Monoclonal antibodies ICO-20 against human thymocyte antigen T10

Monoclonal antibodies (Mab) Ig G2a isotypes reacting in indirect immunofluorescence assay with 68.7 +/- 4.1% of thymocytes, 7% of T-cells and not determining the antigen on other blood cells were obtained. Mab ICO-20 reacted in complement-dependent cytotoxic test. The antigen was expressed on colony-forming cells of granulocyte-macrophage row. Mab ICO-20 reaction with 100% of thymocytes was defined by flow cytometry. Antigen molecular mass is 45000 Dalton. The antigen was expressed on blast cells of patients with ALL and AML. Mab ICO-20 reaction was more more often with T-cell ALL.     

Panel of monoclonal antibodies to CD38 antigen

The panel of monoclonal antibodies (MCA) ICO-16, ICO-17, ICO-18, ICO-19, ICO-20, ICO-27, ICO-28 IgG2a isotypes to CD38 antigen was obtained. MCA discovered the antigen with 45 kD molecular mass, expressed on the surface of 100% thymocytes, 43-53% lymphocytes, 32-46% monocytes. All obtained MCA blocked the binding each other with thymocyte of man. MCA reacts in complement-dependent cytotoxic test. The antigen CD38 is expressed on blast cells of patients with T-cells subset of ALL.     

ICO-45 monoclonal antibodies to the new epitope of antigen CD38

Monoclonal antibodies (MoAB) ICO-45 discovered the antigen with 45 kD molecular mass, expressed on the surface of 77% thymocytes, 68% monocytes, 95% granulocytes, 46% T-lymphocytes, 59% non-T-lymphocytes. MoAB ICO-45 blocked T-lymphocyte blast transformation and inhibited the respiratory burst of monocytes and granulocytes of the peripheral blood.     

Detection of an antigen, associated with cells during the blast crisis of chronic myeloleukemia, in a cytotoxic test using xenogenic antibodies]

The immune antimyeloblast serum (AMS) was obtained from horses immunized with white blood cells from patients suffering from chronic myeloid leukemia (CML) at the blast crisis stage; the serum was completely absorbed with normal red blood cells and white blood cells (WBC). The absorbed antiserum remained cytotoxic to blast cells from 20 of 42 patients with CML at the blast crisis stage. AMS failed to react with the WBC from patients with CML in its chronic phase, and from patients with other types of leukemia Morphological studies indicated a possibility of identification of the antigen associated with myeloblasts from the blood of patients with CML blast crisis, by means to AMS.     

Cytophotometry of granulocytes in chronic granulocytic leukemia patients. Part I. Cell cycle distribution, S-phase transition and quantitative cytochemistry

In the chronic phase of CGL the proportion of granulocytes in S + G2 was lower (18.7 +/- 1.3% in marrow and 16.7 +/- 2.4% in blood) than in normal bone marrow (42.4 +/- 2.9%) as studied by Feulgen-DNA cytophotometry. During the blast crisis the percentage of S + G2 blasts was 39.3 +/- 8.4 in marrow and 38.7 +/- 7.8 in blood which was much higher than in acute myeloblastic leukemia patients (10.8 +/- 1.4 and 5.1 +/- 1.0). Thymidine labelling index values were lower than the percentage of cytophotometrically detected S-phase cells: up to 28% of cells with Feulgen-DNA content corresponding to S-phase did not incorporate 3H-thymidine. The rate of DNA synthesis remained constant during the S-phase but 3H-thymidine uptake increases towards the end of the S-phase. Morphometric parameters and quantitative cytochemical (PAS, Sudan, myeloperoxidase activity) characteristics of polymorphonuclear neutrophils were altered during the chronic phase of the disease but remained in the normal range during the blast crisis. Mature neutrophils in the blast crisis are assumed to originate from normal granulocyte progenitors.     

