Immunization of Mice for Monoclonal Antibody Production

Immunization of Mice for Monoclonal Antibody Production

JE Liddell University of Cardiff, Cardiff, UK

Advanced
doi:10.1038/npg.els.0003760




Monoclonal antibody production requires the immunization of laboratory mice with an immunogenic protein and test sampling of antiserum. For smaller, less immunogenic proteins and peptides, conjugation of the molecule to a carrier protein is necessary before immunization.




Introduction

In many countries, the use of animals for experimentation is controlled by law. Establish what your local rules are by consulting senior staff in your animal breeding facility before attempting the following protocol. In the UK, for example, it is necessary for the work to be covered by a Government Project licence and a Personal licence held by the person carrying out the work. In ad***ion, the work must be performed in licensed premises. See also: Monoclonal antibodies; Monoclonal antibodies: therapeutic uses; Monoclonal antibodies: diagnostic uses


The only animals, to date, that can be used for conventional monoclonal antibody production are mice and rats. Human tissue can also be used but is beyond the scope of this protocol. Any strain of mice can be used for immunizing but Balb/c mice are preferred because most myeloma cells are derived from Balb/c mice. This simplifies the production of ascitic fluid at the final stages of monoclonal antibody production, as cells can grow compatibly in Balb/c mice. The alternative would be to make ascitic fluid in F1 hybrids of Balb/c and the immunizing strain. See also: Mice as experimental organisms


Most molecules above a molecular weight of 1000 are immunogenic if the host recognizes them as foreign. The animal will make antibodies to epitopes exposed to the immune system but some will be more immunogenic than others. Antibodies may also be made against impurities in the immunogen preparation. It is therefore advisable to use as pure a sample as possible in order to maximize exposure to the desired immunogen and minimize the need to purify the final antiserum.


It is usually necessary to use an adjuvant, which is a substance coinjected with the immunogen to augment the immune response. Cells, bacteria and viruses can be injected without adjuvant as they are so immunogenic.


Small proteins, haptens and synthetic peptides will need to be conjugated to a carrier protein. Common carrier proteins are keyhole limpet haemocyanin (KLH), bovine serum albumin or immunoglobulin (Ig), and common coupling reagents are glutaraldehyde and carbodiimide. The protocol for coupling protein to KLH with glutaraldehyde is given in step 2. Alternatively, synthetic peptides can be synthesized on a polylysine branch to produce a polymer.


In order to maximize the slow release of immunogen, the preferred site of injection is subcutaneous (Table 1). A mouse is too small for intramuscular and intradermal injections to be acceptable for general applications. An intravenous tail vein injection without adjuvant is desirable, 4 days before fusion of spleen cells. This is earlier than the peak time of appearance of antibodies in the serum but will ensure that antibody-producing cells are at the best stage of proliferation to promote high fusion efficiencies. See also: Monoclonal antibodies.



Recommended concentrations of various immunogens are given in Table 2. For monoclonal antibody production, the need to use pure immunogen is not so important because postfusion selection of antibodies will reduce the likelihood of getting undesirable crossreactions. Indeed, the process of purifying immunogen may sufficiently alter the protein to destroy epitopes present in the native form.

For further detailed descriptions of the production of polyclonal antisera the reader is referred to Harlow and Lane (1988) or Liddell and Cryer (1991).



Step 1: Equipment and Solutions

Equipment

For immunization and blood sampling

1-mL syringe
21g needle
Balb/c mice
Centrifuge
Cotton wool
Scalpel
Small capped tube to hold total volume (e.g. Eppendorf tube)
Small glass tube
Vortex mixer

For conjugation to carrier protein

Reaction tubes
Dialysis tubing

Reagents

For immunization and blood sampling

Complete Freundõ?Ts adjuvant
Incomplete Freundõ?Ts adjuvant

For conjugation to carrier protein

Peptide
Glycerol for storage
Sodium chloride (NaCl)
Potassium dihydrogen orthophosphate (KH2PO4)
Disodium hydrogen orthophosphate (Na2HPO4.12H2O)
Potassium chloride (KCl)

