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At least 721 records · Page 40Linked to original sources
PubMed1995

Feasibility study of a drug immunoassay based on micellar electrokinetic capillary chromatography with laser induced fluorescence detection: determination of theophylline in serum.

This paper presents principle and first results of a novel competitive binding immunoassay for monitoring of theophylline in human serum. The assay is based upon short time incubation of a mixture of antiserum, containing the antibody raised against theophylline, fluorescein labelled theophylline (tracer) and serum prior to injection of a few nanoliter of this mixture onto a fused-silica capillary for subsequent separation and analysis of free tracer and the antibody-tracer-complex by micellar electrokinetic capillary chromatography with laser induced fluorescence detection. Quantitation based upon multi-level calibration using the height of the peak produced by the free tracer is shown to provide theophylline serum levels which are in agreement with those obtained by a commercial fluorescence polarization immunoassay and with those determined by micellar elektrokinetic capillary chromatography with direct serum injection and on-column UV absorption detection.

Binding, Competitive↗
PubMed1988

Site-specific linkage of methotrexate to monoclonal antibodies using an intermediate carrier.

The site-specific conjugation of methotrexate, 4-amino-N10-methylpteroylglutamic acid, to a monoclonal anti-carcinoembryonic antigen (CEA) antibody, using an intermediate amino-dextran carrier system, resulted in a ratio of 30-50 molecules of MTX per molecule of immunoglobulin. The immunoreactivity of the conjugate was analyzed using flow cytometry or a competitive binding assay, which showed that the conjugate has significant retention of the antigen-binding activity. The pharmacokinetic behavior of the immunoconjugate in BALB/c mice and tumor localization in hamsters indicated that the conjugate remained in the circulation with higher concentration than free antibody, and could recognize the tumor as efficiently as the unconjugated antibody. The high degree of drug incorporation with retained immunoreactivity makes this method preferable to direct antibody conjugation.

Animals↗
PubMed1990

Generation and characterization of monoclonal antibodies to prostate secretory protein.

Monoclonal antibodies (MAbs) were produced against a highly purified preparation of prostate secretory protein (PSP) isolated from normal seminal plasma. Fifteen antibodies were selected for further evaluation based on their strong reactivity and specificity for PSP. All the MAbs had a specificity for prostate epithelial cells and none reacted to any of a variety of normal tissues as determined by immunoperoxidase staining. Six of the MAbs were selected for further immunohistochemical evaluation based on their ability to recognize different antigenic determinants. Using competitive binding immunoassays, a variety of overlapping specificities were observed with at least 2 distinct epitopes identified. Although some staining variability was noted, the 6 antibodies, in general, gave the same pattern of tissue reactivity. Both the normal prostate and the benign prostate hyperplastic ductal epithelial cells stained intensely, with 78 to 100% and 50-100% of the cells staining, respectively. The number and often the staining intensity of the tumor cells decreased as the tumor became more undifferentiated. Approximately 40 to 100% and 15 to 70% of the tumor cells stained in the moderately-differentiated and well-differentiated carcinoma tissues, respectively, whereas either no staining was observed or less than 20% of the tumor cells stained in the poorly-differentiated and undifferentiated tumors. Most of the metastatic prostate tumors showed either no staining or scattered staining in a few cells (i.e., less than 20%).

Adenocarcinoma↗
PubMed1991

Production of a recombinant human T-cell leukemia virus type-I trans-activator (tax1) antigen and its utilization for generation of monoclonal antibodies against various epitopes on the tax1 antigen.

