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Variations in fusion protocols for rat systems. Variations in fusion protocol for human systems. Pre-selection of lymphocytes of pre-defined specificity. Pre-selection of B lymphocytes. Positive selection. Negative selection. Pre-selection of T lymphocytes. Transformation followed by fusion. Mouse human fusions of transformed human cells. Human-human fusions of transformed human cells. Selection and cloning XI Early feeding and assay of fusions.

Failure of fusions. Failure where clonal growth is good. Failure with no clonal growth. How to cope with a fusion which is too successful. Cloning of hybridomas. Cloning by limiting dilution. Cloning in soft agar. Cloning by the Fluorescence Activated Cell Sorter. Cloning of human cells. Additional considerations. Failure of cloning. Failure with no growth. Failure with clonal growth.

Continuation of cloning. Chapter 9. Antibody production and purification 9. Maintenance of cell stocks. Expansion of hybridomas in vitro. Expansion of hybridomas in vivo. Failure in expansion. Storage of antibody. Concentrating the antibody. Purification of the antibody. Purification with Protein A. Purification with DEAE-linked reagents.

Purification utilising affinity chromatography. Purification of an IgM monoclonal antibody. Purification of an IgA monoclonal antibody. Failure of purification. Characterisation of monoclonal antibodies. Determination of antibody class. Class determination by Ouchterlony. Class determination by electrophoresis of antibody labelled in vivo.

Light-chain analysis. Proof that the antibody is monoclonal. Epitope analysis. Determination of overlapping epitopes. Determination of antibody specificity among a group of antigens Analysis of epitope specificity by antigen modification. Analysis of epitope specificity by Western blot. Failure to Western blot. Variations in Western blotting. Determination of antibody affinity. Karyotype analysis of hybridomas. Animal handling techniques. Addresses of suppliers and manufacturers. Protocols f o r polyacrylamide gel electrophoresis PAGE. Subject Index.

Introduction The technique of cell hybridisation or fusion has been applied to a large number of problems in the biomedical sciences. It has proved to be a major tool in determining which human chromosome codes for a specific gene function by means of the analysis of the isoenzyme patterns produced by a panel of clones from human-rodent fusions Ruddle and Kucherlapali, In addition, it has been used to characterise the dominant or recessive nature of malignancy by means of fusion of normal cells with malignant cells Harris, ; Croce and Kaprowski, In the early days the fusion agent, or fusogen, was an inactivated virus, most commonly sendai virus Harris and Watkins, However, in recent years the use of chemical fusogens such as polyethylene glycol PEG has become more common Pontecorvo, The production of monoclonal antibodies remains only one of the many applications of the technique and it is likely that cell hybridisation will be used extensively in the long term for the immortalisation of many other differentiated cell functions and that the extensive practical experience developed in the optimisation of hybridoma production may lead to the expansion of the application of cell fusion techniques in other areas Ringertz and Savage, The theory of monoclonal antibody production is based on the clonal selection hypothesis of Macfarlane Burnet Burnet, Each mammalian B lymphocyte has the potential to make a monospecific antibody.

Recent research in immunogenetics has shown that the origin of this specificity is complex and involves not only the extensive rearrangement of the DNA sequences in the chromosomes which code for the antibody chains but also some degree of somatic mutation during clonal development reviewed by Tonegawa, The broader immunological background can be found in several recent textbooks McConnell, Munro and Waldman, ; Roitt, ; Weissman, Hood and Wood, Weir is the most comprehensive methodology text.

The first report of hybridoma production was in fact in Sinkovics et al. Since then there has been exponential growth in the literature relating to monoclonal antibody production and utilisation. The experimental problem encountered in monospecific antibody production relates to the fact that plasma cells which secrete antibody are terminally differentiated lymphocytes with a finite lifespan.

They cannot normally be grown in culture. However, tumours of such cells can be found in most animals and, in particular, can be readily induced in mice with the aid of mineral oils. The tumour cells secrete an antibody of single, unknown and almost certainly unwanted specificity but can grow indefinitely in culture. Consequently, if such tumour cells can be fused with a lymphocyte which makes antibody of the required specificity, the progeny may have the eternal growth capacity of one parent together with the specific antibody production capacity of the other Kohler and Milstein, , Essentially a refinement of the technique involves the selection of a mutant strain of the tumour parent line which does not itself secrete antibody so that the production capacity of the progeny is directed to the specific antibody.

A further sophistication, which is more essential, is to select a parent line which is in some way vulnerable to the cell culture conditions so that it can not survive unless it has participated in a Ch. The general procedures of monoclonal antibody production in mice. The commonest way of achieving this is to use a parent tumour line which lacks either the enzyme thymidine kinase TK or hypoxanthine phosphoribosyl transferase HPRT.

These are enzymes of the salvage pathway of nucleic acid metabolism and are essential to cells growing in the presence of aminopterin which blocks the main pathways of nucleotide synthesis. After the fusion the cells are therefore usually grown on medium containing Hypoxanthine, Aminopterin and Thymidine HAT Littlefield, ; Chapter 3 in which any parent tumour cells which have not participated in a fusion will die. The hybrid cells are grown in culture plates and assayed for the production of the required antibody after a suitable period of time, usually days.

Suitable clones are then selected for expansion and subcloning so that eventually the required antibody may be produced in large amounts. It is the purpose of this volume to enable the reader to undertake these procedures in his own laboratory and produce an antibody of the required immunoglobulin class, specificity and affinity. At present, the choice of animal is limited to mouse, rat or man.

Several recent reviews and books specifically directed towards monoclonal antibody production have been published Galfre and Milstein, ; Goding, ; Fazekas De St Groth and Scheiddeger, ; Kennet et al. The procedures are undergoing constant modification and the Addendum at the end of the book is intended to detail the more significant of those which occur between the time of going to press and publication.

Comparison of monoclonal antibodies and con- ventional antiserum Monoclonal antibody production consumes very much more time and money than the production of conventional antiserum. It is sensible therefore to consider what advantages may be realistically gained by the use of this technique and whether conventional antiserum may not Ch. Table 1. These are analysed in more detail in the sections which follow. Specifcity: advantages The antigenic determinant is the particular site on the antigen which may be a very large molecule responsible for binding the antibody.

Conventional antiserum polyclonal will not only have antibodies to several determinants Fig. Consequently extensive cross reaction may occur between antibodies and two proteins which have similar determinants. This may be avoided completely by the use of monoclonal antibodies which have been selected for their ability to bind to a determinant unique to the required antigen Fig. The most Fig. The reaction of polyclonal serum with an antigen. At determinant A are two antibodies of the same class but different specificities and affinities.

