Monoclonal antibodies for the in vitro detection of small cell lung cancer metastases in human bone marrow.

Three rat monoclonal antibodies were selected for the immunodetection of small cell lung cancer metastases in bone marrow and other hematologic samples. By membrane immunofluorescence, they define three distinct surface antigens here termed lung cancer-associated antigens or LCAs. The latter are widely expressed on small cell lung cancer and non-small cell lung cancer cells/cell lines, but not detectable on a variety of normal and transformed bone marrow, blood and lymphoid cells. Anti-LCA1 (IgM) is similar to the many anti-lacto-N-fucopentaose III IgM antibodies rasied against human tumors. In contrast, anti-LCA2 (IgG2b) and anti-LCA3 (IgG2a) define surface proteins of 29, 32, 41 and 98 kilodaltons, respectively, that have not been reported earlier. These three reagents have immunodiagnostic potential, since in combination they label all 49 lung cancer cell lines tested. Their ability to detect lung cancer metastases in patient's bone marrow samples is documented in an accompanying paper.

Fc epsilon receptors on rat B-lymphocytes: specificity and binding kinetics.

The binding kinetics of radiolabelled rat IgE to Fc epsilon receptors (R) of rat B-cells have already been studied in IgE-stimulated animals. The receptors expressed after Nippostrongylus brasiliensis infection or 2 injections of 5 mg IgE/100 g body wt were class-specific: IgE binding was not hindered by rat IgM, IgD, IgA, IgG1, IgG2a, IgG2b and IgG2c in immunocompetitive-binding assays. The rat B-cell Fc epsilon R were not species-specific, since mouse IgE competes with rat IgE for binding to these receptors. The apparent number of Fc epsilon R on rat mesenteric lymph node B-cells varied with temp and was 1.1-2.4 X 10(5) at 4 degrees C and 5.9-7.7 X 10(5) at 37 degrees C. The experimental Ka was not influenced by temp and had an average value of 1.38 X 10(8) M-1. At 4 degrees C the IgE binding to B-cell Fc epsilon R had an association rate of 4.9 X 10(3) M-1 sec-1 and a dissociation rate of 4.65 X 10(-5) sec-1. After a very strong stimulation produced by injecting 5 mg IgE/100 g body wt every 24 hr for 5 days, the equilibrium binding curve became diphasic, indicating a probable heterogeneity of the B-cell Fc epsilon R.

Rat AL2, AL3, AL4 and AL5 monoclonal antibodies bind to the common acute lymphoblastic leukaemia antigen (CALLA gp 100).

Four distinct rat monoclonal antibodies against the common ALL antigen (CALLA, gp 100) were obtained in a single fusion. Rat AL2, AL3, AL4, AL5 and the previously reported mouse J5 monoclonal antibodies identified the same subsets of leukaemic cells. AL2 and AL3 reacted weakly with terminal transferase-positive cells in normal bone marrow and foetal liver, as well as with a minority of mature granulocytes in blood. Immunoprecipitation experiments and competitive binding assays demonstrated that the four rat antibodies and J5 bound to the same glycoprotein of approximately 100,000 mol. wt. This set of rat monoclonal antibodies directed against CALLA has not only a diagnostic usefulness but may also be of therapeutic value.

Alteration of membrane barrier in stripped rough microsomes from rat liver on incubation with GTP: its relevance to the stimulation by this nucleotide of the dolichol pathway for protein glycosylation.