A 42-kDa recombinant protein, PX141, consisting of the trans-activator protein encoded by human T-cell leukemia virus (HTLV-1) (tax1 antigen) and the amino-terminal fusion peptide of 12 amino acid residues of the alpha-peptide encoded by the plasmid pUC19 was produced. In order to investigate the immunogenicity of the tax1 antigen, mice were immunized with the purified PX141 and 4 anti-tax1 monoclonal antibodies (MAbs) designated TAXY-1, TAXY-6, TAXY-7 and TAXY-8 were generated, and their reactivity was characterized along with another anti-tax1 MAb, Lt-4. Immunoblot assays showed that all the MAbs reacted with the PX141, the native tax1 antigen expressed in various HTLV-1-infected cell lines and the gp68 of MT-2 cells expressing the tax1 amino acids 94-353. Immunoblot assays using recombinant, truncated tax1 antigens, XD59 (expressing amino acids 180-338) and XD128 (expressing amino acids 1-47 and 286-353) showed that: (1) TAXY-1 and Lt-4 did not react with either antigen; (2) TAXY-6 and TAXY-8 reacted with only XD128: and (3) TAXY-7 reacted with both. In addition, TAXY-1, but not the other MAbs, reacted with a putative tax antigen of an STLV-I-infected cell line, designated RfM26-I. Competitive binding assays showed that TAXY-6 and TAXY-8 did not compete against each other. Sera from HTLV-I-infected humans interfered with the binding of all of these anti-tax1 MAbs. These results indicate that the tax1 antigen and the PX141 express at least 5 distinct epitopes recognized by human and mouse antibodies.

Amino Acid Sequence↗
PubMed1991

Structure-function analysis of interleukin-5 utilizing mouse/human chimeric molecules.

Interleukin-5 (IL5) is a T cell derived glycoprotein that stimulates eosinophil production and activation. In the mouse, but apparently not in the human, it is active on B cells. The murine and human IL5 polypeptides exhibit 70% sequence similarity and yet display distinct species-specific activity. Whilst mouse and human IL5 are equally active in human cell assays, human IL5 is 100-fold less active than murine IL5 in mouse cell assays. Two restriction sites were utilized to divide the human and mouse sequences into three fragments. Hybrid molecules consisting of all combinations of these fragments were constructed and expressed. In the human cell assays [using bone marrow or the erythroleukaemic cell line (TF-1)] all the hybrid proteins generated activity comparable to that of the human and mouse IL5. This implies that replacing different domains does not result in detrimental effects to the tertiary structure of the molecule. In the mouse cell assays [using bone marrow or the pro-B cell line (B13)] the hybrids clearly identified the importance of residues in the C terminus for biological activity. The changing of only eight residues in this region of human IL5, to those of mouse IL5, resulted in the hybrid producing biological activity comparable to mouse IL5. In addition, competition binding assays showed that this region probably interacts with the receptor.

Amino Acid Sequence↗
PubMed1991

Immunoassays for parathyroid hormone 1-84 in the diagnosis of hyperparathyroidism.

The two most frequent causes for hypercalcemia are primary hyperparathyroidism and hypercalcemia associated with malignancy. Elevated or inappropriately high PTH serum levels are the hallmark of hyperparathyroidism. Sensitive immunometric assays for the secreted, biologically active, intact parathyroid hormone molecule, PTH-(1-84), employ two populations of region-specific antibodies, take advantage of saturation kinetics rather than competitive binding, and have many technical advantages over conventional radioimmunoassay. Approximately 90% of patients with primary hyperparathyroidism have elevated serum levels of PTH-(1-84) by immunometric assay; the remainder have inappropriately elevated values of PTH for the serum calcium concentration. Clinical correlation studies comparing measurements of PTH using antisera that recognize the carboxyl, midregion, or amino terminus of PTH with PTH levels determined by immunometric assays demonstrate elevated values in equivalent numbers of hyperparathyroid individuals. Immunometric assays for PTH-(1-84) have their greatest value in separating patients with hyperparathyroidism from those with hypercalcemia of malignancy. In earlier studies using region-specific antisera, there was virtually always an overlap of serum PTH levels in hyperparathyroidism and hypercalcemia associated with malignancy. In contrast, analysis of results using PTH-(1-84) immunometric assays in several hundred reported patients shows a complete separation of PTH values. Clinical judgment, combined with measurement of PTH in the setting of hypercalcemia, can lead to the diagnosis of hyperparathyroidism with confidence in essentially all patients.

