Characterization of mouse macrophage differentiation antigens by monoclonal antibodies

Monoclonal rat antibodies to mouse macrophage antigens were prepared. For immunization phagocytic cells in the spleens of mice recovering from sublethal irradiation were used. Specificities of the monoclonal antibodies obtained were determined on cells of normal mouse cell populations as well as on cells of a panel of mouse cell lines. In an attempt to monitor expression of differentiation-related antigens two models of in vitro-induced macrophage differentiation were used: differentiation of cells of the myeloblast line Ml; CSF-1-induced differentiation of bone marrow cells. The results obtained clearly show that during maturation from undifferentiated to highly differentiated cells of the macrophage lineage expression of antigens recognized by the MIV 38, MIV 55, MV 87, and MV 114 monoclonal antibodies is enhanced. At the same time, expression of antigens recognized by the MIV 52, MIV 113, and MIV 116 monoclonal antibodies diminishes at a similar rate. The suitability of these monoclonal antibodies for the characterization of differentiation states of mouse macrophages is discussed.     

Mouse monoclonal antibodies specific for endothelial cells

Summary Balb/c mice were immunized with a human endothelial cell pool. Spleen cells were then fused with a NS-0 hybridoma cell line. A number of hybridomas secreted antibodies that reacted with the immunizing endothelial cell pool as well as with every other tested umbilical cord vein~derived human endothelial cell. These monoclonal antibodies also stained pig, rabbit and ox aortic endothelial cells indicating their specificity for this cell type. Five of 16 monoclonal antibodies additionally reacted with human fibroblasts (HFIB). The produced monoclonal antibodies did not recognize FVIIIRAG or MHC determinants. They can therefore be regarded as additional and reliable markers for endothelial cells in vitro.     

Detection of platelet activation using activation specific monoclonal antibodies

Platelets may become activated in a number of clinical disorders and participate in thrombus formation. Blood tests reflecting in vivo activation are therefore potentially useful in evaluating patients with thrombotic diseases. Three types of monoclonal antibodies have been described that react preferentially with activated platelets. Antibodies against a 53-kD lysosomal granule protein, and antibodies that recognize a 140-kD alpha-granule protein, are two types expressed on the platelet surface during secretion. A third type is not dependent on secretion and recognizes activation-dependent changes in the configuration or microenvironment of the platelet glycoprotein IIb/IIIa complex. Several procedures were used to detect platelet activation, using radiolabeled or fluorescent antibodies. In a number of disorders, changes in platelets, reflecting activation, could be detected. For the study of in vitro and in vivo platelet activation, these tests may be useful, but further studies are needed to confirm the power and efficiency of this approach compared to other routine tests.     

Production of monoclonal antibodies to antigens of specific mouse T-suppressors

F1(MSU X WAG) rats were immunized with anti B6 BALB/c specific suppressor T cells (SSTC), purified by absorption/elution technique, with the following fusion of splenocytes to NS-I myeloma cell line. Hybrids were screened for their ability to affect SSTC, cytotoxic T lymphocytes (CTL) and producers of macrophage migration inhibition factor (MIF-producers) all triggered by in vivo priming with allogeneic cells. Two hybridoma cell lines--C1 and C4 inactivated SSTC by approximately 50%, leaving CTL and MIF-producers intact. C4 were also active in vivo, if injected as ascitic fluid from nu/nu mice, though to a lesser extent than in vitro.     

Acrylic microspheres in vivo. X. Elimination of circulating cells by active targeting using specific monoclonal antibodies bound to microparticles

The elimination from the blood of 51Cr-labelled mouse erythrocytes modified with trinitrophenyl (TNP) groups was followed in mice. After 24 hours, when a stable concentration of the labelled erythrocytes has been attained, monoclonal anti-TNP-antibodies were given intravenously, either in free, soluble form, or bound to microparticles containing immobilized protein A. The anti-TNP-antibodies induced a rapid elimination of the TNP- and 51Cr-labelled erythrocytes. Over the 8-hours time period studied, the elimination rate was significantly faster when the antibodies were administered bound to the particles. After the elimination of the target cells, the radioactivity was found in the liver, spleen and bone marrow. These results and relevant control experiments indicate that a solid carrier 1. can be directed to a specific target cell with a specific antibody and 2. can induce a rapid elimination of the target cell from the circulation.     

