摘要:

Different monoclonal antibodies detecting the leucocyte common antigen have been obtained, for CD45 (GRT2,GRT3 and GRT4) and for CD45R (GRT22). Several epitopes have been defined with these monoclonal antibodies (MAbs).We have performed a comparative study with CD45 and CD45R MAbs on NK and T cell proliferation.Common epitopes of CD45 antigen were found to be involved in blocking of NK activity but not CD45R restricted determinants. In the cell proliferation assays, fresh human peripheral blood mononuclear cells were stimulated with PHA and soluble CD3 MAbs. CD45 and CD45R MAbs failed to demonstrate the capacity to modify the mitogenic response when optimal and suboptimal doses of PHA were used. In contrast, both (CD45 and CD45R) MAbs led to a significant rise in anti CD3 response. A CD18 (GRF1) was used as control and inhibited both PHA and CD3 T cell proliferation as well as NK activity.We think these results can be explained on the basis of different activation pathways (PHA versus anti CD3) and the accessory signals induced by these MAbs as recorded only in anti CD3 induced mitogenesis.

ISSN:0272-457X    

DOI:10.1089/hyb.1989.8.1

年代:1989

数据来源: MAL

摘要:

Biochemical and functional aspects as well as features of cellular distribution of the differentiation groups CD11a and CD18 were studied in the course of a detailed characterization of two new monoclonal antibodies which recognize the α (GRS3) and β (GRF1) chains of the LFA-1 antigen. Both MAbs inhibited homotypic aggregation of an EBV cell line. In contrast, only GRF1 (anti-β chain) was able to inhibit granulocyte aggregation as well.Different myeloid-monocyte antigen modulation was noted in PMA induced macrophage differention of the U937 and HL60 cell lines. PMA treated HL60 cells showed increased expression of αM (CD11b) and αX (CD11c) antigens but no change in HLA-DR or CD14 antigen expression. No variation in the expression of LFA complex antigen (CD11a,b and c,or CD18) was observed on U937 cells, which on the other hand presented de novo expression of HLA-DR and CD14 anti

ISSN:0272-457X    

DOI:10.1089/hyb.1989.8.13

年代:1989

数据来源: MAL

摘要:

We have recently identified a family of suppressor factors produced by certain human T-T cell hybridomas that we developed (references 1 and 2) and by the Jurkat T cell line. These suppressor factors significantly inhibited proliferative responses to mitogens and allogeneic cells in mixed lymphocyte culture and antibody production by human peripheral blood mononuclear cells. We investigated and report here the effect of these suppressor factors on certainin vitromurine immune responses. Suppressor factors produced by certain of these hybrids, such as 153, 160, 170 and by the Jurkat T-cell line were able to inhibit: (1) proliferative responses to mitogens of mouse thymocytes and splenocytes; (2) proliferative responses of mouse splenocytes to allogeneic cells in mixed lymphocyte cultures; (3) primaryin vitroantibody responses of mouse spleen lymphocytes to sheep erythrocytes; (4) primaryin vitroantibody responses of mouse spleen lymphocytes to a T-cell independent antigen (TNP-Ficoll). Inhibition of murine immune responsesin vitroby these suppressor factors was regular and reproducible and it was observed in a large number of experiments. In contrast, suppressor factors produced by the 169 and by the 77(38F3) hybrids did not suppress the murine immune responses. The basis for these differences are not known at the present. The ability of human suppressor factors to inhibit effectively mouse immune responses provides an additional opportunity for the characterization of the properties of these factorsin vivousing mouse models of human disease.

