Production of anti-CD14 monoclonal antibodies using CD14 expressing COS cells as immunizing antigen.

CD14 is a leukocyte surface molecule expressed on monocytes but not on lymphocytes. Recently, CD14 molecule was demonstrated to function as a receptor for endotoxin. CD14 specific monoclonal antibody (MAb), therefore, can be used to identify monocytes and study the host defense mechanism to bacterial endotoxin. To produce MAb against CD14 protein, in this study cDNA encoding CD14 protein and COS cell expression systems were used to prepare CD14 expressing COS cells. The CD14 transfectants were then used as antigen for mouse immunization. The spleen cells of the immunized mouse were then fused with myeloma cells by conventional hybridoma technique. By using this strategy, 5 hybridroma clones secreting antibody specific for CD14 molecule were generated within one fusion. The generated CD14 MAbs were strongly positive with monocytes, weakly positive with neutrophils but negative with lymphocytes. In addition, the generated CD14 MAb blocked the binding of lipopolysaccharide (LPS) to the CD14 molecules. These CD14 MAbs could be used to enumerate peripheral blood monocytes as well as using referent CD14 MAb. We, therefore, introduce an alternative method for preparation of antigen for production of monoclonal antibody. This type of antigen is a very effective antigen for the production of monoclonal antibodies against cell surface molecules.

Production of monoclonal antibody to CD4 antigen and development of reagent for CD4+ lymphocyte enumeration.

A hybridoma secreting monoclonal antibody (mAb) specific to CD4 protein was generated. This monoclonal antibody, named MT4, was proved to be specific to CD4 protein as it reacted with CD4-DNA transfected COS cells, CD4+ cell lines and CD4+ lymphocytes. Furthermore, MT4 mAb inhibited the binding of standard CD4 monoclonal antibodies to CD4 proteins on CD4+ cells. To develop a home made reagent for CD4+ lymphocyte determination by flow cytometry, fluorescein isothiocyanate (FITC) was conjugated to MT4 mAb. To evaluate the developed reagent, 30 HIV infected and 30 healthy individuals were determined for CD4+ lymphocytes by using both a commercial Simultest reagent kit and home made FITC labeled MT4 mAb simultaneously. The study has shown that both percentages and absolute CD4+ lymphocyte counts obtained from both reagents were equivalent. The correlation coefficient for regression analysis was 0.995 and 0.996 for percentages and absolute CD4+ lymphocyte counts, respectively. The results suggest that home made FITC labeled MT4 reagent is an acceptable alternative reagent for monitoring CD4+ lymphocytes in blood samples by flow cytometry.

Production of mouse anti-CD4 antibodies by DNA-based immunization.

The intramuscular injection of plasmid DNA encoding an antigenic protein has been developed recently as a tool for immunization. DNA-based immunization was shown to generate immune responses against the encoded antigen in diverse animal species. In this report, we present the use of DNA-based immunization for the production of antibodies to CD4, a human leukocyte surface molecule. Mice were injected intramuscularly with eukaryotic expression vector containing cDNA encoding CD4 protein, termed CD4-DNA, and were subsequently assayed for anti-CD4 antibody production by indirect immunofluorescence. Sera collected from 2 of 3 inoculated mice reacted with CD4 expressing transfected COS cells and Sup-T1 cells. Anti-CD4 antibody activity was abolished by adsorption with CD4 molecule expressing cells. CD4+ cell depleted lymphocytes were also used to confirm the specificity of the anti CD4 antibodies present in immune serum. CD4-DNA immune serum reacted with approximately 1/3 of freshly isolated lymphocytes but to very few cells in the CD4+ cells-depleted preparation. CD4-DNA immunized sera was used to enumerate CD4+ cells in the peripheral blood of 6 healthy donors and 2 AIDS patients. The number of CD4+ cells estimated by DNA immunized sera was very similar to estimates using standard anti-CD4 monoclonal antibody Leu3a. DNA-based immunization is therefore capable of raising antibodies to human leukocyte surface antigens. This technology may be useful for producing antibodies to other cell surface antigens in mice or other animals.

Separation of human suppressor and helper T cells by concanavalin A-coated sheep erythrocytes.