Monoclonal antibodies against mouse gamma-interferon inhibit tumoricidal macrophage activation by T lymphocytes

A monoclonal antibody, AN-18.17.24, specific for murine interferon-gamma (IFN-gamma) was produced by immunizing Wistar rats with IFN-gamma secreted by a T-cell lymphoma, L12-R4, upon stimulation with phorbol myristic acetate (PMA). Antiviral activity as well as tumoricidal activation induced by PMA-stimulated L12-R4 cell supernatant or by Con A-stimulated normal spleen cells were neutralized at the same extent by AN-18 monoclonal antibody. Moreover, depletion experiments showed that inhibition of tumoricidal macrophage activation must be ascribed to the direct binding of the IFN-gamma molecule by AN-18 MAb and not to the interference of the monoclonal antibody with the cell surface IFN-gamma receptor. These studies conclusively demonstrate that in supernatants of T lymphocytes stimulated with polyclonal activators IFN-gamma was the only molecule responsible for macrophage activation in tumor cell killing.     

Neutrophil activation detected by monoclonal antibodies

Monoclonal antibodies have been produced against three neutrophil-associated membrane proteins (p 90, p 170, and p 70) expressed at different maturation stages of the cells. The reactivity of the antibodies against p 90 (B13.9) and p 170 (CLB-gran 10), as measured by quantitative flow cytofluorometry, increased after stimulation of the neutrophils by the calcium ionophore A23187, by phorbol myristate acetate, or by the chemoattractant formylmethionyl-leucyl-phenylalanine in combination with cytochalasin B. This increase is regulated independently of the simultaneously increased expression of the C3bi receptor, because neutrophils of a patient deficient for the C3bi receptor showed a normal increase in membrane expression of p 90 and p 170. Neutrophil cytoplasts were not inducible to increased membrane expression, suggesting that the cytoplasts lack the internal pool of these proteins. The reactivity of the antibody against p 70 (CLB-gram 5) was not affected by activation. The antibodies B13.9 and CLB-gran 10 may be useful to detect neutrophil activation.     

Differentiation antigens of mouse teratocarcinoma stem cells defined by monoclonal antibodies

Three differentiation antigens of mouse teratocarcinoma stem cells are defined using a panel of ten IgM-class monoclonal antibodies raised against teratocarcinoma F9 cells. TEC-01 and four other antibodies define an antigen that corresponds to SSEA-1. TEC-02 antibody defines an antigen that is expressed on teratocarcinoma stem cells, parietal yolk sac cells PYS-2, unfertilized eggs including the zona pellucida and blastocysts. It is absent from all mouse adult tissues tested. Three other antibodies exhibit binding properties similar to TEC-02. TEC-03 antibody defines an antigen that is expressed on teratocarcinoma stem cells, PYS-2 cells and mouse blastocysts. It is absent from all mouse adult tissues except for lungs.     

Growth inhibition of murine mammary carcinoma by monoclonal IgE antibodies specific for the mammary tumor virus

Summary Two IgE-producing hybridomas were established from spleen cells of Balb/c mice, which had been immunized with mouse mammary tumor virus (MMTV). These IgE monoclonal antibodies (mAbs) reacted specifically with the major envelope glycoprotein (gp36) of MMTV, as established by the immunoblot assay and by passive cutaneous anaphylaxis. The effect of the IgE mAbs (produced by clone A8) on the growth of the MMTV-secreting mammary adenocarcinoma H2712 was investigated in syngeneic C3H/HeJ mice. The mice were inoculated s.c. with either 10⁵ (100 × LD₅₀) or 10⁶ (1000 × LD₅₀) tumor cells and received repeated i.p. injections of 25 µg anti-gp36 IgE mAbs at 4-day intervals for 8 weeks. This treatment prevented the development of subcutaneous tumors in 50% of the animals. Similar protection was observed when the tumor cells (10⁵/animal) were injected i.p. 4 days prior to the beginning of the i.p. treatment consisting of injections of 25 µg mAbs at 4-day intervals for 6 weeks. However, these mAbs did not protect C3H/HeJ mice against the MMTV-negative MA16/c carcinoma cells. Hence, these results support the view that IgE-mediated cytotoxic mechanisms may play an immunologically specific antitumor surveillance role and that laboratory-induced antitumor IgE mAbs have the potential of specific therapeutic agents for in vivo destruction of tumor cells.     

Inhibition of macrophage invasion by monoclonal antibodies specific to Leishmania (Viannia) braziliensis promastigotes and characterisation of their antigens

Monoclonal antibodies that specifically recognise Leishmania (Viannia) braziliensis promastigotes were produced and termed SST-2, SST-3 and SST-4. SST-2 recognises a conformational epitope present in a 24-28 kDa doublet and in a 72 kDa component, as verified by Western blotting. Indirect immunofluorescence showed that the antigen recognised by SST-2 is distributed homogeneously on the parasite surface. SST-3 recognises a flagellar glycoprotein of approximately 180 kDa. The reactivity of this mAb was abolished by sodium m-periodate treatment, indicating that SST-3 reacts with a carbohydrate epitope of the 180 kDa antigen. SST-4 recognises a conformational epitope of a 98 kDa antigen. SST-2, SST-3 and SST-4 were specific to L. (V.) braziliensis promastigote forms. Indirect immunofluorescence did not show reactivity of SST-2 or SST-3 with amastigotes of L. (V.) braziliensis, or with promastigotes of Leishmania (Viannia) panamensis, Leishmania (Viannia) guyanensis, Leishmania (Viannia) naiffi, Leishmania (Viannia) lainsoni, Leishmania (Leishmania) amazonensis, Leishmania (Leishmania) major, or Leishmania (Leishmania) chagasi. We also evaluated the involvement of SST-2, SST-3 and SST-4 antigens in parasite-macrophage interaction. Fab fragments of SST-3 and SST-4 significantly inhibited the infectivity of L. (V.) braziliensis promastigotes to mouse peritoneal macrophages.     

