CH12 09/26/2012 11:27:25 Page 105

12Production of Antibodies

Elek Moln�arPhysiology & Pharmacology, University of Bristol, UK

12.1 Basic ‘how-to-do’ and ‘why-do’ sectionAntibodies (also known as immunoglobulins (abbreviated ‘Ig’); see Figure 12.1)

are host proteins produced in response to the presence of foreign molecules in the

body. They are gamma globulins that are present in blood or other bodily fluids of

vertebrates and used by the immune system to identify and neutralize foreign objects,

such as bacteria and viruses. The highly specific interaction of an antibody with an

antigen forms the basis of all immunochemical techniques. Antibodies can bind to a

wide range of chemical structures (e.g. proteins, peptides, nucleic acids, carbohy-

drates, lipids, small chemical groups) and can discriminate among related com-

pounds. Small molecules such as peptides may not be immunogenic, and will not

usually produce antibodies if they are injected into an animal.

To make antibodies against small molecules, they must be coupled to a large

protein to form a hapten (partial antigen)-carrier complex (Harlow & Lane, 1988,

1998). The region of an antigen that interactswith an antibody is defined as an epitope.

An epitope is not an intrinsic property of any particular structure, as it is defined

only by reference to the binding site of an antibody. Antibodies bind to complemen-

tary antigens by three-dimensional recognition. The antibody-antigen complex is

stabilized by non-covalent bonds (e.g. van der Waals attraction, hydrogen bonds,

salt bridges, hydrophobic interactions, electrostatic forces, ion pairs).

The binding of antibodies to antigens is reversible, and the interaction will

conform to an equilibrium reaction. Because antibodies can recognize relatively

small regions of antigens, occasionally they can find similar epitopes on other

molecules. This forms the molecular basis for cross-reaction, which is a process

that is exploited during the production of various antibodies (Harlow & Lane,

1988).

Essential Guide to Reading Biomedical Papers: Recognising and Interpreting Best Practice, First Edition.

Edited by Phil Langton.

� 2013 by John Wiley & Sons, Ltd. Published 2013 by John Wiley & Sons, Ltd.

CH12 09/26/2012 11:27:25 Page 106

When an antigen is introduced into a na€ıve animal, the first step in the generation

of the primary response is phagocytosis of the antigen. Antigen-presenting cells

degrade the antigen and display fragments of it on the cell surface. Helper cells

associate with antigen-presenting cells by binding to the antigen fragment. Binding

of a helper T cell to an antigen-presenting cell leads to proliferation of helper T

cells. B cells also process antigens, but they do so in an antigen-specific manner.

Binding of helper T cells to B cells is required for a strong antibody response and it

also leads to proliferation of the B cells. Differentiation of B cells leads to the

production of higher affinity antibodies. At the end of the primary response, the

antigen is cleared, leaving primary memory cells. Subsequent injections of antigen

induce a more potent response.

There are numerous factors that influence the strength and specificity of an

antibody response:

1. Immunogenicity.

2. Preparation and presentation of the immunogen.

3. Techniques for injecting the immunogen to animals.

Papaincleavage

sites

Heavy chain

-S-S-

-S-S-

Light chain Fab

Carbohydrate

siteEffector

CC

Fc

Variable

Constant

Figure 12.1 Schematic representation of antibody structure. Each antibody consists of fourpolypeptides: two large heavy chains and two small light chains connected by disulphide bonds(-S-S-) to form a Y-shaped molecule. While the general structure of antibodies is very similar, theamino acid sequence in the two upper ends of the Y varies greatly among different antibodies.These variable regions give the antibody its specificity for binding antigens. Therefore, eachantibody molecule has two antigen binding sites. Digestion of the antibody with a protease(papain) yields three fragments, of which two (Fab) are identical and bind antigen, and a third(Fc), which comprises the effector domain. Antibodies are divided into five major classes (IgA,IgD, IgE, IgG and IgM), based on their constant region, structure and immune functions. Courtesyof Professor Elek Moln�ar.

