Monoclonal Technique Research Articles

Sponge aggregation factor: in situ localization by fluorescent monoclonal antibody techniques.

The aggregation factor (AF) from sponges mediates a heterophilic interaction of homologous cells. Applying electron microscopical means, we succeeded only very rarely in identifying the 90 S AF particle in tissue sections from Geodia cydonium. By means of a fluorescent antibody technique, we have now localized the cell binding domain of the AF in situ. Previous studies in this laboratory have led to the identification of the 47-kDa cell binding protein of the AF, using the monoclonal antibody (mab) 5D2-D11 [Gramzow M, Bachmann M, Zahn RK, Uhlenbruck G, Dorn A, Müller WEG, J Cell Biol, 102: 1344-1349, 1986]. This mab and mab 7D5, directed against a 92-kDa protein in the AF complex, were chosen for the fluorescent studies. By using mab 5D2-D11, the plasma membranes of cells from different regions in the sponge could be brightly stained. However, mab 7D5 reacted only very weakly with the sponge surfaces. By applying the immuno-blotting technique it was furthermore demonstrated that the cell binding protein is present both in the associated form with AF complex and in a free state. Moreover, it was established that the 47-kDa binding protein is not present in homologous glycoconjugates, lectin, or collagen; these components are known to be involved in cell-matrix interaction.

Basally located epithelial cell surface component identified by a novel monoclonal antibody technique

Tubular aggregates of glandular epithelial cells (gland fragments) were isolated from human endometrium by collagenase digestion of surrounding stroma, thus exposing the basal surfaces of the cells. Using these aggregates as immunogen, monoclonal antibodies could be derived that recognized basally located antigens. One such antibody, G71, is described, that binds to a basal epithelial cell antigen present in a variety of human epithelia. Epitope-bearing molecules in the range M r 60 000–180 000 are present in two of the tissues studied, amnion and endometrium. The epitope is associated with areas of epithelial cell-extracellular matrix contact.

The immunobiology of basal cell carcinoma: an in situ monoclonal antibody study.

A semi-quantitative, immunoperoxidase monoclonal antibody technique was used to study the mononuclear cells surrounding 32 basal cell carcinoma specimens from 30 patients. Tumours were analysed in subgroups based on recurrence, size and ulceration. T cell counts were high (greater than 3 out of 4) for all groups while T helper/inducer and T suppressor/cytotoxic cell counts were equal (approx. 2). Macrophage counts were low for all groups, about I X 2, while B cell and Ia positive cell counts were high (greater than 3). T/B cell and T helper/suppressor cell ratios approached one for the tumours as a whole as well as the sub-groups. The relative importance and contribution of cell mediated vs. humoral immunity in keeping basal cell carcinomas in check is discussed.

Respiratory tract infections: Goals for 1995

Goals to be identified for 1995, a decade hence, in the prevention, diagnosis, and management of respiratory tract infections may conveniently be divided into diagnostic goals and goals in therapy and prophylaxis. Major diagnostic goals for bacterial, viral, and mycoplasmal infections of the respiratory tract focus on the development of systems to identify microbial components, such as specific antigens or segments of DNA, using monoclonal antibody techniques or DNA probes for hybridization. Sputum cultures, in the traditional sense, should ultimately become obsolete. Management goals include the development of algorithms to identify patients who should be hospitalized, in contrast to those who can safely be treated on an outpatient basis. New antibiotic drug development should include drugs active against methicillin-resistant staphylococci, broad-spectrum beta-lactam drugs that are orally active against gram-negative bacilli, and drugs that can be used parenterally on a once-daily basis in settings other than the acute care hospital. There are certainly needs to enhance the present spectrum of antiviral drugs and to develop therapeutically useful immunomodulators. There are promising prospects for vaccine development, including live attenuated influenza virus vaccine, parainfluenza virus vaccine, respiratory syncytial virus vaccine, and a Mycoplasma pneumoniae vaccine. With major research support, such vaccines could possibly be fully developed by 1995. Finally, of greatest importance is the need to achieve greater utilization of existing vaccines, that is, inactivated influenza vaccine and the current 23-valent pneumococcal vaccine. A legitimate goal for 1995 would be to achieve 70 percent or greater utilization of these vaccines within the recommended target populations.

Biological Markers: An Overview

Biological markers (biomarkers) are materials present in abnormal amounts in body fluids of patients with cancer as a product of the disease. They can aid in the clinical management of patients if their levels are indicative of the presence of a neoplasm and proportional to the total tumor burden. The development of a useful biomarker requires a variety of detailed investigations including analytical methodology, study of possible interfering substances, physiologic variations, determination of normal and disease control values, determination of the sensitivity with reference to gross tumor burden, and evaluation of monitoring capabilities during therapy. Research interest continues in the search for better biomarker candidates, particularly in the application of monoclonal antibody techniques, the possible use of multiple biomarkers, and the area of tissue analysis for specific markers.