Production of monoclonal antibodies against mouse molar papilla cells

To develop markers for the analysis of the molecular mechanisms of dental papilla cells differentiation, 10 monoclonal antibodies were produced against trypsin-isolated mouse molar dental papilla cells. These antibodies identify matrix components, cell membrane associated antigens and intracellular-constituents. Changes of the staining patterns were correlated with a typological hierarchy of dental papilla cells and with terminal differentiation of odontoblasts.     

Complement activation by human monoclonal antibodies to human immunodeficiency virus.

It has been shown that the incubation of human immunodeficiency virus (HIV) with polyclonal antibodies from HIV-infected persons and complement results in complement-mediated neutralization due, at least in part, to virolysis. The current study was performed to determine whether any of a panel of 16 human monoclonal antibodies to HIV could activate complement and, if so, which determinants of the HIV envelope could serve as targets for antibody-dependent complement-mediated effects. Human monoclonal antibodies directed to the third variable region (V3 region) of HIVMN gp120 induced C3 deposition on infected cells and virolysis of free virus. Antibodies to two other sites on HIVMN gp120 and two sites on gp41 induced few or no complement-mediated effects. Similarly, only anti-V3 antibodies efficiently caused complement-mediated effects on the HIVIIIB isolate. In general, the level of C3 deposition on infected cells paralleled the relative level of bound monoclonal antibodies. As expected, pooled polyclonal antibodies from infected persons were much more efficient than monoclonal antibodies inducing C3 deposition per unit of bound immunoglobulin. Treatment of virus or infected cells with soluble CD4 resulted in increases in anti-gp41 antibody-mediated virolysis and C3 deposition but decreases in anti-V3 antibody-mediated virolysis and C3 deposition. In general, virolysis of HIV was more sensitive as an indicator of complement-mediated effects than infected-cell surface C3 deposition, suggesting the absence of or reduced expression of functional complement control proteins on the surface of free virus. Thus, this study shows that human monoclonal antibodies to the V3 region of gp120 are most efficient in causing virolysis of free virus and C3 deposition on infected cells. Elution of gp120 with soluble CD4 exposes epitopes on gp41 that can also bind antibody, resulting in virolysis and C3 deposition. These findings establish a serologically defined model system for the further study of the interaction of complement and HIV.     

Methods to label monoclonal antibodies for use in tumor imaging

The use of radiolabeled monoclonal antibodies as a diagnostic tool in nuclear medicine has grown rapidly over the past several years. Early studies utilized antibodies labeled with radionuclides of iodine (i.e. ¹²⁵I and ¹³¹I) although these radionuclides are not ideal for use in tumor imaging. Recent advances and the development of new chemical methods has made it possible to label antibodies with other radionuclides (i.e. ⁷⁷Br, ¹¹¹In and ^99mTc). The advantages and disadvantages associated with all of the different radionuclides and labeling methods will be discussed.     

Identification of an antigen specific to Trypanosoma congolense by using monoclonal antibodies

Using indirect immunofluorescence assays on acetone-fixed smears of a series of different parasites, we have shown that two monoclonal antibodies bind specifically to Trypanosoma congolense organisms. The antibodies bind to both bloodstream trypomastigotes and procyclic culture forms of the parasite and are thus not stage specific. Immunoprecipitation and immunoblot analysis showed that both monoclonal reagents bound a protein of approximately 31,000 m.w. This antigen appeared to be located on the plasma membrane of T. congolense, but the epitope was not exposed on the surface of living bloodstream or procyclic organisms. The antigen was detectable on acetone-fixed organisms or in trypanosome lysates in enzyme-linked immunosorbent assays and may therefore by useful as a species-specific marker in field assays for epidemiologic and clinical investigations.     

