Microcytotoxic HLA typing of cells directly isolated from blood by means of antibody-coated microspheres.
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Antibodies, Monoclonal↗
Biomedical subjects
T Lea
Publications and source records attributed to T Lea.
Immunofluorescence staining of agarose-embedded cells. A new technique developed for immunological characterization of markers on a small number of cells.
A simple new method for the immunofluorescence staining of small numbers of cells is described: cell suspensions are mixed with low-temperature-gelling agarose at 37 degrees C and 2 microliter samples of agarose containing cells are dispensed onto multitest microslides precoated with agarose. The cells are subsequently stained by immunofluorescence techniques. Alternatively, the cell slides can be stored in liquid nitrogen until immunofluorescence staining is carried out. Since cells are entrapped within the agarose matrix, cell loss is prevented during staining and washing procedures. The method permits staining of as few as 250 cells for each marker, thus enabling simultaneous characterization of several separate cell markers in cerebrospinal fluid or other body compartments from which comparatively few cells are obtainable.
Antigens, Surface↗
Complement activation in rheumatoid arthritis evaluated by C3dg and the terminal complement complex.
Complement activation was evaluated in plasma and synovial fluid from patients with rheumatoid arthritis. The activation fragment C3dg and the fluid-phase terminal complement complex were used as indicators of initial and terminal activation, respectively. Considerable activation of the whole complement cascade was demonstrated in most synovial fluid samples and in one-third of the plasma samples. No correlation was found between the level of activation products in synovial fluid and the level in plasma. Therefore, both compartments must be examined in order to evaluate local and systemic complement activation in rheumatoid arthritis.
Arthritis, Rheumatoid↗
The terminal complement complex in sera deficient in the eighth component of complement (C8).
The terminal complement complex (TCC) was quantified in sera from patients with a genetic deficiency of C8 alpha-gamma or C8 beta. The individual sera contained only trace amounts of TCC compared with a normal serum pool. The content of TCC increased after mixing the two sera, which was consistent with reconstitution of C8 activity. Only a moderate increase in TCC was obtained after zymosan activation of the individual sera, whereas activation of the mixture resulted in high amounts of TCC. C8 was demonstrated in the TCC of both deficient sera. These results may indicate that functional C8 is present in trace amounts despite the genetic deficiency, and that the terminal pathway may function to some extent although not enough to be detectable in less sensitive assays.
Antibodies, Monoclonal↗
Characterization of human mononuclear cells after positive selection with immunomagnetic particles.
We have investigated the possibility to employing magnetic monodisperse polymer particles for positive selection of human peripheral blood mononuclear cell populations. By carefully titrating the ratio between particles and cells we succeeded in isolating a number of cell populations that could be cultivated subsequently in vitro for functional studies. The success of the procedure is partly dependent on the properties of the monoclonal antibodies used to sensitize the cells. Provided these antibodies do not react with membrane structures involved in the transduction of activating signals, highly purified, quiescent cell populations can be recovered in a single fractionation step. In most instances particles will detach from the isolated cells by overnight culture, and the particles can then be removed from the system by a suitable magnet. T lymphocytes, subpopulations of T lymphocytes, and B lymphocytes have been isolated in this way and studied in a variety of functional assay systems. Comparison with cells obtained after negative selection clearly demonstrates the usefulness of this technique, especially if the membrane marker selected for it is not directly engaged in the activation processes.
Antibodies, Anti-Idiotypic↗
HLA class I and II typing using cells positively selected from blood by immunomagnetic isolation--a fast and reliable technique.
