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Biomedical subjects

B Solomon

Publications and source records attributed to B Solomon.

At least 19 recordsLinked to original sources
PubMed1992

Affinity purification of antibodies using immobilized FB domain of protein A.

A continuous method for the efficient digestion of protein A into active fragments (FB, Mr = 7000) using immobilized trypsin was developed. These fragments originate from almost identical five-repeated monovalent Fc-binding units of 58 residues each. The fragments obtained were found to be similar to the recently described genetically engineered fragment B. Antibody-binding characteristics of the FB domain and also of intact protein A, immobilized on to adipic dihydrazide-modified Eupergit CB6200 beads, were investigated. Based on the experimental data obtained, a high-performance liquid chromatographic column containing C30N Eupergit C-immobilized FB domain was prepared and its performance in antibody purification was compared with that of Eupergit C-immobilized intact protein A.

Amino Acids↗
PubMed1992

Calcium-modulated conformational affinity chromatography. Application to the purification of calmodulin and S100 proteins.

The purification of proteins by affinity chromatography is based on their highly specific interaction with an immobilized ligand followed by elution under conditions where their affinity towards the ligand is markedly reduced. Thus, a high-degree purification by a single chromatographic step is achieved. However, when several proteins in the crude mixture share affinity to a common immobilized ligand, they may not be resolved by affinity chromatography and subsequent "real" chromatographic purification steps may be required. It is shown that by using properly selected gradient elution conditions, the affinities of the various proteins towards the immobilized ligand may be gradually modulated and their separation may be achieved. This is exemplified by the isolation and separation of a group of Ca(2+)-activated proteins, Calmodulin, S100a and S100b, from bovine brain extract, using a melittin-Eupergit C affinity column which is developed with Ca(2+)-chelator gradients. As expected, separation of the three proteins into individual peaks, eluted in order of increasing affinity to the matrix, was obtained. Sigmoid selectivity curves calculated from the elution volumes under different elution conditions for each of the proteins were obtained, illustrating the chromatographic behaviour of the gradient affinity separation system.

Animals↗
PubMed1992

Microalbuminuria immunoassay based on antibodies covalently conjugated to Eupergit C-coated beads.

OBJECTIVE: To develop a reliable, simple, and sensitive assay for microalbuminuria, based on covalent attachment of anti-HSA to oxirane-bearing polymethylmethacrylate beads (Eupergit CB6200). RESEARCH DESIGN AND METHODS: Anti-HSA antibodies were coupled to CB6200 beads by reaction of their amino groups with the oxirane groups of the matrix. The capability of the beads to bind HSA from standard solutions or urine was evaluated and compared with the state of the art ELISA test. RESULTS: The new bead immunoassay is sensitive and linear in the range of 1-25 mg/L, which is considered the low microalbuminuria range. When HSA levels in urine were tested, the intra- and interassay CV values ranged between 2.7 and 3.9% and between 5.6 and 6.6%, respectively. The long-term storage stability of the antibodies covalently bound on the beads was higher than of the same antibodies adsorbed on ELISA plates. After 16 wk of storage, the CV was about 7.3% with the bead assay, compared with 14% obtained for the ELISA test under the same experimental conditions. CONCLUSIONS: A new procedure for microalbuminuria assay was developed, with Eupergit CB6200 beads as a solid support for covalent binding of the first antibody. Accuracy, sensitivity, reproducibility, and precision of the bead immunoassay were similar to those of commonly used immunoassays, as exemplified by the analysis of HSA in 53 clinical urine samples. The bead assay retains a low degree of variability over long storage periods, and the beads may be reapplied after a simple acid-washing procedure.

Albuminuria↗
PubMed1991

Differences and similarities in the treatment of diffuse goiter in Europe and the United States.

In two separate studies, members of the American Thyroid Association (ATA), and the European Thyroid Association (ETA) were surveyed by questionnaire on their management of Graves' disease. The aim was to determine how expert clinical thyroidologists employ three different therapies which are available for this disorder. Herein we summarize, compare and contrast similarities and differences in the results of these surveys. For the index patient, radioiodine (RAI) was the therapy of choice for 69% of ATA respondents but only 22% of ETA respondents. In contrast, only 30.5% of ATA respondents chose antithyroid drugs as first line therapy compared to 77% of ETA respondents. There was consensus on the relative lack of a role for thyroidectomy. The implications of these differing approaches to the treatment of hyperthyroidism due to Graves' disease are discussed.

