Binding Capacity Assay Research Articles

Effects of cytokines on insulin-like growth factor-binding protein secretion by muscle cells in vitro

The insulin-like growth factors (IGF-I and IGF-II) stimulate muscle cell proliferation and/or differentiation (depending upon the culture conditions). They also increase IGF-binding protein (IGFBP) levels in muscle cell conditioned media and in some instances there is a direct correlation between the apparent rate of IGFBP secretion and muscle cell proliferation. We have investigated the effect of other cytokines on muscle cell IGFBP conditioned media levels using rat skeletal (L6), mouse myocytes (BC3H-I) and porcine vascular smooth (pVSM) muscle cells in vitro to determine if this relationship is maintained. IGFBP levels in conditioned media (CM) were measured by an [125I]IGF-I binding capacity assay and by western blot analysis. Immunoblots indicated that BC3H-1 and L6 cells secrete IGFBP-5 (31-32,000 M(r)), L6 cells secrete IGFBP-4, and pVSM cells secrete IGFBP-2 (34,000 M(r)). Both L6 and BC3H-1 cells responded to transforming growth factor beta 1 (TGF-beta 1), in a dose-dependent manner, with suppressed conditioned media levels of IGFBP-4 and IGFBP-5 but TGF-beta 1 did not affect IGFBP-2 levels in pVSM cell media. TGF-beta 1 (5 ng/ml) suppressed IGFBP levels (CM [125I]IGF-I binding capacity) in L6 and BC3H-1 cell media by 48% and 61%, respectively. IGFBP-5 levels, in BC3H-1 cell media, were decreased by treatment with either basic fibroblast growth factor (bFGF) or epidermal growth factor (EGF). Neither treatment affected IGFBP-2 levels. In contrast in L6 myoblasts, bFGF increased media levels of IGFBP-4 and IGFBP-5; L6 cells were not responsive to EGF. Insulin increased IGFBP-4 and IGFBP-5 levels in L6 and BC3H-1 cell media. This stimulatory effect was markedly suppressed by either TGF-beta 1 (L6 and BC3H-1 cells) or bFGF (BC3H-1 cells). L6 and BC3H-1 cell CM IGFBP levels were also suppressed 34% and 84% by 5 U/ml thrombin, respectively. The inhibitory activity of thrombin was specific, i.e. reversible by hirudin and was not due to direct IGFBP proteolysis. Since suramin and staurosporine increased media levels of the IGFBP, this suggests that constitutive secretion of TGF-beta 1, bFGF, or EGF might provide a tonic suppressive mechanism for controlling IGFBP secretion. Thus, our results support the conclusion that the secretion of IGFBP-4 and IGFBP-5, but not IGFBP-2, by muscle cells was suppressed by several cytokines. Depressed IGFBP secretion may play a key role in determining muscle cell responsiveness to either the mitogenic or differentiation stimulating activity of the IGFs.

A Leu → His substitution at residue 93 in human corticosteroid binding globulin results in reduced affinity for cortisol

A steroid binding capacity assay and a radioimmunoassay were both used to measure corticosteroid binding globulin (CBG) in serum samples from 22 patients with sepsis. An ∼ 50% discordancy between two values in one patient suggested the presence of a CBG variant with reduced affinity for cortisol, and this was confirmed by Scatchard analysis. We therefore used the polymerase chain reaction to amplify exons that encode for human CBG from the genomic DNA of this patient. This revealed two mutations within the coding sequences: one of which results in a Leu → His substitution at residue 93 and another which encodes a Ser → Ala substitution at residue 224 of the human CBG polypeptide. To assess the impact of each substitution on the steroid binding affinity of CBG, each mutation was introduced separately into a normal human CBG cDNA, and the normal and mutated cDNAs were expressed in Chinese hamster ovary cells. Scatchard analysis of the CBG produced in culture indicated that the His93 mutation ( K d = 2.24 ± 1.75 nM) reduced the cortisol binding affinity of CBG (mean ± SD) significantly ( P < 0.024) when compared to normal CBG ( K d = 0.64 ± 0.31 nM), while the Ala224 mutation ( K d = 0.63 ± 0.33 nM) did not influence cortisol binding affinity. We therefore conclude that residue 93 may play an important role in determining the structure of the CBG steroid binding site.