At determinant B are two antibodies of different class, specificity and affinity. At determinant C are two antibodies of different class, one being an IgM but the same affinity and specificity. At determinant D are two antibodies of different class but the same specificity and affinity. Potential cross-reactions with two largely dissimilar antigens. Antiserum raised to antigen 1 will only show partial cross-reaction when tested with antigen 2 as determinant A is shared but the other two determinants are not.

Monoclonal antibodies to determinants B and C will not cross-react at all with antigen 2. Similarly, a pair of polypeptide hormones which have one chain in common and the other unique may readily be differentiated for use in radioimmunoassay, or two steroid hormones which differ by a single chemical grouping may be assayed separately.

At its simplest, the high specificity of a monoclonal antibody reduces non-specific background crossreactivity so that techniques such as immunohistochemical localisation may be very much improved. The high degree of specificity that may be obtained with a monoclonal antibody has opened up the possibility of tumour immunotherapy, either with antibody by itself, or antibody coupled to drugs or toxins, and successful treatments of leukaemias with murine monoclonal antibodies have already been reported Section 1.

While such applications emphasise the immense potential of the specificity of monoclonal antibodies, they also emphasise the importance of understanding the possible cross reactions that may unexpectedly occur with such a tool. Specifcity: disadvantages There are two main areas in which the specificity of a monoclonal antibody may be less precise than expected and where indeed crossreactivity may be greater than that experienced with normal serum Fig.

Firstly, the determinant may be present on other molecules which have not been tested in the screening procedure. An obvious example is with two proteins which have the same prosthetic group. With conventional serum, there would be antibodies directed at determinants not common to both proteins and cross-reactivity could be very slight.

The selection and testing of the monoclonal antibody should in principle be able to eliminate this difficulty but may be a complex process. While it is possible to test the antibody against antigens which are known or suspected to share common determinants, elimination of those antibodies which may display cross-reactivity to a particular cell type is a lengthy procedure.

Many cell surface antigens are carbohydrates in nature and the same antigenic sites may be present on two cell types which are structurally dissimilar and functionally distant in the body Gerson et al. While such cross-reactivity may not represent a major problem in diagnostic and preparative use, it clearly presents a major potential hazard in therapeutic use, and extensive preliminary experiments are essential. It must be emphasised that the extent to which cross-reacting determinants on molecules which have no other structural similarity exist is only now coming to light since before the discovery of monoclonal antibodies there was no methodology sensitive enough to measure it see Lane and Koprowski, for a review.

It is also possible for an antibody selected as totally specific in one assay to cross-react extensively in another assay, especially where fixatives have been used in a primary immunocytochemical assay Milstein et al. A second type of cross-reactivity which may occur with monoclonal antibodies can be found where the same antibody reacts with two Ch.

Potential cross-reaction of single antibodies with two dissimilar determinants. In 1, the antibody is totally specific for determinant A. In 2 , it has equal affinity and specificity for both A and B. In 3, it has greater specificity and probably affinity, see text for B but also cross-reacts with A. The conventional assumption that all antibodies are monospecific may therefore not be correct Richards et al. Cross-reactivities of the two types should not affect the majority of uses of monoclonal antibodies.

However, they do emphasise the importance of the selection procedures being oriented as closely as possible towards the final application of the monoclonal antibody. A typical example may be in general viral or bacterial diagnosis where the antibody reacts with a single determinant not present on all strains of the pathogen. Similarly, a monoclonal antibody cannot be used in a routine clinical radioimmunoassay of a protein which exhibits polymorphism throughout the population, unless it is certain that the antibody is directed towards an invariant determinant. Their standard nature and low background make monoclonal antibodies very attractive reagents for this type of routine immunodiagnostic use and paradoxically, it is therefore likely that the best way of obtaining a suitable reagent will be to mix two or more monoclonal antibodies directed against different epitopes on the antigen.

In this way the chances of not detecting the antigen are minimised while the standard nature and low background are preserved. Further hazards of monoclonal antibody specificity relate to their very precise assay requirements. These are discussed in detail in Chapter 2. None preclude the use of the antibody, but rather influence the possibility of detecting them on the initial screening procedure. Potential cross-reaction with two largely similar antigens.

Conventional serum raised to antigen 1 will cross-react very extensively with antigen 2 as it has determinants A, B and C in common. However monoclonal antibodies directed to determinant D will not recognise antigen 2. Affinity: advantages Since each antigenic determinant may elicit the production of a series of antigens of variable affinity, it is possible by monoclonal antibody technology to select out an antibody of the required association constant, rejecting those of higher or lower avidity.

This has obvious value in techniques such as radioimmunoassay where a high avidity antibody is generally required for maximum sensitivity and in most cases a high affinity monoclonal antibody is required and selected. However, preparative procedures often employ an antibody of Iower affinity deliberately.

For example, the antigen may be a protein vulnerable to denaturation by the extreme conditions required to elute it from a preparative affinity column and purification would be better handled with a low affinity monoclonal antibody where more gentle elution procedures could be applied. The association constant between a monospecific antibody and its determinant is quantitated by the rate constants for association and for dissociation. These two parameters may vary independently of each other according to the experimental conditions.

It is therefore possible in principle not only to select a monoclonal antibody of a chosen avidity but also to select one with a desired association or dissociation rate. The former might be more suitable for preparative procedures than the latter. Affinity: disadvantages It has often been suggested that monoclonal antibodies will never be able to replace conventional antisera as the essential high affinity of an antiserum lies in the cooperative effects between multiple types of antibody.

There is some basis for this view. The mechanism of such enhancement is not clear but it is unlikely to involve allosteric changes in antigen structure. It is possible that circular complexes involving two antibody molecules of different specificity and two antigen molecules have greater stability Fig. Alternatively there may be more subtle interactions between the Fc regions of the antibodies. It should however be made clear that not all pairs of monoclonal antibodies directed at different epitopes on the same antigen have synergistic effects Fig.

Furthermore many monoclonal antibodies of high avidity can have been produced so such effects either cannot be general or cannot be significant. Possible cooperative effects of antibodies. In a the geometry of the antigen-antibody complex is such that the two antibodies can act cooperatively stabilising the binding of each other. As a result the two antibodies together have higher affinity than would be expected from a mixture.

