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The use of monoclonal antibodies for therapeutic purposes and diagnostic imaging has become an important pillar of internal medicine in recent years. Currently, the application of antibodies against cell surface receptors and cytokines makes specific therapies possible, in particular against chronic and malignant diseases. The production of antibody preparations increasingly involves gene and biotechnological processes while continuing to adhere to the basic principle of specific antigen-antibody binding. Despite the complex antigen structure of antibodies, severe treatment side effects remain the exception. This is all the more remarkable considering the increased use of human or humanized and functionally optimized antibodies. In the coming years, numerous new monoclonal antibodies will extend the therapeutic and diagnostic possibilities for diverse clinical applications.

The use of culture supernatant (added in a final concentration of 25% v/v) from chicken embryonal fibroblasts (CEF) for stimulation of established hybridoma cell lines has been shown. The proliferative effect on hybridoma cells from different origins was heatproof and was possible with a reduced serum part (FCS) in the culture medium. After a chromatografic separation the supernatant sections were tested and the positive effect was much lower in comparison to the total conditioned media.

Neutralizing and non-neutralizing monoclonal antibodies against parapoxviruses (PPV) were generated by immunizing BALB/c-mice with gradient-purified PPV Orf D-1701 or purified envelopes. Epitope specificity studies identified three distinct epitopes localized in the virus envelope. These antigenic sites allowed a differentiation between orf and stomatitis papulosa viruses. For a rapid diagnosis of parapoxviruses transmission-electron microscopy, immunofluorescence- or immunoperoxidase-staining, antigen capture ELISA, and polymerase-chain-reaction were used.

A simple procedure for the production of pure monoclonal antibodies (mab) in dialysis tubing has been used. Hybridomas which produce pancreatic islet cell reactive monoclonal antibodies were grown in dialysis tubing containing serum free medium. The dialysis tubing was inserted into a flask with medium containing 7.5% foetal calf serum. The flask was placed on a roller and medium was changed every two days. The optimal time for harvesting the mab could be shown to be after 10 days of culture with a 4 fold increased immunoglobulin concentration in comparison to a conventional hybridoma culture. Immunoglobulin concentrations up to 110 mg/l and cell yields of 1.3 x 10(6)/ml have been obtained. The low concentration of contaminating low molecular weight proteins in the supernatant facilitated or saved purification of mab.

Murine monoclonal antibodies (mabs) are produced in either mouse ascites or bioreactors (spinner culture, stirred-tank reactor, airlift reactor, hollow-fiber reactor). Human mabs are produced solely in bioreactors. Encapsulation represents a special technology. Hybridoma cells have to be adapted prior to growth in bioreactors. Of crucial importance is the construction of over-producing cell lines by cell- and gene-technological methods. Manipulated cell lines often produce modified mabs.

Human lymphocytes were derived from the spleen of a 15-year-old female patient after partial splenectomy. High cell yield and percentage of human B-lymphocytes (over 30%) gave useful conditions for their fusion to mouse myeloma cells (P3 X63/Ag8/653). Fusion efficiency (cultures with growth) was 96%. From 288 initially seeded cultures 176 showed IgM and only one IgG production as determined by enzyme immunoassays. Twenty primary cell lines were cloned with differing results and 17 heterohybridoma cell clones with high IgM production rates could be established for more than 6 months in vitro. The present results show the possibility to produce human monoclonal antibodies in heterohybrids derived from human spleen lymphocytes.

The aim of this work was to optimize the antigen-specific prestimulation of human lymphocytes for further hybridization to produce human monoclonal antibodies. In vitro stimulation of human peripheral blood lymphocytes with purified Tetanus Toxoid (TT) resulted both in an increased amount of secreted specific antibodies and in an enhancement of the portion of anti-TT antibodies in the level of total immunoglobulin secretion. Both IgG and IgM antibodies were found. Donors who had not been boostered in vivo with TT for more than 10 years also showed an antibody response to the antigen in vitro. Five days after in vivo boostering we found in lymphocyte cultures derived from 3 donors an enhanced antibody synthesis in the response to TT and also an increased portion of specific antibodies in the spontaneously secreted immunoglobulin in unstimulated control cultures.

Hybridoma cells injected intraperitoneally into mice induce formation of ascites tumors producing ascites fluid with high levels of monoclonal antibodies. Several parameters affect the growth of the immunoglobulin-producing tumors in vivo. In the present study the average ascites tumor formation rate of 10 different hybridomas could be increased from 32% (n = 338 mice) to 77% (n = 112 mice) by only one whole body irradiation of paraffin-pretreated-Balb/c mice. Production of monoclonal antibodies was better in males because significantly (p less than 0.01) increased volume of ascites fluid. From the increased tumor formation rate in irradiated mice it is suggested that in non-irradiated recipients the tumor growth rate was lowered by immunological reactions against hybridoma cells provoked by cell surface neoantigens revealed by cell fusion and/or tumor-associated antigens of the myeloma parent cells as well as by altered antigen pattern caused by possible mutations in the myeloma cell line and/or Balb/c/K strain.