Prognostic Significance of Quantitative Levels of HIV p24 Binding Capacity in HIV Infection

Human immunodeficiency virus, type 1 (HIV-1), produces a chronic infection with a long latency before clinical disease. We followed 214 untreated subjects for 12-42 months to study the natural history of HIV infection: 110 were classified as asymptomatic, 11 as AIDS-related complex (ARC), 15 as AIDS with Kaposi's sarcoma (KS), 31 as AIDS with opportunistic infections (AIDS/OI), and 47 were HIV-seronegative controls. The quantitative capacity of serum to complex HIV p24 antigen, termed the p24 binding capacity (p24 BC), and quantitative levels of HIV p24 antigen in serum were determined at regular intervals. For people in all diagnostic groups, a p24 BC below 31 ng/ml was more closely associated with progression to AIDS/OI than was p24 antigen positivity; 94% of AIDS/OI, 86% of ARC, 56% of AIDS/KS, and 19% of asymptomatic subjects had p24 BC less than 31 ng/ml during the study period, while 67% of AIDS/OI, 27% of ARC, 61% of AIDS/KS, and 20% of asymptomatic subjects were p24 antigenemic. Prospective analysis of 47 asymptomatic seropositive men followed for 3 years, who showed actuarial progression rates to ARC at 4%, 13%, and 23% and to AIDS at 5%, 8%, and 8% at 1, 2, and 3 years, indicated that entry levels of p24 BC below 31 ng/ml were as strongly associated with progression to ARC/AIDS as was p24 antigenemia (p = 0.0003 vs. p = 0.008). The p24 binding capacity assay is a new and convenient methodology to measure immunocomplexing antibody to HIV p24 and is a powerful indicator of progressive HIV disease.

An enzyme-linked immunosorbent assay (ELISA) for human corticosteroid-binding globulin

A specific and sensitive enzyme-linked immunosorbent assay (ELISA) of the “sandwichtype” for human corticosteroid-binding globulin was developed. A polyclonal rabbit anti-CBG antibody is immobilised to a microtitre plate. Following incubation of standards and samples a second monospecific rabbit anti-CBG antibody, labelled with alkaline phosphatase, is added. After colour development the microtitre plate is read at 405 nm wavelength. The assay shows good agreement to CBG binding capacity assay and commercially available RIA.

Noninterference of isosulfan blue on estrogen-receptor activity.

Because a 1% sterile solution of methylene blue used for occult breast tumor localization has been shown to interfere with the estrogen-receptor protein (ERP) binding-capacity assay, isosulfan blue in a 1% injection was studied as a potential alternate stain. Cytosols derived from ERP-positive lyophilized powders and human breast tissue were evaluated with and without varying levels of treatment with isosulfan blue. No modification of the ERP-specific binding capacity was found with this stain. The use of isosulfan blue for localization of occult breast tumor is suggested when an ERP binding capacity assay is anticipated.

Time-resolved immunofluorometric assay of sex-hormone binding globulin.

A time-resolved immunofluorometric assay (trlFMA) for human sex-hormone binding globulin (SHBG) is described in which antibody-coated tubes or microliter strip-wells and a europium (Eu) chelate-labeled monoclonal antibody are used. The trlFMA sensitivity is similar to that of other SHBG immunoassays, and other analytical variables compare favorably with an SHBG immunoradiometric assay (IRMA) kit and a steroid binding capacity assay: the interassay coefficient of variation (CV) is less than 8% and the intra-assay CV is less than 6% for concentrations between 6 and 200 nmol/L. The reference intervals (means +/- SD) for SHBG concentrations (nmol/L) in serum from 10 men, 10 women, and 10 pregnant women were 23 +/- 12, 65 +/- 39, and 439 +/- 122, respectively. In 14 hirsute women the mean +/- SD serum SHBG concentration (37 +/- 21 nmol/L) was significantly lower (P less than 0.01) than the mean for an age-matched, nonhirsute female comparison group. The trlFMA is technically simple, requires no centrifugation or separation reagent, and takes a counting time of only 1 s. In addition, the Eu-label is nontoxic, presents no waste-disposal problems, and has a long shelf life.