Evaluation of immune-enhancing effects of ibuprofen in an immunodeficiency model.

Three children and one adult with chronic mucocutaneous candidosis with documented deficient cellular immunity to Candida antigen were evaluated as a model to study the specific cellular immune-enhancing potential of the prostaglandin synthetase inhibitor ibuprofen. Oral ibuprofen failed to have any consistent effect during sequential 4-week on and off cycles on the following parameters: delayed hypersensitivity skin testing; lymphocyte transformation to Candida antigen; T-cell subsets as determined by monoclonal antibody techniques; production of human immune interferon in response to staphylococcal enterotoxin A (SEA). Two patients showed a trend toward enhanced lymphocyte transformation to PHA while taking ibuprofen. In two patients who were studied 8-10 weeks after discontinuation of oral ketoconazole therapy, clinical recurrence of CMC was not prevented by oral ibuprofen therapy.

The effect of hormone therapy on peripheral blood leukocytic subset distribution in stage D prostatic cancer patients.

The distribution of mononuclear cell types found in the peripheral blood of patients bearing carcinoma of the prostate were compared by stage and to a control group using monoclonal antibody techniques. Patients with lower stage disease (A, B) had no significant alteration in subset distribution when compared to a control group, while those with higher stage disease (D) had significant deviations. Stage D patients had a decreased representation of helper-inducer T cells and an increased representation of suppressor-cytotoxic T cells, with an overall reduction in the total T cell content. In addition elevated levels of monocytic, granulocytic and null cells were recognized by the polyspecific OKM1 antibody. These differences were in part reversible following hormonal therapy. Such alterations in the ratios between the various T cell populations could be useful in patient staging and treatment selection.

Transferrin receptors on circulating monocytes in hereditary haemochromatosis

In patients with hereditary haemochromatosis (HH) abnormal functional properties of the macrophage system have been observed. The present study is a preliminary report of increased transferrin receptor expression on monocytes from 12 patients with HH. There was no correlation between the degree of iron overload and the transferrin receptor expression on the monocytes. The results obtained thus indicate that the observed increase in transferrin receptors is not a secondary phenomenon due to systemic iron overload but could be an expression of a primary inborn error of iron metabolism in HH. The functional aspects of the receptors were not evaluated as they were analyzed by means of monoclonal antibody technique.

Detection of minimal residual disease in acute leukemia: Possibilities and limitations

Studies are presented on the detection of ‘minimal residual leukemia’ using a monoclonal antibody (MCA) and fluorescence-activated cell sorting (FACS). As a preclinical model the BN rat acute myelocytic leukemia was used (BNML). The MCA Rm124 (IgM) strongly binds to BNML cells as measured by fluorescence intensity of a second-layer antibody (goat anti-mouse IgM fluorescein isothiocyanate). Only weak cross-reactivity occurred with normal mature granulocytes. It appeared possible to detect as low as 1 leukemic cell per 10,000 normal marrow cells, both in artificial mixtures and in marrows obtained after in vivo remission-induction chemotherapy with cyclophosphamide. Furthermore, an example is given of describing the kinetics of growth of leukemia in the liver, based on serial determinations of leukemic cells with the MCA technique. The population doubling time ( T d) in the liver calculated in this way did not significantly differ from that derived from time-consuming and expensive bioassays. Finally, the extrapolation of these techniques developed in a preclinical model to studies in human acute leukemia is discussed.

THROMBOCYTE AGGREGATES IN RENAL ALLOGRAFTS

It has been demonstrated previously that thrombocytes have a role in renal allograft rejection. The purpose of the present studies was to use fine-needle aspiration biopsy techniques during the early postoperative period to determine whether the specific localization of thrombocytes was correlated with the ultimate outcome of renal transplantation. The thrombocyte morphology and location were evaluated using monoclonal antithrombocyte antibody techniques. The results show that loose aggregates, often in combination with granulocytes, are nearly invariably seen in acute blast-cell-dominated reversible rejections; these aggregates disappear from the graft when the inflammation is overcome. In irreversible rejections in which inflammation continues and increases in intensity, the early loose aggregates are followed and accompanied by aggregation of thrombocytes on the graft vascular endothelial cells. The result suggests that thrombocyte aggregation into the graft is a two-stage phenomenon, and that only thrombocyte-graft endothelial cell aggregates are an adverse prognostic sign indicating an unfavorable course of rejection.

