protein's conformational structure and sites of antigenicity assigned a value to each amino acid based on their physiochemical properties to generate a scale of propensity [5]. Prediction of secondary protein structure based on amino acid sequence was one of the first propensity scales to have been developed by Chou and Fasman [3]. Hydrophilicity and antigenicity indices for epitope prediction followed shortly after. The bioinformatics information that is available today is vast and extends beyond the indices and scales sited in this text. A variety of new systems have been developed over the years alongside evaluations and adaptations of historical methods. Recently, epitope prediction resources in the public domain frequently combine the historical propensity scales with novel mathematical algorithms, an example being the BepiPred method to predict linear B-cell epitopes [3].

Developments in drug discovery have prompted bioinformatic advances for epitope prediction to generate antibodies with high affinity and specificity for therapeutic and diagnostic use. Historical systems provided a general protein model to predict antigenic locations. However, a method for determining antigenic sites specific to individual antibodies from epitope–paratope amino acid composition has recently been presented [6]. Furthermore, advances in antibody modelling are facilitating computerized antibody design [7].

Although these developments are a significant improvement for epitope prediction and offer time saving over peptide scanning experiments, further refinement is required before researchers become solely reliant on these models. In the following sections, the general principles are presented that provide a guide to the basic peptide immunogen design, utilizing public domain resources. Consideration of peptide candidates at this stage of antibody production can help generate a specific antibody for the immunochemistry end user. Custom antibody and peptide suppliers are widely available and may offer consultation for advanced immunogen design.

Considerations for Peptide Immunogen Design

Research the Protein

UniProt (http://uniprot.org) is a proteomic database providing protein sequence and function information. Comparing your protein sequence against the UniPort entry is a good practice. Information on isoforms, protein topology and post-translational modifications is provided alongside tissue expression and cellular localization, which will assist identifying a good peptide candidate.

For proteins highly conserved across family members or that contain isoforms, an alignment of the primary sequences is recommended to help avoid or minimize unwanted cross-reactivity. This will highlight regions of the sequence that are distinct from related proteins. Similarly, if cross-reactivity with a particular species is required for your experiments, performing a sequence alignment of the target and secondary species will identify conserved regions as candidates for further investigation. UniProt has alignment functionality. An additional alignment program available in the public domain includes Clustal Omega provided by EMBL-EBI (http://ebi.ac.uk).

It is highly advisable to record the expected size of the protein, tissue expression and subcellular localization. This information will be useful when comparing candidate immunogens against unrelated proteins for cross-reactivity. Cross-reactivity with proteins of similar size (and in particular for immunochemical assays, incorrect tissue expression and cellular localization) is concerning because doubt will be cast over the specificity of antibodies to the correct antigen. Care at the peptide selection stage will reduce the likeliness of cross-reactivity. However, there are proteins that are yet to be discovered and curated, so it is impossible to remove all the potential for cross-reaction. It is recommended in these instances to employ additional assays, for example Western Blot, where the proteins differ in molecular weight, to help verify specificity.

Identifying Candidate Regions

A requirement for a good peptide immunogen is for it to originate from an external exposed (hydrophilic) region of the protein, as this increases the chances of recognizing the tertiary protein structure. This information can be obtained from the tertiary protein structure. If tertiary structure is unavailable (e.g. for a novel protein), then hydrophilicity can be used as a measurement of potential surface exposure. Hydrophilic peptides ensure solubility, which is a prerequisite for synthesis and immunization. The Immune Epitope Database (IEDB) (http://iedb.org) is a public domain resource for B-cell epitope prediction providing hydrophilicity (Parker), secondary structure (Chou and Fasman) and BepiPred (Larsen) analyses for a given protein. Hydrophilic amino acids include serine (S), cysteine (C) and threonine (T) [2].

N- and C-terminal regions are likely to protrude through the surface of the protein, making them as good potential candidates. The C-terminal region is frequently not conserved between species. If species cross-reactivity is desired, performing a sequence alignment is essential before selection.

Protein topology is particularly informative for membrane proteins. A proteomic database will provide locations of transmembrane regions, which should not be selected for an immunogen, since this region is often inaccessible to an antibody. The same applies to cleaved regions, such as pro-peptides and signal peptides, since these will not be present on the mature form of the protein (unless, of course, you specifically require an antibody to recognize these). Instead, select candidates within extracellular domains or cytoplasmic loops. Selection of these regions mimics the presentation of the native protein. Therefore, the probability of generating antibodies suitable for a variety of assays is greater and increases the likelihood for the recognition of fixed protein, such as in immunochemical staining.

Specific Amino Acid Properties

Ideally, candidates should contain immunogenic residues such as proline (P) and tyrosine (Y), which provide a structural motif likely to be present in the native protein. The ring-like structure formed provides rigidity towards the N terminus. This provides greater exposure to the immune system when present inside the host organism compared to a coiled peptide. The position of these residues within the peptide sequence will dictate how well their property is translated.

