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1.
A solid-phase immunoassay has been developed for human immunoglobulin (Ig) E. The specific binding of 125I-labeled protein A (125I-PA) to the Fc region of rabbit IgG anti-IgE served as a quantitative measure of specific anti-IgE antibody bound to the IgE beads under optimal assay conditions. Inhibition of antibody binding by known amounts of standard IgE was reflected in a decreased binding of 125I-PA. The degree of inhibition of 125I-PA binding was related to the amount of fluid-phase IgE present and gave a standard curve which was used to determine the concentration of IgE in test samples. The sensitivity of this method and a double antibody radioimmunoassay (RIA), which was developed using the same IgE preparation and anti-IgE antibody, was approximately the same. These assays gave similar results when used to determine levels of IgE in normal human sera that had been absorbed with protein A—Sepharose to remove components responsible for specific and nonspecific interference in the assays. 相似文献
2.
《Prostaglandins and medicine》1979,2(3):167-175
The sera of rabbits that were injected with indomethacin covalently linked to human albumin bound increasing amounts of [14C]indomethacin during the course of immunization. The serum-binding components chromatographed with the γ-globulin fraction and were precipitated with goat-anti-rabbit-γ-globulin. When measured by radioimmunoassay with [14C]indomethacin under optimal conditions, 34 ng (95 pmoles) of unlabeled indomethacin inhibited [14C]indomethacin binding 50%. The antibodies reacted most effectively with indomethacin and the desmethylated analogue, 1-(p-chlorobenzoyl)-2-methyl-5-hydroxyindole-3-acetic acid, but not 2-methyl-5-methoxy-indole-3-acetic acid.An immunoassay based on the use of [125I]Protein A as tracer for IgG antibody was developed for quantitative determination of indomethacin at the picogram level. Indomethacin, immobilized by covalent linkage to a solid sup port, bound the rabbit anti-indomethacin. Protein A, labeled with [125I], measured the levels of the bound IgG antibody. Fluid phase indomethacin competed with solid-phase indomethacin for the anti-indomethacin which resulted in decreased anti-indomethacin and consequently decreased [MI]Protein A on the immobilized indomethacin-anti-indomethacin complex. The serologic specificity with the immobilized ligand immunoassay was the same as that found with the [14C]indomethacin radioimmunoassay, but the sensitivity for detection of indomethacin was increased over 300-fold. 相似文献
3.
《Bioscience, biotechnology, and biochemistry》2013,77(10):2012-2014
A protein which bound to 125I-labeled peptidoglycan (PGN) was isolated from hemolymph of silkworm larvae. The N-terminal amino acid sequence and the molecular weight of the protein were in accord with those described for Promoting Protein (PP) from the silkworm. The binding of the protein to [125I]PGN was competitively inhibited by various β-glucans. The binding kinetics of PGN and chitin to the protein were analyzed in a biosensor. 相似文献
4.
Antigen-coated plastic tubes were used with 125I- or enzyme-labeled staphylococcal protein A in a general immunoassay method for antigens and haptens. Protein A reacts with immunoglobulin G (IgG) regardless of antibody specificity at sites distal to the antigen combining site and does not inhibit the immune reaction. It therefore serves as a general tracer and its use eliminates the need to purify and to label individual components for each assay. Macromolecular antigens were bound to polystyrene or polypropylene tubes by direct passive adsorption. Haptens with free carboxyl groups were bound covalently to poly-l-lysine and these conjugates passively adsorbed to the tube surface. Optimal assay conditions were established for the quantitative determination of immunoglobulins and the folate derivatives, methotrexate and 5-methyltetrahydrofolate, using 125I-labeled protein A or protein A labeled with alkaline phosphatase. The method has been used to estimate levels of IgG, IgA, IgM, and IgE in serum in volumes up to 1 ml. 相似文献
5.