In b the position of the determinants is such that cooperative binding does not occur. Very small amounts of antibody are available at the early stages so that the determination of the association constant is an unrealistic aim. A strongly positive result may indicate an antibody of high affinity or simply the presence of a clone secreting large amounts of antibody.

A second procedure involving quantitative estimation of the amount of immunoglobulin secreted can be employed in such situations Chapter 2; Section Clones which give a strong reaction with the antigen but have little immunoglobulin are then selected for subcloning. Affinity and specificity: interrelationships It will be apparent from the preceding sections that affinity and specificity are closely linked in the selection precedures for monoclonal antibodies. It is possible to isolate a monoclonal antibody which is highly specific in the assay system used for the screening procedure only to find that it cross-reacts extensively when it is used in a more sensitive assay system.

Conversely an apparently non specific antibody may be made specific under more stringent experimental conditions where only epitopes specific to the antigen are recognised Mossman et al. The importance of the selection procedure is again emphasised and if this does not bear a close relationship to the ultimate use of the final monoclonal antibody produced, undesirable cross-reaction may occur. Standardisation As a standard reagent, a monoclonal antibody is undoubtedly superior to polyclonal serum. The latter is variant with animal and bleed so that much experimental effort must go into the standardisation of each serum sample.

The animals themselves are mortal and a regular supply of purified material for immunisation is essential. The only possible difficulty could lie in antigenic variation within the population as is discussed in Section 1. Human hybridomas may be propagated by either method since it is possible, though difficult, to produce them in milligram quantities in nude athymic mice.

The majority of monoclonal antibodies will grow in ascitic fluid. In principle tissue culture should yield antibody free of contaminating immunoglobulins, although the foetal calf serum employed may have up to 1. Other contaminating proteins from the foetal calf serum will of course also be present but these may readily be removed by affinity chromatography.

Ascitic fluid may have a small amount of contamination from mouse serum, the irrelevant immunoglobulin content being more difficult to remove as it it is from the same species. While the yield of conventional serum in terms of antibody may be of the same order of magnitude the contamination with irrelevant immunoglobulin is very much greater and the monoclonal system is therefore again superior. Purification requirements In the production of conventional antiserum there are two conventional methods of preparing antibodies specific to a single antigen.

The most common is to purify the antigen before immunisation of the animal. However, it is also possible to absorb the serum produced so that antibodies which cross-react with undesired components of the original mixture may be removed. Antigen purification may be a very Ch. In theory, monoclonal antibody production does not require a pure antigen since the appropriate antibody is selected from the cloned culture. Thus it is possible to produce an antibody to a minor component of a complex mixture which was itself impossible or very hard to purify by conventional biochemical techniques.

This has indeed been achieved with spectacular success in the case of alpha interferon Secher and Burke, In this respect monoclonal antibodies may be said to be greatly superior to conventional antisera. However two points should be made. The first is that in order to select a clone secreting the specific antibody a certain amount of pure antigen is required at some stage unless a bioassay can be established.

Secondly, in practice most fusion products reflect the immunodominance of the serum antibodies. If the immunising mixture contains a large number of strong antigens then these will tend to mask the minor antigenic components and the number of clones which produce antibody reacting with the minor antigens is greatly reduced. Some sort of preliminary purification of the antigen is therefore highly desirable and one method of accomplishing this is by sequential fusions where the major antigens are selected out and removed from the immunising mixture by their own monoclonal antibodies.

Consequently while it may be true in general to say that no purification procedures are required, this may not be true in practice with any specific antigenic mixture see Chapter 4. Selection of immunoglobulin isotype For certain uses of antibodies it is desirable to select for or against a certain type of immunoglobulin. For example antibodies which specifically do or not react with Fc receptors may be desirable in the final application, or the multivalence of IgM molecules may be considered an advantage in, say, a complement fixation assay. It is, however, becoming apparent that the general properties which were attributed to such antibodies in studies with conventional sera do not always extrapolate to monoclonal ones.

For example not all IgM monoclonal antibodies will fix complement and many IgG ones do not. Furthermore, it is becoming apparent that while the multivalence of IgM antibodies may be considered to increase assay sensitivity, in general such antibodies appear to be of lower affinity Rodwell et al. While typical production costs are shown in Table 1. A mouse or rat monoclonal antibody to a dominant antigen could be produced by an experienced operator in a well equipped laboratory in a few months.

The same operator could be responsible for a variety of antigens provided that the experiments were arranged to minimise overlap. This would also be time saved by the reduced requirements for purifying the primary antigen or absorbing the serum. On the more pessimistic side, a laboratory which had no previous experience and was obliged to purchase the appropriate tissue culture apparatus and develop their own expertise would take considerably longer at much greater expense.

If, in addition, they were obliged to undertake sequential monoclonal antibody production in order to isolate a minor component of a highly antigenic mixture, the time involved and the running costs would be very extensive. If a single hybridoma line is all that is required then collaborative work with a laboratory with the appropriate facilities and experience would generally be the better course of action. More detailed costing information is given in Chapter 5. Applications of monoclonal antibodies The applications of monoclonal antibody technology are generally outwith the scope of this volume and are reviewed in several others McMichael and Fabre, ; Albertini and Ekins, ; Hammerling, Hammerling and Kearney, ; Kennett, McKearn and Bechtol, ; Edwards, ; Yelton and Scharff, The Index Medicus lists several hundred papers each month under the heading of monoclonal antibodies.

It is, however, possible to outline some of the major applications of monoclonal antibody technology at the present time so that the range and scope of the technique may be summarised. Diagnostic uses Antibodies produced in the mouse or the rat are most commonly used for diagnostic purposes as they are more readily produced. Not only is the fusion frequency an order of magnitude higher but the animal can be hyperimmunised with the chosen antigen.

The major advantage of monoclonal antibodies over conventional sera in this application is probably their ready availability for an indefinite period at a standard titre, making direct comparisons between different laboratories comparatively simple. However, their high specificity has added greatly to the accuracy and speed of the diagnosis. Thus antibodies to common serum analytes such as protein hormones or alphafetoprotein are already commercially marketed and are slowly replacing conventional sera.

A very large number of monoclonal antibodies have been produced to a wide range of viruses such as influenza Gerhard et al. Their high specificity has led to accurate identification between similar strains of virus such as Herpes simplex Types I and I1 Pereira et al. On a more basic level of research, it has been possible to make detailed charts of the antigenic drift which occurs in viruses such as influenza.

