Monoclonal antibodies in animal production : their use in diagnostics and passive immunization

One of the landmarks in immunology was the invention and development of monoclonal antibody-secreting hybridomas by Milstein and his coworkers. The enormous promise of monoclonal antibody technology, which became apparent soon after its discovery, may explain the unusual speed with which monoclonal antibodies have been applied to biological and medical sciences.

In animal production monoclonal antibodies are increasingly finding application in the areas of diagnostics, passive immunization and fundamental research. In Chapter 1 of this thesis some applications of monoclonal antibodies within these areas, with emphasis on reproduction, are discussed. It has been concluded that the particular advantages of monoclonal antibodies can firstly and most easily be shown in immunodiagnosis. Once a hybridoma producing a monoclonal antibody appropriate for a particular application has been obtained, large amounts of a homogeneous and reliable reagent are available for as long as they are needed. As ingredients in test kits, monoclonal antibodies have rapidly replaced conventional polyclonal antibodies.

An example of a typical diagnostic test in animal production in which monoclonal antibodies might be used is the milk-progesterone test for confirmation of oestrus and pregnancy diagnosis in cattle. In Chapter 2 the production and characterization of monoclonal antibodies against progesterone have been described in order to standardize an enzyme immunoassay for milk-progesterone. The antibodies differed considerably in their binding affinity for progesterone and showed distinct specificities for a variety of steroids. Results show that, although the technique of monoclonal antibody production selects antibodies specific for a single antigenic determinant, this does not always preclude the possibility of cross-reactivity. Moreover, most antibodies produced have affinities far below the corresponding conventional antisera, as has been discussed in Chapter 1. The monoclonal antibody with the highest association constant and relatively good specificity did not detect progesterone with any greater sensitivity than the conventional polyclonal sera. However, since monoclonal antibodies avoid the dependency upon animals producing high quality antisera and improve test standardization, a commercially available rapid progesterone cow-side test has been designed based on the monoclonal antibody with the best characteristics.

Results of Chapter 2 underline the necessity to evaluate carefully the need of producing monoclonal instead of polyclonal antibodies for a given antigen, as considerable time and effort are required to obtain a monoclonal antibody with suitable properties. Preselection of antibodies on affinity and specificity in an early stage makes the monoclonal antibody technology more efficacious. In our laboratory a cocktail of related steroids was used to enable such an early selection of antibodies against either oestrone or oestrone sulphate. Testing the replacement of oestrone, coupled to horse- radish peroxidase, from the antibodies made it possible to produce high affinity monoclonal antibodies with nearly unique specificities in a relatively short time. Experiments are in progress to develop a test for pregnancy diagnosis in pigs in faecal samples based on those antibodies.

Another application in diagnostics is the use of monoclonal antibodies against a male-specific protein, the H-Y antigen, for sexing bovine embryos before implantation. H-Y is a weak antigen and immunization with H-Y antigen in an inbred strain of mice usually results in production of low titered, low affinity antisera. Because only a low percentage of mice has a good antibody response and their sera run out quickly, monoclonal antibodies were produced (Chapter 3). Male specificity of the antibodies was tested in a variety of assays including enzyme immunoassays based on various sources of soluble H-Y and indirect immunofluorescence assays based on binding of the antibodies to H-Y antigen on the cell surface of male and female cells obtained from a number of tissues and species. Several monoclonal antibodies appeared to be positive in all assays tested, suggesting that the molecule conferring the H-Y antigenicity lacks speciesspecificity and appears to be identical for soluble and membrane-bound H-Y antigen.

The most promising monoclonal antibodies reactive with the H-Y antigen have been evaluated for their efficiency in sexing Day 7 bovine preimplantation embryos (Chapter 4). Although in an indirect immunofluorescence assay a discrimination between male and female embryos could be made, evaluation of the staining patterns was fairly subjective because of non-specific binding of the monoclonal antibodies to the embryonic cells. After modifying the technique in order to reduce non-specific binding, the number of false positives after sexing under these conditions was greatly reduced, which suggests that the monoclonal antibodies detect a male-specific antigen. It was concluded that the occurrence of false-negative embryos might be caused by a weak expression of the H-Y antigen and/or a low affinity of the monoclonal antibody for bovine cell-surface H-Y antigen. The antibodies used in our experiments had been selected on basis of their binding to both soluble and cell-surface H-Y antigen originating from different sources. Currently, monoclonal antibodies which are selected on high affinity for protein structural determinants on bovine cell-surface H-Y antigen are being produced in order to be used in a highly discriminating sensitive fluorescence assay.