Solutions

For immunization and blood sampling

Immunogen solution (e.g.10õ?"100 ẻẳg in 200 ẻẳL of saline)

For conjugation to carrier protein

KLH 1 mg mLõ^'1 (1.0 mol Lõ^'1 KLH = 3 - 106 mg mLõ^'1)
Peptide solution
Phosphate buffered saline (PBS), pH 7.2 for dilution and dialysis (Recipe 1)

Glutaraldehyde (e.g. grade 1, 25% aqueous solution, Sigma or equivalent)
õ?Âl-Lysine (1.0 mol Lõ^'1)
Step 2: Conjugation of Small Peptide to Carrier Protein for Immunization

There are several carrier proteins and coupling methods used for this purpose. The following method describes the conjugation of a small peptide to KLH with glutaraldehyde.


1. Mix a 1 mg mLõ^'1 suspension of KLH with the peptide solution to give a molar ratio of one part carrier protein to 20 to 40 parts peptide (see also Hints and Tips 2.1).
2. Add an equal volume of glutaraldehyde (0.1õ?"2% v/v) dropwise with constant stirring and incubate at room temperature for 2õ?"3 h.
3. Stop the reaction by ad***ion of 1.0 mol Lõ^'1 l-lysine at 1:20 volume and dialyse extensively against PBS.
4. Add an equal volume of glycerol and store at õ^'20°C.


Step 3: Preparation of Stable Antigen/ Freundõ?Ts Adjuvant Emulsion

1. To prepare a 1-mL injection emulsion, place 0.5 mL of Freundõ?Ts adjuvant in a capped Eppendorf tube. Use complete adjuvant for the first injection but incomplete adjuvant for all subsequent boosts. Using a pipette, slowly add the antigen solution dropwise to the adjuvant, vortex mixing between each ad***ion until all the antigen solution is used (see Hints and Tips 3.1).
2. This should form a stable emulsion. If the two solutions separate, continue the vortex mixing and/or draw the solution repeatedly through a syringe and 21g needle.


Step 4: Injection of Mice

1. Draw up 1 mL of antigen/adjuvant emulsion into a 1-mL syringe with a 21g needle. The solution will be very viscous if properly prepared. It should not be possible to pass it through a finer needle.
2. With the thumb and forefinger of one hand, pick the mouse up at the midpoint of the tail. Hold it firmly by the tail but if it is allowed to rest on the barred roof of the cage it will pull away, which will allow you to take a large pinch of skin at the nape of its neck with the other hand. Tuck the tail under the little finger of this hand. This will immobilize the head and minimize the risk of being bitten. A mouse bite can be very painful but if they are handled correctly and confidently the risk should be minimal. Ask an experienced handler to show you the correct way.
3. Insert the needle carefully under the skin held between the fingers and inject up to 0.2 mL. Allow the immunogen to disperse before removal of the needle, otherwise it may leak out. Remove the needle and boost the mouse again in 3õ?"4 weeks time. If complete Freundõ?Ts adjuvant was used in the first injection, Incomplete Freundõ?Ts adjuvant should be used for all subsequent injections to minimize inflammatory reactions at the injection site.
4. It is desirable to test bleed (Step 5) the mice, from 7 days after the second injection, to ensure an antibody response has been achieved before using a spleen for fusion. If the titre is unsatisfactory, more boosts can be given at 3õ?"4-week intervals.
5. Four days before fusion the mouse will need to be boosted once more without adjuvant intravenously through a tail vein.


Step 5: Bleeding a Mouse

1. The recommended blood sampling site in mice is the tail vein (see Hints and Tips 5.1). Swab the tail with alcohol. Cut the vein with a sterile scalpel and collect blood into small glass tubes to promote the maximum yield of serum.
2. Keep the serum at 4°C overnight, centrifuge at 3000 rpm and remove the serum for storage at õ^'20°C. One should expect to get a few hundred microlitres of serum by this method, which is ample for a test bleed (minimum dilution 1:100).


