Biomedical subjects
Publications and source records attributed to U Karsten.
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320 testicular biopsies were evaluated from the years 1974 to 1984 and in (5.9%) cases an orchiopexy in the history of patients were found. The orchiopexy was made in 14 cases on both and in 5 cases on one site. Average age was 8.1 years during the operations and it seems to be very high. The spermiograms of these cases showed severe oligozoospermia and azoospermia. Histological investigation of biopsies demonstrated mild to severe hypospermatogenesis with and without maturation arrest and Sertoli cell and Klinefelter's syndrome. In the testicular biopsy from one patient were found granulomas with foreign material as talc which induced regional fibrosis.
Sera of 100 infertile women and of 100 patients with early pregnancy as control group were investigated for antibodies to zona pellucida by the indirect immunofluorescence. Zonae of oocytes from pigs were used for the procedure. A positive control serum with a high titer was produced by immunization of rabbits with an antigen from pig zonae. The frequency of antibodies to zona pellucida was 29% for infertile patients and 18% in the control group. The titers of these autoantibodies were higher in infertile than in fertile women. There was no increase of these antibodies if the age increased and no relation to the menarche and the ovulation. Antibodies to zona pellucida were demonstrated frequently in patients with disturbances of cycle, free Fallopian tubes, secondary infertility, and ovarian and unknown origin of infertility.
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Hybridomas producing mouse monoclonal antibodies to antigens of the human mammary carcinoma cell line, MCF-7, have been generated by electric field-mediated fusion at a frequency ten times higher than by polyethylene glycol. One of the monoclonal antibodies obtained recognizes a cytoskeletal structure restricted to epithelial cells and carcinomas with a distribution pattern resembling cytokeratin 19.
In experiments aimed at generating monoclonal antibodies against tumour associated antigens, mice are usually immunized with cancer cells (or membrane fractions prepared from it) obtained from surgery or cultured in vitro. In both situations there is the danger of introducing mycoplasmas into the hybridoma cultures even if the myeloma cell line and the mice used for immunization or feeder cell preparation are not infected. Mycoplasmas kill hybridoma cells during HAT selection because they excessively degrade thymidine. The use of azaserine instead of aminopterin in hybrid selection circumvents the necessity for thymidine and therefore may allow survival and growth of hybridomas in the presence of mycoplasmas.
A solid phase radioimmunoassay was developed for the epitope analysis of human alpha-fetoprotein (AFP) using hybridoma culture fluids. The following incubation sequence was performed: anti-mouse Ig, hybridoma culture fluid, normal mouse serum, and a mixture of 125I-labeled AFP and hybridoma culture fluid. Seven epitopes were detected by monoclonal antibodies produced by 15 presumably independent hybridoma clones.
Conventional antisera used as a positive control in antibody tests during hybridoma screening and processing can be replaced to advantage by supernatants of short term spleen cell cultures. Such cultures can easily be initiated from the same spleen cell suspension which is prepared in connection with a fusion experiment. Either supernatants of corresponding cultures from nonimmune animals or medium alone can serve as a negative control. In vitro produced polyclonal standards show very low background binding compared to antisera raised in vivo. They are convenient "ready-to-use" reagents with an antibody titer comparable to that expected in hybridoma culture fluids. There is no need for immunization and bleeding of separate animals. In addition, these supernatants provide an internal control of the immunization efficiency.
A two-site binding enzyme immunoassay for the detection of alphafetoprotein (AFP) was developed by using either a combination of two monoclonal antibodies or of one monoclonal antibody and polyclonal antibodies. The conjugation of the monoclonal antibodies to peroxidase by the periodate method yielded a somewhat higher sensitivity in the enzyme immunoassay (EIA), when compared to conjugates produced by the glutaraldehyde method. The detection limit was 10 micrograms AFP per litre when using only monoclonal antibodies in the assay. Simultaneous incubation of the sample and the monoclonal labelled antibody should be avoided unless using at least two different dilutions of the sample for investigation.
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Monoclonal antibodies specific for human alpha-fetoprotein (AFP) were produced by the hybridoma technique. By using solid-phase immunofluorescence and radioimmunoassays, the antibodies were proven to distinguish 4 different epitopes on the AFP molecule.
A solid phase radioimmunoassay using the incubation sequence: mouse immunoglobulin (Ig) rabbit anti-mouse Ig, monoclonal antibody and 125I-labeled antigen was performed to determine the class and subclass of seven murine monoclonal antibodies against human alpha-fetoprotein. All antibodies belonged to the IgG class. The subclass type was IgG 2a for four antibodies, IgG 1 for two antibodies and IgG 2b for one antibody.
Mouse monoclonal antibodies against the human mammary carcinoma cell line, MCF-7, were produced and tested against a panel of cell lines. They recognize a variety of intra- and extracellular antigens. Four of them did not react with the human rhabdomyosarcoma cell line A-204 used as a discriminating second target during initial screening. One of these, 45-B/B3, is specific for epithelia, and probably recognizes a cytokeratin. This antibody clearly differentiates carcinomas from sarcomas and lymphomas, and epithelial cells in culture from fibroblasts. It is not species-restricted.
In addition to type A virus particles which are common in Balb/c derived mineral oil induced plasmacytomas, type C virus particles have been found in the plasmacytoma cell lines X63-Ag8 and X63-Ag8.653 used in the hybridoma technique. In hybridomas obtained after fusion of X63-Ag8.653 cells with Balb/c spleen cells, C particles were expressed in 5 out of 13 different clones examined, whereas A particles were always present. Virus expression and antibody production do not exclude each other.
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A solid-phase immunofluorescence assay (SIFA) is presented which allows the demonstration of antibodies against soluble antigens. The test is performed by successively incubating nitrate cellulose discs in microtitration plates with anti-Ig, the sample to be tested and FITC-labeled antigen. The fluorescence intensity of the discs is then measured by using a microfluorometer. The assay is simple, needs a small amount of material and allows to test a large number of samples.
By using FITC- and 125I-labeled human serum albumin (HSA) the reactivity of anti-HSA hybridoma antibodies was compared in solid-phase immunofluorescence assay (SIFA) and radioimmunoassay (SRIA). A considerable number of hybridoma antibodies had been detected which are positive in SIFA but not in SRIA. Since this cannot be explained by a difference in the sensitivity of the assays it seems likely that these monoclonal antibodies detect antigenic determinants which contain tyrosine as an essential part.
The superhelical properties of nuclear DNA from malignant cells of human origin (leukemic cell lines Reh and D-562, mesothelioma cell line, and peripheral blood monocytes from acute monocytic leukemia) were investigated in neutral sucrose gradients with ethidium bromide. As compared with the universal superhelical density of nuclear DNA from normal cells, the DNA released from malignant cells showed a substantially higher negative superhelix content, equivalent to an increased deficiency in right-handed DNA duplex turns. In regard to analogous results obtained from other malignant cell systems, the extremely underwound structural state of nuclear DNA appears to be a feature common to malignant cells.