John J. Langone 《Analytical biochemistry》1980,104(2):347-354
A sensitive and specific solid-phase radioimmunoassay for 5-methyltetrahydrofolate (5-MTHFA) has been developed. 125I-Labeled staphylococcal Protein A (125I-PA) was used as the tracer molecule for rabbit IgG antibodies bound to 5-MTHFA immobilized on polyacrylamide beads. The dose-dependent inhibition of antibody binding by fluid-phase drug was reflected in decreased binding of 125I-PA. This inhibition, determined in the presence of known amounts of 5-MTHFA, served as the basis for quantification of 5-MTHFA in test samples. An early bleeding was relatively specific: 4.5 ng 5-MTHFA inhibited immune binding by 50% compared to 7700 ng folinic acid or 1200 ng tetrahydrofolate. Other folic acid analogs, including methotrexate, failed to inhibit significantly. The assay using a later bleeding was more sensitive since 1.6 ng 5-MTHFA gave 50% inhibition (detection limit 0.2 ng), but folinic acid cross-reacted significantly. Absorption with immobilized folinic acid markedly enhanced the specificity of this antiserum and resulted in a 15–20% increase in maximum inhibition by 5-MTHFA. The assay could be carried out in the presence of 0.025 ml human serum or urine without affecting the standard curve, and was used to determine levels of 5-MTHFA in serum of drug-treated rabbits. 相似文献
6.
《Biochemical medicine and metabolic biology》1993,49(3):392-397
The kinetics of oxidatively modified high-density lipoprotein (HDL) in vivo were investigated. 125I-labeled oxidized (Ox) I-IDL and 131I-labeled native (N) HDL were injected simultaneously into control and WHHL rabbits. The fractional catabolic rates of 125I-labeled Ox-HDL were significantly greater than those of 131I-labeled N-HDL in both control (2.52 ± 0.36/day vs 0.94 ± 0.02/day) and WHHL rabbits (4.07/day vs 1.32/day). Oxidized HDL was catabolized faster than native HDL and was taken up primarily by the liver, spleen, and kidney. 相似文献
7.
A general method is proposed for the direct estimation of the degradation in various tissues of macromolecules that are metabolized by a lysosomal mechanism. This involves coupling to the macromolecule a small molecule that is cleaved from it only after entry into the lysosome, that is not metabolized but is “trapped” in the lysosome, and that therefore accumulates as a direct function of the amount of macromolecule degraded. The feasibility of the method was shown using low density lipoprotein and serum albumin doubly labeled with covalently bound [14C]sucrose and 125I. Uptake by normal fibroblasts, measured in terms of 14C accumulated in the cells, correlated very closely with uptake measured in terms of 125I-labeled metabolites in the medium plus 125I in the cells. 相似文献
8.
J. Zapf M. Mäder M. Waldvogel D.S. Schalch E.R. Froesch 《Archives of biochemistry and biophysics》1975,168(2):630-637
125I-labeled nonsuppressible insulinlike activity—soluble in acid/ethanol (NSILA-S) binds to cultured chicken embryo fibroblasts and to an extract obtained by treating the fibroblasts with Triton X-100. Binding to intact cells and to the cell extract is timetemperature, and pH-dependent and shows saturation kinetics. The apparent intrinsic association constant for binding of NSILA-S to intact cells is 109 M?1, the number of binding sites per cell approximately 6000. Cold NSILA-S preparations of different purity displace bound 125I-labeled NSILA-S according to their biological potency. Moreover, cold NSILA-S displaces bound 125I-labeled NSILA-S in concentrations in which it also stimulates thymidine incorporation into fibroblast DNA. Insulin displaces bound 125I-labeled NSILA-S only at concentrations above 1 mU/ml. Glucagon, ACTH, human growth hormone and inactivated NSILA-S are ineffective. The displacement curves obtained with human serum are similar to those obtained in the presence of cold NSILA-S. 相似文献
9.
Wendy A. Wallner Michael J. P. Lawman Michael D. P. Boyle 《Applied microbiology and biotechnology》1987,27(2):168-173
Summary The interaction of bovine immunoglobulins with staphylococcal Protein A and a group C streptococcal bacterial Fc receptor (FcRc) were compared. The isolated group C streptococcal receptor was reactive with both bovine IgG1 and IgG2. The reactivity of the streptococcal FcRc with IgG2 was approximately 40 fold greater than that observed with IgG1. By contrast, protein A reacted only poorly with bovine IgG2 and no detectable reactivity was observed with IgG1. A two stage competitive binding assay to measure bovine IgG in serum and secretions using 125I-labeled protein A as tracer was developed. This assay was found to be sensitive and reproducible and was used to measure serum IgG levels in cattle of differing ages and breeds. 相似文献
10.