Step 6: Testing Antiserum

1. There are many different ways of testing the antiserum for its binding to antigen. Select an assay format according to availability of antigen, assay hardware and the final application of the assay.
2. As a general rule, the antiserum should be tested as a series of doubling dilutions starting at, for example, 1:100 for ELISA, 1:10 for immunohistochemistry and 1:2 for immunodiffusion. Parallel dilutions of normal or nonimmune serum should also be tested to control for nonspecific binding. Nonimmune serum should be a pool of samples from several animals or a sample taken for an individual animal before starting immunization.

Hazards

The hazards associated with the chemicals/apparatus used in this protocol are detailed in Table 3.

Hints and Tips

Step 2

2.1

KLH is not very soluble in aqueous solutions. Dissolve as much as possible and measure the protein present in a spectrophotometer (optical density at 280 nm). Adjust other concentrations. Large polymers may form but this is not necessarily a problem for immunization.


Step 3

3.1

Practise the technique with an unimportant protein solution before wasting valuable antigen and test by adding a drop of emulsion to a beaker of water. If the drop remains as a discrete drop on the surface and doesnâ?Tt spread, then the emulsion is of the correct consistency.


Step 5

5.1

When giving an intravenous tail vein injection it will help to place the mice in a warm room for 30 min to dilate the vein.



Troubleshooting

These questions assume that the assay con***ions are working correctly. For assay troubleshooting refer to the relevant assay.


1

Does the antiserum give a negative response in the test assay?


YES. Go to 2.


NO. Go to 3.


2

Is the assay working correctly?


YES. Go to 5.


NO. Go to 4.


3

Does the antiserum give a low response in the test assay?


YES. Go to 6.


NO. Go to 7.


4

Refer to troubleshooting questions under the relevant assay protocols.


5

Is the immunogen from the same or a closely related species to the host?


YES. Go to 8.


NO. Go to 9.


6

Try giving two more boosts 3â?"4 weeks apart and testing a new antiserum sample. If the response is still low, see 9.


7

If the antiserum gives a high value but the controls are also high, refer to the assay troubleshooting section.


8

You may find that the screening test will enable you to pick up less prominent antibodies that are not so noticeable in the serum. Alternatively, it may be worth considering immunizing a rat. The rat spleen can then be fused with a rat myeloma line or a mouse myeloma to produce a heterohybridoma.


9

Try increasing the dose of immunogen, conjugating to another carrier protein or other methods of increasing immunogenicity.



Originally published: October 2002


References

Harlow E and Lane D (1988) Antibodies: A Laboratory Manual. Cold Spring Harbor, NY, USA: Cold Spring Harbor Laboratory Press.

Liddell JE and Cryer A (1991) A Practical Guide to Monoclonal Antibodies. Chichester, UK: Wiley

Monoclonal Antibodies

Heddy Zola Child Health Research Institute, Adelaide, Australia

Advanced
doi:10.1038/npg.els.0001205




Monoclonal antibodies are antibodies with a unique specificity, generally made by cloning cells containing a particular antibody gene to produce a population of identical cells derived from a single cell which all produce the same antibody. This should be contrasted with polyclonal antibodies found in the serum of immunized animals, which consist of a very diverse mixture of antibodies against many different molecules.




Introduction

Vertebrates have evolved an immune system that protects them from invading microorganisms. A major component of the immune response *****ch organisms is the production of antibody molecules. These molecules possess a binding site for structures on the surface of the invading organism. In order to be effective, the immune system must be capable of making antibodies which can bind the enormous diversity of molecular structures expressed by viruses, bacteria and other parasitic organisms, and must be capable of coping with mutations in these organisms. This challenge is met by the immune system in two ways. First, B lymphocytes are capable of generating a diverse set of antibody structures (estimated at 1011) by permutation and combination of a limited number of gene elements. Secondly, and uniquely in the body, the genetic elements which code for the antigen-binding structure of antibody are subjected to a high rate of mutation, coupled with a process that allows selection of cells which make antibody that bind the antigen strongly. This process results in antibodies that bind their antigens with high specificity and high affinity. See also: Antibodies