Hydrogen bonds form between polar side chains of amino acids, such as glutamine (Q). This reduces solubility, and in the presence of too many hydrophobic residues it may cause the peptide to precipitate out of the solution. A good combination of hydrophilic and hydrophobic residues is preferred.

Glycine (G) is the most abundant amino acid. It is also the smallest and creates flexibility in the polypeptide chain. Conformational changes occur in the presence of multiple glycines across the peptide sequence. This should be avoided. Methionine (M) is usually the first amino acid in the protein sequence and can be cleaved off when the protein is processed into its mature form. Methionine can also undergo oxidation. For this reason, restrict the number of methionine residues within the peptide sequence if unavoidable. Cysteines (C) form disulfide bonds under oxidation and provide stability to the tertiary protein structure. Disulfide bonds between cysteine residues are common in structural proteins like keratin along with proteins that function in harsh environments, for example the digestive system. Consecutive serine (S), threonine (T), alanine (A) and valine (V) residues impede peptide synthesis. Avoid them where possible or consult a peptide supplier for a strategy to overcome the synthesis issues.

Review Potential Cross-Reactivity

Candidate peptides identified using the aforementioned guidelines must be checked for cross-reactivity with unrelated proteins, for reasons previously stated. Cross-reactivity with related proteins will already have been addressed by performing a sequence alignment. A Basic Local Alignment Search Tool (BLAST) algorithm, such as NCBI blastp (http://blast.ncbi.nlm.nih.gov/Blast.cgi), identifies and annotates locations of similarity between protein sequences. The precise nature of the epitope is unknown; therefore, gauging the potential for these regions to cross-react is purely subjective. As a guide, avoid five of more consecutively shared amino acids with unrelated proteins present in the species of interest, particularly proteins of similar molecular weight, tissue expression or localization. The BLAST results may identify cross-reactive regions within the peptide sequence, and reduction or elimination of these is possible by extending or shortening the sequence. Restricting the BLAST search to the organism of interest will aid interpretation of results. If species cross-reactivity is desired, then a BLAST against the species proteins is required to inform the final peptide selection.

Refining Selected Candidates

A length of 15–20 amino acids is common for peptide immunogens and allows for the generation of multiple epitopes across the peptide sequence. For small proteins, it may not be possible to implement the guidelines to the best ability and obtain a sequence of 15 residues. Consult with a peptide supplier in these situations and establish a strategy for increasing the overall length to facilitate an immune response without compromising antibody specificity.

Considerations for Post-Translational Modification Peptide Immunogens

Unlike unmodified peptide immunogens where the entire protein sequence is reviewed and analysed before a final peptide sequence is selected, the sequence used for a modified peptide is dictated by the site of the post-translational modification. The modified residue is usually positioned at the centre of the sequence to ensure that the epitope incorporates the modification. As a general guide, ensure that the modified residue is flanked by five amino acids on either side. Increasing the number of flanking residues beyond seven is not recommended, since the likelihood of the resulting antibodies recognizing an epitope lying in the flanking region is increased and, therefore, risks antibodies favouring the unmodified form of protein dominating the modified form. An unmodified version of the peptide sequence should be synthesized to remove from the sera (by affinity purification), any resulting antibodies that recognize the unmodified form, and to serve as a specificity control in immunochemical experiments.

Peptide Carrier Protein

Peptides are immunogenic, but the antibody response raised when used on their own is low, since they are typically around 15–20 amino acid residues in length (<6–10 kDa). Therefore, peptides are typically conjugated to a larger, immunogenic carrier molecule in order to generate a significant amount of antibody [8]. The process of peptide conjugation is discussed in more detail later in this chapter. Conjugation to KLH is commonly via a cysteine (C) residue in the peptide sequence. If a cysteine residue is absent, one must be added to either end of the sequence prior to peptide synthesis.

There are many types and variations of carrier proteins that are commercially available, such as ovalbumin (OVA) and catonized BSA (cBSA). However, the most popular and widely used carriers in antibody production are bovine serum albumin (BSA) and keyhole limpet hemocyanin (KLH).

BSA makes a suitable carrier protein due to a number of beneficial characteristics. It is stable and readily soluble in aqueous buffers. It is large enough in size (67 kDa) and sufficiently complex to be immunogenic. BSA has numerous carboxyl groups and primary amines, which are suitable for coupling peptides using a broad range of cross-linking reagents. A potential downside to the use of BSA as a carrier protein is that immunochemical experiments commonly use BSA as a reagent to block non-specific protein–protein interactions. Therefore, if antibodies that recognize the peptide–BSA complex are present in the final antibody preparation, false-positive/general background staining may occur [9].

KLH is an effective carrier protein for several reasons. It is large and complex (2–3 × 106 Da) and has numerous epitopes and a wealth of lysine residues for coupling peptides. KLH is isolated from the mollusk Megathura crenulata, which is phylogenetically distant from mammals, ensuring a good host immune response [8]. This means that KLH shows a higher immunogenicity compared with BSA. A downside to the use of KLH is limited solubility in aqueous buffers. However, due to the popularity and extensive use as a carrier, commercial product variants (mcKLH) are available. These are purified, lyophilized and pre- activated forms, which are opalescent blue when in solution.

