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81.
82.
Antisperm antibodies are implicated as one causative factor of infertility, but the target antigens have not been identified. Immune responses to sperm antigens are qualitatively variable even within a single mouse strain. We took advantage of this variability and immunized individual female mice to allogeneic sperm to reflect their natural exposure during mating. We determined the ability of the individual sera to inhibit in vitro fertilization and to bind to sperm antigens separated by electrophoresis. Compared to preimmune sera, four of five immune sera significantly inhibited in vitro fertilization. The serum from individual mice bound variable panels of sperm antigens. By comparing the panels, we identified two polypeptides with molecular weights of 40,000 and 44,000 that were bound by all sera. We propose that these molecules may be good candidates for further investigation of the immunoprophylaxis of pregnancy. 相似文献
83.
Three stable hybridoma cell lines (AF8, BC11, CE2) have been produced that secrete antibodies specific for cathepsin B. These have been characterized by ELISA, SDS-PAGE immunostaining, immunoprecipitation and immunofluorescent staining. CE2 immunoprecipitated native cathepsin B with retention of enzymic activity, but failed to cross-react with the alkali-denatured enzyme. BC11 bound only to the denatured form of cathepsin B and AF8 cross-reacted with both native and denatured cathepsin B. However, unlike CE2-immunoprecipitated enzyme, activity could be detected only after dissociation of the antigen-AF8 antibody complex. No cross reaction was found with any lysosomal protein includihg the cysteine proteinases, catbepsins H and L.Abbreviations ELISA
Enzyme-linked immunoadsorbent assay
- EIP
Enzyme immunoprecipitation
- PAGE
polyacrylamide gel electrophoresis
- Ep-475
L-trans-epoxysuccinyl-leucylamido (-methyl) butane
- Z
benzyloxycarbonyl
- NMec
N-methylcoumarin
- PEB
phosphate-EDTA-Brij 35
- IAA
iodoacetic acid
- PBS
phosphate-buffered saline
- DMEM
Dulbecco's Minimal Essential Medium
- FITC
fluorescein isothiocyanate 相似文献
84.
Summary A vasopressin anti-idiotype antibody was generated by immunization with purified IgG of a primary vasopressin antiserum. The anti-idiotype antibody immunostained neurons in the supraoptic and paraventricular nuclei of the hypothalamus of normal and Brattleboro rats. The distribution of immunostained perikarya in these hypothalamic nuclei together with the staining of fibers in median eminence and neural lobe was similar to that observed in normal rats with anti-vasopressin and suggests strongly that vasopressinergic neurons are being stained. Absorption studies with vasopressin and a vasopressin-binding receptor protein further indicate that a receptor associated with vasopressinergic neurons is recognized by the anti-idiotype antibody.Supported by NIH grants ES03239, NS18626 and NSF grant BNS-8310914. D.T.P. is the receipient of RCDA award NS00869 相似文献
85.
Gabriella Goldberg Daria Mochly-Rosen Sara Fuchs Yoram Lass 《The Journal of membrane biology》1983,76(2):123-128
Summary Monoclonal antibodies directed against the cholinergic binding site of the acetylcholine receptor were found to alter the ion channel properties in cultured chick myoballs. Time and dose dependent reduction in acetylcholine sensitivity was observed. Noise analysis experiments indicated a decrease in the mean single channel conductance and an increase in the mean single channel open time. 相似文献
86.
By coupling Anisakis larval hemoglobin with cyanogen bromide-activated Sepharose, a stable and sensitive antigen for the indirect fluorescent antibody test for anisakiasis was prepared. Using this antigen, cross-reaction with other helminth infections was minimized and the test was greatly simplified.Furthermore, the results obtained indicate that this technique may be useful not only in the sero-diagnosis of anisakiasis but may also be applicable to the diagnosis of other helminthic diseases. 相似文献
87.