The potential of antibody as a tool in medicine has long been recognized. Antibodies made in animals have been used to assay for the presence of hormones (in pregnancy tests for example), and even to neutralize toxic substances (as in the use of antibodies against snake venom proteins, made in horses, to treat human victims of snake bite). Antibodies made in animals have a number of limitations. The product of a single B cell is multiple copies of antibody with a unique binding site. However, when we immunize an animal and subsequently bleed it we obtain, in the serum, a mixture of antibodies produced by the animal in response to the many antigenic molecules it encounters. Antisera made in animals are thus variable and of limited overall specificity. See also: Antibody function


In 1976, Georges Kohler and Cesar Milstein, working in Cambridge, England, developed a procedure to isolate and propagate the individual B cells making antibody against the antigen of interest. They fused cells obtained from the spleen of an immunized mouse with myeloma cells. Myeloma is a tumour of antibody-producing cells, and myeloma cell lines are available in which the cells multiply rapidly and produce large amounts of antibody â?" though not usually of a specificity that is of use to us. Some of Kohler and Milsteinâ?Ts fusion products (â?~hybridomasâ?T) retained these properties of indefinite propagation and high antibody secretion rates but made antibody coded for by the antibody genes of the mouse spleen cells. The mixture of hybridomas still would make a variety of antibodies, but this mixture could be separated out by cloning â?" that is, individual cells isolated and allowed to proliferate into separate populations, or clones. Then it was a matter of screening the many clones to see which ones made antibody against the antigen of interest. See also: Milstein, Cesar


This is the basis of the production of monoclonal antibodies. With relatively minor changes, this procedure has spawned an industry and revolutionized many aspects of medical diagnosis and research. Monoclonal antibodies have also been used as therapeutic agents, and the potential for further therapeutic applications is exciting.



Generation of Hybridomas

The process of generating hybridomas centres on the fusion reaction between immune spleen cells and myeloma cells, but the generation of these two components is critical and complex, involving several stages. The generation of hybridomas is illustrated schematically in Figure 1.

Strategies

Ideally the target antigen, the molecular structure against which the antibody is to be made, will be available in pure form and in adequate quantity (a few milligrams). However, this ideal is often not achievable. Antibody against a complex multimolecular structure such as a virus or a blood cell may be desired, or the molecule may be known but it may not be possible to purify it in sufficient quantity. Monoclonal antibodies provide excellent reagents to use in purifying molecules from complex mixtures, so it may be that the antibody is wanted in order to be able to purify the antigen.


Monoclonal antibodies against individual components of a complex mixture can be made by devising a selection strategy that will identify hybridomas making antibody against the antigen in question. The most effective strategy depends on the situation. Differential screening of hybridoma clones, against the mixture containing the antigen of interest and against another mixture, lacking the antigen but otherwise as similar as possible, is widely used. For example, in making antibodies that will specifically identify a particular peptide growth factor produced in culture, a culture lacking the stimulus that elicited the production of the growth factor may be a useful control. In most cases these strategies will not identify antibodies against the antigen with certainty, but will allow the selection of a group of hybridomas that are likely to include clones making antibody against the antigen of interest. Supplementary studies, for example western blotting to characterize the molecular weight of the molecule detected and functional inhibition studies to neutralize biological activity, will then be needed to identify the hybridomas secreting the antibodies in question.


The principal consequence of these considerations is that a screening strategy must be in place before the immunization of mice.


Immunization

Virtually all hybridomas are made using immune cells from mice. Immunization protocols vary widely depending on the nature of the antigen, but generally involve a priming dose injected subcutaneously with adjuvant to provide a strong immune stimulus, followed about 4 weeks later by a booster dose, often given intravenously without adjuvant. It is sensible to use an immunization protocol which has been described in the literature for a similar antigen.


The myeloma cell line

A small number of mouse myeloma lines are available. The earlier lines are able to make their own antibodies, so that the hybridoma can make the light and heavy chains of both the myeloma and the spleen cell fusion partner. The light and heavy chains are made independently and assemble in the cell, so such a hybridoma can make a variety of antibody molecules, only one of which will have the desired binding sites. To avoid this heterogeneity, myelomas have been selected which have lost the ability to make their own light and heavy chains.


