Monoclonal Antibody: a Major New Development in Immunology

3. F o r m . Sci. SOC.(1980), 20,

163

Received 18th February 1980

Monoclonal Antibody: a Major New Development in lmmuno~ogy S. M. FLETCHER and M. J. DAVIE* Home O@e Central Research Establishment, Aldermaston, Reading, Berkshire, England, R G 7 4PN

A recently developed technique-lymphocyte hybridization-has made possible the isolation and large-scale culture of a single antibody-producing lymphocyte. The 'antiserum' obtained in this way is a pure preparation of a single immunoglobulin species, and not a highly complex mixture as is present in conventional antisera. In this brief review we &scribe the technique and its product, and discuss some of its possible applications in forensic serology and toxicology. Introduction Quality assurance poses particularly difficult problems for the forensic serologist. The variable quality and erratic availability of some antisera (e.g. rhesus antisera and anti-Leb) mean that from time to time poor reagents have to be tolerated for want of better. Also, the selection of antisera for use in both absorption-inhibition and absorption-elution methods on the basis of saline agglutination titre is clearly insufficient in the light of the general opinion that the two techniques require antisera of different avidity characteristics. Full investigation of the properties of antisera on the lines suggested by Kind (1963), Bargagna and Pereira (1967), Lincoln and Dodd (1973) and Lang (1976) would probably provide the guidelines for choice but would certainly involve far more work than any forensic laboratory could normally contemplate. Commercial antisera for species identification pose another problem. Cross reactions vary considerably from batch to batch and these need to be checked for each new batch put into service. It would be a different matter if specially selected antisera were available in very large quantities. Full evaluations would then be needed only rarely and these in theory could be done by a central laboratory. Unfortunately the nature of the present antibody sources rules this out. However an alternative source of antibody is now available. A new technique, developed over the past five years, now makes possible the isolation and long-term culture of modified lymphocytes derived from single antibody-producing cells. Such cultures produce large amounts of completely homogeneous antibody-so-called 'monoclonal antibody' (MCA). The cells can be stored under liquid nitrogen indefinitelyassuring more of the same whenever needed-and in theory, at least, antibody of any type, specificity and affinity could be prepared in this way. What follows is a description of the MCA technique with a discussion of some potential applications of MCA in forensic serology and toxicology. Polyclonal Antibody Modern immunology is firmly based on the clonal, or germ-line, theory of antibody production. According to this theory there exists a large number of individual 'clones' of lymphocytes within the body, each of which is programmed to produce, upon appropriate stimulus, antibody with a different specificity. The structure and specificity of each antibody is determined by the *Present address: Home Office Forensic Science Laboratory, Washington Hall, Euxton, Chorley, Lancashire, England, PR7 6HJ.

genes of the clone and is not affected in any way by antigens. Any antigen which possesses a spatial arrangement of chemical structures complementary to those in the binding site of the antibody will trigger the process leading to proliferation and antibody secretion by a particular clone. I n practice when a single antigenic compound such as a protein is injected into a n animal it triggers the proliferation of several clones by virtue of the variety of stereo-chemical structures distributed over its surface. Thus even highly purified antigens induce the production of a number of different antibodies. The composition of this population of antibodies depends primarily on the 'clonal repertoire' of the animal, and is known to vary considerably within a species. This 'polyclonal' response to highly purified antigens largely explains the wide variation in the relative proportions of different immunoglobulin classes and specificities observed in sera from experimental animals immunized with the same antigen. Since different antibodies exhibit a wide range of binding affinities, antisera with different proportions of the same antibodies show different overall affinity for the same antigen. This explanation underlies the differences observed in antisera used for blood grouping. For example, some ABO blood grouping antisera, which perform satisfactorily in the absorptioninhibition method of stain grouping, give poor results in the absorption-elution method. This is probably due to the presence of a high proportion of very avid antibodies (Kind, 1963). Again, for saline agglutination methods it is clear that in many instances the type of antibody required is IgM and that IgG of the same specificity causes interference. For the techniques just quoted it would be of great advantage if it were possible to isolate only the antibody type most suitable for each method. This is equally true for most immunochemical techniques in current use. Affinity chromatography has been used to fractionate antisera with considerable success for this purpose in many areas, but the technique has several limitations which restrict its application. Monoclonal Antibody A more fundamental approach towards obtaining homogeneous antibody has been the selection of individual antibody-synthesizing cells from spleens of immunized animals in a n attempt to isolate individual clones. Unfortunately normal lymphocytes cannot be grown in tissue culture and this fact, until recently, has limited this approach to the research laboratory. However, there has been a recent breakthrough which enables isolated lymphocytes to be propagated (Kohler and Milstein, 1975; 1976; Kohler et al., 1976, 1978; Pontecorvo et al., 1977). This has been achieved by fusing isolated lymphocytes with plasmacytoma cells. Plasmacytomas are tumour cells derived from small lymphocytes (plasma cells) which occur spontaneously in many animals. One of their characteristics is the secretion of incomplete antibody molecules, either light chains or heavy chains, rarely both. Mouse plasmacytomas (MOPC) have been extensively studied and such cell lines have been propagated in the laboratory for many years. When a plasmacytoma cell suspension is incubated with a suspension of normal spleen lymphocytes in medium containing polyethylene glycol a number of fusions of the two cell types occur. I n the resulting 'hybridoma' the chromosomes of the two parent cells undergo rearrangement and some nuclear material is rejected from the cell. Many of the fusions fail to produce viable hybridomas but in a mixture of lo8 MOPC cells and lo7 spleen cells about lo3 stable hybridomas are produced. Purification of hybridomas from the parent cells is achieved by using a n enzyme-deficient mutant of a n MOPC cell line; the deficiency is overcome in hybridomas by the genetic material from the normal lymphocyte. Once the hybridoma population has been isolated the suspension is distri-