ISSN:0272-457X    

DOI:10.1089/hyb.1989.8.25

年代:1989

数据来源: MAL

摘要:

Six monoclonal antibodies (MAbs) to pertussis toxin (PT) have been generated and characterized. Five of these MAbs (3CX4, 3C4D, 6D11C, 6FX1, and X2X5) interact with determinants on the catalytic subunit (S1) of PT, and one (6DX3) is specific for subunit S4. The MAbs are divided into three groups based upon their ability to neutralize the effects of PT in a Chinese hamster ovary (CHO) cell assay. Three of the MAbs (3CX4, 3C4D and 6D11C) had high neutralization titers, one MAb (6FX1) displayed weak neutralizing activity, and two MAbs (X2X5 and 6DX3) had no neutralizing ability. The combination of one of the high titer MAbs (3CX4) with the low titer MAb (6FX1) resulted in a synergistic enhancement of neutralizing capability. F(ab')2 fragments prepared from MAb's 3CX4 and X2X5 displayed activities in the CHO-cell assay which were identical to the native MAb's. The ability of the MAbs to neutralize PT in the CHO-cell toxin neutralization assay correlated with their ability to inhibit thein vitroADP-ribosylation of PT. A competition ELISA method demonstrated that this panel of MAbs recognizes at least four separate epitopes on the PT molecule. Biotin-conjugated MAbs were shown to be useful reagents to probe the interaction of pertussis toxin with fetuin.

ISSN:0272-457X    

DOI:10.1089/hyb.1989.8.37

年代:1989

数据来源: MAL

摘要:

A hybridoma cell line that secretes monoclonal antibody, MAb-ER-Br-1-15-4-18 is established. The MAb is highly specific for estrogen receptor (ER) from human breast tumor cells. In order to raise the antibody, the ER was first isolated from human breast tumor. Mice were immunized with the partially purified ER and the fusion of the spleen cells from the mouse, showing the highest serum titer, with the cells of the NS-1 mouse myeloma line, produced hybrid cells which continuously secreted antibodies specific for ER. Three of the hybridoma cultures which tested strongly positive were cloned using limiting dilution method and one of the cell lines was selected for further study. The recovery of the MAb from the cell culture was done by ammonium sulfate precipitation followed by dialysis and then hydroxylapatite liquid chromatography using linear gradients. The purity of the antibody was checked by polyacrylamide gel electrophoresis. The MAb was isotyped and found to be IgG1. When checked against other antigens, the MAb showed a minimal crossreactivity to ER from rabbit uterus and none to ovalbumin or rat liver ferritin. Further experiments showed that the MAb recognized the ER bound to the hormone and ER in the nucleus of breast tumor cells.

ISSN:0272-457X    

DOI:10.1089/hyb.1989.8.53

年代:1989

数据来源: MAL

摘要:

T23 are hybrids derived from the fusion between an IL-2-dependent mouse cell line, C10 and the rat lymphoma C58NTD. Supernatants from exponentially growing T23 cells induce the growth of CTLL2, an IL-2-dependent cell line, suggesting that these hybrids secrete interleukin 2. Addition of recombinant IL-2 to slowly growing T23 cells increases the rate of growth. Using an125I IL-2 binding assay, a low number of cell surface IL-2 receptors were detected. T23 hybrids contain mouse but not rat IL-2 receptor genes as revealed by Southern blot analysis. These receptors are functional because the growth of exponentially growing hybrids is inhibited by an anti-mouse IL-2 receptor antibody. These data suggest an autocrine-like mechanism as responsible for the growth of these T cell hybridomas.

ISSN:0272-457X    

DOI:10.1089/hyb.1989.8.61

年代:1989

数据来源: MAL

摘要:

A hybrid hybridoma cell line secreting a bispecific monoclonal antibody (MAb) was constructed by fusing horseradish peroxidase (HRPO)-immunized mouse splenocytes with previously established mouse hybridomas secreting anti-human lymphotoxin (hLT). This cell line was grown in ascitic fluid in mice to obtain large quantities of hybrid M Abs and a bispecific antibody, reacting with both HRPO and hLT, was separated from the monospecific antibody or other inactive immunoglobulin populations by hydroxylapatite chromatography. Sodium dodecylsulfate-polyacrylamide gel electrophoresis demonstrated that the bispecific antibody molecule contained two different types of heavy and light chains of both anti-HRPO and anti-hLT origin. The bispecific antibody was used to establish one-step enzyme immunoassays (EIAs) employing competitive and sandwich systems. The simple sandwich EIA was able to detect 1-100 U/ml of hLT and there was good correlation (r=0.96) between hLT concentrations measured by the one-step EIA and a bioassay using L929 cell-lysis.