Since myeloma cells grow continuously, a method is required to allow selective growth of hybridomas and suppress growth of the parent myeloma. The selection method used most widely depends on the use of myeloma cell lines which have lost the ability to make nucleotides by the salvage pathway. The main biosynthetic pathway can be blocked with the drug aminopterin, so that the myeloma cells cannot make DNA or RNA and die. Hybridomas, on the other hand, have the enzymes for the salvage pathway and can grow, provided they are supplied with the substrates for the pathway, hypoxanthine and thymidine. The selective system is named after the three substances that are added to the culture medium, HAT (hypoxanthine, aminopterin and thymidine). See also: Nucleotide synthesis via salvage pathway


Myeloma cells are available for the production of hybridomas with rat, human and chicken lymphocytes, but most work has been carried out using the mouse, and extensive efforts to make human hybridomas for therapeutic purposes have yielded limited success. Heterohybridomas, using mouse myeloma and lymphocytes from a second species, have also had limited success. Interest has turned to the use of libraries of human antibody genes and genetic engineering methods when human antibodies are required.


The fusion process

The reaction at the heart of the field of monoclonal antibodies, the fusion reaction, is surprisingly simple. Myeloma cells, taken from culture, are mixed with spleen cells isolated from an immunized mouse, at a ratio usually of one myeloma cell to 10 spleen lymphocytes. The cells are centrifuged together to form a pellet and resuspended in a small volume of a viscous solution of polyethylene glycol. After 1 min the suspension is gradually diluted, care being taken not to break up small aggregates of cells, some of which will form the hybridomas. The cell suspension is washed and resuspended in culture medium. After a few hours in a tissue culture incubator (which provides a physiological pH and temperature), the cells are dispensed into small tissue culture wells at a concentration which, from experience, is likely to yield single hybridoma colonies in the wells. The cells are then allowed to grow over the next 7â?"14 days, with occasional changes in medium. See also: Cell culture: basic procedures


There are many minor variations of this procedure, and many critical points. Polyethylene glycol is rather an inert chemical and probably stimulates fusion passively, by allowing cells to stick together and excluding water from the junction. The cells should be in good con***ion before the fusion â?" the myeloma cells should be growing exponentially and the lymphocytes not subjected to traumatic processes during preparation. This requires a gentle procedure for disaggregating the spleen and for removal of red cells. The fusion procedure itself is critical; inadequate or over vigorous resuspension of cells will reduce yields. Postfusion dilution, washing and plating out again need to be carried out with an understanding of the objective â?" at this stage the cells are not yet fused stably; there are doublets with adhering and perhaps partly fused membranes, and these should not be disrupted. See also: Lymphocytes

Growth and selection

Growth of hybridomas occurs gradually over the first 2 weeks after fusion. The yield may be improved by adding interleukin 6 (IL-6), which acts as a hybridoma growth factor. Other ad***ives, found empirically to improve hybridoma yields, such as feeder cells or a variety of commercial supplements, are thought to act through IL-6. Cytokines have overlapping functions, so it is likely that other growth factors help hybridoma growth. See also: Interleukins


After a few days in culture, small colonies of hybridoma cells are seen using an inverted microscope and, if all goes well, these colonies expand to the point where they are visible by eye and begin to affect the pH of the medium, turning the indicator dye yellow. About 1 week after the initial fusion the unfused myeloma cells should be dead, and it is then helpful to gradually supplement the cells with medium lacking aminopterin. Since hypoxanthine and thymidine are used up while aminopterin accumulates, the HAT medium should be replaced with HT medium.


Once the culture wells are showing visible colonies and the medium is turning yellow it is time to test the supernatant for antibody. At this stage there will be many colonies to test and maintain, perhaps several hundred, depending on the scale of the experiment. A priority is therefore to reduce the number of cultures by eliminating negative cultures. It is possible to test simply for immunoglobulin production, but this usually eliminates very few. A more useful test is to screen for binding to the antigen by a simple assay capable of being run daily on large numbers of samples.


Selected cultures must be cloned, because there is no guarantee that they arose from a single cell. Cloning is usually repeated several times over the first few weeks, because the fusion products are still genetically unstable and may produce loss mutants, which grow but do not produce antibody. Clones are grown into larger culture vessels to produce quantities of cells and antibody for more extensive evaluation.