Adult↗
PubMed1991

Differences and similarities in the diagnosis and treatment of Graves' disease in Europe, Japan, and the United States.

In three separate studies, members of the American Thyroid Association (ATA), the European Thyroid Association (ETA), and the Japan Thyroid Association (JTA) were surveyed by questionnaire on their management of Graves' disease. The aim was to determine how expert clinical thyroidologists employ diagnostic procedures and the three different therapies that are available for this disorder. In this report, we identify, summarize, compare, and contrast similarities and differences in the results of these surveys in these three different regions of the world. In general, ATA members used fewer diagnostic tests than did their European or Japanese colleagues. For the index patient, radioiodine was the therapy of choice for 69% of ATA respondents but only 22% and 11% of ETA and JTA respondents, respectively. In contrast, only 30.5% of ATA respondents chose antithyroid drugs as first-line therapy compared to 77% of ETA and 88% of JTA respondents. There was consensus on the relative lack of a role for thyroidectomy except for narrow indications. The implications of these differing approaches for the diagnosis and treatment of hyperthyroidism due to Graves' disease are discussed.

Adolescent↗
PubMed1991

Thermostabilization of carboxypeptidase A by interaction with its monoclonal antibodies.

The effect of interaction of carboxypeptidase A (CPA) with three monoclonal antibodies, each with a different epitope (CP 10, CP 9, and CP 8), on the heat stabilization of enzymes is described. These monoclonal antibodies bind to CPA with a relatively high binding constant (approximately 10(8) M-1) and do not affect its catalytic properties. Intact carboxypeptidase A lost more than 95 and 90% of its esterase and peptidase activities within 120 min at 50 degrees C. The monoclonal antibodies increased the thermal stability of the enzyme by 90 and 60%, as compared with the peptidase and esterase initial activities, respectively. Binding of these monoclonal antibodies, alone or in pairs, to the enzyme epitopes that are supposedly involved in heat denaturation of CPA result in stabilization of the conformation of the enzyme. The effect of thermostabilization by monoclonal antibodies was more pronounced with respect to peptidase activity than to esterase activity, indicating that these activities follow different reaction mechanisms. Since properly selected monoclonal antibodies can be prepared against virtually any enzyme, their immunocomplexation may provide a general and convenient method for stabilization of the enzyme conformation to heat denaturation, without affecting the catalytic properties.

Antibodies, Monoclonal↗
PubMed1990

Effect of polyethylene glycol on the non-specific adsorption of proteins to Eupergit C and agarose.

Non-specific adsorption of serum proteins to Eupergit C (EC) and agarose during the process of immunoaffinity chromatography often leads to contamination of the specifically eluted antigens to be purified. This effect was studied by application of serum samples to a beta-mercaptoethanol-blocked EC (EC-beta ME) column followed by analysis of proteins eluted with various elution buffers. Inclusion of polyethylene glycol (PEG 400 or 1500) in the loading buffer reduced the non-specific adsorption of proteins to EC but had an adverse effect on agarose. Covalent attachment of amino-PEG to EC and to epoxy-activated Sepharose mimicked the effect of PEG in solution with EC and resulted in a marked reduction in non-specific adsorption of serum proteins. Inclusion of PEG in the loading buffer during immunopurification of a serum protein (immunoglobulin G) or seminal plasma protein (human decidua protein hDP71) resulted in a marked improvement in the purity of these proteins eluted from the respective columns by ammonium acetate (pH 10).

Adsorption↗
PubMed1990

Eupergit C as a carrier for high-performance liquid chromatographic-based immunopurification of antigens and antibodies.

An immunoaffinity purification system using C30N and C1Z Eupergit C beads was developed and optimized. Poly- and monoclonal antibodies were purified using immobilized antigens and antigens were purified using immobilized antibodies. Antigens were used that possess enzymic activities and the efficiency of antigen binding was determined from the enzymic activity of the matrix-bound immunocomplexes. High-performance immunoaffinity purification using Eupergit C beads proved to be highly specific, reproducible, free from protein leakage and possessed a low degree of non-specific adsorption of tissue proteins. These characteristics of the system were illustrated by the isolation of immunoglobulin G from serum and of human decidua proteins from the decidua tissue and from seminal plasma. These proteins were obtained at high purity in a single purification step, as shown by sodium dodecyl sulphate-polyacrylamide gel electrophoresis.