Characterization of mouse monoclonal antibodies to human interferon-gamma

Mouse monoclonal antibodies (mAb) to human interferon-gamma (HuIFN-gamma) were characterized. The mAbs studied--E4-18, G4-15, and SAT-1--which are all IgGl-type, reacted to all HuIFN-gamma molecular species, both glycosylated and non-glycosylated. Affinity constants calculated of E4-18 and G4-15 didn't have considerable differences for both kinds of HuIFN-gamma (1-3 x 10(8) liter/mol), but SAT-1 had a difference--a higher value (10(10) liter/mol) for the former than for the latter (8 x 10(8) liter/mol). In epitope specificity, the results suggested that E4-18 and G4-15 recognized an overlapped region remote from the region of SAT-1. Competition experiment using synthetic peptides suggested that epitope of G4-15 is around N9-26 of the HuIFN-gamma sequence. Those mAbs could be used for sandwich radioimmunoassay of HuIFN-gamma using double mAbs in two combinations, one (G4-15/E4-18) based on dimer forms of HuIFN-gamma and the other (SAT-1/E4-18) based on epitope difference. The mAbs are all neutralizing antibodies in which SAT-1 neutralized at a lower concentration than did G4-15, and at a much lower one than did E4-18. The receptor binding of HuIFN-gamma was inhibited by mAbs G4-15 and SAT-1. Efficacy of G4-15 and SAT-1 for the inhibition correspond with that for neutralization.     

Approaches to improve tumor accumulation and interactions between monoclonal antibodies and immune cells

Monoclonal antibodies (mAb) have become a mainstay in tumor therapy. Clinical responses to mAb therapy, however, are far from optimal, with many patients presenting native or acquired resistance or suboptimal responses to a mAb therapy. MAbs exert antitumor activity through different mechanisms of action and we propose here a classification of these mechanisms. In many cases mAbs need to interact with immune cells to exert antitumor activity. We summarize evidence showing that interactions between mAbs and immune cells may be inadequate for optimal antitumor activity. This may be due to insufficient tumor accumulation of mAbs or immune cells, or to low-affinity interactions between these components. The possibilities to improve tumor accumulation of mAbs and immune cells, and to improve the affinity of the interactions between these components are reviewed. We also discuss future directions of research that might further improve the therapeutic efficacy of antitumor mAbs.     

Marked enhancement of macrophage activation induced by synthetic muramyl dipeptide (MDP) conjugate using monoclonal anti-MDP antibodies

Activation of peritoneal exudate macrophages of mice to inhibit the in vitro proliferation of tumor target cells was achieved with low concentrations of N-acetyl-l-alanyl-d-isoglutamine (MDP for muramyl dipeptide) conjugated to a synthetic carrier. Addition to the cultures of monoclonal anti-MDP or anti-carrier antibodies renders a thousandfold-smaller concentration of the conjugate highly effective in activating macrophages. This synergistic effect was observed neither with a control monoclonal antibody of different specificity nor with an F(ab)₂ fragment of the monoclonal anti-MDP antibody. Other controls, such as addition to the cultures of the carrier alone with its specific monoclonal antibodies, also demonstrated that there exists a requirement for the presence of MDP in the conjugate. The possible uses of such a system as well as the underlying mechanisms are discussed.     

Enhanced immunization techniques to obtain highly specific monoclonal antibodies

Despite fast advances in genomics and proteomics, monoclonal antibodies (mAbs) are still a valuable tool for areas such as the evolution of basic research in stem cells and cancer, for immunophenotyping cell populations, diagnosing and prognosis of diseases, and for immunotherapy. To summarize different subtractive immunization approaches successfully used for the production of highly specific antibodies, we identified scientific articles in NCBI PubMed using the following search terms: subtractive immunization, monoclonal antibody, tolerization, neonatal, high-zone tolerance, masking immunization. Patent records were also consulted. From the list of results, we included all available reports, from 1985 to present, that used any enhanced immunization technique to produce either polyclonal or monoclonal antibodies. Our examination yielded direct evidence that these enhanced immunization techniques are efficient in obtaining specific antibodies to rare epitopes, with different applications, such as to identify food contaminants or tumor cells.     

Development of monoclonal antibodies specific to urochordate intracellular epitopes

Sixteen monoclonal antibodies (MAbs) specific to 2 urochordate genera (Botryllus schlosseri and Botrylloides) intracellular epitopes were generated in mice immunized with a mixture of fresh and paraformaldehyde-fixed cells obtained from animal's blood and cells from dissociated organs. Hybridoma clones were selected by ELISA tests and immunohistochemistry assays on paraffin-embedded animal tissues. Five MAbs were tested for reactions with different zooidal organs and cell compartments; 7 MAbs were tested, separately, on 5 different botryllid colonies (3 Botryllus and 2 Botrylloides). The results revealed high polymorphism. Whereas some of the MAbs recognized, specifically, only part of the botryllid genotypes tested, others recognized only part of the cellular compartments. These MAbs will be used as an important tool in the study of botryllid ascidian immunology and developmental biology, revealing the first wide panel of MAbs specific to urochordate intracellular antigens.