ISSN:0272-457X    

DOI:10.1089/hyb.1989.8.73

年代:1989

数据来源: MAL

摘要:

Purification of murine IgG monoclonal antibodies from ascitic fluid by precipitation with caprylic acid was compared to that: 1) by sequential precipitation with caprylic acid and (NH4)2SO4; 2) by affinity chromatography on either antiidiotypic monoclonal antibodies or antimouse IgG xenoantibodies; and 3) by sequential (NH4)2SO4precipitation, ion exchange chromatography and high pressure liquid chromatography (referred to as HPLC). In terms of yield of antibodies, precipitation with caprylic acid is comparable to sequential precipitation with caprylic acid and (NH4)2SO4, but superior to affinity chromatography on antibodies and to HPLC. In terms of purity of antibodies, precipitation with caprylic acid is less efficient than the other three methods. It should also be noted that precipitation with caprylic acid is associated with a reduction in the affinity of some antibodies and is not suitable to purify murine IgA and IgG3.

ISSN:0272-457X    

DOI:10.1089/hyb.1989.8.85

年代:1989

数据来源: MAL

摘要:

An IgG human-human monoclonal hybridoma antibody, AML-19, reactive with human myeloid cells of non-malignant and malignant origin has been produced by fusion of blood mononuclear cells from a patient with acute myeloid leukemia (AML) and the human B-lymphoma cell line RH-L4. The monoclonal antibody (MAb) AML-19 was purified from hybridoma supernatant by primarily anion-exchange chromatography, in order to separate the AML-19 MAb from contaminating immunoglobulin (Ig), e.g. bovine Ig and MAb derived from the parental fusion partner, and followed by immunoaffinity chromatography. This purification method gave the highest yield and purity of the AML-19 MAb. The isoelectric point (pI) of the MAb was estimated to be 5. Inhibition assays indicate an apparent dissociation constant (Kd) corresponding to 4 x 10-9M and an affinity constant (Ka) to 2.5 x 108M-1to K562 erythroleukemia cells. Scatchard plot demonstrated a linear slope as a manifestation of monoclonality and a low number of AML-19 specific epitopes, estimated to 1500 per cell.

ISSN:0272-457X    

DOI:10.1089/hyb.1989.8.97

年代:1989

数据来源: MAL

摘要:

A variety of modified electrofusion protocols designed to improve the efficiency of hybridoma production have recently appeared in the literature. We undertook to maximize the number of antibody secreting murine hybridomas by optimizing the temperature and fusion strength parameters of the conventional electrofusion technique. Anti-DNP secreting hybridomas were generated by fusing SP2/0 to immunized mouse splenic lymphocytes using an unmodified electrofusion protocol consisting of washing in a weakly conducting sorbitol fusion medium supplemented with bovine serum albumin, calcium and magnesium ions. This was followed by dielectrophoretic alignment and application of 3 short duration, high intensity field pulses in helical chambers. Optimal efficiencies of hybridomas were generated by the application of 2000 V/cm pulses at 25° c (2.45 hybridomas x 10-4splenocytes) and as many as 63% of resulting hybridomas secreted anti-DNP monoclonal antibodies, the majority of which were IgG's. These data show that modification of the electrofusion protocol by pretreatment of fusion partners with proteolytic enzymes or the use of antigen bridging is not required for the successful and efficient production of specific monoclonal antibodies by electrofusion

ISSN:0272-457X    

DOI:10.1089/hyb.1989.8.107

年代:1989

数据来源: MAL