Antibody-producing human-human hybridomas. III. Derivation and characterization of two antibodies with specificity for human myeloid cells

Peripheral blood mononuclear cells from a patient with acute myeloid leukemia (AML) and spleen cells from a patient with chronic myeloid leukemia (CML) were fused with HAT-sensitive human B lymphoma cells (RH-L4) in attempts to generate human monoclonal antibodies (Mab) against antigens with high specificity for myeloid leukemia cells. Forty-seven of 246 hybridomas secreted Ig that bound to AML cell surface constituents, as determined by FACS analysis of viable cells that were FITC-stained with the human Mab as the first-step reagent and FITC-conjugated rabbit anti-human Ig as second-step. Two of the 47 human Mab (one from each patient and designated AML-19 and CML-20, respectively) bound to both autologous and allogeneic myeloid leukemia cells. No significant binding was observed to cell surface constituents on human bone marrow cells, granulocytes, lymphocytes, erythrocytes, thymocytes, monocytes, lymphoblastic leukemia cells, fibroblasts, malignant B and T lymphocytic cell lines, and murine bone marrow cells. Both human Mab were IgG and were cytotoxic to myeloid leukemia cells in the presence of complement. About 70% of peripheral blood cell samples from 46 AML patients contained AML-19- and CML-20-positive cells, but the reactivity pattern had no correlation to the morphologic FAB classification of the samples. The promyelocytic HL60 cell line and the K562 cell line reacted with the two antibodies. Dot blot analysis of binding of AML-19 and CML-20 to cellular extracts immobilized on nitrocellulose paper showed that both human Mab in this assay also reacted with normal bone marrow cells. This was supported by microscopic immunofluorescence because both human Mab stained intracytoplasmatic structures in normal bone marrow cells, but both intracytoplasmatic and cell surface components stained in myeloid leukemia cells. Moreover, immunoblotting demonstrated that both human Mab in leukemia cells reacted with two cellular proteins with Mr approximately 14,500 and 18,000, and in normal bone marrow cells with a molecule with Mr approximately 20,000. Immunoprecipitation of cell membrane molecules with both the AML-19 and CML-20 antibody precipitated from leukemic cells only the molecule with Mr approximately 18,000 and no components from normal bone marrow cells. It is concluded that myeloid leukemogenesis may result in generation of cell surface expression of either new or abnormally processed molecules that are immunogenic in the autochthonous host. These molecules may also be useful as markers in diagnosis of myeloid leukemia.     

Monoclonal antibodies to lymphocytes of rainbow trout (Oncorhynchus mykiss)

Monoclonal antibodies (Mabs) to lymphocytes of rainbow trout have been developed by immunisation with synthetic peptides, prepared from selected parts of the alpha- and beta-gene sequences of the T-cell receptor (TCR). Mab 1C2 (TCR beta immunisation) identified lymphocytes in blood (11%), spleen (18%) and in thymus (9%) in flow cytometry analysis (FCM). Immune complexes of lymphocytes coupled to Mab 1C2 was used for further immunisations resulting in numerous supernatants reactive with lymphocytes in FCM, of which Mabs 7A5 and 8H4 were selected for further characterisation. Mab 7A5 identified 31% of lymphocytes in blood and 9% in the spleen. Mab 8H4 labelled 61% and 85% of lymphocytes in the same organs. Mab 8H4 reacted with the majority of the lymphocytes in the thymus (98%). Mabs 1C2, 7A5 and 8H4 recognised surface markers on both Ig(-) and Ig(+) lymphocytes in peripheral blood and in spleen in double staining experiments. An increased proportion of Ig(-) lymphocytes were identified when Ig(+) lymphocytes were eliminated by immunomagnetic separation. No cross-reactivity of Mabs 1C2, 7A5 or 8H4 to anti-thrombocyte Mabs was detected. Mab 1C2 captured molecules of about 40 and also of 55-60kDa, in an immunoprecipitation assay. Mab 7A5 recognised an antigen of approximately 75-80kDa and Mab 8H4 identified proteins of about 70, 100 and 150kDa. Immunohistochemical staining by Mab 8H4 of fixed thymus, revealed a strong labelling of lymphoid cells in the outer zones of thymus. The 8H4 positive lymphoid cells surrounds circular structures, which were not labelled by Mab 8H4. These distinctly appearing structures have a similar shape as nurse cells described in mammals.     