At this stage cells should be cryopreserved, so that if anything goes wrong there is a seed culture to go back to. Many things can go wrong, including contamination with bacteria or fungi, so cells should be cryopreserved as soon as there is evidence that the colony may be producing a useful antibody. However, cryopreservation requires a few million cells, so cannot be performed until the culture has been sufficiently expanded. See also: Cryopreservation of cells


Cryopreservation and long-term maintenance

The amount of work required to establish a hybridoma is considerable; the final product is unique and may not be reproduced exactly in a subsequent fusion. It is therefore essential to establish a secure õ?~bankõ?T for hybridomas. This is achieved by storing ampoules of hybridomas in liquid nitrogen. The procedures for freezing down cells and for thawing them out to re-establish them in culture are straightforward; the critical issues are essentially administrative. It is important to freeze down at least 5õ?"10 ampoules, to validate the õ?~depositõ?T by reconstituting one ampoule, to maintain adequate records and to lay down more ampoules when stocks get low. It is wise to store a set of ampoules in a separate laboratory.


Hybridomas may be maintained in culture essentially indefinitely, by õ?~splittingõ?T the culture every 2õ?"3 days, maintaining the cell concentration at between 2õ?"3 - 105 and 106 cells per mL. However, it is more usual to grow them only when fresh antibody is required, and re-establish them from cryopreserved stocks when needed. If they are maintained for long periods it is necessary to check antibody production regularly, because loss of production may occur.


Antibody production

The amount of antibody secreted in small cultures such as microwells is adequate for screening. However, once a hybridoma has been found to make a useful antibody larger amounts will be needed. Cells may be grown in conventional culture flasks and supernatant harvested 2õ?"3 times per week; such supernatants generally contain antibody at 1õ?"5 ẻẳg mLõ^'1. This concentration is adequate for many assays; however if the antibody needs to be purified, for example to conjugate it to an enzyme or fluorochrome, larger amounts and higher concentrations are needed. These have generally been prepared by growing the hybridoma as an ascitic tumour in mice, yielding ascitic fluid with antibody at concentrations of 1õ?"5 mg mLõ^'1. This procedure is increasingly unacceptable for ethical reasons, as alternatives become available. Furthermore, the ascitic fluid contains, in ad***ion to the antibody made by the hybridoma, antibodies made by the mouse against environmental antigens. The antibody is no longer truly monoclonal and monospecific, and significant difficulties in interpretation may result. Fermenters and mini-fermenters are available for the production of monoclonal antibodies in culture at any scale from a few milligrams to the gram amounts required for clinical trials. While there are still significant difficulties and uncertainties, and production costs are high, these methods are gradually replacing ascites production in mice.


A number of methods are available for antibody purification in good yield and purity. For many purposes, however, conventional culture supernatant will work well, without the need for purification.

Applications

Monoclonal antibodies have a range of applications which take advantage of the specific binding of their target antigen (Figures 2 and 3).

Analytical applications

Antibody may be used to detect an antigen, for example in forensic applications, in microbiological testing of foodstuffs, and in diagnostic testing of blood samples for toxins or infectious organisms. The antibody-based test for the presence of antigen may be rendered quantitative, providing an assay for antigen. Antibody-based assays are widely used in medicine to determine levels of growth factors, hormones, blood cells or malignant cells; the applications are essentially unlimited. Still in analytical mode, monoclonal antibodies may be used to locate antigen. Antibodies are widely used in conjunction with colour-forming labels and microscopy to localize antigens in tissue sections. This is known as immunohistochemistry. See also: Immunohistochemical detection of tissue and cellular antigens; Monoclonal antibodies: diagnostic uses


