Zhu Hui-fen

Studies on monoclonal anti-idiotypic antibodies to human B cell leukemia.

SummaryMurine immunocytoma cell line, NS-1, was fused with spleen cells of Balb/C mice which had been stimulated by tolerogenic disaggregated human gamma globulin and immunized by purified serum IgM from the patient with chronic B cell leukemia (B-CLL). 10 hybridoma cell lines secreting monoclonal anti-idiotype (anti-Id) antibodies to human CLL were obtained. The McAbs were subclasses belonging to IgM and of IgG mouse. Specificity and biologic characters of the monoclonal anti-Id antibodies from culture fluid or ascites were assayed by ELISA, indirect mixed ELISA sandwich, ELISA inhibition, immunofluorescence (IF) and IF inhibition. The study also proved that monoclonal anti-Id antibodies could react with homologous IgM, but not with Ig from normal donors or a panel of patients with myeloma. The results of IF and IF inhibition assay showed that monoclonal anti-Id antibodies were bound to lymphocytes of patient with B-CLL. Their reactivity was inhibited by homologous IgM, but not by lymphocytes of patients with ALL or lymphoma. Monoclonal anti-Id antibodies were heterogenous reactive patterns with cell lines in vitro.

Studies on monoclonal anti-isotypic and anti-idiotypic antibodies against leukemia and myeloma: I. preparation and characteristics of monoclonal anti-isotypic antibodies to igm from B chronic lymphocytic leukemia

SummaryFive hybridomas producing McAbs against human serum IgM-isotype were obtained by fusing the myeloma NS-1 cell and the RALB/C murine spleen cell which had been immunized by serum IgM of patient with B chronic lymphocytic leukemia (B-CLL). These McAbs only reacted with human IgM, not with other immunoglobulins in ELISA and immune double diffusion test. An approximate positive rate of peripheral blood lymphocyte (PBL) was got when these McAbs and the McAbs aginst B cell were tested by indirecl immunofluorescent assay. The positive rate was similar to that obtained by direct immunofluorescent test. Immunoblotting showed that the molecular weight of the antigen to these McAbs was 70–90 Kd, indicating that the antigen was IgM. The practical value of the McAbs against human IgM was also discussed.

Determination of IL-2 and cytotoxicity of killer cells in MTT colorimetry

SummaryThe activity of interleukine 2 (IL-2) in culture supernatants of lymphokine-activated killer (LAK) cells and tumor infiltrating lymphocytes (TIL) as well as cytotoxicity of LAK cells on cultured leukemic cells were determined by MTT colorimetry. The results showed that higher activity of IL-2 in culture supernatant of LAK and TIL cells was found it could be used to support the culture of IL-2 dependent cell lines. The significant cytotoxicity of LAK cells on leukemic cell lines could be found in vitro, and it was consistent with the ratio of effector cells to target cells. The number of living leukemic cells is consistently related with the concentration of formazan metabolite of MTT. It suggested that the numbers of living cells and cytotoxicity of LAK cells could be estimated by determination of formazan metabolite OD value.

Expression of anti-CD4 human/murine chimeric antibody and their killer tumor activity

SummaryFrom the mouse hybridoma cell line secreting an anti-CD4 monoclonal antibody (McAb), total RNA was prepared. The VH and VL genes were amplified by RT-PCR with family specific primer pairs. The PCR products were cloned into pGEM-T vectors, then tranfected into JM109. The VH and VL genes were analyzed by automatic DNA sequencer. According to Kabat classification, the VH and VL genes belong to the mouse Ig heavy subgroup I (A) and k chain subgroup I, respectively. The VH and VL genes were subcloned into pγ1-Expr and pk-Expr respectively, then transfected into XL2-Blue. The VH-pγ1 and VL-pk were transfected by electroporation into mouse myeloma cell X63Ag8. 653. The transfectoma cells were selected by G418 screening, and then supernatant of cultured transfectoma were analyzed by ELISA and immunofluorescence techniques. We have acquired transfectoma cells secreting anti-CD4 chimeric antibodies. These chimeric antibodies are able to kill tumor cells specificallyin vitro.From the mouse hybridoma cell line secreting an anti-CD4 monoclonal antibody (McAb), total RNA was prepared. The VH and VL genes were amplified by RT-PCR with family specific primer pairs. The PCR products were cloned into pGEM-T vectors, then tranfected into JM109. The VH and VL genes were analyzed by automatic DNA sequencer. According to Kabat classification, the VH and VL genes belong to the mouse Ig heavy subgroup II (A) and kappa chain subgroup III, respectively. The VH and VL genes were subcloned into p gamma 1-Expr and p kappa-Expr respectively, then transfected into XL2-Blue. The VH- p gamma 1 and VL- p kappa were transfected by electroporation into mouse myeloma cell X63Ag8. 653. The transfectoma cells were selected by G418 screening, and then supernatant of cultured transfectoma were analyzed by ELISA and immunofluorescence techniques. We have acquired transfectoma cells secreting anti-CD4 chimeric antibodies. These chimeric antibodies are able to kill tumor cells specifically in vitro.