Biotechnology: Present and Future Roles in the Pharmaceutical Industry

Biotechnology is the integration of a number of scientific disciplines including microbiology, genetics, biochemistry and chemical engineering. It uses living organisms, or systems or products from these organisms to make or modify useful products. New biotechnology comprises genetic engineering, protoplast fusion and monoclonal antibody techniques, powerful new “tools” designed to generate efficient bioprocesses and products for the pharmaceutical industry. The following areas of biotechnology are highlighted: human insulin, interferons and other growth factors, neuroactive peptides, blood products, antibiotics, enzymes, monoclonal antibodies, vaccines and oncogenes.

Evaluation of Immune‐enhancing Effects of Ibuprofen in an Immunodeficiency Model

Abstract: Three children and one adult with chronic mucocutaneous candidosis with documented deficient cellular immunity to Candida antigen were evaluated as a model to study the specific cellular immune‐enhancing potential of the prostaglandin synthetase inhibitor ibuprofen. Oral ibuprofen failed to have any consistent effect during sequential 4‐week on and off cycles on the following parameters: delayed hypersensitivity skin testing; lymphocyte transformation to Candida antigen; T‐cell subsets as determined by monoclonal antibody techniques; production of human immune interferon in response to staphylococcal enterotoxin A (SEA). Two patients showed a trend toward enhanced lymphocyte transformation to PHA while taking ibuprofen. In two patients who were studied 8–10 weeks after discontinuation of oral ketoconazole therapy, clinical recurrence of CMC was not prevented by oral ibuproten therapy.

Chronic myelogenous leukemia

Chronic myelocytic leukemia (CML) is a well-known myeloproliferative disorder, with typical clinical and hematological features, which develops into a lethal blastic crisis within an average of 4 years. In the last few years much has been learned about the cell responsible for the leukemic deviation, chromosomal abnormalities both in chronic phase and in blastic transformation, biochemical and cytochemical behavior of leukemic cells, and the immunology of the disease. On the contrary, polychemotherapy has brought but little progress in CML treatment. However, thorough research on prognostic factors at the onset of the disorder allows a more rational approach to the treatment, and in the near future interesting progress in bone marrow transplantation is to be expected. Also, monoclonal antibody techniques will reveal further knowledge on the origin and differentiation of leukemic cells and the relationship between the chronic phase and the blastic deviation.

A set of actin-filament associated proteins characterized by quantitative two-dimensional gel electrophoresis.

Our laboratories have been using two-dimensional gel electrophoresis and monoclonal antibody techniques for the study of contractile and structural proteins. The resolving power of the two-dimensional gels, combined with computer methods for quantitation and pattern matching, have been used in previous studies to analyze the proteins of cultured L6 muscle cells as they differentiate from myoblasts to myotubes (453,497,498). Highly specific monoclonal antibodies have made possible the identification and biochemical characterization of major and minor structural proteins. Matsumura et al (1983) have used the monoclonal antibodies to tropomyosins to identify 5 isoforms that are present in several rat cell lines (499). These results confirm and extend the earlier identification of tropomyosin isoforms in L6 myoblasts (453). Matsumura et al (1983) have further shown that actin-filament assemblies can be purified from non-muscle and muscle cells as ordered bundles cross-linked in a highly regular fashion by monoclonal antibodies to tropomyosin. (500).KeywordsMyosin Light ChainLabel PeriodUnphosphorylated FormTropomyosin IsoformsMinor Structural ProteinThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Review of melanoma antigens recognized by monoclonal antibodies (MAbs). their functional significance and applications in diagnosis and treatment of melanoma

The introduction of monoclonal antibody techniques has led to a rapid advance in information concerning antigenic structures in melanoma cell membranes. These have been classified according to the extent of their expression on cells of other tissues, but it is evident that a more precise classification based on their biochemical nature is possible. Several monoclonal antibodies appear to define antigens restricted to melanoma cells and fetal tissues. Many antibodies recognize antigens shared with gliomas and nevi, whereas other groups can be defined which recognize antigens on melanocytes or other carcinomas. One of the commonly detected antigens was shown to be a high molecular weight (MW) proteoglycan which may be involved in reactions with other cells and the intercellular matrix. A second antigen was shown to be a ganglioside which may have receptor functions in cells. A third was shown to be a glycoprotein with iron transport functions. The latter antigen and the large MW proteoglycan have been a focus of attention for in vivo targeting studies in treatment and diagnosis. The ganglioside, large MW proteoglycan and a melanocarcinoma antigen may be detected in the circulation of patients and are being evaluated for monitoring of disease activity in patients with melanoma. Several monoclonals may be of value in histological evaluation of melanoma, e.g. diagnosis of preneoplastic lesions, metastatic lesions of unknown origin and identification of cell structures related to metastatic behaviour in the host. Further studies should help to define cellular structures recognized by the immune system in humans.

Pharmacological modification of immunoregulatory activity of lymphocytes: facts and potential.