Antibodies↗
PubMed1991

Mutational analysis of leucine 47 in human epidermal growth factor.

Seven site-specific mutants (including changes to other hydrophobic, charged, and heterocyclic amino acids) of leucine 47 of human epidermal growth factor (EGF) were generated by protein engineering and characterized for their activity in three assays: radioreceptor competition binding in membrane fractions, the stimulation of the EGF receptor's tyrosine kinase activity, and the stimulation of thymidine uptake in tissue culture cells. K1/2 (concentration required for half maximum response) values for each of the mutants are reported in the three assays. The results show that the native leucine residue is quite important for EGF activity. Substitutions are tolerated to different degrees, depending upon hydrophobicity and size of the side chain. Substitution with ionic residues led to the most drastic reduction in activity. One-dimensional nuclear magnetic resonance spectroscopy, at physiological pH, of several of the mutants did not detect any major structural perturbations which would account for the loss of activity. The results suggest that the side chain of leucine 47, because of its charge neutrality, size, and hydrophobicity, is highly important, although not absolutely essential for the interaction of EGF with its receptor. A striking finding was the lower (compared with wild type) Vmax values of the mutants in the tyrosine kinase reaction, but these low Vmax mutants, in cell culture experiments, were able to stimulate at high concentrations a growth response equivalent to wild type EGF.

Base Sequence↗
PubMed1991

Effect of different test designs of immunoassays on "hook effect" of CA 19-9 measurement.

Determining serum CA 19-9 using regular Centocor kit frequently requires numerous repeats when specimens contain highly elevated levels of CA 19-9. We found that the number of repeats could be reduced if the Centocor kit with an extended concentration range was used. Using an extended Centocor kit could also eliminate falsely low CA 19-9 results. However, when a Biomira RIA kit, based on a competitive binding principle, was used, falsely low CA 19-9 values could be completely avoided and, in most cases, only a single repeat was usually required to arrive at the true result. This is because the level of CA 19-9 in the specimen can be approximated from the radioactivity count. Therefore, when designing an immunoassay for tumor markers, one needs to take into consideration the concentration range of the tumor marker in the specimens if one would like to avoid repeats and falsely low values.

Antigens, Tumor-Associated, Carbohydrate↗
PubMed1993

Development of radioimmunoassays on microplate: application for CA-GI and CA-Br tumor markers.

By taking advantage of a newly available microplate counter for radioactivity and the organic solvent-resistant, pigmented microplates, we have successfully established radioimmunoassays (RIA) for both CA-GI and CA-BR on microplate for routine clinical use. In the process of assay development, we found that both pigmented PicoPlate, made of acrylonitrile, and polystyrene Microlite 2 can be coated with antialpha fetoprotein (AFP) and antinerve growth factor (NGF) and used for setting up immunoassays for AFP and nerve growth factors. There were no problems following a test format of either competitive binding or sandwich design. Microlites 2 is recommended over PicoPlate because Microlites 2 is made of polystyrene, which is less expensive and separable into 8-well strips or even single wells. Single-well separation allows for the use of regular gamma counters in case Topcount is unavailable. We also found that the sensitivity of these tests was not significantly affected even though Topcount counts the weaker beta emissions. Similar dose-response curves could also be generated between original Biomira tube assays and assays using PicoPlate or Microlite 2 coated with protein antigens CA-Br and CA-GI. Excellent correlations were also obtained between the microplate assays and the Biomira tube assays for CA-GI and CA-Br using groups of serum specimens from cancer patients. We recommend the development of various RIAs on the microplate: it requires less reagents and less sample handling by the technologists and it can be essentially automated.

Antibodies↗
PubMedDate not supplied

A powerful cooperative interaction between a fusogenic peptide and lipofectamine for the enhancement of receptor-targeted, non-viral gene delivery via integrin receptors.