This paper describes a new cell isolation and HLA typing technique, which permits cell separation and HLA class I or class II typing to be performed in 70 min. Magnetic monodisperse microspheres (Dynabeads TM) were coated with monoclonal antibodies (MAbs) specific for the CD8 T cell antigen or for HLA class II monomorphic epitopes. They could then be used to obtain HLA class I or class II positive cells directly from ACD blood in approximately 15 min by the use of magnetic separation. The cells (attached to the microspheres) were subsequently used in microcytotoxic HLA typing (total incubation time of 55 min) using acridin orange/ethidiumbromide to stain viable (yellow) and dead (red) cells. It was found that this immunomagnetic (IM) HLA typing technique was specific, has a sensitivity superior to that observed for conventional microcytotoxicity assays and gave low background staining. IM HLA-ABC typing of 50 healthy donors and 10 patients and IM HLA-DR typing of 25 healthy donors and 30 patients gave results corresponding well with that obtained independently by conventional HLA typing (concordancy rates 92-100%). Furthermore, the IM HLA typing technique permitted reliable HLA class II typing of blood cells from six patients where conventional HLA class II typing was impossible. The IM HLA typing technique also enables HLA class I and II typing to be quickly and reliably performed on cells from ACD blood of cadaveric donors.
Antibodies, Monoclonal↗
Effect of nephritic factor on C3 and on the terminal pathway of complement in vivo and in vitro.
Plasma samples from patients with nephritic factor (NeF) were examined for their C3 converting activity. C3, C3dg, C5 and the fluid phase terminal complement complex (TCC) were quantified. All patients had evidence of C3 activation with low plasma C3 and high C3dg. Some patients had normal C5 and normal TCC levels, and thus no evidence of terminal pathway activation in vivo; others, with slower C3 conversion in vitro, had low C5 levels with TCC either elevated or in the upper normal range, suggesting in vivo activation of the terminal pathway. These observations were confirmed by in vitro experiments using purified NeFs. It is concluded that considerable activation of C3 may occur in vivo without a simultaneous activation of the terminal pathway, and that NeF is heterogeneous with regard to its ability to activate complement.
Adult↗
Monoclonal antibodies recognizing a neoantigen of poly(C9) detect the human terminal complement complex in tissue and plasma.
The terminal complement complex (TCC), consisting of C5b, C6, C7, C8, and C9, contains neoantigens that are absent from the individual native components. Neoantigens are present both in the membrane-bound (MAC) and the fluid-phase (SC5b-9) complex. The present study describes production of monoclonal antibodies against neoantigens of both forms of the TCC. A convenient screening and detection system, based mainly on enzyme-linked immunosorbent assays, crossed immunoelectrophoresis with autoradiography, and affinity chromatography with subsequent sodium dodecyl sulphate-polyacrylamide gel electrophoresis including immunoblotting, is described in detail. Two monoclonal antibodies were specific for a neoantigen located in the poly(C9) moiety of the TCC. One of these antibodies, MCaE11, was used for immunohistochemical detection of MAC in tissue and for quantification of the fluid-phase TCC in ethylenediaminetetraacetic acid plasma.
Antibodies, Monoclonal↗
Quantification of the terminal complement complex in human plasma by an enzyme-linked immunosorbent assay based on monoclonal antibodies against a neoantigen of the complex.
The fluid-phase terminal complement complex (TCC), consisting of the components C5b, C6, C7, C8, C9, and the S-protein, has recently been detected in normal human plasma by using antibodies against native terminal complement components. Increased amounts of TCC were then found in several patients with in vivo activation of complement. We now describe a sensitive, specific, and reliable enzyme-linked immunosorbent assay for quantification of the TCC, based on monoclonal antibodies against a neoantigen of the complex. The results indicate that the TCC is present in normal human plasma and in increased amounts in patients with complement activation in vivo, thus confirming previously obtained results. The assay is easy to perform and can be used for examination of large numbers of plasma samples.
Antibodies, Monoclonal↗
Magnetic monosized polymer particles for fast and specific fractionation of human mononuclear cells.