Binding characteristics and affinity labeling of protein constituents of the human IM-9 lymphoblast receptor for substance P

The neuropeptide substance P (SP) stimulates human T-lymphocyte function in vitro. Human blood T-lymphocytes and cultured human IM-9 B-lymphoblasts express 7,000-10,000 and 25,000-30,000 substance P receptors per cell, respectively. The specific binding of 125I-SP is retained in IM-9 lymphoblast membranes solubilized in 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS) at a detergent-to-protein ratio of 1.0. In addition, specific and reversible SP binding to soluble IM-9 cell membrane proteins is demonstrated by gel filtration. The saturation of binding of 125I-SP to both intact and solubilized IM-9 cell membranes attained a steady state after 40-50 min at 4 degrees C. Scatchard analysis of the concentration dependence of 125I-SP binding to IM-9 cell membranes revealed a KD of 0.87 +/- 0.8 nM (mean +/- S.D., n = 4), which is similar to that observed in intact cells, and a density of receptors of 21 +/- 3 fmol/mg of membrane protein (mean +/- S.D.). Binding of 125I-SP to solubilized membranes demonstrated a KD of 0.75 +/- 0.33 nM (mean +/- S.D., n = 3) and a density of receptors of 3.7 +/- 1.5 fmol/mg of membrane protein (mean +/- S.D., n = 3). Affinity cross-linking of 125I-SP by disuccinimidyl suberate to intact IM-9 cells and membranes revealed specifically labeled proteins of Mr 58,000 and 33,000 in cells, and 58,000, 33,000, and 16,000 in membranes by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under both reducing and nonreducing conditions. Competitive effects of substituent peptides of SP on cross-linking and 125I-SP binding to membranes demonstrated that the SP receptor recognized the carboxyl-terminal domain of the peptide. Membranes from cells preincubated in vitro for 12 h at 37 degrees C with 10(-8) M SP demonstrated a decrease in SP receptor density to 13 +/- 2 fmol/mg (mean +/- S.D., n = 2), and a parallel diminution in the specific labeling of membrane proteins of Mr 58,000 and 33,000. These observations suggest that solubilization in CHAPS preserves the binding characteristics of the IM-9 lymphoblast receptor for SP, and that affinity cross-linking techniques identify by sodium dodecyl sulfate-polyacrylamide gel electrophoresis membrane proteins that are specifically labeled by SP.     

Control of culture environment for improved polyethylenimine-mediated transient production of recombinant monoclonal antibodies by CHO cells

In this study we describe optimization of polyethylenimine (PEI)-mediated transient production of recombinant protein by CHO cells by facile manipulation of a chemically defined culture environment to limit accumulation of nonproductive cell biomass, increase the duration of recombinant protein production from transfected plasmid DNA, and increase cell-specific production. The optimal conditions for transient transfection of suspension-adapted CHO cells using branched, 25 kDa PEI as a gene delivery vehicle were experimentally determined by production of secreted alkaline phosphatase reporter in static cultures and recombinant IgG4 monoclonal antibody (Mab) production in agitated shake flask cultures to be a DNA concentration of 1.25 microg 10(6) cells(-1) mL(-1) at a PEI nitrogen:DNA phosphate ratio of 20:1. These conditions represented the optimal compromise between PEI cytotoxicity and product yield with most efficient recombinant DNA utilization. Separately, both addition of recombinant insulin-like growth factor (LR3-IGF) and a reduction in culture temperature to 32 degrees C were found to increase product titer 2- and 3-fold, respectively. However, mild hypothermia and LR3-IGF acted synergistically to increase product titer 11-fold. Although increased product titer in the presence of LR3-IGF alone was solely a consequence of increased culture duration, a reduction in culture temperature post-transfection increased both the integral of viable cell concentration (IVC) and cell-specific Mab production rate. For cultures maintained at 32 degrees C in the presence of LR3-IGF, IVC and qMab were increased 4- and 2.5-fold, respectively. To further increase product yield from transfected DNA, the duration of transgene expression in cell populations maintained at 32 degrees C in the presence of LR3-IGF was doubled by periodic resuspension of transfected cells in fresh media, leading to a 3-fold increase in accumulated Mab titer from approximately 13 to approximately 39 mg L(-1). Under these conditions, Mab glycosylation at Asn297 remained essentially constant and similar to that of the same Mab produced by stably transfected GS-CHO cells. From these data we suggest that the efficiency of transient production processes (protein output per rDNA input) can be significantly improved using a combination of mild hypothermia and growth factor(s) to yield an extended "activated hypothermic synthesis".     