BACKGROUND: Following receptor-mediated endocytosis, vector/DNA complexes require assistance to exit endocytic vesicles in order to avoid degradation in the lysosomes. Overcoming this barrier is a major challenge for the development of receptor-targeted, non-viral gene delivery. METHODS: The fusogenic peptide of influenza virus haemagglutinin, lipofectamine and chloroquine were tested singly and in combination in various doses for promoting in vitro gene transfer by an integrin-targeted, non-viral DNA vector (polylysine-molossin). RESULTS: The fusogenic peptide and lipofectamine both individually promoted integrin-targeted gene delivery. However, the combined use of these agents was particularly effective, even at concentrations where neither agent singly had any effect on promoting gene delivery by polylysine-molossin. This optimal combination was effective on several cell lines and primary cell cultures. On the HuH7 cell line, it was approximately five-fold more effective than optimal chloroquine concentrations for integrin-targeted gene delivery and four to five times more effective than commercially available polyethylenimine. With the beta-galactosidase reporter gene, 60-65% of HepG2 cells and 75-80% of HuH7 cells were positive. The surface charge of polylysine-molossin/DNA/lipofectamine/fusogenic peptide complexes was approximately the same as that of polylysine-molossin/DNA complexes. The size distribution of the complexes suggested that competitive binding of polylysine-molossin and lipofectamine to DNA influenced the overall efficacy of this approach. CONCLUSIONS: Although the mechanisms are not clear, the combined use of very low doses of two membrane-destabilizing agents results in high levels of receptor-targeted gene delivery.

Amino Acid Motifs↗
PubMed1984

Monoclonal antibodies directed against human Ia antigens detect an evolutionary conserved epitope on guinea pig Ia antigens with unique functional properties.

Three murine monoclonal antihuman Ia antibodies were found to cross react with guinea pig Ia antigens. Sequential immunoprecipitation studies demonstrated that these antibodies recognized a subset of guinea pig Ia antigens. In competition binding studies, the antihuman Ia antibodies competed with each other, but not with a number of murine monoclonals raised directly to guinea pig Ia antigens. When the antihuman Ia monoclonals were tested for their ability to inhibit the proliferative response of primed guinea pig T cells to antigen-pulsed macrophages, they completely inhibited the response of strain 2 T cells to the complex antigen ovalbumin while having no effect on the response to the copolymer L-glutamic acid, L-lysine. In contrast, the monoclonals raised against guinea pig Ia had much less dramatic effects on the response of uncloned T cells to these antigens. These results suggest that the cross-reactive monoclonals may recognize an evolutionary conserved epitope on Ia that has a regulatory function in T-cell activation.

Animals↗
PubMed1982

Synthesis and characterization of iodoazidobenzylpindolol.

A high affinity beta-adrenergic ligand, iodoazidobenzylpindolol, was synthesized and characterized. The absorption spectrum of this compound changed markedly upon photolysis, consistent with decomposition of the azide group. This compound has a KD of 5--7 x 10(-10) M for the duck erythrocyte ghost beta-adrenergic receptor when measured in a competitive binding assay.

Affinity Labels↗
PubMed1992

Concanavalin A microspheres for a self-regulating insulin delivery system.

A self-regulating insulin delivery system based on the competitive binding of glucose and p-succinylamidophenyl-alpha-D-glycopyranoside-insulin (SAPG-insulin) to crosslinked concanavalin A (Con A) microspheres (MSs) has been investigated. Con A MSs prepared by a water-in-oil emulsion technique demonstrated properties of binding to glucose and SAPG-insulin (binding constants of 1.25 x 10(3) and 1.28 x 10(4) M-1, respectively) that are similar to the literature values of these properties for unmodified Con A. The retained binding capacity (number of binding sites per molecule) of Con A MSs, produced with 20% of the epsilon-amino groups crosslinked, was approximately 28% that of intact Con A. As a new device, Con A MSs loaded with SAPG-insulin was enclosed in a heat-sealed, surface-modified, porous poly(vinylidene difluoride) membrane pouch. This system showed a pulsatile release pattern for insulin with a short lag time in response to glucose challenges of 50-500 mg/dL. The pattern of release of SAPG-insulin from the devices was studied with varying design parameters, such as surface area, membrane pore size, and loading content of SAPG-insulin.