Work in this area has clearly emphasised some of the problems of overspecificity referred to above in that variants which do not react with a single monoclonal antibody are readily isolated and such an antibody would clearly be unsuitable for wide ranging diagnostic purposes. Already, panels of monoclonal antibodies which are selected so that all known variants of a viral infection may be detected have been produced Richman et al.

Antibodies designed to perform a similar function with bacterial diseases are plentiful though less common. Their applications are broadly similar and the high specificity should prove particularly helpful in the analysis of bacterial spores which tend to show extensive cross-reactivity in conventional immunological analysis. In addition, monoclonal antibodies to bacterial toxins have been generated Remmers et al.

The considerable potential of monoclonal antibodies in the study of parasitic diseases has already been widely exploited in the most common ones such as malaria Yoshida et al. The advantages here are not only the obvious diagnostic ones but also that the study of the disease itself may be undertaken in greater depth since many different surface antigens may be expressed at varying stages of the life cycle of the parasite see Rowe, for a review. Immunological tissue typing has been a diagnostic area in which large amounts of reliable reagents at high titre have long since been required.

Until recently such identification has depended on sera from multiple blood transfusion recipients, multiparous women or volunteers, and extensive adsorption has been required so that low and variable titres are produced. Monoclonal antibodies which may be used in tissue typing are now being produced by several laboratories Brodsky et al. Tumour diagnosis is undoubtedly the field in which there has been most interest in monoclonal antibodies at the present time.

There has Ch. However, antibodies to tissue or cell type specific antigens are more readily generated and these have great potential in the detection of tumours and their metastases. Monoclonal antibodies have been generated against a wide variety of lymphocyte cell surface antigens reviewed in Janossy, ; Janossy et al. In addition, monoclonal antibodies of high affinity will probably prove superior in the detection of tumour markers in the serum of affected patients.

The range of immunochemical techniques involved in this major area of application is wide including immunocytochemistry McGee et al. It is evident that the monoclonal antibodies are not only used for detection of the primary tumour but also for the monitoring of the progress of the disease and of the effects of therapy. It is, however, worth noting that unexpected cross-reactivities may confuse the results. Thus monoclonal antibodies which showed intitial apparent total specificity for certain differentiation stages of T lymphocytes were subsequently shown to cross react extensively with Purkinje neurones Gerson et al.

The techniques largely developed for the detection of primary and secondary tumour growth may of course be used in the study of other diseased states where abnormal tissue or serum components are a characteristic feature and there has been considerable interest in the fields of cardiology Haber et al. There are diagnostic applications in which human monoclonal antibodies are the preferred tool and may become more widespread in their use as their production becomes easier.

The advantage in some of these applications is self evident such as in scintigraphy where there is a danger of antispecies antibodies being produced in the patient if frequent scans are required. In tumour diagnosis this can give an indication of the extent to which the patient has been able too mount a response to the disease at different stages of progression and there is much information to be obtained by amplifying this response in vitro. There is also considerable interest in the potential amplification and analysis of the immune response in autoimmune disease Schoenfeld et al.

Small serum samples do not readily yield a high enough titre for the full spectrum of antibodies produced by the patient to be identified but monoclonal antibody production can again amplify the response in vitro. The range of epitopes within a group of patients may also be studied.

In this context it is interesting to note the increasing use of Epstein Barr virus to transform the peripheral blood lymphocytes. EB virus has the considerable advantage of transforming most of the B lymphocytes in a sample thus increasing the probability of detecting those lymphocytes producing autoantibodies which are as yet uncharacterised Kozbor and Roder, ; Steinitz et al. Therapeutic uses The majority of therapeutic applications o- monoclonal antibodies to date have involved those raised in a mouse Ritz et al.

This is largely because of the considerable problems that have been encountered in the production of human monoclonal antibodies and it is likely that as these difficulties are resolved human antibodies will have much wider use. Murine antibodies have the obvious disadvantage of being foreign to the human system and likely therefore to lose their efficacy on continual application as a host response is mounted.

In addition, the host response in itself may be harmful to the patient leading to serum sickness. While there are comparatively few situations in which the treatment with the monoclonal antibodies may be undertaken externally, the treatment of bone marrow allogeneic or autologous transplants is one such area in which murine monoclonal antibodies have Ch. Tumour therapy with monoclonal antibodies has largely been confined to lymphoid tumours since antibodies to lymphocyte cell surface markers of specific stages of differentiation were the first to be developed.

However, therapy with a human anti-glioma monoclonal antibody has also been reported Philips et al. This may prove to be a special case since the regression of the tumour may be due to feedback through the immune network Miller et al. One of the current complexities of assessing the effects of monoclonal antibodies in tumour therapy is the fact that the tumour is usually one related to the cells of the immune system and is being treated by products of other cells of the immune system.

The mechanisms by which such therapy may be effected are not fully resolved but probably involve opsonisation, activation of the complement system and blocking of target cell function Fig. Comparatively few murine antibodies fix human complement but many rat ones are reported to do this Clark et al. If a murine antibody is used in human therapy it may also operate by stimulating a host response to the foreign antibody bound to its target cell.

Therapy of this type is not always successful and any remission obtained may be temporary Fig. One reason for this in the case of murine antibodies may obviously be host rejection. However, in addition the cells may undergo antigenic modulation and lose the target antigen. Another possibility is the emergence of an unreactive subpopulation of tumour cells, It is also possible that the antibody will be rendered inactive because it combines with free antigen released from the tumour cells and fails to reach them in consequence Hamblin et al.

A further major problem may simply be access of the antibody to the tumour cell, particularly if the tumour is a solid one or if the antibody is of the IgM class. These toxic materials may be cytotoxic drugs which may be transported either covalently linked to the antibody Hurwitz et al. In this last case, much interest has been shown in the possible use of ricin or diphtheria toxin covalently linked to the antibody and such systems can be shown to be selectively cytotoxic to a chosen population of cells in mixed cell culture Krolick et al.

However, therapy with such powerful reagents is obviously not to be undertaken lightly particularly in light of the unexpected cross-reactivities which can occur with cell surface antigens, all of which would have to be extensively screened Section 1. Human monoclonal antibodies are likely to prove of considerable value in immunosuppression in heart and kidney transplant recipients as well as in the prevention of both graft versus host and host versus graft disease in bone marrow transplantation.

Antibodies specifically directed to cytotoxic T cells may have considerable value in these conditions. This type of antibody may also have therapeutic potential in the control of autoimmune disorders. While it may be possible to use murine antibodies for some of these purposes, the length of the period of therapy and amounts of antibody required may preclude the use of a foreign antibody because of the extent of the host rejection of the antibody itself.