The rapid expansion in our understanding of immunoregulatory mechanisms which has occurred during the last 5 years has largely been due to the delineation of lymphocyte subpopulations through the application of monoclonal antibody techniques. In man the OKT and Leu series of monoclonal antibodies have been most extensively used for the study of T-lymphocyte differentiation and identification of immunoregulatory lymphocyte subpopulations [1]. During intrathymic differentiation of T lymphocytes there is sequential appearance and disappearance of antigens upon thymocytes and maturing T cells giving rise to the notion that the antigens detected by the OKT and Leu monoclonal antibodies represent 'differentiation antigens' [2]. While most thymocytes express the T10, T6, T4 and T8 antigens on their surfaces, the TI0 and T6 antigens are lost during late intrathymic differentiation and the T3 'pan T-cell' antigen is expressed. Moreover, the T4 and T8 antigens segregate during late intrathymic maturation such that only mature T cells with the T3+T4+ or

Developmental changes in unique cell surface antigens of chick embryo spinal motoneurons and ganglion cells

The monoclonal antibody technique was used to investigate neuronal heterogeneity and its developmental changes in the chick embryo trunk especially at the thoracic level. We report here four monoclonal antibodies (called SC 1, SC 2, SC 3, and SC 4) that bound to cell surface antigens. These antigens appeared to be proteins or glycoproteins because of their susceptibility to trypsin. In the spinal cord, antibody SC 3 stained all cells, but antibody SC 1 specifically stained motoneurons and ventral epithelial cells. The staining of motoneurons by antibody SC 1 was transient. It appeared at early stages (stage 16–17; Hamburger and Hamilton), but decreased markedly in intensity at older stages (stage 30–31). Antibody SC 2 did not stain cells in the spinal cord. It stained only neurons in the dosal root and sympathetic ganglia. Antibody SC 4 stained only cells derived from the neural crest at the early stages (stage 16–20). At later stages, it stained a wider population of cells, including sensory neurons, Schwann cells, and cells in the central nervous system. In the dorsal root ganglion, antibodies SC 1 and SC 2 stained only neuronal cells whereas antibodies SC 3 and SC 4 stained both neuronal and glial cells. The dorsal root ganglionic antigens recognized by these antibodies were not expressed concurrently but appeared in a developmental sequence. Staining with antibodies SC 3 and SC 4 appeared first, then SC 1, and finally SC 2. Among these four antigens, the antigens common to both neuronal and glial cells appeared earlier than the neuron specific antigens. Thus, our monoclonal antibodies revealed heterogeneities in cell surface neuronal molecules and their transient and sequential appearance during embryonic development.

Use of monoclonal antibody and colloidal gold in E.M. localization of von Willebrand Factor in megakaryocytes and platelets

The subcellular localization of Factor VIII/von Willebrand protein (VIII R:Ag) is studied with monoclonal antibody and gold immunocytochemical technique. Monoclonal antibody against purified VIII R:Ag is brightly fluorescent on megakaryocytes and platelets. In E.M., gold immunolabeling is performed on thin cell sections of human megakaryocytes and platelets. Different embedding materials are used to preserve the antigenicity: Epon embedded megakaryocytes show a high concentration of VIII R:Ag in α-granules using 4F9 monoclonal antibody. In comparison, lowicryl K4M embedded material does not improve the same specificity, only a few platelets granules were stained. This subcellular localization, in full agreement with biochemical results appears visualized for the the first time in E.M.

Clinical uses of monoclonal antibodies

Immunization of animals for the purpose of generating antisera for clinical and experimental use results in the production of antibodies with multiple specificities directed at the parent antigen. Furthermore, if the immunogen contains contaminants of a highly immunogenic nature, large amounts of irrelevant antibody will be generated and which must subsequently be absorbed. The unpredictable nature of heteroantisera can be overcome by adopting the monoclonal antibody technique which allows one to produce standardized antibody with well defined and selected specificity toward a given epitope on a macromolecule. Cross-reactions are not eliminated by monoclonal antibodies.

Multiple sclerosis as a disease of immune regulation.

ConclusionsMost available evidence suggests that infection with enveloped viruses of the myxo-, paramyxo-, herpes-, and poxvirus families may occasionally induce immunization to neural antigens, myelin antigens in particular, and a consequent encephalomyelitis localized to the white matter. In individuals with a genetically determined abnormality of immune regulation, this process may not be suppressed and may give rise to the recurrent or long-lasting periods of new lesion formation which we call multiple sclerosis. Exacerbations may be provoked by further virus infections or by childbirth. The responsible white matter antigen(s) have not been firmly identified and the immunoregu-latory abnormality is unknown. The character of the lesions appears to be determined by multiple simultaneous immune responses to different antigens and multiple superimposed immunopathologic effects.Much of what is presented in the present review may require revision as the further application of monoclonal antibody techniques ...