An enzyme-linked immunosorbent assay (ELISA) for human sex hormone-binding globulin

An enzyme-linked immunosorbent assay (ELISA) of the “sandwich-type” for sex hormone binding globulin (SHBG) has been developed. A rabbit anti-SHBG antibody (RAb) is immobilized to the microtitre plate. After incubation with standards and samples a second monospecific rabbit anti-SHBG antibody, labelled with alkaline phosphatase is added (RAb ∗). Following further washing substrate is added, colour developed and the plate read at 405 nm wavelength on a standard ELISA plate reader. The assay is not influenced by the presence of steroids at the binding site, and shows good agreement to SHBG binding capacity assay and commercially available IRMA. Its sensitivity, specificity and precision allows its use in the routine laboratory. The SHBG ELISA has been used to measure SHBG concentrations in sera of normal men, women, pregnant women, and women receiving high-dose medroxyprogesterone acetate as a treatment of metastatic breast cancer.

Immunoturbidimetric assay of transferrin: effect of iron and need for serum blanks

The introduction of specific antisera has led to the replacement of total iron binding capacity assays with direct immunochemical measures of transferrin. However, some controversy exists over the effect of the degree of iron saturation on antigen-antibody interactions in these immunochemical assays. This study describes a simple automated immunoturbidimetric assay of transferrin, and includes a reference range and an evaluation of the effects of common potential interferents, including iron, bilirubin, lipemia, and hemoglobin. The measured and the calculated TIBC are also compared for 78 healthy individuals and 51 patients with anemia.

Proposal for the standardization of the serum unsaturated iron binding capacity assay, and results in groups of subjects with normal iron stores and with prelatent, latent, and manifest iron deficiency

An effort has been made to standardize the indirect iron saturation excess method for the determination of the serum unsaturated iron binding capacity (UIBC) and thus to relinquish the direct adsorption methods for the assay of the serum total iron binding capacity (TIBC) which give falsely high results due to unspecific binding of the saturating iron to serum proteins. In order to eliminate the interfering effects of hydrolytic polymerization of iron(III) on the saturation of apotransferrin in serum and on the colorimetric determination of the unbound iron excess at pH 8.3, conditions have been studied for the preparation of the iron-nitrilotriacetate-complex (Fe(NTA) 2) solution at pH 8.3 with respect to its reactivity with the reductant sodium ascorbate and with the chromogen bathophenanthroline-disulfonate in photometric standards and in samples containing iron-saturated serum. The validity of the results for the UIBC thus obtained has been investigated (1) by direct spectrophotometric titration with Fe(NTA) 2 of the apotransferrin in serum by measuring the absorbance of transferrin at 470 nm in 50-mm cuvettes, and of the UIBC using the modified indirect iron saturation excess assay, both of which gave the same saturation points, and (2) by the correlation of the TIBC obtained from serum iron determinations and the UIBC, with the transferrin concentration measured by the radial immunodiffusion assay. Results of UIBC determinations are presented along with serum iron concentration, TIBC, and transferrin saturation in groups of subjects with normal iron stores and prelatent, latent, and manifest iron deficiency.

Changes in Physiologically Free Circulating Estradiol and Testosterone during Exposure to Levonorgestrel*

The biological activity of circulating sex steroids is dependent upon the relative binding to (SHBG) sex hormone-binding globulin. Only that fraction which is not specifically bound to the high affinity binding protein is available to the receptors or for metabolism. (D-Ng) Levonorgestrel, a synthetic gestagen used for contraception decreases the hepatic production of SHBG. Volunteers wearing contraceptive vaginal rings containing (E2) estradiol and d-Ng were studied to determine the changes in sex steroid binding which occur when d-Ng is administered in a sustained release fashion. The % of steroids not specifically bound to SHBG was measured by fractionating the serum proteins with ammonium sulfate precipitation. A binding capacity assay was used to quantitate SHBG. During the normal menstrual cycle in control subjects, the unbound fractions of both E2 and (T) testosterone remained constant at about 25% of total E2 and 10% of total T. During treatment with d-Ng, however, the % of unbound E2 increased to about 80% of the total, and that of T increased to about 55% of the total, significantly greater than that in the control cycles (p 0.01). SHBG was constant during the normal menstrual cycle, averaging 85.9 + or - 1.92 nM but was suppressed during the administration of d-Ng to 10.0 + or - 2.6 nM. When SHBG concentration was greater than 50 nM, the % binding of both E2 and T were independent of the concentration of this binding protein. When SHBG was suppressed below 50 nM, the % binding of E2 and T was directly related to the concentration of the binding protein. The affinity of SHBG for d-Ng allows competition with E2 and T for SHBG-binding sites at concentrations of SHBG below 50 nM. The increase in physiologically free E2 and T, therefore, may be a result of both the suppression of SHBG concentration by d-Ng as well as the competition between d-Ng and endogenous sex steroids for the decreased number of available binding sites on SHBG.