Binding, Competitive↗
PubMed1991

Receptors for epidermal growth factor and steroid hormones in primary colorectal tumors.

The presence of epidermal growth factor, estrogen, and progesterone receptors (EGFR, ER, and PR) was investigated by a competitive binding assay in 43 colorectal adenocarcinomas and 32 normal colorectal mucosa specimens. EGFR were expressed in most of the tumor specimens analyzed at levels comparable with normal mucosa. There was no correlation between EGFR and tumor localization, tumor size, tumor stage, and grading. Among tumor specimens, 13.9% and 6.9% expressed very low but detectable ER and PR levels, respectively. No statistically significant difference was found between steroid hormone receptor levels in the tumor and normal mucosa specimens, and neither was there any correlation of ER and PR with the pathological findings. Our results suggest that the EGFR system may play a role in regulating the growth of colorectal tissues. Further studies should demonstrate whether, despite the lack of correlation with histopathological parameters, EGFR expression may have a biological significance in human colorectal cancer.

Adenocarcinoma↗
PubMed1993

LY207320 (6-methylene-4-pregnene-3,20-dione) inhibits testosterone biosynthesis, androgen uptake, 5 alpha-reductase, and produces prostatic regression in male rats.

LY207320 is an in vitro inhibitor (estimated IC50 = 0.06 microM) of steroid 5 alpha-reductase that catalyzes the conversion of testosterone (T) to dihydrotestosterone (DHT). In contrast, LY207320 was only moderately active against rat prostatic 5 alpha-reductase in vivo (32% inhibition at 50.0 mg/kg single dose). LY207320 did, however, inhibit the in vivo uptake of [3H]-T by the prostate. The antiprostatic and endocrine effects of this agent were evaluated following daily (21 days) administration to castrated, androgen-supplemented castrate, and intact rats. LY207320, which has modest progestational competitive binding activity, does not bind to rat prostatic androgen or uterine estrogen cytosolic receptors. In the castrated male rat, subcutaneously (s.c.) administered LY207320 had no androgen agonist activity, as evidenced by a lack of accessory sex organ weight gains. Administration of s.c. LY207320 to intact rats for 21 days at doses greater than 5.0 mg/kg-day produced significant (P < 0.05) reductions of seminal vesicle and ventral prostatic weights (maximal regression = -65% and -40% from control values, respectively at 50.0 mg/kg-day). The compound had no regressive activity on male accessory sex organs when administered orally. LY207320 did not alter circulating prolactin, LH, or corticosterone levels, but at high doses (> or = 50.0 mg/kg-day), lowered circulating T[-67% from intact control levels (P < 0.05)]. Histological analysis of the rat ventral prostates (RVPs) in LY207320-treated rats was consistent with an androgen-deprived state. Decreased circulating androgens and prostatic regression are associated with inhibition of testicular 17 alpha-hydroxy/C17,20-lyase enzyme activity (IC50 = 0.06 microM). These findings support the contention that LY207320 is a physiological antagonist of androgen action in male rats, and that its effects are mediated primarily through inhibition of testicular androgen production rather than accessory sex organ 5 alpha-reductase.

Aldehyde-Lyases↗
PubMed2004

A 4.2 kDa synthetic peptide as a potential probe to evaluate the antibacterial activity of coumarin drugs.