Preparative applications

Antibodies may also be used preparatively to purify molecules or cells from crude mixtures. Immunoaffinity-based preparative techniques are very powerful compared to more tra***ional biochemical purification methods, although in general a successful purification procedure will combine both affinity-based and conventional methods. Antibody-based purification methods are useful from the laboratory scale, where the aim is to purify nanogram to milligram quantities of biological substances from complex mixtures such as serum, to production of therapeutic substances, such as the blood-clotting factor VIII from large volumes of blood. The use of antibody to identify particular cell types in complex mixtures has been extended to preparative methods to purify these cells. One approach is to link antibody to magnetic particles. A magnet is then used to physically separate cells which bear the antigen from cells which do not. A more powerful technique uses the fluorescence-activated cell sorter, in which a cell may be identified on the basis of antibody tagged with fluorescent dye and physically separated from the other cells (Figure 3). Because the absolute differentiation of a particular cell type from all other cells may require several different markers, cell sorters can sort on the basis of seven, or more commonly four, parameters simultaneously. See also: Immunofluorescence


Therapeutic applications

Potential therapeutic applications of antibodies include the neutralization of toxins, the removal of infectious agents from the circulation, and the destruction of body cells mediating disease, including autoimmune cells and cancer cells. See also: Autoimmune disease: treatment


Polyclonal antisera have been used for many years in the treatment of snake-bite and infections where the major threat to life is a toxin, such as tetanus. These con***ions are relatively rare (bacterial infections such as tetanus generally being prevented by immunization), so that an individual is unlikely to need repeated treatment. The immune system recognizes antibody made in another species as a foreign protein and will make an antibody response against the protein. This means at best that second or subsequent treatment with antibody from the same species as the first treatment will be of limited effectiveness, because the protein is cleared rapidly; at worst the resulting immune reaction can take the form of a life-threatening anaphylactic shock. See also: Antiserum; Venoms


Polyclonal antisera against human lymphocytes were developed in the 1970s to treat patients who were rejecting organ grafts. The principle was that the host immune response was responsible for the organ graft rejection; antibodies against key components of the immune system should suppress the rejection. These antisera have largely been superseded by a monoclonal antibody called OKT3, directed against a molecule expressed on human T cells and involved in T cell function. OKT3 has been highly successful in reversing rejection episodes. The injected antibody is a mouse protein and is immunogenic, but the response is muted because the patients are immunosuppressed, both by the antibody itself and by other immunosuppressive therapy used to reduce the graft response. Nevertheless OKT3, successful though it has been, has been perceived as limited in effectiveness partly by its immunogenicity. See also: Graft rejection: mechanisms; Immunosuppression: use in transplantation


There have been some promising results with other monoclonal antibodies in human therapy, but these must be set against a number of failed clinical trials and generally a lack of sustained success. This situation is contrasted with the highly promising results in animal models, where antibodies can destroy established tumours, induce a state of permanent tolerance to transplanted organs, reverse autoimmune disease, and rescue animals from acute toxicity caused by, for example, bacterial endotoxin. Therapeutic development of monoclonal antibodies does not appear to have realized its full potential. There are a number of promising products in clinical trial and a small number approved for use, but it is too early to be sure their success will be sustained. See also: Tumour antigens recognized by antibodies


Major factors which have limited the clinical applications of monoclonal antibodies have been:


immunogenicity;
difficulty and cost of production on an adequate scale;
unwanted biological activity õ?" due for example to direct effects on cells of the immune system;
limited binding affinity, which necessitates the injection of large amounts of antibody in order to achieve a therapeutic effect;
lack of direct functional action, requiring conjugation of drugs or other biologically active materials; and
limited penetration into the target tissue õ?" especially dense, poorly vascularized tumour tissue.

There is currently a mood of optimism that many of these difficulties will be overcome by antibody engineering to tailor the properties of the antibodies. See also: Monoclonal antibodies: therapeutic uses


Antibody Engineering

Antibody is a protein which can be modified in a variety of ways: removing specific functional portions, reducing the overall size of the molecule, changing critical amino acids to increase affinity, linking to other functional molecules such as drugs, radioisotopes or toxins to add õ?~teethõ?T. It is generally easier in the long term to modify the DNA rather than the protein õ?" this needs to be done once only, and the modified gene is then expressed to produce the modified protein.


