A solid-phase immunoassay for human immunoglobulin E: use of 125I-labeled protein A as the tracer

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.     

Promoting Protein,a Silkworm Hemolymph Protein Promoting in Vitro Replication of Nucleopolyhedrovirus,Binds to β-Glucans

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.     

Immunoassay using 125I- or enzyme-labeled protein A and antigen-coated tubes

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.     

Immunoassay of 5-methyltetrahydrofolate: Use of 125I-labeled Protein A as the tracer molecule for specific antibody

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.     

In Vivo Kinetics of Oxidatively Modified HDL

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.     

A new approach for assessing cumulative lysosomal degradation of proteins or other macromolecules.

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.     

Specific binding of nonsupressible insulinlike activity to chicken embryo fibroblasts and to a solubilized fibroblast receptor.

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.     

Reactivity of bacterial Fc receptors with bovine immunoglobulins: implications and limitations for quantitative immunochemistry

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.     

Relationship of prolactin receptors to concanavalin A binding

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 (K_d) 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 (K_d ≈ 6.6 · 10^?8 M). At high lectin concentrations, low affinity (K_d ≈ 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.     

Comparative Inhibitory Effects of Antigen and Antibody in the Staphylococcal Enterotoxin Solid-Phase Radioimmunoassay System

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.     

The development of a radioimmunoassay for 12-L-hydroxyeicosatetraenoic acid

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.     

Specific Deposition of Passively Transferred Monoclonal Antibodies against Herpes Simplex Virus Type 1 in Rat Brain Infected with the Virus

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.     

Consequences of 125I-labeled insulin degradation by hepatocytes on the interpretation of receptor binding studies

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.     

Comparison of Direct and Indirect Solid-Phase Microradioimmunoassays for the Detection of Viral Antigens and Antiviral Antibody

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.     

Immunological studies on the Purkinje cells from rat and mouse cerebella. I. Evidence for antibodies characteristic of the Purkinje cells.

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.     

Production and characterization of antibody blocking epidermal growth factor: Receptor interactions

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.     

Binding of HMG-T to trout testis chromatin

When ¹²⁵I-labeled HMG-T was incubated with trout testis nuclei under conditions of pH and ionic strength approximating those $\text{in vivo}$, 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.     

Determination of Serratia protease by radioimmunoassay

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.     

Binding and degradation of 125I-labeled insulin by a clonal line of rat pituitary tumor cells

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.     

Identification of albumin-synthesizing polysomes from mouse liver and a mouse hepatoma cell line.

Albumin-synthesizing polysomes from mouse liver and mouse hepatoma cells in in tissue culture have been localized on sucrose gradients with ¹²⁵I-labeled antimouse serum albumin used as a marker. Competition studies show that the ¹²⁵I-labeled antibody binds specifically to albumin-synthesizing polysomes from both tissues. The ¹²⁵I-labeled polysomes from liver and hepatoma cells have identical sedimentation properties on sucrose gradients, which indicates that the polysomes range in size from 9–14 ribosomes. This is comparable in size to polysomes from rat liver and Morris hepatoma. One significant difference between these albumin-synthesizing polysomes is that those extracted from hepatoma cells bind 70% less antibody than equivalent amounts of polysomes from liver cells. Since the level of albumin synthesis in the hepatoma cells is comparable to the level of albumin synthesis $\text{in vivo}$, this difference in antibody-binding capacity is not likely to be due to differences in polysomal content, but appears to be a characteristic difference between hepatoma and normal mouse liver cells.