Measurement of androgen receptor in cytosols from normal, benign hypertrophic and cancerous human prostates.

The binding of R 1881 in cytosols from the normal, benign hypertrophic and cancerous human prostates was examined in the presence of molybdate and triamcinolone acetonide. The addition of 10 mM Na2MoO4 resulted in an increase in the stability of the binder without any change in Kd. Triamcinolone acetonide added to the incubation of cytosol with R 1881 modified the inhibition pattern by other steroids, and the binding in the presence of triamcinolone acetonide exhibited the characteristics of androgen receptor. The complex of cytosol and R 1881 formed in the incubation in the presence of triamcinolone acetonide was sedimented at 8.5S. Kd of the binding to R 1881 of the normal and pathological prostates was almost identical, but maximum binding sites of the androgen receptor were larger in benign hypertrophic and cancerous prostates than in normal tissues. For the assay of binding capacity in needle biopsy specimens, one point determinations were performed using 2.5 nM R 1881 as the ligand. The number of binding sites obtained by this method were well correlated with those obtained by the Scatchard plot in various prostatic tissues.

Rapid Fluorometric Assay of Bilirubin and Bilirubin Binding Capacity in Blood of Jaundiced Neonates: Comparisons with Other Methods

The concentrations of total blood bilirubin, albumin-bound bilirubin, and the reserve and total bilirubin binding capacities of 35 neonatal blood samples (28 patients) were determined by automated front-face fluorometry ((hematofluorometer). These values were compared to results of diazo determinations, Sephadex gel filtration, and peroxidase-oxidation methods. Total blood bilirubin level by fluorometry agreed well with the total plasma bilirubin level by diazotization (r = .96, sigma = 1.7 mg/100 ml). Albumin-bound bilirubin concentrations by fluorometry also correlated well with diazo values (r = .95, sigma = 1.9 mg/100 ml) and were slightly lower than the total blood bilirubin concentrations. Values for total bilirubin binding capacity determined by fluorometry agreed well with results obtained for the same specimens by Sephadex gel filtration (n = 28, r = .97, sigma = 1.8 mg/100 ml) and by peroxidase-catalyzed oxidation (n = 25, r = .97, sigma = 1.7 mg/100 ml). The agreement among the results obtained by the three methods indicates a well-defined in vitro end point at which available primary or "tight" binding sites on albumin are saturated with bilirubin. In this clinical experience the coefficient of variation of results with the hematofluorometer was 8.4% for total blood bilirubin and 6.5% for total binding capacity. A comparison of "sick" with "well" infants revealed that the fraction of bilirubin not bound to albumin was significantly different for these two groups. The assays made with the hematofluorometer are quick (10 to 15 minutes) and require only a small quantity (approximately 150 microliters) of blood.

Mechanisms of inhibition of active transport ATPases by mercurials

Inhibition by methylmercury and mercuric chloride of Mg,Ca ATPase and Na,K ATPase activities in human erythrocyte ghosts was correlated with the binding capacity of ghosts for the mercurial. Full inhibition was always reached below saturation of binding capacity, and half-inhibition at levels as low as 10% saturation. Under such conditions, concentrations of free inhibitor were negligibly low, and existing mathematical models of inhibition were not applicable. New inhibitor partition equations were introduced to model the mechanisms of action of mercurials. Up to 7 methylmercury groups were calculated to bind to one Na,K ATPase molecule at non-inhibitory sites, while only one reacted with the inhibitory site. Mg,Ca ATPase showed simple one-hit inhibition (one mercurial per enzyme); further washing of ghosts, however, unmasked a second binding site (cooperative two-hit inhibition). Affinities of mercurials to sites of inhibition were calculated relative to other ligands in erythrocyte membranes: the ratios ranged from 3 : 1 to 50 : 1. The results demonstrated the use of binding capacity assays and inhibitor partition equations to measure and compare the susceptibilities of membrane-bound enzymes to poisoning by mercurials.