106 CH12 PRODUCTION OF ANTIBODIES

CH12 09/26/2012 11:27:26 Page 107

12.2 Primary antibodiesPrimary antibodies may be polyclonal or monoclonal (Lipman et al., 2005).

12.2.1 Polyclonal antibodies

The simplest method of producing primary antibodies against a foreign molecule is

to immunize an animal (usually a rabbit, goat or guinea pig) against it. If it is a large

molecule such as a protein, a peptide sequence from the protein is chosen for:

1. its accessibility (it is no use using a sequence that in vivo is inaccessible to the

antibody due to its tertiary structure or its position in the hydrophilic (middle)

region of a membrane); and

2. its unique sequence, as far as can be determined from sequence databases

(sequences present in many different molecules are not good).

Repeatedly injecting the peptide, or other antigen, into the animal will trigger the

immune system and produces antibodies against it. Blood serum which contains

antibodies (usually IgG isotypes) is called antiserum. The animal may produce high

concentrations of several antibodies against different (possibly overlapping) anti-

genic regions. Additionally, there will be very low concentrations of other anti-

bodies that the animal may have been producing in the background, unrelated to the

foreign antigen. The serum thus has high titres of a ‘polyclonal primary antibody’,

which is several antibodies directed against antigens.

The primary goal is to have a high antibody titre and high antibody affinity (binds

strongly to antigens). IgG is the most desirable antibody, because of its favourable

binding properties, its stability, its high concentration in serum and because it

is simple to purify from antisera. First serum samples with antibodies are available

6–8 weeks after the start of the immunization.

Advantages Relatively easy to make. Also, even if one of the epitopes re-occurs in

a different molecule, the strength of staining for that moleculewould beweaker than

for the original injected antigen, due to the several different antibodies directed

against different epitopes on the same antigen.

Limitations

1. It is not possible to know which epitope binds the antibody.

2. Due to individual variations of immune-responses induced in various host

animals, the effectiveness and properties of polyclonal antibodies are highly

variable, even for the same immunization protocol using the same antigen.

Therefore, antibodies obtained from different host animals need to be

12.2 PRIMARY ANTIBODIES 107

CH12 09/26/2012 11:27:26 Page 108

validated individually and used separately. This inherent variability of

polyclonal antibodies is often responsible for fundamentally different

results obtained with different batches of custom-made or commercially

supplied antibodies.

12.2.2 Monoclonal antibodies

These are produced in vitro from single clones of (usually mouse) antibody-

producing B-cells fused with myeloma cells. Each clone produces only one

antibody (hence ‘monoclonal’). An appropriate clone is selected, and this produces

antibody against a single epitope. The fused cell is called a hybridoma cell, and it

possesses the antibody-producing ability of the B-cell with the infinite life of the

myeloma cell.

Advantages

1. High specificity for a single known epitope.

2. The hybridoma cells can be grown in a nutrient media and the antibodies can

be harvested and purified from the supernatant.

3. Hybridoma cells can be frozen in liquid nitrogen and resurrected and grown

when additional antibody is needed.

4. Monoclonal antibodies insure a consistent antibody with theoretically endless

supply.

Limitations

1. The antibody may bind equally well to the epitope if it occurs in a different

molecule.

2. Antibodies take 6–8 months to develop if the fusion of an appropriate antibody

producing B-cell is successful.

12.3 Secondary antibodiesSecondary antibodies are antibodies used for the detection of the primary antibody

in various immunochemical experiments (Primers 13, 14, 15, and see Figure 12.2).

Secondary antibodies may be polyclonal or monoclonal, and they are available with

specificity for whole Ig molecules or antibody fragments such as Fc or Fab regions.

They are usually directed against the species-specific region on the antibody; hence,

primary antibody produced in rabbit would require a secondary anti-rabbit antibody.

The secondary antibody is usually conjugated to an enzyme (Figure 12.2), fluoro-

chrome or colloidal gold particle to enable its visualization and detection (see

Primers 7 and 8).

108 CH12 PRODUCTION OF ANTIBODIES

CH12 09/26/2012 11:27:26 Page 109

12.4 Choice of antigens for antibody preparation;their advantages and limitations

There are four main strategies:

1. Complete native protein

2. Synthetic peptide

3. Synthetic peptide using MAPS (multiple antigen presentation system)

4. Bacterially derived fusion protein

12.4.1 Complete native protein

Antigen Purified native protein from tissue or transfected cells.

