Studies of 125I trace labeling of immunoglobulin G by chloramine-T.
TL;DR: Among the parameters which were found to influence the individual and over-all reactions and which were studied to establish optimal conditions were the concentrations of oxidant, iodide, and protein as well as pH, time, temperature, ionic strength, and particular batch of protein.
About: This article is published in Immunochemistry.The article was published on 1970-11-01. It has received 216 citations till now. The article focuses on the topics: Iodide & Population.
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TL;DR: The ability of the hybridoma IgG to inhibit mouse FcRII was independent of the major histocompatibility complex and the monoclonal 2.4G2 IgG antigenic determinant was not present on rat, guinea pig, rabbit, or human F cR-bearing cells.
Abstract: To investigate the antigenic relationship between the macrophage and lymphocyte Fc receptors (FcR), a monoclonal antibody capable of blocking mouse macrophage Fc receptors was selected. Hybrids were formed by fusing the P3U1 mouse myeloma and spleen cells from a rat immunized with the mouse macrophage-like cell lines J774 and P388D1. The Fab fragment of the monoclonal IgG secreted by clone 2.4G2, inhibited the trypsin-resistant Fc receptor II (FcRII), which is specific for immune aggregates of mouse IgG1 and IgG2b, but had no inhibitory effect on the trypsin-sensitive Fc receptor I (FcRI), which binds monomeric IgG2a and erythrocytes coated with IgG2a. Thus, the monoclonal 2.4G2 IgG appeared to be specific for macrophage FcRII. Further evidence that the 2.4G2 IgG was directed against FcRII came from binding studies of the monoclonal antibody to J774 cells and a series of independently isolated variants which do not express FcRII. These variants of J774 bound 5% as much of the monoclonal antibody as the parent line, which bound 600,000 molecules of 2.4G2 IgG per cell. The antigenic relatedness of mouse lymphocyte FcR to mouse macrophage FcRII was demonstrated by the binding of 2.4G2 IgG to FcR-bearing lymphoid cell lines and the inhibition of the lymphocyte FcR by the monoclonal antibody. Preincubation of spleen cells and peritioneal cells with 2.3G2 IgG likewise inhibited rosette formation with ox erythrocytes coated with rabbit IgG. The ability of the hybridoma IgG to inhibit mouse FcRII was independent of the major histocompatibility complex. The 2.4G2 IgG antigenic determinant was not present on rat, guinea pig, rabbit, or human FcR-bearing cells.
1,263Â citations
TL;DR: The 1/20/5-D-4 monoclonal antibody appears to recognize a common determinant in their polysaccharide moieties, consistent with several biochemical analyses showing the absence of keratan sulfate in proteoglycan synthesised by this tissue.
385Â citations
TL;DR: The effects of various treatments on nucleoid integrity were investigated and structures resembling nuclei but depleted of protein may be released by gently lysing cells in solutions containing non-ionic detergents and high concentrations of salt.
Abstract: Structures resembling nuclei but depleted of protein may be released by gently lysing cells in solutions containing non-ionic detergents and high concentrations of salt. These nucleoids sediment in gradients containing intercalating agents in a manner characteristic of DNA that is intact, supercoiled and circular. The concentration of salt present during isolation of human nucleoids affects their protein content. When made in I-95 M NaCl they lack histones and most of the proteins characteristic of chromatin; in 1-0 M NaCl they contain variable amounts of histones. The effects of various treatments on nucleoid integrity were investigated.
326Â citations
TL;DR: From the rapidity of association and dissociation, it appears that the surface of the macrophage is in a dynamic equilibrium with IgG2a molecules in the cell's immediate microenvironment.
Abstract: The binding properties of surface receptors of immunoglobulins on mouse macrophages were studied with mouse myeloma proteins and normal peritoneal macrophages, thioglycollate-stimulated macrophages, and a macrophage cell line, P388D1. Primary cultures of mouse embryo fibroblasts served as controls. IgG2a proteins were bound strongly;IgG2b was bound weakly (one-twentieth as well as IgG2a);IgM, IgA, and IgG1 were not bound significantly. The number of binding sites per cell for IgG2a was 4 X 10(5) for thioglycollate-stimulated cells and 1 X 10(5) for normal and P388D1 cells. Binding was exothermal: with decreasing temperature the equilibrium (association) constants increased and dissociation rate constants decreased (at 37degreesC the respective values were 2 X 10(7) M-1 and 0.26 min-1, the latter value corresponds to a half time for dissociation of 2.6 min). From the rapidity of association and dissociation, it appears that the surface of the macrophage is in a dynamic equilibrium with IgG2a molecules in the cell's immediate microenvironment. The receptors for IgG2a are clearly specific for determinants in the immunoglobulin constant domain: two IgG2a proteins with greatly different isoelectric points (determined by isoelectric focusing) were bound with the same affinity to the same receptors; moreover, the Fc fragment was bound and Fab fragments were not. The Fc receptors for IgG2a proteins were readily eliminated by exposing macrophages briefly to trypsin. The receptors were regenerated during subsequent cultivation in serum-free medium; regeneration was inhibited totally by cycloheximide and partially by actinomycin D.