Affinity and avidity of antibodies to the random polymer (T,G)-A-L and a related ordered synthetic polypeptide

The affinity values of antibodies to the random synthetic antigen poly-(Tyr,Glu)-poly( dl Ala)-poly(Lys) [known as (T,G)-A-L] and to the ordered (Tyr-Tyr-Glu-Glu)-poly( dlAla)-poly(Lys) [known as (T-T-G-G)-A-L] were measured. Determinations of the association constants were performed by antigen binding capacity assay, ABC, using the whole antigen, and by equilibrium dialysis, using a radioactive conjugate of the ordered peptide T-T-G-G which was found to represent the major determinant of the random (T,G)-A-L. The affinity values of antibodies elicited by high responder mice to (T,G)-A-L and to (T-T-G-G)-A-L were found to be similar. However, a difference of two orders of magnitude was found between the values obtained by the two methods. This difference is partially explained by probability analysis. Upon immunizing low responder (H-2 k ) mice with complexes of the antigens with methylated bovine serum albumin (MBSA), an increase in their antibody response was observed. The affinity values of these antibodies were found to be similar to those of high responder antibodies after immunization with either the antigens complexed to MBSA or with the antigens alone. On the basis of these data we conclude that low responder (H-2 k ) B cells have the potential to produce antibodies of the same specificity and quality as the high responders to the random (T,G)-A-L and to the ordered (T-T-G-G)-A-L, and that the genetic defect in these mice should be located at the level of the B-cell acceptor for the T-cell signal.

The measurement of total and unsaturated iron-binding capacity in serum.

An inter-laboratory investigation into the assay of serum iron binding capacity by the magnesium carbonate adsorption method was undertaken. The source of MgCO3, storage temperature of whole serum, lyophilization and storage temperature of lyophilized serum were identified as sources of inter-laboratory variation. Protocols for the clinical assay of UIBC and TIBC are given which have achieved satisfactory inter-laboratory precision.

The nature and functions of specific immune response genes and their products.

Antibodies produced by inbred mouse strains immunized with the random synthetic polypeptide poly (Tyr,Glu)-poly(DLAla)--polyLys denoted (T,G)-A--L were found to be specific mainly to the ordered peptide Tyr-Tyr-Glu-Glu. Low responder H-2k mice, upon immunization with either the random (T,G)-A--L or the ordered (T-T-G-G)-A--L coupled to methylated bovine serum albumin (MBSA), produce antibodies with comparable titers to those observed in high responder H-2b mice following immunization with the antigens alone or with their complexes with MBSA. A comparison of the above antibodies have led to the conclusion that low responder mice, upon immunization with the synthetic antigens complexed with MBSA, produce antibodies of the same specificity and quality as those of high responders (as shown by the isoelectric focusing technique) and they also have the same affinity and heterogeneity as antibodies of H-2b mice (measured by equilibrium dialysis and antigen binding capacity assay). Anti-idiotypic sera to anti-T,G)-A--L antibodies of C3H.SW (H-2b,Ig-1a) mice were raised in guinea pigs. C3H.SW anti-(T,G)-A--L antibodies from different pools cross reacted idiotypically. Anti-(T,G)-A--L antibodies of CWB (H-2b, Ig-1b) mice did not react with the anti-idiotypic serum suggesting linkage between the genes coding for idiotypes and allotypes. C3H/DiSn (H-2k, Ig-1a) anti-(T,G)-A--L antibodies elicited by immunization with (T,G)-A--L complexed to MBSA reacted with the anti-idiotypic serum to the same degree as C3H.SW anti-(T,G)-A--L antibodies, confirming the similarity between the high and low responder anti-(T,G)-A--L antibodies. C3H.SW (H-2b) mice as well as C3H/DiSn (H-2k) mice were found to be capable of producing an antigen specific factor from "educated" T cells which replaces the helper effect of T cells in the process of antibody production. On the other hand B cells of H-2k mice were not triggered by a factor of either high or low responder specific T cells. The activity of a C3H.SW (T,G)-A--L specific T cell factor was removed after passage on a Sepharose column coupled to the anti-idiotypic serum prepared against C3H.SW anti-(T,G)-A--L antibodies, suggesting similarity between the antigen specific T cell factor and the B cell recognition system. A (T,G)-A--L specific factor produced by C3H/DiSn (H-2k, Ig-1a) "educated" T cells reacted with the anti-idiotypic serum as well. Thus, C3H.SW high and C3H/DiSn low responder (T,G)-A--L specific T cell factors cross react at the level of their binding site for antigen.