Advantages

1. Immunization with the native protein.

2. Can be done without knowing the amino acid sequence of the protein.

Limitations

1. Specific protein purification protocol is required.

2. Lack of subtype/subunit specificity.

Enzyme conjugated secondary antibody

Enzyme

Substrate

Detectablesignal

Primary antibody

Target protein

Figure 12.2 Schematic representation of primary and enzyme-conjugated secondary antibodyreactions. After the primary antibody is bound to the target protein, a complex with enzyme-linkedsecondary antibody is formed. The enzyme (e.g. alkaline phosphatise, horseradish peroxidise), inthe presence of a substrate, produces a detectable signal. This approach is commonly used intechniques such as ELISA, immunoblotting, immunocytochemistry and immunohistochemistry.Courtesy of Professor Elek Moln�ar. A full colour version of this figure appears in the colour platesection.

12.4 CHOICE OF ANTIGENS FOR ANTIBODY PREPARATION 109

CH12 09/26/2012 11:27:26 Page 110

12.4.2 Synthetic peptide

Antigen Chemically synthesized peptide, usually 11–18 amino acids in length

with a molecular weight of around 1–2 kDa.

Advantages

1. Great predetermined specificity.

2. Can be prepared immediately after determining the amino acid sequence of a

protein.

Limitations

1. Short sequence reduces probability of reactivity with native protein.

2. Small size requires coupling to a carrier protein for antibody production.

3. The obtained antibodies must be affinity-purified.

12.4.3 Multiple antigen presentation system (MAPS)

This is used with synthetic peptides to improve antibody production (Molnar et al.,

1993; Fujita & Taguchi, 2011).

Antigens MAPS peptides are synthesized as multiple copies on a branching lysyl

matrix using solid-phase synthesis. Molecular weight of peptide complex is around

13–15 kDa for a 15 amino acid sequence.

Advantages

1. Preparation of the immunogen is quicker because no elaborate peptide

purification is necessary.

2. No requirement for carrier protein.

3. Most of the immunogen consists of the amino acid sequence against which the

antibody is to be raised.

4. Quantification of the immunizing dose is easier.

5. Higher anti-peptide antibody titres.

6. Much smaller quantity is required when compared with the unconjugated

monomeric peptide for coating enzyme-linked immunosorbent assay (ELISA)

plates.

110 CH12 PRODUCTION OF ANTIBODIES

CH12 09/26/2012 11:27:26 Page 111

Limitations

1. C-terminus of the peptide is attached to lysyl matrix and is not free, as in the

native protein.

2. In some experiments, antisera to MAPs containing C-terminal peptides

showed no cross-reactivity with the native protein.

3. Antibodies must be affinity-purified using monomeric peptide.

12.4.4 Bacterially derived fusion protein

Antigen Long peptides representing significant portion of the native protein (see

Figure 12.3; Pickard et al., 2000).

Advantages

1. the large antigen has a great probability of adapting the conformation of the

native protein.

2. Unlimited supply of antigen.

Limitations

1. Nucleotide sequence is required.

2. Lack of subtype/subunit specificity.

3. Technically demanding.

12.5 Purification of antibodiesThe purity of the immunoglobulins is often critical, because other substances in the

source material (e.g. antiserum, cell culture supernatant) may interfere with the

detection process. For example, background labelling due to non-specific binding

can interfere with the detection of the target antigen.

Antibodies can be purified by affinity chromatography or via precipitation.

Antibodies are affinity purified with a protein A or protein G column that binds

to the Fc portion of most immunoglobulins. Saturated ammonium sulphate precipi-

tation is also frequently used to separate immunoglobulins (Harlow & Lane, 1988,

1998). Purification of specific antibodies can be achieved by immobilizing the

antigen to a solid support surface in a way that the epitopes are available for

antibody binding (Huse et al., 2002; Figure 12.3). Following washing to remove

non-specific proteins and contaminants, a low pH or high salt solution is used to

12.5 PURIFICATION OF ANTIBODIES 111

CH12 09/26/2012 11:27:26 Page 112

GST fusion protein

GST Targetsequence

Immunized rabbit Whole antiserum

S-transferaseGlutathionAnti-target Ig

Anti GST Igcolumn -

Non-specific Ig

GST

Fusion protein affinity column

Elution buffer

Non-specificIg flow through

GST Targetsequence

Eluted specificantibody

R4R3 R2 R1 Rabbit No.