buted into a 96-well culture plate and the cells cultured for a few days. T h e supernatants from each well are then tested for antibody content and specificity. The well or wells with the most promising antibody are then distributed into another 96-well culture plate and the same procedure repeated until a single hybridoma clone producing the desired specific antibody is isolated. Once a n individual clone is isolated it can be used for antibody production. The clone may be grown 'in vitro' and the culture supernatant withdrawn at regular intervals. Alternatively it is possible to inject mice subcutaneously with a suspension of the clone. Within 2-3 weeks large solid tumours appear and clonal antibody is present in the serum at levels up to 2mg/ml. At this concentration lml of mouse serum would contain as much specific antibody as several litres of conventional antiserum. The antibody can be separated from other serum components by simple means giving a relatively pure preparation.

Advantages and Disadvantages of MCA One important advantage of this technique is that the cloned hybridoma can be stored indefinitely in liquid nitrogen for future use, whereas the conventional experimental animal is decidedly mortal. Another is that from a single fusion experiment one can select any of a large number of antibody specificities and affinities-so that highly purified antigens are unnecessary. More important, the availability of homogeneous immunoglobulin means that quality control of routine diagnostic immunological reagents now becomes practicable since contaminating specificities and affinity variations are eliminated. Not only will current techniques benefit but it will be possible to produce modified antibody by linking to solid supports, to enzymes, to fluorophores, etc., much more easily and efficiently. All these advantages are purchased at the cost of more work than is needed with conventional antibody production. The most time-consuming stage is the testing of culture supernatants to identify the type of antibody present; and the design of such assay procedures poses questions about the desired specificity and affinity which may, initially, be difficult to answer. Nevertheless the increased production costs are more than offset by the gains in quantity and quality of the product. Applications The MCA technique is only five years old and its application so far has been largely restricted to the area of cellular immunology that gave birth to it. MCAs have been produced against human B-lymphocyte antigens (Trucco et al., 1978), histocompatibility antigens of the mouse (Lemke et al., 1977) and man (Barnstable et al., 1978; Trucco et al., 1978), rat tissue antigens (Williams et al., 1977), human P2-microglobulin (Trucco et a]., 1978), sheep erythrocytes (Kohler and Milstein, 1976), mouse immunoglobulins (Pearson et al., 1977; Galfre et al., 1977), influenza virus (Koprowski et al., 1977), and human group A erythrocytes (Barnstable et al., 1978). Some MCAs to individual HLA-D antigens have been prepared and attempts are being made to produce anti-Rhesus D MCA for prophylaxis of haemolytic disease of the newborn. Quite a number of MCA are now available commercially. Without doubt the use of monoclonal antibody will bring about a revolution in immunological methods of diagnosis and analysis. Some developments in the clinical field may have important applications in forensic work but there are a number of potential applications of purely forensic interest. Some of these are listed below. Routine Blood-group Serology Antiserum supply. Sera in current use could be replaced by a secure supply of one of consistently homogeneous quality. I t is probable that MCA could be

tailored to suit techniques requiring antibody of different characteristics (e.g., low affinity for A/E; high affinity for A/I). MCA might be used in the production of antisera at present difficult or impossible to obtain. Antisera to many Gm and Km markers currently unobtainable could give useful splits (e.g., Gm 17, 21, 23 and Km 3) and antisera to some Gm markers could be useful for racial discrimination. Anti-H MCA is another possibility. New Techniques in Serology HLA typing. The lymphocyte toxicity test for HLA is unsuited to forensic science laboratories and the samples they handle. Anti-HLA MCA should enable a more practical method to be developed. Enzyme and protein polymorphism. An abundant and relatively cheap supply of MCA would enable immunological staining methods for enzyme visualization to be developed. Labelled MCA. The homogeneity of MCA makes it much simpler to derivatize efficiently with radio-isotopes, enzymes and fluorochromes. This is an obvious advantage for future developments in labelled antibody methods such as Enzyme Linked Immunosorbant Assay (ELISA). Immobilized MCA. The use of MCA has considerable potential for the recovery of specific antigens of interest or for the removal of interfering compounds (e.g., Hb) from samples or extracts. Theoretically it should be possible to carry out a large number of analyses on even a small sample in this way. New polymorphisms. The dissection of an immune response by the MCA method has enormous potential for the resolution of antigen mosaics. It is certain that some of the antigens currently regarded as homogeneous will be shown to be subdivisible and this will bring improvements in discriminating power. I t may also be possible by this technique to isolate MCA to new antigens characteristic for race, sex (e.g., Y antigen) or other commonplace characteristics, and for different body secretions. - - ..- - - - - LV

Drug antisera for immunoassay. Required specificities should be easier to achieve by the MCA approach than by conventional methods. Mixed conjugates could be used for injection and the amount ofwork involved in producing anti-drug MCA should be no more than the methods in current use. Immobilized MCA. The homogeneity of MCA will make for more efficient immobilization on solid particles for solid-phase immunoassays. Such immunoassays are easier to automate, an important feature for future developments. Immobilized MCA may bring the extraction of drugs by immunological means within reach. This could be extremely useful in cases with small sample volumes, (e.g. Road Traffic Act (RTA) blood samples for drug analysis).

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