Antibodies↗
PubMed1990

Enzymic oxidation of monoclonal antibodies by soluble and immobilized bifunctional enzyme complexes.

Site-specific modification of monoclonal antibodies was achieved by oxidation of the carbohydrate moieties of antibodies which are located remote from the antigen binding sites. Sialic acid and galactose are terminal sugars of these carbohydrate chains. Concomitant treatment of the antibodies with neuraminidase and galactose oxidase generated aldehyde groups in the oligosaccharide moieties of immunoglobulins which reacted selectively with amino or hydrazide groups of the matrix. Subsequent immobilization of neuraminidase and galactose oxidase on Eupergit C-adipic dihydrazide proved to be an efficient and selective system for the enzymic oxidation of the monoclonal antibodies without impairing their immunological activity. Oriented immobilization of enzymically oxidized monoclonal antibodies on hydrazide or amino Eupergit C derivatives thus leads to the formation of antibody matrix conjugates which possess high antigen-binding activities.

Antibodies, Monoclonal↗
PubMed1990

Oriented immobilization of periodate-oxidized monoclonal antibodies on amino and hydrazide derivatives of Eupergit C.

Amino and hydrazyno derivatives of Eupergit C were prepared by reaction of the beads with hexamethylene diamine (HMD) and adipic acid dihydrazide (ADH), respectively. Monoclonal antibodies (mAbs) against carboxypeptidase A (CPA) and horse radish peroxidase (HRP) were prepared, and those that did not inhibit the respective enzymatic activities were selected. The carbohydrate moieties of these antibodies were oxidized by reaction with sodium periodate and then coupled onto the modified beads. The oxidation and coupling reactions were optimized to achieve highly active matrix-conjugated antibodies. Thus, antibody-matrix conjugates that possessed antigen-binding activities close to the theoretical value of 2 mol antigen bound/mol immobilized antibody were obtained.

Acrylic Resins↗
PubMed1990

Prospective computer-assisted voice analysis for patients with early stage glottic cancer: a preliminary report of the functional result of laryngeal irradiation.

In January 1987 we began a prospective study aimed at evaluating objective parameters of vocal function for all patients treated with RT for early glottic cancer. All patients underwent vocal analysis using a voice analyzer interfaced with a computer. This allowed for the determination of percent voicing (%V) (normal = presence of phonation = 90-100%V). Other parameters such as breathiness (air turbulence or hoarseness) and strain (vocal cord tension) were also measured. Patients were recorded before RT, weekly during RT, and at set intervals after RT. There have been 25 patients studied. Eighteen (18) are evaluable at 9 months after treatment. All patients were male and ranged from 45-84 years old. Fourteen (14) and T1 lesions and received 66 GY/33 fractions to their larynx and 4 had T2 tumors and received 66-70 Gy/33-35 fractions. To date, all patients are locally controlled. Three distinct patterns of %V changes have been encountered. However, all patients demonstrated normal phonation pattern by 3 months after RT, and this is sustained at 9 months follow-up. In addition, 94% of patients have had significant decrease in breathiness after RT, which objectively documents diminished hoarseness. In 83%, breathiness is normal after RT. Most patients have had increased strain after RT, which documents increased vocal cord tension. However, strain remained within normal limits in 89%. Our preliminary analysis suggests that the majority of patients irradiated for early glottic cancer demonstrate a decrease in breathiness and an increase in strain after RT, and enjoy a resultant voice that has normal phonation maintained at 9 months after RT. Our data also demonstrate three distinct phonation patterns. Further follow-up will allow us to determine the prognostic significance, if any, of these patterns, and to continue to follow objective vocal parameters on larger numbers of patient.

Aged↗
PubMed1990

Enzyme-linked immunosorbent assays for determination of plasma aldosterone using highly specific polyclonal antibodies.

Two enzyme-linked immunosorbent assays were established and compared for the estimation of plasma aldosterone. In the first method immobilized aldosterone-protein complexes on the ELISA plates compete with aldosterone to be determined for the binding of certain amount of anti-aldosterone antibodies. The sensitivity of this method depends on the protein carrier used to conjugate with aldosterone. In the second method, anti-aldosterone antibodies adsorbed on ELISA plates compete for binding of known amount of the enzyme-labeled aldosterone and aldosterone to be determined. The highly specific rabbit anti-aldosterone antibodies were obtained by injection of aldosterone-oxime thyroglobulin. The detection limit of aldosterone in both methods ranged between 2-20 pg. The proposed assays are suitable for the determination of aldosterone in biological fluids compared with other reported ELISA assays, as well as with RIA.