Immunoaffinitypurified antibody

Antiserum withoutpurification

205

R4R3 R2 R1

11697

66

45Mol

. wt x

10-

3 Targetprotein

29

Figure 12.3 Purification of antibodies raised against a glutathione S-transferase (GST) fusionprotein. The antiserum is first pre-adsorbed using an affinity column coupled with the GST protein.Any antibodies against GST are removed by this column. Specific antibodies to the target sequenceflow into the secondaffinity column,which is coupled to aGST fusionprotein containingaminoacidsequences of the target epitope. Both columns allow the non-specific antibodies to flow through.Specific antibodies directed against the epitope region of theGST fusionprotein are captured in thesecond column and eluted using a low pHbuffer (0.1 Mglycine-HCl, pH 2.5; red arrows). The elute isimmediately neutralised by the addition of 1M Tris-HCl (pH 8.0). Immunoblots illustrate reactionspecificity of rabbit polyclonal antibodies before and after immunoaffinity chromatographypurification. Full experimental details are available in Pickard et al. (2000). Courtesy of ProfessorElek Moln�ar. A full colour version of this figure appears in the colour plate section.

112 CH12 PRODUCTION OF ANTIBODIES

CH12 09/26/2012 11:27:26 Page 113

transiently relax the interaction between the epitope and the antibody so that the

antibodies can be recovered. The pH is then returned to neutral, and the purified

antibody fraction is dialyzed into the appropriate buffer to regain its original

conformation (Harlow & Lane, 1988, 1998; Figure 12.3).

12.6 Required controlsThedevelopment of immune response in the animal is analyzed in comparisonwith the

serumobtainedbefore the first inoculationwith the antigen. It is essential to obtain pre-

immune serum from each animal. The specificity of the primary antibody is usually

established using immunochemical techniques (e.g. immunoblotting, ELISA, immu-

noprecipitation, etc.), immunocytochemistry and immunohistochemistry. It is impor-

tant to prove that the obtained antibody cross-reacts readily and specifically with the

native protein in the investigated tissue or protein extracts. Ideal control samples are:

1. transfected cells that express a high concentration of the investigated protein;

and/or

2. tissue obtained from transgenic animals, which lack the target antigen.

Primary antibodies should be pre-absorbed with excess of the corresponding

antigenic peptide or protein. Under these conditions, all immunoreactivity should

disappear. While this control will show that the antibody binds to the antigen, it does

not exclude the possibility that it does not bind to anything else.

12.7 Common problems and pitfalls in executionor interpretation

While there is a vast amount of literature available for each step of the antibody

production process, and no shortage of advice for individual antibody produc-

tion strategies, the final outcome is very uncertain. Most common problems

include:

� Lack of immune response,

� While the antibody strongly interacts with the antigen used for the immuniza-

tion, there is no cross-reactivity with the native protein.

� Non-specific cross-reactions with unrelated proteins.

� Limited supply of polyclonal antibodies.

� Individual variations of immunoresponse in different animals.

� High costs.

� Time-consuming.

12.7 COMMON PROBLEMS AND PITFALLS IN EXECUTION OR INTERPRETATION 113

CH12 09/26/2012 11:27:26 Page 114

The success rate can be improved somewhat by using:

� more than one antigen derived from the investigated target;

� different antigen presentation methods;

� different immunization protocols;

� larger numbers of animals;

� different species.

While huge number of antibodies are available commercially for a range of

targets, and these are used extensively in various experimental approaches, investi-

gators have to navigate through a complex system to find out which particular

antibodies are appropriate for a specific experiment. At present, there is no universal

validation for antibodies (Bordeaux et al., 2010). An antibody that works in one

system may perform poorly in other (see Primers 13 & 15 for more details).

Therefore, researchers need to know exactly how these antibodies have been

produced and characterized to determine whether an immunoreagent will work

in the assay they are using.

Using the same antibody to investigate isoforms of target proteins in different

species needs to be considered particularly carefully, due to potential inter-species

differences in the corresponding epitope regions that may prevent specific immu-

nochemical interactions. Therefore, if this information is available, sequences of the

epitope regions need to be compared carefully before an antibody is used in a

species different from the one it was originally developed for.