Apart from using carrier proteins to prepare peptide immunogens, an alternative is to synthesize peptides as multiple antigenic peptides (MAPs). MAPs are complexes created from multiple copies of the peptide immunogen coupled with a core structure via lysine residues. The advantage of MAPs is that as they do not require a carrier protein, antibodies are only generated against the peptide and not the highly immunogenic carrier protein. The use of MAPs is, however, limited to peptides that contain a good T-helper-cell epitope due to lack of affinity maturation and class switch to IgG. The purity of the MAP is also difficult to determine during peptide synthesis.

Conjugating Peptide Immunogens to Carrier Protein

Peptides can be conjugated to a carrier protein by several methods. The methods outlined in the following use EDC (1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride) and sulfo-SMCC (sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate) cross-linkers.

EDC is a zero-length cross-linker that allows for carboxyls to be covalently attached to primary amines. Depending on the method of coupling, the peptide will conjugate to the KLH in a number of different orientations. This will lead to the generation of antibodies against the peptide sequence presented to the immune system. EDC is suitable for conjugating peptides, which have no terminal cysteine or where a specific peptide orientation is not required.

Sulfo-SMCC is a heterobifunctional cross-linker that possesses both an amine reactive NHS (N-hydroxysuccinimide) ester (amino group directed) and sulfhydryl reactive maleimide group (thiol directed), which are connected at opposite ends by a cyclohexane spacer arm. A peptide with a terminal cysteine will conjugate to the KLH with high specificity allowing for a desired epitope (located away from the terminal cysteine) to be presented favourably to the immune system. Sulfo-SMCC is suitable for conjugating peptides that have a terminal cysteine and where specific peptide orientation is desired. A downside of sulfo-SMCC is that antibodies may be raised against the linker.

To prepare an immunogen, the KLH is first reacted with sulfo-SMCC, as the abundance of lysine residues will react with the NHS–ester linker end to form stable amide bonds at pH 7–9. As the conjugation is performed via the primary amines, buffers containing Tris and glycine should be avoided because they will compete [9]. Any excess cross-linker should be separated from the conjugated KLH by gel permeation chromatography. To complete the immunogen, a fully reduced sulfhydryl-containing peptide will react with the maleimide linker end to form stable thioether bonds at pH 6.5–7.5 [8].

Glutaraldehyde is an amine to amine homobifunctional cross-linker as it contains two aldehyde groups, which react with both surface lysines on the carrier protein and amino groups on the N terminus of peptides. The coupling is not as controlled compared with that of heterobifunctional linkers and there is a greater chance of polymerization (as with EDC). The reaction is dependent on pH, temperature and ionic strength [8]. Glutaraldehyde is best suited to coupling peptides that do not contain internal lysine residues or where a terminal lysine is preferred over that of a cysteine. If a peptide contains internal lysine residues, then interpeptide links are formed that can cause the formation of large multimeric complexes and lead to the peptide not being presented properly. As the conjugation is performed via the primary amines, buffers containing tris and glycine should be avoided because they will compete [9].

Once conjugated to an appropriate carrier molecule, the antigen is now ready for immunizing into a suitable animal model.

ANTIBODY PRODUCTION

Once the antigen has been identified, sourced and prepared for immunization (commonly in the form of a synthesized peptide), it is administered into the host animal via injection. Routes of administration include subcutaneous and intradermal. The presence of the foreign molecule stimulates the host's immune response, whereby B cells undergo proliferation into plasma and memory B cells. This process usually takes several days post-immunization and is described as the lag phase. Initially, plasma B cells synthesize IgM subclass antibody. IgM population declines as a result of the T-cell-dependent class switch of IgM producing plasma cells to produce IgG. The first exposure to an antigen is described as the primary response, and as shown in Figure 1.2, it can take several days to generate sufficient antibody to be effective against the antigen. The memory B cells produced during primary exposure enable a quicker response to a subsequent exposure to the same antigen, termed the secondary response. This is the fundamental principle behind vaccinations and the method used to produce large quantities of antibody for commercial use.

Figure 1.2 Typical Primary and Secondary Responses to an AntigenUpon first exposure to an antigen, a series of B-cell divisions must occur before enough antibody is present to be effective. This lag phase is significantly reduced upon subsequent exposure to the same antigen. Memory B cells generated from the primary response are available to rapidly divide, thus reducing the time required to produce antibodies and generating larger quantities.

Polyclonal Antibody Production

Commercial polyclonal antibodies cover a diverse range of proteins from neuroscience to cancer and metabolism targets. The reason for this is because the polyclonal production process is not complex and becomes cost-effective when generating large quantities of antibody. Unfortunately, the main limitation