Magnetic monodisperse polymer particles were developed and the necessary conditions established to use them for both quantification and fractionation of human peripheral blood mononuclear cell populations. The particles consist of a styrene divinylbenzene core into which magnetite has been deposited by an in situ oxidation process. Thereafter the core has been coated with a hydrophilic polymer containing epoxy and hydroxyl groups. The particles have strong nonspecific binding capacity for protein and can be coated with the appropriate antibodies by physical adsorption only. However, the hydroxyl groups on the outer polymer also make covalent coupling possible. After appropriate blocking they can be used in a rosette assay for quantification of mononuclear leukocytes previously sensitized with monoclonal antibodies. Furthermore, a suitable magnet makes it possible to deplete the cell suspension efficiently of the rosette-forming cells. We have thoroughly investigated the functional properties of human mononuclear cells depleted of T lymphocytes by this technique. Our results show that T cells are virtually completely eliminated, as demonstrated by flow cytometry and various functional assay systems.
Antibodies, Monoclonal↗
Enrichment of human epidermal Langerhans dendritic cells.
Langerhans cells (LC) were enriched from a human epidermal cell (EC) suspension by a rosette-forming technique. First EC were sensitized with the monoclonal antibody OKT6, and then they were mixed with ox erythrocytes (ORBC) coated with affinity-purified rabbit IgG anti-mouse IgG antibodies. Rosette-forming LC were then separated from non-rosetting EC by means of Percoll flotation. After hypotonic lysis of the ORBC, the cells obtained had a viability greater than 90%, and 67-98% of the viable cells were OKT6-positive. In functional studies the enriched cells were strongly stimulatory for allogeneic lymphocytes and were able to function as antigen-presenting cells for the T-cell response to the soluble antigen purified protein derivative.
Animals↗
Detection and quantification of the terminal C5b-9 complex of human complement by a sensitive enzyme-linked immunosorbent assay.
An enzyme-linked immunosorbent assay for detection and quantification of the terminal complexes (SC5b-9 and membrane attack complex) of human complement is described. We separate the complex from the native complement components, to use antibodies against the native components in a 'double-antibody sandwich' technique. It is thereby possible to detect the terminal complement complex in solution without the requirement of specific antibodies against the neoantigens. The results show that the assay is both sensitive and specific. Evidence is presented that a terminal complement complex occurs in a normal plasma pool. The terminal complement complex may be valuable for evaluating both the physiology and pathophysiology of the complement system in vivo.
Antibody Specificity↗
Differentiation antigens in human T-cell activation: evidence that anti-VH antibodies react with a membrane structure on human T lymphocytes distinct from the antigens detected by monoclonal antibodies of the OKT and Leu series.
The effect of a panel of monoclonal antibodies and heteroantibodies on T-cell proliferation in various assay systems has been examined. The antibodies tested were directed against T-cell differentiation antigens, HLA-DR antigens, and structures defined by an anti-human VH antiserum. As the test cell system highly purified subpopulations of T-cell growth factor (TCGF)-dependent T-cell lines activated either by mitogen or antigen were used. A survey of the data indicates the following: (1) Mitogenic and antigenic triggering of T lymphocytes are mediated through partly different membrane structures. (2) Antigenic stimulation by purified protein derivative (PPD) as well as polyclonal activation induced by OKT3/anti-Leu 4 monoclonal antibodies can be inhibited by heteroantibodies raised against human immunoglobulin VH fragments thus pointing to a possible connection between the antigens detected by these antisera. (3) There does not seem to be differences between the two major subpopulations of T lymphocytes (i.e., helper/inducer and suppressor/cytotoxic cells) as to how they respond to antigens or mitogens in the investigated assay systems. (4) A clear distinction was found between T blasts specific for PPD and allogeneic cells as compared to cytotoxic T cells (CTL), as the T4 and T8 antigens seem to be functionally important for antigen recognition among CTL but not for the blasts proliferating in response to PPD and allogeneic cells. (5) An inhibitory effect of OKT3/anti-Leu 4, OKIa1, and anti-HLA-DR on TCGF-dependent growth was detected, possibly indicating a steric relationship between these antigens and TCGF receptors on mitogen-induced T blasts. (6) Soluble factors obtained after incubating adherent cells with OKIa1 and anti-HLA-DR antibodies seemed to have an inhibitory effect on overall T-cell proliferation stressing the importance of studying the T-cell activation process at different levels in these kinds of experiments. (7) The results further suggest a complexity in the build up of antigen receptors on the various T-effector cells, perhaps also involving receptors for growth factors, HLA-DR antigens, and receptors for the latter.