The diagnosis and classification of leukaemic diseases is still primarily based on morphological and cytochemical criteria. Interpretational difficulties occur quite frequently, especially in acute leukaemias. Subjective morphological cell-type characterization--which is acquired only after many years of experience--is rather unsatisfactory in the long run. Therefore in order to obtain uniform results in all haematological areas, new methods are needed. In principle, immunological cell-type characterization represents an alternative method. By applying requisite antibodies practically any cell component can be demonstrated and quantitated. Up to recently the major obstacle to a more rapid development of immunological cell typing was the lack of specific antisera, but with the introduction of hybridoma technology it became possible to obtain monoclonal antibodies and thus, markedly improve immunological cell typing virtually overnight. We have, therefore, used this new technology for the production of monoclonal antibodies to human leucocyte antigens. This paper describes six of the cell-type specific monoclonal antibodies obtained, their suitability for diagnostic purposes and the classification of human leukaemias. With the help of these antibodies the majority of human leukaemias can now be typed. Without claiming completeness and in full awareness of the fact that a number of problems remain to be solved, we nevertheless believe that the presented data point to the possibilities opened up by this technology for leukaemia diagnosis in the future.

Hybridomas were produced which secrete antibodies against cell surface antigens of neoplastic bovine lymphocytes. The monoclonal antibodies of three hybridomas react only with leukemic cells but not with other tested cellular and soluble antigens. Four hybridomas secrete antibodies which probably recognize virus-encoded structures on outer membranes of leukemic cells.

Rare plasmocytomas without paraprotein (frequency less than 1%) can present a diagnostic trap. Three observations give grounds for drawing attention to misinterpretation of symptoms and the problematic nature of the diagnosis. In one patient, a monoclonal immunoglobulin (kappa light-chains) was detectable in the bone-marrow myeloma cells (non-excretory plasmacytoma). In a further patient no paraprotein could be found in the myeloma cells (non-secretory plasmacytoma) despite repeated investigations. An additional unusual feature in a third patient was the simultaneous presence of chronic lymphocytic leukaemia; his plasmocytoma was first detected at autopsy.

Working on the isolation of allergens from crude pollen extracts we were looking for a simple method to produce monoclonal antibodies (mab) specific for a major allergen of cultivated rye (S. cereale) with an isoelectric point (pl) of 5.9. We used immunization with allergen isolated from nitrocellulose blots. Allergen was obtained by isoelectric focusing (IEF) of a rye pollen extract followed by immunoprint and detection of allergen bound to nitrocellulose by a monoclonal antibody (mab). Fusion with myeloma cell line P3X63Ag8.U1 led to production of 7 different mab specific for the protein with pl 5.9. The method seems to be a simple way to produce mab specific for a certain component of a crude protein mixture as a pollen extract.

Monoclonal antibodies are useful tools for characterizing components causing type I allergy. Three aspects in production and characterization of monoclonal antibodies are discussed: 1. production of monoclonal antibodies raised against a special allergen of rye pollen extract. 2. investigation of monoclonal antibodies raised against peptide fragments of ovalbumin, 3. analysis of epitopes on allergenic components of timothy pollen extracts. The results indicate that monoclonal antibodies raised against certain allergens are useful for isolation of allergenic components and epitope mapping.

Hybridomas that secrete monoclonal antibody to Toxoplasma gondii have been developed. Two groups of 10 female BALB/c mice each were immunized either over a shorter (71 d) or longer (117 d) period at first with Toxoplasma lysate antigen and afterwards with intact tachyzoites of the RH strain. Higher titres of antibody were obtained with the long-period immunization. The fusion experiments have shown that both schemes of immunization approximately result in the same number (16 and 14% respectively) of hybridoma cell lines producing antigen-specific monoclonal antibodies. Hybridoma cultures secreting antitoxoplasma monoclonal antibodies were screened parallel by indirect immunofluorescence antibody test (IFAT) and enzyme immunoassay (EIA). 16 of the hybridoma cell cultures produced positive results only in the IFAT, 112 reacted only in the EIA and 21 were positive in both tests. The monoclonal antibodies 5B10, 5G6 and 1B2, which are positive in the IFAT form a chemical compound with the major antigens on the surface of RH tachyzoites. The patterns of fluorescence produced by these monoclonal antibodies are in conformity with those produced by using polyclonal sera of Toxoplasma gondii infected hosts (mouse, rabbit, man).