As discussed in greater details in Primer 13 (Immunocytochemistry and immu-

nohistochemistry), antibodies display ‘method specificity’. This means that some

antibodies may work well in one protocol (e.g. immunoblotting – Primer 14) but fail

to produce specific labelling in another (e.g. immunohistochemistry, Primer 13 or

immunoprecipitation, Primer 14). This is due to the different presentation of

epitopes under different experimental conditions. For example, proteins are

denatured and unfolded for immunoblotting and epitopes are fully accessible for

immunochemical interactions.

In contrast, fully folded proteins are used for most immunoprecipitation and

immunolocalization experiments, where epitope regions may not be readily acces-

sible on the surface of the target protein and antibodies are unable to bind to these

regions. Fixation of samples for immunocytochemistry and immunohistochemistry

experiments can significantly alter the structure of the epitopes, which can interfere

with antibody binding (see Primer 13 for details).

There is a growing need for high-quality antibodies andmore robust validation data.

Furthermore, it is often disappointing that only few of the commercially available

antibodies seem to work in independently performed tests. Without appropriate

validation, misinterpretation of non-specific immunoreactions can lead to

114 CH12 PRODUCTION OF ANTIBODIES

CH12 09/26/2012 11:27:26 Page 115

erroneous conclusions. The necessity ofmore rigorous specificity tests is becoming

more widely accepted. Verification of immunoreaction specificity is particularly

important in immunohistochemical experiments, where only few antibodies pass

correctly applied specificity criteria (Blow, 2007; Pradidarcheep et al., 2008;

Michel et al., 2009).

12.8 Complementary techniques� Primer 13: Immunohistochemistry

� Primer 14: Immunoprecipitation

� Primer 15: Immunoblotting (Western blotting)

Cited work, further reading and resourcesBlow, N. (2007). The generation game. Nature 447, 741–744.

Bordeaux, J., Welsh, A.W., Agarwal, S., Killiam, E., Baquero, M.T., Hanna, J.A.,

Anagnostou, V.K. & Rimm, D.L. (2010). Antibody validation. BioTechniques 48,

197–209.

Fujita, Y. & Taguchi, H. (2011). Current status of multiple antigen-presenting peptide

vaccine systems: Application of organic and inorganic nanoparticles. Chemistry Central

Journal 5, 48.

Harlow, E. & Lane, D. (1988). Antibodies: a laboratory manual. Cold Spring Harbor

Laboratory Press, Cold Spring Harbor, NY.

Harlow, E. & Lane, D. (1998). Using antibodies: a laboratory manual. Cold Spring Harbor

Laboratory Press, Cold Spring Harbor, NY.

Huse, K., B€ohme, H. & Scholz, G.H. (2002). Purification of antibodies by affinity

chromatography. Journal of Biochemical and Biophysical Methods 51, 217–231.

Lipman, N.S., Jackson, L.R., Trudel, L.J. & Weis-Garcia, F. (2005). Monoclonal versus

polyclonal antibodies: distinguishing characteristics, applications, and information

resources. ILAR Journal 46, 258–268.

Michel, M.C., Wieland, T. & Tsujimoto, G. (2009). How reliable are G-protein-coupled

receptor antibodies? Naunyn-Schmiedebergs Archives of Pharmacology 379, 385–388.

Molnar, E., Baude, A., Richmond, S.A., Patel, P.B., Somogyi, P. & McIlhinney, R.A.J.

(1993). Biochemical and immunocytochemical characterization of antipeptide antibodies

to a cloned GluR1 glutamate receptor subunit: Cellular and subcellular distribution in the

rat forebrain. Neuroscience 53, 307–326.

Pickard, L., No€el, J., Henley, J.M., Collingridge, G.L. & Molnar, E. (2000). Developmental

changes in synaptic AMPA and NMDA receptor distribution and AMPA receptor subunit

composition in living hippocampal neurons. Journal of Neuroscience 20, 7922–7931.

Pradidarcheep, W., Labruy�ere, W.T., Dabhoiwala, N.F. & Lamers, W.H. (2008). Lack of

specificity of commercially available antisera: better specifications needed. Journal of

Histochemistry and Cytochemistry 56, 1099–1111.

CITED WORK, FURTHER READING AND RESOURCES 115