Aldosterone↗
PubMed1990

Current trends in the management of Graves' disease.

Members of the American Thyroid Association were invited to participate in a survey of the management of Graves' disease. One primary case and several variations were provided, which differed in respect to age, sex, goiter size, severity, etc. The questionnaire was based on the format used in a similar survey of members of the European Thyroid Association. The aim of the survey was to determine 1) how expert thyroidologist employ diagnostic procedures for this disorder, and 2) the choice of therapy of the three treatment options and its manner of implementation. Questionnaires were sent only to clinically active members. The overall response rate was 62%. Data analysis was possible on 52% of members surveyed and was performed using SPSS and a specific Fortran program. In the laboratory evaluation of the primary case a radioiodine uptake, scan, serum total T4, and basal TSH were requested by 92%, 47%, 83%, and 66%, respectively, with 84% of respondents using an ultrasensitive TSH assay. For management of the primary case, radioiodine treatment was the first choice of 69% of the respondents. Antithyroid drugs were used briefly (3-7 days) before 131I by 28%, whereas 41% said they would employ thioureas after 131I. Of those using 131I, 66% tailored the dose to achieve euthyroidism as the goal of therapy, while 34% aimed for hypothyroidism requiring T4 replacement. Only 30% of respondents chose thioureas as a first line of treatment (72% propylthiouracil; 28% tapazole). The duration of drug therapy was a predetermined fixed interval for 80% of the respondents, with 90% treating for 1-2 yr. Other specific trends in diagnostic approach and therapeutic preferences were identified for the eight variations on the primary case problem.

Adrenergic beta-Antagonists↗
PubMed1990

Vocal changes in patients undergoing radiation therapy for glottic carcinoma.

A prospective evaluation of vocal changes in patients receiving radiation therapy for T1 and T2 (AJC) glottic carcinoma was undertaken in January 1987. Vocal analysis was performed prior to radiotherapy and at specific intervals throughout the radiation treatment program. The voicing ratio was extrapolated from a sustained vowel phonation using the Visipitch interfaced with the IBM-PC. Preliminary observations suggested three distinct patterns of vocal behavior: 1. reduced voicing ratio with precipitous improvement within the course of treatment, 2. high initial voicing ratio with reduction secondary to radiation induced edema, with rapid improvement in the voicing component after the edema subsided, and 3. fluctuating voicing ratio during and following treatment. Enrollment of new patients and a 2-year follow-up of current patients was undertaken.

Aged↗
PubMed1989

Localization of a highly immunogenic region of carboxypeptidase A recognized by three different monoclonal antibodies and their use in the detection of subtle conformational alterations in this enzyme region.

Three murine monoclonal antibodies (mAb 100, 104, and 121) elicited against carboxypeptidase A (CPA) were prepared and characterized. All three mAbs recognize the same or partially overlapping sites of CPA. This is corroborated by the lack of antibody additivity in the ELISA assay carried out in the presence of pairs of mAbs, the similarity in molecular weight of the immunocomplex formed between CPA and one of the mAbs in the presence of another, and also a competition experiment in which one of the mAbs was labeled enzymatically. The three mAbs do not affect the enzymatic activity of CPA. Even at high concentrations, they do not recognize carboxypeptidase B (CPB) in spite of the similar tertiary structure and the 50% homology in amino acid sequence with CPA. This antigenic determinant is located on one of the four cyanogen bromide fragments of CPA. On the basis of the known sequences of the two enzymes, criteria which predict high antigenicity, and experimental data using synthetic peptides, such a determinant was found to be located within the amino acid sequence from residues 209 to 218 of the CPA molecule. The mAbs prepared detect conformational alterations in the above enzyme epitope when the enzyme is exposed to various conditions. The binding of the mAbs to CPA adsorbed onto a polystyrene plate is characterized by apparent binding constants higher by 1 or 2 orders of magnitude than those characterizing the interaction of the mAbs with CPA in solution. The mAbs also readily detect both conformational alterations of CPA on treatment with urea and subtle, reversible conformational alterations on removal of zinc from the active site of the enzyme.

Amino Acid Sequence↗