Properties of a panel of monoclonal antibodies which react with the human T cell antigen receptor on the leukemic line HPB-ALL and a subset of normal peripheral blood T lymphocytes

Five Mab raised against the T cell antigen receptor of the human T cell line HPB-ALL which react with a subpopulation of normal peripheral blood T cells are described. Three Mab, 3D6, 1C1, and 1C2, react with 3 to 5% of normal PBL and stimulate proliferation of the cells with which they react. An increase in the number of cells which react with all five Mab occurs. Two Mab, 2D4 and 65, react with subsets of the cells which bind 1C1, 1C2, and 3D6 and divide the family into four subgroups, 2D4+ 65+, 2D4+ 65-, 2D4- 65+, and 2D4- 65-. Functional T cell clones in all four subfamilies have been observed. Cytolytic function can be correlated with the TcR phenotype expressed because all of the Mab which react with a particular clone inhibit its ability to lyse a specific target. The epitopes recognized by the panel are closely related because all five block each other's binding to HPB-ALL. In addition, the determinants recognized by 3D6, 1C1, and 1C2 on normal lymphocytes are probably very closely related because all clones examined react with all three Mab.     

Recombinant human colony stimulating factor-granulocyte/macrophage and -granulocyte, but not macrophage induce the development of a respiratory burst in primary human myeloid leukemic cells in vitro

Respiratory burst develops in myeloid blast cells if they differentiate functionally along the monocytic or granulocytic lineage. Using the nitroblue tetrazolium (NBT) assay we studied the effects of recombinant human granulocyte/macrophage colony stimulating factor (rhuGM-CSF), rhuG-CSF and rhuM-CSF on development of respiratory burst activity in primary blast cells from patients with myeloid leukemia. Assessing suspension cultures containing cells from patients with acute myeloid leukemia (AML, n = 13) or myeloid-blast crisis (myBC) of chronic myeloid leukemia (CML, n = 5) it was found that the percentage of NBT positive cells was increased by at least 20% as compared to control cultures by rhuGM-CSF in 6/17 cases, by rhuG-CSF in 7/17 cases and by rhuM-CSF in 0/16 cases, representing in 'responders' a mean increase of 267% and 270% in the absolute number of NBT positive cells by rhuGM-CSF and rhuG-CSF, respectively. Morphological examination of cultured cells from 'responders', as compared to controls, showed decreased blast cell content but generally no evidence of terminal differentiation. The demonstration of Auer rods in NBT positive cells indicates that respiratory burst developed in a leukemic clone. These findings may be of physiological, pathophysiological and clinical relevance.     

Calibration of fluorescence intensities to quantify antibody binding surface determinants of cell subpopulations by flow cytometry.

Quantitative indirect immunofluorescence analysis by flow cytometry was used to determine the mean number of antibody binding sites per cell in a small subpopulation of rat brain cells expressing low levels of a cell surface differentiation antigen recognized by monoclonal antibody (Mab) RB13-6 (Kindler-R?hrborn et al.: Differentiation 30:53-60, 1985). For these non-disjunct distributions of fluorescence intensities, the cut-off border between antigen-positive and antigen-negative cells was defined by a statistical test. To eliminate the influence of accidental disturbances leading to incorrect statistical decisions, the curves for antigen-negative cells were fitted according to cell number and shape. The flow cytometer was calibrated with the use of a clonal cell line for which the average number of Mab RB13-6 binding sites per cell had previously been determined by radioimmunoassay and Scatchard-plot analysis. Using this analytical procedure, both the proportion of Mab binding brain cells and the mean number of Mab binding sites per Mab binding cell could be determined as a function of developmental stage.     

Production and characterization of a monoclonal antibody specific for the human lymphocyte low affinity receptor for IgE: CD 23 is a low affinity receptor for IgE