The Assay of Iron‐poly (sorbitol‐gluconic acid) complex (Ferastral®) and its Separation from Transferrin in Serum

A new iron-poly (sorbitol-gluconic acid) complex (Ferastral) has been studied. A method of assay is described. The iron complex may be separated from serum transferrin using a Sephadex DEAE A50 column. This binds the iron complex and elutes iron-transferrin which can then be assayed. It is shown that the assay of serum transferrin unsaturated binding capacity using excess 59FeCl2 and MgCO3 adsorption, is valid in the presence of Ferastral. Serum unsaturated iron binding capacity may therefore be used to follow the binding of Ferastral iron by transferrin. These methods may be used to follow the distribution of iron in plasma after an intramuscular injection of Ferastral.

“SINGLE-POINT” SEPHADEX GEL FILTRATION ASSAY FOR ESTIMATION OF RESERVE BILIRUBIN BINDING CAPACITY

The sephadex G-25 elution method for assay of reserve bilirubin binding capacity has proven to be a valid and useful technique for evaluating the danger level of hyperbilirubinemia in the newborn. A major disadvantage of the method is the relatively large volume of serum required from very small newborns who usually have high hematocrits. A modification of the method has been developed which requires only 0.5 ml total serum. A concentration of bilirubin is chosen to add to half the sample such that the binding capacity will be closely approached but not exceeded, and the optical density of this eluted sample is compared to that of the patient's own serum blank. The bilirubin binding capacity is thus expressed as “greater than” or “less than” the concentration of bilirubin added. Results comparing the same sera using the “single-point” method and the classical 3 or 4 point assay show excellent agreement, thus confirming the “single-point” assay as a valid and useful alternate method when sample size is limited.

Comparison of in vitro bile acid binding capacity and in vivo hypocholesteremic activity of cholestyramine.

SummaryMethods for in vitro assay of cholate binding capacity and for in vivo assay of hypocholesteremic activity of preparations of cholestyramine, an anion exchange resin, were developed and compared. In the in vitro assay, 20 ml of 0.3 M phosphate buffer at pH 6.0, 20 or 40 mg of the resin and 40 mg of sodium cholate were mixed and shaken for 30 minutes; the filtrates were assayed for cholate spectrophotometrically, and the cholate bound by the resin determined by difference.In the in vivo method, a hypercholesteremic diet was fed to day-old chicks for 2 weeks; graded levels of the anion exchange resin were added to the diet during the second week. Plasma cholesterol levels were then determined as an indicator of the activity of the resin.The relative activities of the various preparations of cholestyramine and other anion exchange resins compared very well when tested by the two methods of assay. Addition of excipients in preparations of cholestyramine for clinical use had no effect on cholate binding...

Rapid Charcoal Assay for Intrinsic Factor (IF), Gastric Juice Unsaturated B12 Binding Capacity, Antibody to IF, and Serum Unsaturated B12 Binding Capacity

Abstract The principle of the dialysis assays for intrinsic factor (IF) and antibody to IF was integrated with the demonstration that protein-coated charcoal adsorbs only free and not bound B12, in a single rapid method for 4 separate assays: (1) assay of IF, (2) assay of gastric juice unsaturated B12 binding capacity, (3) assay of serum antibodies to IF and (4) assay of serum unsaturated B12 binding capacity. The method is sensitive, accurate, reproducible, and can easily be completed within an hour. The simplicity and ready availability of reagents and equipment lends itself to ready adoption in any clinical laboratory using radioisotopes. The charcoal particle is considered as a solid microsponge, and its coat of albumin, other protein, carbohydrate, or other large molecule as a molecular sieve surrounding the sponge. The whole constitutes a system of "instant dialysis," with a wide range of applications in the separation of large from small molecules.