Mark E. Costlow Patricia E. Gallagher 《Biochimica et Biophysica Acta (BBA)/General Subjects》1979,587(2):192-201
Concanavalin A, which binds to specific carbohydrate determinants on the cell surface, was used to investigate the binding of prolactin to its receptors in liver membranes from female rats. The binding of 125I-labeled ovine prolactin to receptors was sharply inhibited by concanavalin A. This effect was reversed by the competitive sugar α-methyl-D-mannopyranoside and thus required the presence of specifically bound lectin. Concentrations of concanavalin A of up to 50 μg/ml caused a progressive decrease in the apparent affinity of the prolactin receptor for hormone. When higher concentrations were used, the number of available binding sites decreased. Concanavalin A-resistant receptors, about 30% of the total, had the same dissociation constant (Kd) as the controls. The binding of 125I-labeled concanavalin A in the same membrane preparations showed the presence of two distinct types of concanavalin A binding. At low concentrations, the lectin bound with high affinity (Kd ≈ 6.6 · 10?8 M). At high lectin concentrations, low affinity (Kd ≈ 6.7 · 10?5 M) binding predominated. Since high affinity concanavalin A binding was saturated at 50 μg/ml, this class of binding most likely alters the affinity of the prolactin receptor for hormone; low affinity concanavalin A binding may mask prolactin receptors, making them inaccessible to the hormone.Binding sites for concanavalin A and prolactin appear to be independent but closely related since (i) concanavalin A did not displace bound prolactin from its receptor, and (ii) detergent-solubilized 125I-labeled prolactin-receptor complexes bound to concanavalin A-Sepharose and were eluted by α-methyl-D-mannopyranoside. 相似文献
11.
Comparative Inhibitory Effects of Antigen and Antibody in the Staphylococcal Enterotoxin Solid-Phase Radioimmunoassay System
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A solid-phase radioimmunoassay employing 125I-labeled enterotoxins and polystyrene tubes coated with specific antibody has been developed for assaying the relative concentrations of antibodies to staphylococcal enterotoxins A and B. Competitive binding occurs between tube-bound antibody and free antibody for binding sites on 125I-labeled enterotoxin. The sensitivity of the system is affected by the amount of antibody on the walls of the tubes, the concentration of 125I-labeled enterotoxin added to the system, and probably by the relative binding affinities of the bound and unbound antibodies. Antibody, 0.01 to 0.07 mug/ml, inhibited the uptake of 125I-labeled enterotoxin by 20%. Both the antibody and antigen solid-phase radioimmunoassay inhibition systems can be appropriately represented by either of the following two models: Loge (Y/1 - Y) = alpha0 + alpha1 LogeX and LogeY = beta0 + beta1 LogeX, where Y is bound activity, X is antigen or antibody concentration for inhibition, and alpha0, alpha1, beta0, and beta1 are regression coefficients. Estimates from the first model were slightly more precise for the antibody system, whereas the reverse was true for the antigen system. 相似文献
12.
Lawrence Levine Iftekhar Alam Hilda Gjika Thomas J. Carty Edward J. Goetzl 《Prostaglandins & other lipid mediators》1980,20(5):923-934
Antibodies directed toward 12-L-hydroxyeicosatetraenoic acid (12-L-HETE) were generated in rabbits by immunization with conjugates of 12-L-HETE and human serum albumin. The concentration of antibodies was determined by incubating immune plasma with 12-L-HETE that had been covalently linked to a solid support, washing the 12-L-HETE support, and measuring the quantity of bound antibodies by reaction with [125I] Protein A. The addition of 0.5 ng-10 ng of fluid-phase 12-L-HETE to the standard mixture of solid-phase 12-L-HETE and anti-12-L-HETE plasma inhibited by 21–80% the binding of antibodies and consequently of [125I]Protein A to the solid support. The 12-OH function positioned between two double bonds was the immunodominant determinant of this antigen-antibody reaction, but the carboxyl function also was recognized. This radioimmunoassay was used to detect and quantitate 12-L-HETE resolved by high pressure liquid chromatography. 相似文献
13.
Koh-ichi Tamada Shigeki Fujinaga Rihito Watanabe Ryoko Yamashita Yoshinao Takeuchi Mitsuru Osano 《Microbiology and immunology》1995,39(11):861-871
The kinetics of human monoclonal antibody (anti-gB) to herpes simplex virus type 1 (HSV-1) were investigated after intravenous injection of anti-gB into an HSV-1 encephalitis animal model. Immunohistochemical study revealed specific deposition of passively tansferred anti-gB in the hippocampus and thalamus of the infected rat brain, and it bound to the same neurons in which HSV-1 antigen was positively stained. To examine the macroscopic distribution of anti-gB in the infected brain, we undertook an 125I-labeled anti-gB injection study, and the same distribution of 125I-labeled anti-gB deposition was observed by brain semimicroautoradiography as in the immunohistochemical study. These results suggest that anti-gB easily permeates the capillary wall and is deposited in the inflammatory site where HSV-1-specific antigen is detectable. The use of radioisotope-labeled anti-gB injection and external brain imaging could lead to a noninvasive diagnostic tool for the early detection of HSV-1 antigen in cases of suspected HSV-1 encephalitis. 相似文献
14.