Normal human peripheral blood mononuclear leukocytes (PBML) were activated by concanavalin A (Con A). Con A-activated and non-activated T cells were separated by E (AET) rosettes (2-aminoethylisothiouronium hydrobromide treated sheep erythrocyte rosettes). Purified T cells were rosetted with Con A-coated sheep red blood cells (Con A-SRBC) at 37 degrees C resulting in Con A-SRBC rosetted and non-rosetted T cells. The Con A-SRBC rosetted T lymphocytes in the T lymphocytes from Con A-activated and non-activated PBML were 44.4 +/- 5.4 percent and 16.0 +/- 7.5 percent (Mean +/- S.D.) while the Con A-SRBC non-rosetted T lymphocytes were 55.6 +/- 5.4 percent and 84.0 +/- 7.5 percent respectively. The Con A-SRBC rosetted and non-rosetted T cells were separated by Ficoll-Hypaque gradient centrifugation. Functional studies of Con A-SRBC rosetted and non-rosetted T cells were performed by in vitro tests using pre-amplified reverse hemolytic plaque assay for measuring numbers of immunoglobulin G (IgG) secreting cells and ELISA quantitation of IgG concentration. Both techniques were used to assess the suppressor and helper functions of the Con A-SRBC rosetted and non-rosetted T cells. The Con A-SRBC rosetted cells obtained from T cells of Con A-activated PBML showed strong suppressor activities to normal PBML in both pre-amplified reverse hemolytic plaque assay and sandwidh ELISA of IgG concentration, while the Con A-SRBC non-rosetted T cells demonstrated strong helper activities to normal PBML in both assay systems.(ABSTRACT TRUNCATED AT 250 WORDS)

Agar plating technique for enumeration of IL-2 producing cells in human peripheral blood mononuclear leukocytes.

An agar plating technique for detection and enumeration of IL-2 producing cells from human peripheral blood mononuclear leukocytes (PBML) has been developed. This method is based on the principle that PHA-stimulated PBML, as effector cells, secrete interleukin 2 (IL-2) into soft agar containing mouse 3-day Con A blasts as IL-2 dependent responder cells. The IL-2 dependent Con A blasts proliferating around the IL-2 producing cells form colonies or clusters of cells and are easily visualized under a dissecting microscope. The development of IL-2 producing cells was optimum when 1 X 10(6) cells/ml PBML were stimulated with 2 micrograms/ml PHA-P for 4 hours, and when 2.5 X 10(5) cells were co-cultured with 6 X 10(6) Con A blasts in soft agar for 5 days. The average number of IL-2 producing cells in 10 normal healthy controls were 754 +/- 94 (mean +/- S.E.M.) cells/10(6) PBML. The numbers of IL-2 producing cells and the levels of IL-2 produced were highly correlated (r = 0.929). The subpopulation of lymphocytes in the colonies was shown to be mostly murine T-cells, since they were mostly Thy 1.2 positive, CD3 negative and surface immunoglobulin negative. This technique is very simple to perform and provides an accurate and straightforward means to enumerate IL-2 producing cells from human PBML in a variety of human immunologic disorders.

Enumeration of interleukin 2-producing cells from rat spleen.

A method for the enumeration of IL2-producing cells from rat spleen has been developed. Rat spleen cells were stimulated with concanavalin A (Con A), washed, then mixed with IL2-dependent cells (3 day Con A blasts) and plated in soft agar. Clusters of IL2-dependent cells formed around IL2-producing cells, giving colonies which were easy to count under a dissecting microscope. All experimental factors influencing development of colonies of IL2-producing cells surrounded by IL2-dependent cells were evaluated and set up. Optimum number of effector cells was 2.5 x 10(5) cells/culture, while optimum number of IL2-dependent cells was 6 x 10(6) cells/culture. Optimum concentration of Con A for IL2 stimulation was 40 micrograms/ml with an optimal stimulation time of 10 hours. Optimum incubation time for development of IL2-producing cell colonies was 5 days. The number of IL2-producing cells by this technique had a good correlation with the level of IL2 in the cell culture fluid (r = 0.885). When colonies were aspirated from agar and stained by Wright stain, a big purple stained cell at the center was surrounded by small cells in almost all colonies examined. All cells from colonies were fluoresed with anti-mouse Thy 1.2-fluorescein conjugate. However, they were negative with anti-mouse Ig-fluorescein conjugate. The number of IL2-producing cells was 816-2080 cells/10(6) of rat spleen cells with mean +/- S.E.M. = 1404 +/- 154/10(6) cells.

Regulation of immunoglobulin secretion by T lymphocytes in human malaria.

In vitro studies were carried out on the nature of immunoglobulin synthesis and secretion by peripheral blood mononuclear leukocytes (PBMLs) and on the function of T and B cells from malaria patients. The mean values of secreted IgG and IgM concentrations of 22 malaria patient PMBLs were significantly higher than those of 20 normal PBMLs. When the suppressor T cell activity and the function of B cells in response to suppressor T cells were assayed by the cell co-culture technique, it was found that there was a decrease in suppressor T cell activity and the B cell function in response to normal suppressor T cells in malaria patients. The defects of these T and B cell functions may play some role in the immunological abnormalities seen in some malaria patients.