Possible mechanisms of antibody mediated tumour cell destruction. Opsonisation leads to the binding of phagocytic cells X which can engulf the tumour cell.

[PDF] Monoclonal Antibody Technology: The Production and Characterization of Rodent and Human

At the moment donors of serum are increasingly hard to obtain and a human monoclonal antibody has been produced by the Epstein Barr virus transformation method. It is currently undergoing clinical trials Crawford et al. Other possible uses of human monoclonal antibodies are in the therapy of viral diseases such as hepatitis and in the treatment of snake venom or other forms of poisoning. In the long term there may be possibilities of specific immune therapy for autoimmune disease such as the use of anti-idiotype antibodies Fig.

Alternatively monoclonal antibodies directed to the products of genes controlling the immune response may have therapeutic value. However these applications are little developed at the present though trials on animal systems have been undertaken Newsome-Davis, ; Waldor et al. Preparative uses The earliest most successful preparative use of monoclonal antibodies was in the purification of alpha interferon Secher and Burke, In principle, it should be possible to immobilise any monoclonal antibody on an affinity column and use it to obtain large quantities of the required antigen from a crude mixture Fig.

To perform such an experiment it should not again in principle be necessary to purify the original immunising antigen though a selection assay must Fig. Possible mechanisms to the account for the failure of antibody mediated tumour cell destruction. Either this has been internalised or a subpopulation of tumour cells without the antigen have been selected.

Cells bearing the antigen will thus return after initial therapy. Possible mechanisms of tumour therapy with toxins. Possible mechanisms of anti-idiotype therapy in autoimmune disease. The use of a monoclonal antibody for the purification of a minor antigen in a complex mixture. In practice this is not always the most effective approach since the major antigens which dominate the serum antibodies also dominate the lymphocyte population used in a cell fusion experiment and if an antibody to a minor antigen is required it is not readily isolated.

In theory this problem can be circumvented by performing a sequential series of fusion experiments. In the early ones the antibodies to the major antigens can be isolated and used to remove these antigens from the mixture which is used to immunise subsequent mice so that a large panel of antibodies to minor antigens may be prepared Fig. However, comparatively few of Ch. Other possible ways of handling this problem involve the introduction of more complex immunisation and screening schedules so that the unwanted antibodies are not selected either because tolerance has been induced or the selection procedure is timed to avoid them for example selection for IgM a short period of time after boosting a mouse extensively preimmunised with the unwanted major antigen.

One undoubted preparative advantage of monoclonal antibodies is the possibility of selection of an antibody of chosen affinity for purification. Where conventional sera are employed, the affinity may be so high as to make recovery of the antigen from the preparative column extremely difficult without the use of extreme denaturing conditions which cause irreversible damage. A monoclonal antibody of submaximal affinity would obviously be of value in such a circumstance Section 1.

Preparative uses of monoclonal antibodies are in the early stages as yet in comparison to diagnostic uses. However in the long term they may be of considerable value in many branches of industry.

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Basic research The potential of monoclonal antibodies in basic research is considerable though much less widely discussed than the more immediately obvious applications. In principle they can resolve a single protein from a complex mixture or indeed a single epitope responsible for a specific function of a complex macromolecule. They have already been widely used in basic enzymology, in nucleic acid structural studies, and in the analysis of hormone receptors. It is customary now for any group working on a macromolecule to both clone the genes coding for it and make monoclonal antibodies to it sometimes without a clear objective for their application.

One field of research in which monoclonal antibodies may prove of particular value is in the study of chromosomal proteins. As hybridoma production becomes a more routine laboratory technique it is likely that this aspect of their application will expand considerably. T cell hybridomas The role of the T lymphocyte in the immune response is still poorly understood in comparison to that of the B cell.

The response is cellular in that the receptor which recognises the antigen is on the surface of a T lymphocyte. However, it does not recognise the antigen alone but rather the antigen which has been processed by a macrophage or dendritic cell. Individual T cells may have a highly specific response to the antigen, recognising only a small number of, say amino acid residues in the same way as B cell derived antibodies do, and the macrophage in some way processes the antigenic molecule to present these residues on the cell surface.

However, an additional complication is that the T cell cannot recognise this small region of antigen alone but must do so in combination with the histocompatibility antigens on the surface of the presenting cell. There is still dispute as to whether the antigen fragment and histocompatibility antigen actually form a composite recognition site, or are recognised separately by two parts of the same receptor or even different receptors though this is unlikely.

After recognition of the antigen, clonal development of the appropriate cell line occurs. There are many functional subsets of T cells and the T cell progeny thus produced may have a variety of functions and it is clear that T cells are not just a single group but rather a large family of lymphocytes, each of which has a different response still to be understood. Most established T cell lines with known antigen recognition and known functions, secrete lymphokines which attract and activate scavenger cells to the site of the invasion by foreign material.

However, some T cells develop cytotoxic functions for cells Ch. T cell hybridomas are still rare but it is possible to grow T cells from immunised animals in culture for short periods of time and stimulate them with antigen and T cell growth factor TCGF Beezley and Ruddle, ; Haas and Von Boehmer, Alternatively a T cell hybridoma may be made by fusing the lymphoid tissue or the enriched T cell population from the lymphoid tissue of immunised animals with a T cell leukemia partner analogous to the B cell myeloma lines but derived from a T cell tumour, usually made HAT-sensitive.

T cells which respond by expressing some sort of function such as the secretion of a lymphokine may then be cloned by the Fluorescence Activated Cell Sorter Section 6. T cell clones isolated in this way have immense potential for both basic and applied research. Firstly, they may be used to isolate the elusive T cell receptor. This is present in such small quantities on the cell surface that large numbers of cells are necessary. This is possible with a T cell hybridoma and the T cells which produce a response such as lymphokine secretion specific to a particular specific antigen and histocompatibility antigen may then be used to immunise a mouse to produce B cell hybridomas.

Antibodies secreted by these which block the T cell function are then used to purify the T cell receptor and the handful of experiments of this type which have been performed suggest that this is a heterodimer of molecular weight around 90 in both mouse and human systems e. Kappler et al. The nature of the dual recognition of both specific and histocompatibility antigens may then be analysed.

Secondly, the B cell antibodies to the T cell receptors may then be used to isolate the RNA coding for these receptors. This is, to date, one of the major unsolved problems in immunogenetics. A third contribution of T cell hybridomas to basic research lies in the analysis of the functions of different T cell clones. The differential functions of helper, suppressor, cytotoxic and lymphokine-releasing T cell clones can then be dissected. The implications of such information for basic research are obvious since it is the T cell response which is thought to be the major contribution to defense against viral infection, tumours and transplantation.