Michael McCaleb David B. Donner 《Biochimica et Biophysica Acta (BBA)/Molecular Cell Research》1982,720(2):147-153
The association of 125I-labeled insulin with hepatocytes was assayed by filtration or microcentrifugation. Assay by centrifugation resulted in a greater amount of retained radioactive label throughout the course of association of 125I-labeled insulin with hepatocytes. Similarly, saturation experiments assayed by microcentrifugation suggested greater binding than filtration. During dissociation, cells isolated by centrifugation released a greater amount of rapid-dissociating radioactive label. Control experiments of [3H]-inulin exclusion with cell pellets, which were isolated during microcentrifugation, demonstrated that the difference between the methods was not due to extracellular trapping of radioactivity. Therefore, the data suggested that there was more low-affinity retention when binding was assayed by centrifugation than filtration. The integrity of the 125I-labeled insulin extracted from hepatocytes was determined by column chromatography. A substantially greater proportion of the extracted radioactivity was fragments of 125I-labeled insulin in cells isolated by centrifugation. It is suggested that the extensive washing of the cells during filtration removes more fragments than does centrifugation. During dissociation, the low-affinity component of radioactivity, which was observed in the centrifugal assay, resulted from the transient retention of insulin fragments. The extensive degradation of insulin, which was assayed by either method, and the differences observed between these methods, should be considered in the interpretation of binding experiments with cells. 相似文献
15.
Comparison of Direct and Indirect Solid-Phase Microradioimmunoassays for the Detection of Viral Antigens and Antiviral Antibody 总被引:14,自引:1,他引:13
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Viral antigens were fixed to the surface of microtiter wells, and serial dilutions of antiviral antibody were added. The amount of antiviral antibody bound to viral antigens was determined by measuring the extent to which the antiviral antibody either inhibited the specific binding of (125)I-labeled antiviral immunoglobulin G (IgG) (direct technique) or enhanced the specific binding of (125)I-labeled anti-IgG (indirect technique). Immune complexes composed of viral antigens and antiviral antibody (human) could be detected by the binding of (125)I-labeled rheumatoid factor. Specific binding was influenced by the concentration of protein in the diluents used during the different steps of the procedure. A high concentration of protein in the diluent used with the viral antigens decreased specific binding, whereas a high concentration of protein in the diluent used with (125)I-labeled anti-IgG increased specific binding by decreasing nonspecific attachment of the labeled anti-IgG. Under the conditions employed, the titer of a given antiviral serum was several hundredfold greater by the indirect than by the direct technique. 相似文献
16.
Antibodies have been raised against an enriched preparation of isolated rat cerebellar Purkinje cells. The immunoglobulins were labeled with 125I and the strength and specificity of the serum determined by a direct binding assay on cerebellar membranes. About 2% of the 125I-labeled IgG bound to an excess of cerebellar membranes. Absorption with heart and liver membranes removed 80.5% of the 125I-labeled IgG binding to cerebellar membranes; absorption with cerebrum membranes removed 13% more; the remaining 6.5% were directed specifically against cerebellar membranes. An enriched 125I-labeled anti-Purkinje antibody population was prepared by absorption and subsequent elution from cerebellar membranes. The absorption pattern with heart, liver, and cerebrum membranes resembled that found with the total population of IgG except that the nonspecific binding was significantly diminished. The Purkinje cell degeneration (pcd) mouse mutant was used to assess the specificity of the serum toward the Purkinje cells. After absorption of the enriched anti-Purkinje antibodies with heart, liver, and cerebrum membranes, the binding of labeled IgG to membranes prepared from pcd/pcd cerebella was about one-fourth that found with control cerebella. The direct immunoperoxidase technique performed on smears of purified Purkinje and granule cells shows that the unabsorbed serum stains both classes of cells, but that after absorption with liver, heart, and cerebrum membranes, only the Purkinje cells react positively. 相似文献
17.