In Chapter 1 has been discussed that it will take some time to fully realize the potential of monoclonal antibodies in the area of passive immunization or immunomodulation. The production costs are still relatively high and the reaction of the animal to the injected antibodies may limit the effects of passive immunization. Some such limitations are illustrated in the experiments in which anti- progesterone murine monoclonal antibodies were administered to cyclic pigs (Chapter 5). Intravenous injection of increasing amounts of anti- progesterone antibodies resulted in a concomitant rise in levels of antibody-bound progesterone. At the same time a significant rise in plasma concentrations of total progesterone was observed immediately after administration of higher doses of antibodies. Therefore, the net effect of progesterone binding by the antibody was relatively small and more or less independent of the quantities of antibody administered. It has been suggested that animals maintain adequate levels of free progesterone in their circulation by resorption of progesterone from a pool present in body tissues. The effects of administration of anti-progesterone antibodies on plasma levels of free progesterone are, however, not only influenced by the proposed compensating effect of resorption, but also by the possible initiation of a humoral response of the pigs to the injected antibodies. In the experiments described in Chapter 5 it is shown that a minimum dose of 32 mg anti-progesterone antibody elicited an antimouse response after the first injection, having an neutralizing effect on the anti-progesterone monoclonal antibodies administered with the second injection. When smaller quantities of antibody were used, an anti-mouse reaction was detected after the second or third injection.

From reports concerning the therapeutic use of monoclonal antibodies in man it is known that such an immune response may be directed partially against the isotypic determinants, partially against the idiotypic determinants of the murine antibodies administered. Although anti- idiotypic responses cannot be excluded as a complicating factor, homologous antibodies might offer some advantages over their murine counterparts in terms of effectiveness for passive immunization.

In Chapter 6 the construction of a bovine-murine heteromyeloma cell line to be used for the production of bovine monoclonal antibodies has been described. It was anticipated that a heteromyeloma would retain the superior fusion characteristics of the mouse myeloma cells and, because of the presence of bovine chromosomes, would be better able to support stable bovine antibody production than interspecies hybridomas produced by fusing mouse myeloma cells with bovine lymphocytes. First (bovine-murine) and second generation (bovine-[bovine-murine]) fusion partners were compared for fusion efficiency and the generated number of antigen- specific antibody-producing clones. In addition, the optimal time- interval between boosting and harvesting of the lymphocytes for fusion and the source of lymphocytes was studied. It could be concluded that fusion of bovine lymph node cells with the second generation heteromyelomas on Day 7 after the final booster injection resulted in the largest number of specific antibody-producing clones. Experiments with a third generation bovine-murine heteromyeloma cell line indicated that fusion efficiency could be further improved (unpublished data). Studies with anti-rotavirus and anti-pregnant mare serum gonadotrophin (PMSG) bovine monoclonal antibodies, produced with the second generation fusion partners, indicate that the heteromyeloma cell lines are very useful for the production of bovine monoclonal antibodies.

The availability of such bovine monoclonal antibodies offers the possibility to compare the efficacy of homologous and heterologous antibodies in cattle after repeated passive immunization. The in vivo immunoneutralization of PMSG by murine and bovine monoclonal antibodies was chosen as a model for such a study (Chapter 7). Results indicate that repeated injection of murine monoclonal antibodies against PMSG (mMCA) alone did not, or only to a small degree, elicit an anti-mouse immune response. The simultaneous administration of mMCA and PMSG resulted in relatively high levels of anti-mouse antibodies after the second injection, leading to a decrease in neutralizing activity of mMCA. The results suggest that the neutralizing activity of mMCA is inhibited more by anti- idiotypic than by anti-isotypic antibodies against mMCA. After repeated administration of the bovine monoclonal antibody against PMSG (bMCA), either alone or in combination with PMSG, no anti-bMCA antibodies could be detected. In addition, no change in plasma levels of bMCA and PMSG, compared with levels after the first injection, was observed. Although it has to be confirmed by further experiments whether our findings can be generalized, the present results suggest that for repeated passive immunization in cattle homologous antibodies are to be preferred above heterologous antibodies. As far as we can see now it is necessary to evaluate carefully the need to produce homologous or heterologous antibodies, dependent on the amounts of antibody to be administered and the number of treatments.

In conclusion, the potential of monoclonal antibodies for diagnostic use, therapy or fundamental research, discussed in Chapter 1, together with the results presented in this thesis indicate that the monoclonal antibody technology will have an important impact on the improvement of animal quality and productivity.