It is likely that T cell hybridomas will have immense therapeutic value over the next decade. The technology of T cell hybridoma production is years behind that of B cell hybridoma production but developing fast see Fathman and Fitch, and Hammerling et al. Both murine Kapp et al. The most recent work is reviewed by Berzofsky General assay requirements The assay system is probably the most critical factor in the generation of a large panel of good hybridomas.

The emerging clones secrete small amounts of antibody and early cloning of positive samples Chapter 8 is a key factor in the production of successful clones. This requires a very sensitive assay indeed and many of the assay systems which work well with serum are not of the required level of sensitivity. Additionally, many conventional assays such as those involving immunoprecipitation, depend on multiple epitopes on the antigen and these are ill suited to hybridoma selection.

It is almost impossible to emphasise too often that the final use of the antibody should wherever possible determine the type of assay employed. If the final assay is known to be relatively insensitive, or hard to use for extensive screening then it may be better to perform the initial screen with a sensitive assay and screen the positive samples by the final application assay. Theoretical considerations The kinetics of antibody-antigen interactions are discussed in detail in classical texts Steward, , Most of the earlier antibody kinetic studies were described by the kinetics of a univalent antigen usually a hapten and divalent antibody.

Like all reactions between proteins they are dependent on the concentration of both reactants and the conditions under which the reaction takes place. In the case of monoclonal antibody assay these are all highly relevant and may be very different from conventional antibody work. A comprehensive understanding of the theoretical background is essential for the production of a large number of hybridomas secreting antibody of the required characteristics. The subject is introduced in Section 1. The number of epitopes on the antigen is much reduced and in the case of a small protein is usually only one.

Thus the effective concentration of the antigen is very low. The antibody will still be bivalent or decavalent in the case of an IgM but its concentration may be very low. This is particularly true in the case of early assay which is advisable in order to detect suitable clones in order to avoid overgrowth by revertants which have lost the appropriate chromosomes. Additionally, however, clones vary greatly in the amount of antibody that they are able to secrete and human clones in particular are likely to secrete very small amounts of specific antibody Chapter 3.

In these circumstances therefore the concentration of effective antibody is likely to be exceptionally low. As a consequence of this, the parameters on the right-hand side of the equation defining the association rate are liable to be several orders of magnitude below those in conventional serum and indeed below those in the serum of the mouse, rat or human used in the testing of the chosen assay.

As a result, the conventional assay time of periods of one or two hours at room temperature or 37 "C have very limited relevance. A longer incubation time of antibody or antigen during Ch. The association rate is usually the parameter which determines the final equilibrium constant see Section 2. However, by assaying for too short a time, or too early when the antibody concentration is low, it is possible to miss an antibody which may be suitable for the required purpose but is secreted in small amounts at early stages or an antibody which is suitable for the required purpose but which is of comparatively low affinity.

The situation defined above is altered by the fact that most antibodies are at least divalent. However, for most assays at initial stages where antibody and antigen concentration are low they may be regarded as univalent. The antigen may also be multivalent as in cell or bacterial surface epitopes. These are covered in Section 2.

Additionally, with conventional reactions there are many complexes formed between different antibodies and their epitopes so that, at any one time, there is still substantial reaction towards complex formation. The dissociation rate is not only the most variable among different antibodies under a defined set of conditions but also the most variable in a single antibody with respect to environmental conditions such as pH, temperature, etc. Mason and Williams, While this sample is small, it suggests that for the antigens described, assay at lower temperatures is more likely to detect positive samples after an overnight period at 4 "C than assays for the same time at higher temperatures, especially if the antibody concentration is low.

Equilibrium concentration of reactants The equilibrium constant for an antibody antigen reaction is the ratio of the forward to the backward rates i. From these it is clear that the concentrations of the reactants and the assay conditions may influence the possibility of detection of a suitable hybridoma. Thus with polyclonal sera it is usually considered reasonable to assume that an optimal equilibrium condition will be achieved by incubation for an hour or two at room temperature, and biological pH, ionic strength, etc.

Monoclonal antibodies at early screening may not be detected unless several of these parameters are varied and it seems more than likely that antibodies to more interesting or relevant epitopes will be detected when it becomes possible to extend screening procedures to cover a variety of parameters. Effect of multivalence All antibodies are at least divalent and IgA and IgM antibodies may have several idiotypes on the same molecule.

Additionally, many antigens have neighbouring epitopes. Multivalent antibodies are usually helpful to screening procedures since the effective dissociation rate can be reduced if enough antigen is present. If one antibody-combining region dissociates from the antigen, the chances of it or its partner reassociating are increased because of their geometric proximity. It is possible that assay selection procedures alone account for the Ch. If the antigen has a large number of epitopes then this can affect the reaction in several ways. Antigens with multiple epitopes are probably most commonly encountered in bacterial systems with symmetrical cell wall structures.

They can also occur in situations in which large amounts of antigen have been bound to a solid surface. If the antibody is in excess in such a system it may bind through only one of its possibly variable regions and consequently it may dissociate more readily than an antibody with two regions anchored. Consequently there are rare situations in which an ELISA assay for example may detect antibodies better if small amounts of material are bound to each well. Specificity and affinity The complex relationship between specificity and affinity is discussed in Section 1.

Much of this information is based on a paper by Mossman et al. Nonetheless, it is assumed that at least in the initial stages, an assay detecting the maximum numbers of positive clones is required. Suitable conditions for obtaining specific responses can then be achieved at a later stage. Practical considerations 2. Numbers of assays The number of plates to be used in the initial fusions and in subsequent clonings is discussed in Chapter 6 and 8.

In a typical fusion procedure utilising 4 x 96 well plates some samples must be assayed in a short period of time.

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Additionally, as discussed in Section 2. With mouse systems it is exceptionally valuable to assay as early as possible in order to decide which cells should be cloned before overgrowth by non-secretors occurs. With rat systems this is less essential and with human ones there is not enough data on established hybridomas to delineate a clear production pathway but the antibody concentration is likely to be low and consequently screening should be comprehensive and also performed more than once i.

The number of assays involved is consequently considerable and individual screening by, say, immunocytochemical techniques, is likely to be either impractical or inevitably less comprehensive. To cope with the numbers a broad screening system which can be easily handled is recommended. If this system is far removed from the final application of the antibody then positive samples detected by the first screen should be assayed by a second directed to the final application, since the number of positives is very much lower than the total number of clones.