Grazyna Adamus Z. Suzanne Zam Scotts Emerson Paul A. Hargrave 《In vitro cellular & developmental biology. Plant》1989,25(12):1141-1146
Summary We present a practical method for the rescue of previosly stable hybridoma clones which increases the proportion of desired
cells in the population before cloning by limiting dilution. When the antibody activity of a culture supernatant was lower
than that previously obtained, a precloning distribution at a density of 10 cells per microtiter well greatly improved the
chances of obtaining a single active clone by subsequent limiting dilution. The Poisson distribution model was used to evaluate
the method. Probabilities calculated clearly demonstrate the advantage of this precloning distribution step when attempting
to isolate a hydridoma cell line that is relatively rare in a population.
This work was supported in part by grants EY 06225 and EY 06226 from the National Eye Institute of the National Institutes
of Health, Bethesda, MD and by an unrestricted departmental award from Research to Prevent Blindness, Inc. 相似文献
88.
Carol L. Williams Vanda A. Lennon Mark R. Pittelkow 《In vitro cellular & developmental biology. Plant》1989,25(5):397-401
Summary A time-dependent redistribution of microfilaments was observed in cultured human keratinocytes using a human monoclonal autoantibody
specific for myosin. Immunofluorescent staining revealed that 5 days after plating keratinocytes in either 0.1 mM or 2.0 mM
Ca++, myosin was distributed uniformly throughout the cytoplasm. At day 6, parallel arrays of myosin-containing microfilaments
were prominent in the cell peripheries. At day 7 the microfilaments formed circumferential rings. The distribution of the
microfilaments was disrupted by cytochalasin but not by colchicine, indicating that this novel distribution of myosin was
not dependent on colchicine-sensitive vimentin intermediate filaments. The time-dependent redistribution of myosin was not
influenced by cell population density, cell shape or cell cycle phase, except for mitotic cells in which myosin was distributed
diffusely through the cytoplasm. If, as suggested by Kolega (9), microfilaments align parallel to the direction of applied
tension, the redistribution of myosin-containing microfilaments in cultured keratinocytes may reflect the increased tension
between cells resulting from increasing strength of cell-cell junctions over time. In sectioned human skin, myosin was localized
in the peripheral cytoplasm of stratified epidermal cells. Tensions arising from the numerous desmosomal junctions between
cellsin vivo could account for this distribution of myosin.
Supported by grant NS-23537 (V. A. L.) from the National Institutes of Health, Bethesda, MD, and by the Mayo Foundation. C.
L. W. is recipient of the Kermit E. Osserman and Blanche McClure Fellowship, 1987, National Myasthenia Gravis Foundation. 相似文献
89.
Alston-Mills Brenda Li Qi Chang Ottinger Mary Ann 《In vitro cellular & developmental biology. Plant》1989,25(10):934-938
Summary Production of antibodies against peptides or poorly antigenic proteins by conventional methods often requires either large
quantities of the native immunogen or some chemical modification to increase their antigenicity. In this study an in vivo
and in vitro immunization protocol has been used to generate monoclonal antibodies against the decapeptide luteinizing hormone-releasing
hormone (LHRH). Two injections of 100 μg of avian LHRH-I into BALB/c mice were given 7 d apart. Dissociated splenocytes were
collected under sterile conditions. They were incubated with 100 μg of the immunogen in 75-cm2 tissue culture flasks in thymocyte-conditioned media. After 5 to 8 d exposure to the antigen, splenocytes were fused with
SP2/O myeloma cells by polyethylene glycol. The cells were plated into 24 wells and then incubated in hypoxanthine aminopterin
and thymidine selective media. After 14 d an initial screening was done by enzyme immunoassay. The positive wells (6/24) were
expanded into 96-well plates and rescreened. Selected lines were cloned out 3 times by limiting dilution and the most positive
expanded for ascites production. The antibody was affinity purified in a protein A column. The antibody cross-reacted with
LHRH-I and II but preferentially to LHRH-I, as shown by competitive assay. A hypothalamic extract from a mature chick showed
a higher response than preparations from whole brain explants of 1- to 3-d posthatched chicks, mature quail, and mature mouse.
This work was funded by the Maryland Agricultural Experiment Station artical no. A4975, contribution no. 8019. 相似文献
90.