Studies on monoclonal anti-isotypic and anti-idiotypic antibodies against leukemia and myeloma: I. study on monoclonal anti-idiotypic antibodies against λ chain protein of myeloma

SummaryWe used A chain extracted from urine of patient with myeloma (IgD λ) as antigen for immunizing BALB/C mice, and 86 hybridoma cell clones secreting monoclonal antibodies (McAb) were obtained after fusing twice and cloning 3–4 times. 15 of these clones secreted monoclonal anti-idiotypic antibodies (anti-Id McAb). The results showed that 12 of 15 anti-Id antibodies reacted only with homologous λ chain and IgD, not with A chain, κ chain, IgG, IgA, IgM, IgD, IgE, albumin and paraglobulin from normal subjects. Indirect immunofluorescent assay demonstrated that positive reaction rate between anti-Id McAbs and peripheral blood lymphocytes or bone marrow cells of the patient with myeloma was up to 23 %. No reaction was observed between anti-Id McAb and peripheral blood lymphocytes or bone marrow cells from normal subjects. Some of these McAbs presented positive reaction with plasmacytoma cell lines cultured in our laboratory.

Immunomodulation of musk-moxa-string therapy in patients with scrofula.

SummaryThe effects of musk-moxa-string therapy on the immune system in man were investigated in 39 patients with scrofula. Before treatment, the numbers of peripheral blood (PB) CD3+ and DC4+ cells and the ratio CD4+/ CD8+ were found to be lower in patients with scrofula than in normal subjects, while those of B cells and DR+ cells were higher. Response of peripheral blood mononuclear cells (PBMC) to phytohemagglutinin (PHA) diminished in patients with scrofula. At month 2–6 of musk-moxa-string therapy the number of PB CD8+ cells showed slight diminution along with significant increaes in CD3+ and CD4+ cells and CD4+/CD8+ ratio in total lymphocytes (P<0.001). In vitro a marked increased blastogenic response to mitogen stimulation with PHA was observed in PBMC of patients with scrofula after treatment (P<0.001). In contrast, B lymphocytes, monocytes, DR+ cells and blastogenic response to concanavalin A and pokeweed mitogen were not influenced by musk-moxa-string therapy.

Preparation of AHTG-DNR conjugates and their antitumor effect in vitro.

Summary100%, 75%, 50%, 25% and 12.5% oxidized dextran T10 (Dex T1o) were used as intermediate carriers for conjugating drug daunorubicin (DNR) and antibody anti-human thymocitic globulin (AHTG), to form different immunoconjugates, AHTG:Dex:DNR. It was demonstrated that the conjugate with 25% oxidized Dex T10 as intermediate carrier linked more DNR molecules than the others. The degree of its substitution was 10–11 moles of DNR per mole of AHTG. Moreover, because the amount to reducing agent sodium borohydride (NaBH4), required for the reduction reaction, was relatively small, its damaging effect on AHTG and DNR was lessened accordingly.The antitumor effect of AHTG: Dex: DNR in vitro was tested by using 24-h cytotoxicity assay, with CEM as target cell. Cytotoxic effect of the conjugate was proven and the LD56 was 10.68 μg/ml. However, it showed only slight cytotoxic effect on non-target cell K562. When 10 min cytotoxicity assay was performed to show the specific tumor-killing effect of the conjugate, it revealed an obvious cytotoxic activity toward CEM, with the LD50 being 14.79 μg/ml, but hardly toward K562. These results suggest that AHTG:Dex:DNR possesses specific cytotoxic effect.

Cd4+ T cell apoptosis induced by anti-CD4 antibodies

SummaryTo explore the inhibitory effects of anti-CD4 human/murine chimeric antibodies on lymphocyte proliferation, CD4+ T cell apoptosis induced by anti-CD4 antibodies was examined. Annexin-V-FITC and PI double stain method was employed to qualitatively and quantitatively determined CD4+ T cell apoptosis induced by anti-CD4 antibodies. Our results showed that anti-CD4 chimeric antibodies could specifically induce CD4+ T cell apoptosis. The ability of anti-CD4 chimeric antibodies to induce CD4+ T cell apoptosis was related with the presence of monocytes. It is concluded that the further cross-linking of anti-CD4 antibodies is important for inducing CD4+ T cell apoptosis.

Construction and analysis of three-dimensional graphic model of single-chain Fv derived from an anti-human placental acidic isoferritin monoclonal antibody by computer.

SummaryA three-dimensional (3D) graphic model of a single-chain Fv (scFv) which was derived from an anti-human placental acidic isoferritin (PAF) monoclonal antibody (MAb) was constructed by a homologous protein-predicting computer algorithm on Silicon graphic computer station. The structure, surface static electricity and hydrophobicity of scFv were investigated. Computer graphic modelling indicated that all regions of scFv including the linker, variable regions of the heavy (VH) and light (VL) chains were suitable. The VH region and the VL region were involved in composing the “hydrophobic pocket”. The linker was drifted away VH and VL regions. The complementarity determining regions (CDRs) of VH and VL regions surrounded the “hydrophobic pocket”. This study provides a theory basis for improving antibody affinity, investigating antibody structure and analyzing the functions of VH and VL regions in antibody activity.

Anti-proliferative effects induced by anti-CD4 human/murine chimeric antibody and murine anti-CD4 monoclonal antibody

SummaryThe effects of chimeric anti-CD4 human/murine chimeric antibody and murine anti-CD4 monoclonal antibody (McAb) on the proliferation induced by anti-CD3 McAb, phytohemagglutinin (PHA), IL-2, and allogeneic cells were studied. The results showed that chimeric anti-CD4 antibody and murine anti-CD4 McAb could inhibit the proliferation induced by the above inducers and the inhibitory effects were related to the dosage of the antibodies.