Time of assay In terms of clonal growth the time of assay should be soon after clones are microscopically visible and again a few days after when clones are visible to the eye Sections 2. Screening should continue for several weeks. The time of the actual assay procedure is a different consideration influenced very much by the antibody concentration as discussed in Section 2. However, it should ideally be for a very much longer period than the conventional serum from the mouse used for fusion to allow for the low antibody concentration as discussed in Section 2.

At least in some cases Mossman et al. It is quite possible to fail to detect a good hybridoma by screening at a single pH. However if the final use of the antibody must be at a certain pH value, there is no point in screening at other pH values. The pH of tissue culture fluid in which cells are growing can vary over almost a whole pH unit. The serum antibody can be detected at most pH values but the monoclonal is not detectable by this assay below p H 7. Courtesy of Dr. Robin Fraser and Dr. Angus Munro. Most assay procedures involve direct transfer of material from the tissue culture wells to the assay plates and thus in any screening procedure, pH becomes a natural variable.

Polyclonal sera usually react over a wide range but monoclonal antibodies may have very defined and discrete optima. If a defined pH is required for the final application then the assay should be buffered accordingly during the incubation of antibody with antigen. This may be particularly relevant for in vivo uses. Where pH adjustments are necessary the nature of the buffer should also be considered as the buffer components themselves may affect the assay i.

Temperature of assay It has already been mentioned in Section 2. The current limited evidence suggests that the best assay conditions for hybridomas favour incubation at 4" rather than at room temperature or higher but ideally both should be attempted unless the final use precludes a certain temperature.

Ionic strength of assay There is no detailed information available on the ionic strength variations in hybridoma assays. However, since a protein-protein interaction is involved, it seems likely that the usual considerations apply. Non-specific binding is more likely to occur at low ionic strengths. If pH variations are used in the assay then all the buffers used should have the same ionic strength. Antibody sampling It has been emphasised in Section 2. Antibodies, like other proteins, will be adsorbed readily on surfaces of glass or synthetic materials. The albumin and possibly the small amount of y-globulin in the fetal calf serum should minimise this, blocking most sites.

Monoclonal antibody production using Hybridoma Technology

However, albumin is a less than ideal blocking reagent since its isoelectric point is several units of pH above that of y-globulins and it is possible that albumin does not therefore occupy all possible blocking sites on sampling material. Any assay system which involves multiple transfers of the antibody from the tissue culture plate to the final assay is therefore not only cumbersome and inconvenient but unwise since antibody may be lost on surfaces. Equipment used for transfer should be pre-treated with bovine y-globulin or serum in order to minimise this. In addition, many sampling devices which allow more convenient transfer or even direct assay are now marketed e.

If these devices are to be immersed in the tissue culture plate, they must, of course, be sterilised as well as blocked. It is also possible to assay using short term tissue culture of hybridomas in antigen coated plates and thus avoid any transfers at all Weetman et al. Types of assay It is difficult to classify the possible types of assay for hybridomas without consideration of the relevant antigen. However, the fact that the assay should ideally be as close as possible to the final use of the monoclonal antibody cannot be overemphasised. The condition of the antigen formaldehyde fixed, radiolabelled, bound to other molecules, accessible or inaccessible in cells should be as similar as possible in the screening assay as in the final use.

The assay conditions Sections 2. Historically, cellular assays were the first to be used for hybridoma detection Kohler and Milstein, In more recent years solid-phase assay systems have become by far the most common. However, liquid-phase assays such as radioimmunoassay are also frequently used. Biological assays are the least common. Solid-phase assays The solid-phase assay system is adaptable to almost any antigen with almost any antibody.

Two major types of assay are shown in Figs. In the ELISA or radioactive binding assay the antigen is bound to the plate and incubated with the monoclonal antibody. A second antibody coupled to an enzyme or isotope is then used to detect the first. Solid-phase binding assays for specific antibody. Solid-phase binding assays for specific antigen IRMA.

The first system is generally used for screening and quantitating antibody and the second one for quantitating antigen. However, if both antibodies are of different species, the second assay may be adapted for screening with the use of a third labelled antispecies antibody. In principle, many types of sandwich assay with increasing layers may be developed but each additional layer adds to the uncertainty of the assay, to the complexity of performing it, and to the risk of antigen leaking from the solid support. Solid-phase assays of both types can be adapted for the use of small molecules by haptenisation.

However, the most common are polyvinyl or polystyrene. The physical nature can be balls Ziola et a]. However, the great majority of assays are performed with a solid phase support which is in the form of a well polystyrene or polyvinyl plate. The plates are generally coated with material which encourages the binding of the antigen.

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This type of plate is marketed by all the major manufacturers Flow, Gibco, Biorad, Dynatech and the performance may vary widely under assay conditions. Some give good positives with sera from immunised animals but also a high background with negative serum and in any preliminary test of plates it is the signal to noise ratio rather than the signal strength which should be evaluated.

While it has been emphasised that maximum sensitivity is necessary for early screening the plates contribute substantially to the cost of hybridoma production and more economical ones of slightly lower sensitivity are frequently employed. The mice were kept in polycarbonate plastic cages containing wood shavings for bedding. Mice were sacrificed 11, 14, 21, and 28 days after injection. Iliac lymph nodes were collected and lymph node lymphocytes from 2 mice were pooled and used for each cell fusion attempt.

Cell number was determined using a blood corpuscle counting chamber after lymph nodes were passed through a mesh stainless steel wire mesh. On days 9 and 10 after cell fusion, culture supernatant was collected and assayed by solid-phase enzyme-linked immunosorbent assay ELISA. Mouse serum was collected at sacrifice and serum antibody titers against ovalbumin were determined by ELISA using the 2-fold dilution method [ 5 ]. Positive wells were defined as wells that showed an absorbance of 0. P values of less than 0. Intramuscular tail base injection Fig.

Enlargement of both the right and left iliac nodes usually occurred, and a range of sizes was observed. Often, two iliac lymph nodes were present on either side of the caudal vena cava; however, sometimes three lymph nodes were present. The injected antigen emulsion was always found in the muscles of the tail base, and often found in the sacral lymph nodes and within cysts in the peritoneal cavity.

A small portion of the antigen emulsion was found in the sacral lymph node. The scale of the graph paper is 1 mm. To find and collect the iliac lymph nodes from age-matched normal mice was more difficult than to find and collect the enlarged iliac lymph nodes of immunized mice, due to the small size of the nodes and their being buried under the retroperitoneal membrane in the normal mice.