In the present study a gamma 1 kappa monoclonal antibody, Mab 25, specific for the receptor for the Fc fragment of IgE on lymphocytes (Fc epsilon RL) was established. This antibody was generated after fusion of spleen cells from mice immunized with the EBV-transformed lymphoblastoid cell line RPMI 8866, which is known to express Fc epsilon RL at high density. Mab 25 inhibits strongly the binding of IgE to RPMI 8866 cells and to other Fc epsilon RL-positive EBV-transformed lymphoblastoid cell lines. A 50% inhibition of IgE binding was observed at a Mab 25 concentration of 10 ng/ml. The binding of IgE was also inhibited by Fab fragments of Mab 25, suggesting that the inhibition is not simply due to steric hindrance or to an eventual binding through its Fc portion. Mab 25 only binds to cell lines expressing Fc epsilon RL. Mab 25 immunoprecipitated a single polypeptide with an apparent m.w. of 42 Kd, pI 4.9. The membrane molecule bound to and eluted from a Mab 25 immunoabsorbent had the same apparent m.w. and pI as the Fc epsilon RL purified from an IgE immunoabsorbent. Additionally, when RPMI 8866 cell lysates were cleared with Mab 25, no Fc epsilon RL could be bound to or eluted from an IgE immunoabsorbent. Mab 25 was found to weakly bind to a minor proportion of blood (1 to 4%), tonsil (2 to 9%) and spleen (4 to 5%) mononuclear cells with a low intensity. By double fluorescence analysis, most of the Fc epsilon RL-positive cells were found to be CD 20-positive B lymphocytes. The staining pattern of Mab 25 and the biochemical characteristics of the antigen detected by Mab 25 were comparable to those of the CD 23 Mab. The four CD 23 Mab MHM 6, PL 13, HD 50, and Tü 1 were found to inhibit the binding of IgE. PL 13 was found to totally inhibit the binding of Mab 25 to RPMI 8866 cells, whereas Tü 1 and MHM 6 only partially inhibited Mab 25 binding. HD 50 was unable to block the binding of Mab 25. The finding that different CD 23/Fc epsilon RL-specific monoclonal antibodies recognizing distinct epitopes have in common the capacity of inhibiting the binding of IgE suggests that upon binding they induce a conformational alteration of the Fc epsilon RL resulting in a loss of the IgE binding capacity. In conclusion, our data demonstrate that the CD 23 antigen is a low affinity receptor for IgE on lymphocytes.     

Evidence for a common leukemia-associated antigen in acute myelogenous leukemia

Summary An antiserum was raised in rabbits to extracts of a pool of acute myelogenous leukemia cells. The immunization protocol used (antibody feedback) gave rise to antisera with marked specificity for AML extracts. After absorption, the antiserum demonstrated essentially no reactivity with cell extracts from 12 individual normal peripheral blood samples, while it reacted positively with 16 individual extracts from AML cells. Reactivity was assayed by the enzyme-linked immunosorbent assay (ELISA). The antiserum was not reactive with extracts from normal PHA-induced blast cells, with extracts of bone marrow cells from six individuals, or with three individual extracts of chronic lymphocytic leukemia (CLL) blast cells. These data indicate that this antiserum is detecting an antigen that is common to AML cells but may not be common to other blast cells.     

A novel cell surface antigen (T305) found in increased frequency on acute leukemia cells and in autoimmune disease states

Monoclonal antibody T305, prepared by immunizing mice with the T-ALL derived cell line RPMI-8402, immunoprecipitates a single chain glycoprotein with m.w. 160,000 daltons (under reducing conditions) or 180,000 daltons (under nonreducing conditions). In immunofluorescence assays, antibody T305 reacted with a subpopulation of T cells in normal blood (22 +/- 6%), thymus (28 +/- 11%), and lymph node (24 +/- 6%). Increased frequency of T cells reactive with antibody T305 was found in peripheral blood of patients with infectious mononucleosis (greater than 80%), graft-vs-host disease after bone marrow transplantation (65 +/- 11%), acquired immunodeficiency syndrome (53 +/- 12%). The T cells in synovial fluid of patients with rheumatoid arthritis had increased frequency of antibody T305 reactive cells (59 +/- 8%) as compared to their peripheral blood (18 +/- 7%). Two color immunofluorescent studies demonstrated that the T305+ T cells predominantly co-stained with antibody Leu 2a (suppressor/cytotoxic subset) in both normals and disease state blood. After cell sorting to obtain T305+ and T305- subpopulations, we demonstrated that a) natural killer and antibody-dependent cellular cytotoxicity activity in normal blood was in the T305+ but not T305- T cells; b) cytotoxic T cells induced by mixed lymphocyte reaction were predominantly T305+; c) T305- T cells could be induced in vitro to express T305 antigen by mitogens or allogeneic B cells; d) the DNA content of T305+ and T305- T cells in normal blood was similar (greater 95% of cells with G0/G1 level); e) after mitogen stimulation, T305 antigen induction on previously T305- cells occurs before S-phase; and f) significantly more [3H]-thymidine after mitogen stimulation was incorporated by originally T305- cells than by originally T305+ cells (p less than 0.001). The T305 antigen was not restricted to T cells because it was also found on myeloid precursors in bone marrow but was not present on polymorphonuclear leukocytes, red blood cells, platelets, muscle, liver, skin, kidney, lung, or brain. Antibody T305 was found on 24/25 cases of acute leukemia (6 T-ALL, 10/11 cALL, 7 AML, and 1 AMOL) but not on 18 cases of chronic leukemia (B-CLL, T-CLL, null CLL, CML). The importance of the T305 antigen is that it is present on a high number of T cells in certain autoimmune diseases and on virtually all acute leukemia cells. Its distribution on immature and in vitro activated cells suggests that it may represent a receptor for signals related to cellular replication or differentiation.     