The membrane barrier of stripped rough microsomes from rat liver is markedly altered on incubation with GTP at 37 degrees C: after 30 min the structure-linked latency of mannose-6-phosphatase was considerably reduced, and esterase and nucleoside diphosphatase were partly released into the suspension medium. This phenomenon was already maximal with 30 microM GTP and was specific for this nucleotide. Similar conditions enhance the dolichol-mediated glycosylation of protein in microsomes incubated with uridine diphosphate N-acetylglucosamine and guanosine diphosphate mannose (Godelaine, D., H. Beaufay, M. Wibo, and A. Amar-Costesec, 1979, Eur. J. Biochem., 96:17-26; Godelaine, D., H. Beaufay, and M. Wibo, 1979, Eur. J. Biochem., 96:27-34). The GTP-induced permeability and glycosylation activities evolved in parallel in rough microsomes subjected to various treatments to detach the ribosomes and were maximal after removal of congruent to 60% of the RNA. In addition, GTP had no effect of this type in smooth microsome subfractions. Triton X-100, in spite of complex inhibitory effects on glycosylation reactions, mimicked the action of GTP by increasing the amount of microsomal dolichylphosphate that reacts with uridine diphosphate N-acetylglucosamine and by enhancing synthesis of dolichylpyrophosphoryl-chitobiose at concentrations greater than 2 mg/ml. Thus, GTP may activate dolichol-mediated glycosylation reactions in stripped microsomes by lowering the permeability barrier that prevents access of sugar nucleotides to the inner aspect of the membrane. The genuine role of GTP in the functioning of the endoplasmic reticulum membrane in situ remains unknown. Because GTP seems to act only on rough microsomes, we hypothesize that this role is somehow related to biosynthesis of protein by the rough endoplasmic reticulum.

Rat hybridoma technology: culturing of rat myeloma cell line IR983F prior to cell fusion.

The nonsecreting IR983F rat myeloma cell line grew exponentially with a mean generation time of 16 h when reset daily at 5 X 10(5) cells/ml in culture medium with horse serum. High yields of rat-rat hybrids were systematically obtained when fusing exponentially growing IR983F cells with syngeneic immune spleen cells. The yields of hybrids secreting specific antibodies varied greatly according to the immunisation procedure. In the best conditions, 22 hybrids secreting anti-human leukocyte antibodies were recovered per 10(7) plated cells.

Quantitative assay and subcellular distribution of enzymes acting on dolichyl phosphate in rat liver.

To establish on a quantitative basis the subcellular distribution of the enzymes that glycosylate dolichyl phosphate in rat liver, preliminary kinetic studies on the transfer of mannose, glucose, and N-acetylglucosamine-1-phosphate from the respective (14)C- labeled nucleotide sugars to exogenous dolichyl phosphate were conducted in liver microsomes. Mannosyltransferase, glucosyltransferase, and, to a lesser extent, N- acetylglucosamine-phosphotransferase were found to be very unstable at 37 degrees C in the presence of Triton X-100, which was nevertheless required to disperse the membranes and the lipid acceptor in the aqueous reaction medium. The enzymes became fairly stable in the range of 10-17 degrees C and the reactions then proceeded at a constant velocity for at least 15 min. Conditions under which the reaction products are formed in amount proportional to that of microsomes added are described. For N- acetylglucosaminephosphotransferase it was necessary to supplement the incubation medium with microsomal lipids. Subsequently, liver homogenates were fractionated by differential centrifugation, and the microsome fraction, which contained the bulk of the enzymes glycosylating dolichyl phosphate, was analyzed by isopycnic centrifugation in a sucrose gradient without any previous treatment, or after addition of digitonin. The centrifugation behavior of these enzymes was compared to that of a number of reference enzymes for the endoplasmic reticulum, the golgi complex, the plasma membranes, and mitochondria. It was very simily to that of enzymes of the endoplasmic reticulum, especially glucose-6-phosphatase. Subcellular preparations enriched in golgi complex elements, plasma membranes, outer membranes of mitochondira, or mitoplasts showed for the transferases acting on dolichyl phosphate relative activities similar to that of glucose- 6-phosphatase. It is concluded that glycosylations of dolichyl phosphate into mannose, glucose, and N-acetylglucosamine-1-phosphate derivatives is restricted to the endoplasmic reticulum in liver cells, and that the enzymes involved are similarly active in the smooth and in the rough elements.