Nine mice injected with antigen emulsion subcutaneously at the tail base were sacrificed 14 days after the injection to confirm the injection site effect. The inguinal lymph nodes were enlarged in all mice; however, the iliac lymph nodes remained normal in size in 7 of the 9 mice.

Monoclonal antibody technology : the production and characterization of rodent and human hybridomas

In the 2 mice with the enlarged iliac lymph nodes, a portion of the emulsion was present in the muscle at the injection site. Although large individual variations were observed, the median value at day 14 remained low but increased rapidly thereafter, reaching a plateau around day Serum titer was defined as the reciprocal of the highest dilution giving an absorbance reading 0. After cell fusion, the cells were cultured in four well culture plates. Hybridoma colonies were confirmed 4 days after cell fusion. There were several well-grown colonies in each well. Histogram of screening results of supernatant for primary hybridomas by ELISA nine days after cell fusion using iliac lymph node lymphocytes from two mice immunized 14 days before.

Wells with absorbance of 0. Fusion was performed using iliac lymph node lymphocytes from two injected mice. Positive wells were also present using lymphocytes obtained at 11 and 28 days, but at a third of the concentration observed using lymphocytes obtained at 14 and 21 days. Timing of cell fusion using the mouse iliac lymph node method. Data points indicate the results of independent experiments, and the line indicates the mean of all the experiments.

The differences in the means between days 11 and 14, days 11 and 21, days 14 and 28, and days 21 and 28 were statistically significant. Relative proportions of IgM and IgG produced by hybridomas using the mouse iliac lymph node method. IgG1 was the main subclass observed using lymphocytes obtained 11, 14, 21 and 28 days after injection.

Only 4. Only two wells producing IgG3 subclass antibodies were observed in a total of 12 fusion experiments in the present study. Two attempts at cell fusion were made for each strain using iliac lymph node lymphocytes obtained 16 days after immunization. The number of positive wells obtained using the present method was 75 to per cell fusion attempt when lymph node lymphocytes were used 14 to 21 days after injection.

These numbers are much higher than those obtained in a previous study [ 5 ] using the mouse spleen method, which generated 9 to 11 positive wells per cell fusion attempt. In addition, the mouse spleen method requires three immunizations over a four-week immunization period. These results clearly indicate that the use of enlarged iliac lymph node lymphocytes from mice injected intramuscularly with an antigen emulsion via tail base is an excellent method by which to prepare hybridomas producing mouse monoclonal antibodies.

It has several advantages. Specifically, only one antigen injection is required and lymph nodes can be used from approximately two weeks after injection. This method saves time, effort, antigen requirements, expense, and has a better chance of success. However, the present study is the first to describe monoclonal antibody production using a mouse iliac lymph node method.

The major reason for this delay has been the small size of mouse lymph nodes, which necessitates 5 or more mice to be injected for immunization for a single cell fusion [ 2 , 8 , 9 ]. Another reason is that high antibody titers in serum have been thought to indicate the potential for success of cell fusion, and high titers are achieved with the mouse spleen method. This generally requires two or three booster injections, requiring at least four weeks from the time of initial injection. This long period of immunization might in fact reduce the number of positive wells obtained and make it difficult to achieve the success of the present mouse lymph node method.

A particular stage of B cell differentiation is thought to be most suitable for cell fusion in order to produce antibody-secreting hybridomas [ 1 , 4 , 12 ]. In the conventional mouse spleen method [ 12 ], the last booster injection is usually made intravenously with antigen in saline 3 or 4 days before cell fusion to induce the splenic lymphocytes into a suitable stage for cell fusion [ 12 ].

Using the present mouse iliac lymph node method, the greatest number of positive wells was observed using lymphocytes obtained 14 to 21 days after injection, and decreased numbers were observed using lymphocytes obtained 28 days after injection. Since the iliac lymph nodes were similar in size 14 and 28 days after injection, and since similar numbers of hybridomas were observed in each well from one fusion attempt to the next, the observed decrease at day 28 in the proportion of positive wells may have been due to a shift of B cells into an unsuitable stage.

IgM was easily obtained when mouse lymph nodes were used two weeks after injection. Individual mouse differences were observed with regard to the production of IgG2a and IgG2b; however, these subclasses were obtained after several attempts at cell fusion. Mirza et al. However, this method remains unpopular since lymph node lymphocytes from five mice are required for a single fusion attempt.

Moreover, lymph node enlargement is less successful than with the current method.

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We tried to immunize mice in the same way as Mirza et al. Of note, the injection site became less obvious several days after injection unpublished data. Davis et al. Although they used iliac lymph node lymphocytes as the source of B lymphocytes for the first time, their method is clearly different from the present mouse lymph node method.

Judging from the results of subcutaneous injection in the present study, subcutaneous injection at the tail base of a mouse induces hypertrophy of inguinal lymph nodes, although enlargement of both inguinal and iliac lymph nodes occasionally occurs. The main target lymph nodes in the Davis et al. The effect of booster injections was not examined in the present study, because booster injections given three or four days prior to sacrifice did not influence the number of positive wells obtained using the rat lymph node method in a previous study [ 5 ].

Typically, booster injections are given at least one week after initial immunization, which would lengthen the immunization period required for this experiment and increase the amount of antigen required. The antigen saved could then be used to immunize more mice. We have previously reported on a rat lymph node method [ 5 ]. This method uses lymphocytes obtained from enlarged medial iliac lymph nodes following hind footpad immunization for cell fusion. Intramuscular tail base injection of antigen into rats to obtain lymphocytes for cell fusion was also examined in the present study, and tail base injection was observed to produce enlarged rat medial iliac lymph nodes, from which lymphocytes for cell fusion were obtained and a number of hybridomas detected by ELISA unpublished data.

Therefore, the present study is the first report indicating that the iliac lymph node method is also useful in rats. The tail base is a more humane site for immunization than the hind footpads. Thus, researchers should use the tail base for immunization when using the rat lymph node method to achieve monoclonal antibody production using lymphocytes from enlarged medial iliac lymph nodes. Given the immunotolerance of rats to various rat antigens, monoclonal mouse antibodies have an important role in research.

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Although this data has not been published, we were able to produce antibodies to a rat antigen using the mouse lymph node method. We produced more than 30 clones of antibodies against rat renal basement membrane collagen, otherwise known as type IV collagen [ 6 ].