Isolation and characteristics of IKO-GM-1 monoclonal antibodies

The hybridoma clone IKO-GM-1 was obtained as a result of fusion of P3X63Ag8.653 cells with splenocytes of BALB/c mice with the aid of 50% polyethyleneglycol. Antigen demonstrated by antibodies IKO-GM-1 expressed on 100% polymorphonuclear neutrophils, 54.4 +/- 3.1% monocytes, 25.9 +/- 2.4% mononuclears of healthy subjects' blood, on 11.1 +/- 1.0% T lymphocytes, on 14.6 +/- 1.6% T lymphocytes bearing Fc-receptor for IgG, on 49.3 +/- 8.2% enriched population of B lymphocytes and O cells. The treatment of healthy donors' mononuclears with antibodies IKO-GM-1 and complement blocked EK cellular activity against Molt-2 cells but not against K-562 cells. Antigen demonstrated by MAT IKO-GM-1 did not express on the colony-forming granulocyte or macrophagal cells. Antigen expressed on blast cells of patients with AMonoL, on those in part of patients with AML and AMML, on leukocytes of patients with chronic ML, on monocytes of a patient with chronic MonoL. Antigen was absent from blast cells of patients with ALL, LSA, on lymphocytes of patients with ChLL.     

Core temperature regulation of heart rate during exercise in humans

The relationship between two abnormalities of exercise physiology in chronic heart failure patients was investigated: chronotropic incompetence and decrease in core temperature. While at rest, 13 heart failure patients had an average sinus heart rate that was significantly higher than seven normals (92 +/- 13 vs. 82 +/- 10 min-1, P less than 0.05). However, during exercise, the trend of increase in sinus heart rate as a function of work load and O2 uptake was significantly greater in normals compared with heart failure (P less than 0.05), and the absolute increase in heart rate at 50 W of cycle ergometry was larger in normals compared with heart failure (38 +/- 17 vs. 22 +/- 13 min-1, P less than 0.05). Differences in core temperature regulation were also observed. In the normals, core temperature increased from 37.13 +/- 0.33 degrees C at rest to 37.37 +/- 0.31 degrees C at 50 W of exercise (P less than 0.01). In the heart failure patients, core temperature decreased from 36.99 +/- 0.33 degrees C at rest to 36.66 +/- 0.39 degrees C at 50 W of exercise (P less than 0.01). As expected, significant differences in hemodynamic and gas exchange variables were observed between the normals and the heart failure patients both at rest and during exercise. A multiple linear regression analysis was performed of heart rate changes as the dependent variable and thermoregulatory and hemodynamic changes as the independent variables to test for their influence on heart rate.(ABSTRACT TRUNCATED AT 250 WORDS)     

A unique antigen expressed on myeloid cells and acute leukemia blast cells defined by a monoclonal antibody