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264Â citations
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TL;DR: The loss of immunological reactivity at high specific radioactivities or at high levels of chemical substitution with STAI/sup 127/!iodine is demonstrated.
Abstract: A simple and rapid method is presented for the preparation of I/sup 131/- labeled human growth hormone of high specific radioactivity (240-300 mu C/ mu g). Low amounts of carrierfree I/sup 131/ iodide (2 mC) are allowed to react, without prior treatment, with small quantities of protein (5 mu g) in a highyield reaction (approx. 70% transfer of I/sup 131/ to protein). The degree of chemical substitution is minimized (0.5- 1.0 atom of iodine/molecule of protein) by the use of carrier-free I/sup 131/ iodide. The I/sup 131/-labeled hormone (up to 300 mu C/ mu g) contains no detectable degradation products and is immunologically identical with the unlabeled hormone. The loss of immunological reactivity at high specific radioactivities or at high levels of chemical substitution with STAI/sup 127/!iodine is demonstrated. (auth)
10,047Â citations
TL;DR: Values greater than 50 per cent can be obtained by adding oxidizing agents to liberate iodine from iodide, but most if not all of these appear to affect adversely the properties of the labelled protein.
Abstract: IN the methods of iodination currently used only the cationic portion of the iodine molecule becomes bound to the ring structure of tyrosine, so that the theoretical efficiency of labelling is 50 per cent. In practice, efficiencies are always lower than this and may be only a few per cent when the ratio of iodine to protein used is less than one atom per molecule. Values greater than 50 per cent can be obtained by adding oxidizing agents to liberate iodine from iodide, but most if not all of these appear to affect adversely the properties of the labelled protein.
2,409Â citations
TL;DR: For years investigators have sought an assay for insulin which would combine virtually absolute specificity with a high degree of sensitivity, sufficiently exquisite for measurement of the minute insulin concentrations usually present in the circulation as mentioned in this paper.
Abstract: For years investigators have sought an assay for insulin which would combine virtually absolute specificity with a high degree of sensitivity, sufficiently exquisite for measurement of the minute insulin concentrations usually present in the circulation. Methods in use recently depend on the ability of insulin to exert an effect on the metabolism of glucose in vivo or in excised muscle or adipose tissue. Thus, the insulin concentration in plasma has been estimated: a) from the degree of hypoglycemia produced in hypophysectomized, adrenalectomized, alloxan-diabetic rats (1); b) from the augmentation of glucose uptake by isolated rat hemidiaphragm (2); or c) from the increased oxidation of glucose-1-C14 by the rat epididymal fat pad (3). Since there have been reports indicating the presence, in plasma, of inhibitors of insulin action (4) and of noninsulin substances capable of inducing an insulin-like effect (5,6), these procedures, while yielding interesting information regarding the effects of various plasmas on glucose metabolism in tissues, are of doubtful specificity for the measurement of insulin per se (5).
2,311Â citations
1,860Â citations
TL;DR: Proteins differ in their susceptibility to iodination by this method, and human gammaG immunoglobulin, for example, is iodinated more than ten times as readily as is human alpha(2)-macroglobulin under the same conditions.
Abstract: 1. A method is described for the trace iodination of immunoglobulins and other serum proteins by a system consisting of lactoperoxidase, hydrogen peroxide and iodide. 2. gammaG immunoglobulin that had been labelled to a specific radioactivity of 5muc/mug. by use of carrier-free [(125)I]iodide gave no evidence of denaturation when analysed by electrophoresis and density-gradient ultracentrifugation. 3. Tryptic hydrolysis and peptide ;mapping' of a completely characterized peptide radioiodinated by this method showed that the [(125)I]iodide was bound to tyrosyl residues. 4. Proteins differ in their susceptibility to iodination by this method. Human gammaG immunoglobulin, for example, is iodinated more than ten times as readily as is human alpha(2)-macroglobulin under the same conditions. 5. Lactoperoxidase catalyses the iodination of proteins much more readily than does horseradish peroxidase.
1,335Â citations