Antibodies, Monoclonal↗
Heterogeneity of human amyloid protein AA and its related serum protein, SAA.
The heterogeneity of the human amyloid proteins SAA and AA was studied. Both proteins could be separated into several fractions by ion-exchange chromatography. Amino acid analysis of the ion-exchange-chromatographed fractions of protein AA showed that the main difference was in the length of the polypeptide. Thus, it seems that the original AA preparation consists of a mixture of AA proteins with length ranging from 66 to 78 amino acid residues. By enzymatic degradation of three different forms of SAA with kallikrein, fragments were formed with a molecular weight very similar to that of protein AA.
Amino Acids↗
T-cell immunoregulatory functions in rheumatoid arthritis patients.
We have studied the immunoregulatory function of T8+ (suppressor/cytotoxic) and Leu3a+ (inducer/helper) T cells from rheumatoid arthritis (RA) patients by measuring the effect of these T-cell subpopulations on the generation of immunoglobulin-secreting cells by normal allogeneic B cells after stimulation with pokeweed mitogen (PWM) in vitro. When T8+ or Leu3a+ cells from blood or synovial tissue from nine patients were substituted for T8+ or Leu3a+ cells, respectively, from normal blood mononuclear cells (MNC), RA T8+ cells showed an increased suppressor activity, whereas RA Leu3a+ cells were, except for one patient, weak augmentors. Unreplaced normal MNC and MNC replaced with allogeneic normal T-cell subpopulations responded equally to PWM. When T8+ plus Leu3a+ cells from the same patient replaced normal T cells, high B-cell responses were detected. Normal T8+ plus Leu3a+ cells generally supported the response to a lower degree. Substitution with two allogeneic T-cell subpopulations did not result in a B-cell response to PWM. Thus, whereas RA T8+ seemed to be strong suppressors and RA Leu3a+ cells weak augmentors by themselves, together they are possibly able to generate a B-cell stimulatory potential that might be of pathogenetic significance in the patients.
Adult↗
A rapid rosette technique for quantitation and separation of mononuclear cell subsets using monoclonal antibodies.
We present a rapid and simple method for simultaneous quantitation and separation of mononuclear cell (MNC) subsets. When lymphoid cells are sensitized with monoclonal antibodies of the OK and Leu series, they rapidly form rosettes with ox erythrocytes (ORBC) coated with affinity-purified rabbit IgG against mouse IgG. Rosette-forming cells (RFC) may then be counted and separated from non-rosetting MNC by Isopaque-Ficoll gradient centrifugation. The yield and viability are close to 100% after ORBC lysis. Adherent cells do not interfere. Isolated T8+ and Leu3a+ cells were further tested: the purity was 97-99%, and the cells were functionally intact with respect to their modulating activity on the generation of immunoglobulin-secreting cells by MNC after stimulation with pokeweed mitogen.
Animals↗
Demonstration of immunoglobulin-secreting cells in a sensitive reversed hemolytic plaque assay.
A sensitive modification of a reversed hemolytic plaque assay demonstrating individual immunoglobulin-secreting cells (ISC) is presented. The test is performed by adding test cells to a monolayer of indicator sheep erythrocytes coated with an IgG fraction of rabbit antiserum against the F(ab')2 portion of human immunoglobulins (Ig). After having added a developing antiserum and complement, hemolytic plaques with a central ISC could be counted in an inverted microscope. In addition, when ISC were killed and fixed in situ in the monolayer, the cells could be further studied by immunofluorescence of their cytoplasmic Ig.
Animals↗
Quantitation of cells secreting rheumatoid factor of IgG, IgA, and IgM class after elution from rheumatoid synovial tissue.
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Arthritis, Rheumatoid↗