A murine hybridoma-derived monoclonal antibody, PM-81, was obtained from a fusion of cells of the NS-1 myeloma cell line with cells from a mouse immunized with the HL-60 promyelocytic leukemia cell line. This cytotoxic IgM monoclonal antibody was specific for myeloid cells. Employing indirect immunofluorescence and flow cytometry, we determined that this antibody reacts strongly with normal human granulocytes, eosinophils, and monocytes but not lymphocytes (including phytohemagglutinin-activated lymphocytes), null cells, red blood cells, or platelets. Moreover, the PM-81 antibody reacts with leukemia cells from 19 of 22 patients with acute myelocytic leukemia of all FAB subclasses, three of three patients with common acute lymphocytic leukemia, four of four patients with chronic myelocytic leukemia (CML) in myeloid blast crisis (terminal transferase (TdT)-negative) but did not react with cells from two patients with CML in lymphoid blast crisis (TdT-positive) or five patients with chronic lymphocytic leukemia. The myeloid cell lines HL-60, K562, KG-1, and U937 were all reactive with PM-81. The lymphoid lines CCRF-CEM and Daudi did not express PM-81 but HSB-2 was positive. The PM-81 antigen was absent on myeloid and erythroid progenitor cells as determined by their insusceptibility to complement-dependent lysis. In addition, only PM-81-unreactive cells were capable of colony formation. Furthermore, the PM-81 antibody does not appear to induce modulation of the antigen to which it binds. Thus, this monoclonal antibody appears to fulfill several criteria for clinical utility in the diagnosis and treatment of both acute myelocytic and acute lymphocytic leukemia.     

Epitope mapping of two major inhalant allergens, Der p I and Der f I, from mites of the genus Dermatophagoides

The repertoire of antigenic sites on two major dust mite allergens, Der p I of Dermatophagoides pteronyssinus and Der f I of D. farinae, was studied using murine (BALB/c) monoclonal antibodies (Mab), polyclonal rabbit IgG antibodies, and human IgE antibodies. Fifty-three IgG Mab were analyzed from six different fusions (five vs Der p I, one vs Der f I). By antigen binding radioimmunoassay (RIA), most Mab were either Der p I or Der f I specific, and only 2/53 bound to both allergens. Epitope mapping studies using cold Mab to inhibit the binding of six 125I labeled Mab to solid phase allergen defined four nonrepeated, nonoverlapping epitopes on Der p I, a single species-specific epitope on Der f I and a cross-reacting epitope present on each allergen. All but one of the 53 Mab bound to one of these six epitopes. Seventy percent (25/35) of anti-Der p I Mab were directed to the same epitope, suggesting that this epitope is immunodominant for BALB/c mice. Similarly, 88% (16/18) of anti-Der f I Mab bound to the same epitope on Der f I. Parallel cross-inhibition curves were obtained using the species-specific Mab, 10B9, and the cross-reacting Mab, 4C1, to compete for binding to Der p I, suggesting that the epitopes defined by these two Mab on Der p I are adjacent to one another. Both murine Mab and polyclonal rabbit IgG antibodies to cross-reacting sites on both allergens were used to inhibit binding of human IgE antibodies to Der p I by using 19 sera from mite allergic patients. Cross-reacting rabbit IgG antibodies strongly inhibited all sera tested (mean 79.5% +/- 7.7) and two Mab, 10B9 and 4C1, partially inhibited (38% +/- 12). However, the four Mab directed against separate species-specific epitopes (including murine immunodominant sites) showed little or no inhibition (less than or equal to 20%). Our results suggest that most of the epitopes defined by Mab are not the same as, or close to, those defined by human IgE antibody. The striking differences in the repertoires of murine IgG and human IgE antibody responses to Der p I and Der f I could be explained by genetic differences or by altered antigen processing and presentation occurring as a result of different modes of immunization in mice and in mite allergic humans.     

Long-term operation of depth filter perfusion systems (DFPS) for monoclonal antibody production using recombinant CHO cells: Effect of temperature,pH, and dissolved oxygen

Recombinant CHO cells of DG44 origin (CS*13-1.00), expressing a chimeric antibody against the S surface antigen of the Hepatitis B virus, were cultivated in single-stage and two-stage depth filter perfusion systems (DFPS) under varying temperature, pH, and oxygen tension conditions to determine their effects on recombinant antibody production. A long-term culture was carried out in a single-stage depth filter for 81 days, during which an occasional clog interrupted the experiment. However, this problem was solved via trypsin injection. The DFPS showed a steady production of monoclonal antibody at a concentration of 100∼150 mg/L. As the cultivation temperature was increased from 33 to 37°C, the monoclonal antibody (Mab) concentration increased from 80.33 to 133.47 mg/L. Likewise, the glucose uptake rate (GUR) and lactate production rate (LPR) also increased. With an increase in pH from 6.95 to 7.61, the Mab concentration increased from 61.64 to 94.31 mg/L. When the oxygen tension was increased from 60 to 80%, the Mab concentration increased from 93.78 to 128.30 mg/L.