The coumarin antibiotics are potent inhibitors of DNA replication whose target is the enzyme DNA gyrase, an ATP-dependent bacterial type II topoisomerase. The coumarin drugs inhibit gyrase action by competitive binding to the ATP-binding site of DNA gyrase B protein. The production of new biologically active products has stimulated additional studies on coumarin-gyrase interactions. In this regard, a 4.2 kDa peptide mimic of DNA gyrase B protein from Escherichia coli has been designed and synthesized. The peptide sequence includes the natural fragment 131-146 (coumarin resistance-determining region) and a segment containing the gyrase-DNA interaction region (positions 753-770). The peptide mimic binds to novobiocin (Ka = 1.4+/-0.3 x 10(5) M(-1)), plasmid (Ka = 1.6+/-0.5 x 10(6) M(-1)) and ATP (Ka = 1.9+/-50.4 x 10(3) M(-1)), results previously found with the intact B protein. On the other hand, the binding to novobiocin was reduced when a mutation of Arg-136 to Leu-136 was introduced, a change previously found in the DNA gyrase B protein from several coumarin-resistant clinical isolates of Escherichia coli In contrast, the binding to plasmid and to ATP was not altered. These results suggest that synthetic peptides designed in a similar way to that described here could be used as mimics of DNA gyrase in studies which seek a better understanding of the ATP, as well as coumarin, binding to the gyrase and also the mechanism of action of this class of antibacterial drugs.

Adenosine Triphosphate↗
PubMed2005

Further analogues of plant peptide hormone phytosulfokine-alpha (PSK-alpha) and their biological evaluation.

Phytosulfokine-alpha (PSK-alpha), a sulfated growth factor of structure H-Tyr(SO3H)-Ile-Tyr(SO3H)-Thr-Gln-OH universally found in both monocotyledons and dicotyledons, strongly promotes proliferation of plant cells in culture. In studies on the structure/activity relationship of PSK-alpha the synthesis was performed of a series of a further 23 analogues modified in position 1, 3 or 4 as well as simultaneously in positions 1 and 3 of the peptide chain. Peptides were synthesized by the solid phase method according to the Fmoc procedure on a Wang-resin. Free peptides were released from the resin by 95% TFA in the presence of EDT. All peptides were tested by competitive binding assay to the carrot membrane using 3H-labelled PSK-alpha according to the test of Matsubayashi et al. Among these peptide analogues, [H-Phe(4-Cl)1]-PSK-alpha (IV), [H-Phe(4-I)1]-PSK-alpha (VII), and [Phe(4-Cl)3]-PSK-alpha (XI) retained 30% PSK-alpha activity. Analogue [Tyr(PO3H2)3]-PSK-alpha (IX) showed 10% of PSK-alpha activity.

Binding, Competitive↗
PubMed1996

The anti-estrogenic activity of selected polynuclear aromatic hydrocarbons in yeast expressing human estrogen receptor.

Polynuclear aromatic hydrocarbons (PAH) represent a large class of chemicals present in environment. We have used yeast strain ER(wt) expressing human estrogen receptor (hER) and an estrogen-sensitive reporter to characterize the estrogenic or anti-estrogenic activities of 21 PAHs. The PAHs did not exhibit estrogenic activity in yeast strain ER(wt). Four of the PAHs, dibenz[a,h]anthracene, 6-hydroxy-chrysene, 2,3-benzofluorene, and benzo(a)pyrene, inhibited estradiol-dependent reporter activity in strain ER(wt). A mutant hER lacking the amino terminus expressed in yeast was inhibited by the four PAHs to a lesser extent than the full-length hER. 6-hydroxy-chrysene, 2,3-benzofluorene, and benzo(a)pyrene, but not dibenz[a,h]anthracene, weakly displaced [3H]estradiol from the hER in a competition binding assay. A strong correlation between the inhibition of [3H]estradiol-binding from the hER and the reduction of hER-mediated transactivation in yeast was not observed. These observations suggest that the PAHs dibenz[a,h]-anthracene, 6-hydroxy-chrysene, 2,3-benzofluorene, and benzo(a)pyrene may antagonize activity in yeast by binding to an anti-estrogen binding site on the hER or by mechanisms independent of the hER.

Binding, Competitive↗