Antibody engineering is a relatively new field which is still developing. Techniques that appear important include:


preparation from existing hybridoma genes of genes coding for small proteins which include the antigen-binding site but omit most of the rest of the molecule, including sequences responsible for some of the biological effects of antibodies;
modification to increase antigen-binding affinity;
preparation of fusion proteins consisting of the antigen-binding site linked directly to, for example, a toxin, an enzyme, a sequence suitable for radioisotope labelling, another antibody sequence, to achieve increased or novel biological activities;
modification to make the sequence more human-like and less immunogenic;
generation of libraries of genes derived from human antibody genes, to avoid the immunogenicity associated with foreign protein; and
preparation of antibody fragments in bacterial culture, to increase yield.

Products of these techniques are undergoing clinical trial and some have been approved for patient use. The next few years will show how successful they will be in practice. See also: Affinity of antigenõ?"antibody interactions; Protein domain fusion



Relative Merits of Polyclonal Antisera, Monoclonal Antibodies and Genetically Engineered Antibodies

The major characteristic of antisera, prepared by immunizing and subsequently bleeding animals, is the heterogeneity of the antibody preparation. This may be a strength or a weakness, depending on the application. An antiserum will contain a mixture of antibodies against the antigen of interest, together with antibodies against other antigens encountered by the animal. The latter may cause confusing reactions, and may need to be removed by absorption. The multiple antibodies against the antigen of interest, reacting with a multiplicity of epitopes and showing a spectrum of binding affinity, can be advantageous, giving a stronger reaction overall than is seen with a single monoclonal antibody.


There are important uses of antibodies where polyclonal antisera work very well. This is particularly true in radioimmunoassays, which have been established for many years; there is no pressing need to change. On the other hand, in immunohistochemistry, change was initially slow in coming, because polyclonal antisera apparently worked very well, until awareness of the increased specificity achievable by monoclonal antibodies became widespread. A polyclonal antibody may react with the antigen of interest in one tissue, but when applied to another tissue, it is not possible to know whether it is reacting with the same antigen. See also: Immunoassay


Monoclonal antibodies must always be preferred when specificity is important. However, monoclonal antibodies can be cross-reactive. A rabbit antiserum may react with the antigen of interest and ad***ionally with a completely dissimilar molecule against which the rabbit made an antibody without being asked to. This type of nonspecificity is unlikely with a monoclonal antibody (unless it was made as an ascitic fluid õ?" see earlier comments). Cross-reactivity of antibodies results from their reaction with epitopes with a similar shape, which may be found on unrelated molecules. This type of cross-reactivity is found with monoclonal antibodies, and must be guarded against.


Apart from specificity, monoclonal antibodies possess a major advantage of reproducibility. Two rabbit antisera against a particular antigen will exhibit differences in affinity and in the mixture of antibodies against different epitopes of the antigen; they will also carry a different set of unrelated antibodies. Even two batches taken from the same rabbit may differ in activity and in cross-reactivity. The specificity of a monoclonal antibody, once established, should not vary. Differences in titre (amount of antibody) are relatively easy to adjust for. There remain subtle variations between batches of monoclonal antibody, depending on denaturation and aggregation during storage and processing, but these are minor compared to the variations between batches of rabbit antiserum.


In laboratory applications the choice between polyclonal and monoclonal antibody may best be summed up as follows: if there is a choice use the monoclonal antibody. However, when there is no antibody available, it will sometimes be quicker to make a polyclonal antiserum; this should be replaced in due course with monoclonal antibody.


For therapeutic applications the situation is sometimes radically different. A polyclonal antiserum against the venom of the Australian king brown snake will neutralize all of the toxic activities of this witchesõ?T brew õ?" neurotoxins, anticoagulants, procoagulants, and others we may not be aware of. To replace this well-tried preparation with a ****tail of monoclonals would be a major undertaking. Polyclonal antisera have an assured future in some therapeutic applications. In others, monoclonals have proved disappointing, but, as discussed above, antibody engineering may greatly extend the therapeutic scope of antibodies.



Originally published: March 1999


Further Reading

Kohler G and Milstein C (1975) Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256: 495õ?"497.

Winter G and Milstein C (1991) Man-made antibodies. Nature 349: 293õ?"299.

Zola H (1987) Monoclonal antibodies: A Manual of Techniques. Boca Raton, FL: CRC Press.

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