Membranes were prepared from the human epithelioid carcinoma cell line A-431 which has approx. 2 · 106 epidermal growth factor receptors per cell. This membrane preparation which retained a high epidermal growth factor binding specific activity was used as an antigen to produce antisera in rabbits. Double-immunodiffusion experiments demonstrated that the immune serum contained precipitating antibodies to several components of detergent solubilized A-431 membranes.The immonoglobulin G fraction of this immune sera inhibited 125I-labeled epidermal growth factor binding to receptors in: (1) intact human and mouse cells; (2) membrane preparations from A-431 cells and human placenta, and (3) solubilized A-431 membranes. Inhibition of 125I-labeled epidermal growth factor binding was observed with divalent and monovalent fragments of immunoglobulin G prepared from the immunoglobulin G fraction. Also, the immunoglobulin G fraction blocked growth factor binding to membranes at low temperature (5°C).Anti-A-431 antibody blocked the induction of DNA synthesis in quiescent fibroblasts by epidermal growth factor in a manner similar to that of anti-epidermal growth factor antibody. Addition of either anti-A-431 or anti-epidermal growth factor antibodies to fibroblasts at times up to 5 h after the addition of epidermal growth factor completely reversed the hormone's mitogenic potential. At later times (after 12 h) addition of either antibody was without effect on the stimulation of DNA synthesis by epidermal growth factor. Anti-A-431 antibody did not block the induction of DNA synthesis in fibroblasts by fibroblast growth factor or serum. 相似文献
18.
Binding of HMG-T to trout testis chromatin 总被引:1,自引:0,他引:1
L Kuehl T Lyness D C Watson G H Dixon 《Biochemical and biophysical research communications》1979,90(2):391-397
When 125I-labeled HMG-T was incubated with trout testis nuclei under conditions of pH and ionic strength approximating those , most of the radioactivity bound to the chromatin. Most labeled non-nuclear proteins which were tested did not bind. Four large cyanogen bromide fragments of HMG-T each bound, suggesting that HMG-T interacts with chromatin along most of its length. Trout testis chromatin contains two populations of HMG-T molecules which differ in their extractability with NaCl solutions; the 125I-labeled protein equilibrated mainly with the more readily extracted population. HMG-T also bound to nuclease-treated chromatin, an observation with important implications for studies in which nucleases are employed to probe chromatin structure. 相似文献
19.
Determination of Serratia protease by radioimmunoassay 总被引:1,自引:0,他引:1
A specific, highly sensitive radioimmunoassay has been developed for the determination of Serratia protease. The radioimmunoassay (RIA) was based upon competition of the protease with 125I-labeled protease for antiprotease, followed by a second antibody to separate bound enzyme from free enzyme. The RIA provided a range of 1 to 10 ng for determining the enzymes under conditions in which the enzymatic activity could not be measured. The assay was completely inhibited in the presence of human plasma. The inhibition resulted from a complex formation of the enzyme with plasma α2macroglobulin. By treatment of the complex with acetone, however, the RIA could be achieved. 相似文献
20.
Robert E. Corin F.Carter Bancroft Martin Sonenberg David B. Donner 《Biochimica et Biophysica Acta (BBA)/Molecular Cell Research》1983,762(4):503-511
Receptor sites for insulin on GH3 cells were characterized. Uptake of 125I-labeled insulin by the cells was dependent upon time and temperature, with apparent steady-states reached by 120, 20 and 10 min at 4, 23 and 37°C, respectively. The binding sites were sensitive to trypsin, suggesting that the receptors contain protein. Insulin competed with 125I-labeled insulin for binding sites, with half-maximal competition observed at 5 nM insulin. Neither adrenocorticotropic hormone nor growth hormone competed for 125I-labeled insulin binding sites. 125I-labeled insulin binding was reversible, and saturable with respect to hormone concentration. 125I-labeled insulin was degraded at both 4 and 37°C by GH3 cells, but not by medium conditioned by these cells. After a 5 min incubation at 37°C, products of 125I-labeled insulin degradation could be recovered from the cells but were not detected extracellularly. Extending the time of incubation resulted in the recovery of fragments of 125I-labeled insulin from both cells and the medium. Native insulin inhibited most of the degradation of 125I-labeled insulin suggesting that degradation resulted, in part, from a saturable process. At steady-state, degradation products of 125I-labeled insulin, as well as intact hormone, were recovered from GH3 cells. After 30 min incubation at 37°C, 80% of the cell-bound radioactivity was not extractable from GH3 cells with acetic acid. 相似文献