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Papers by Richard Sportsman
Real-time quantification of fatty acid uptake using a novel
Procedures for identifying compounds having at least one characteristic selected from a group con... more Procedures for identifying compounds having at least one characteristic selected from a group consisting of a composition (A) MODULATES kinase activity of the insulin receptor; And / or (B) potentiates insulin activation of the insulin receptor; And / or (C) DIFFERENCE stimulation of glucose uptake by insulin CELLULAR; (D) stimulates glucose uptake in cells INDICATING insulin receptor; (E) REDUCES BLOOD GLUCOSE DIABETIC INDIVIDUALS; (F) stimulates phosphorylation of IRS - 1; (G) stimulates the activity of PL KINASE 3; And / or (H) stimulates translocation of GLUT - 4; ALL which are described ASI. Substances that have these characteristics alter the conformation of the scope of Cytoplasmic QUINASA lobules or preferentially bind the points that have been identified as binding sites Modulators CHAIN INSULIN BE RECEIVING. Furthermore, the modulating the activity of the insulin receptor, increasing glucose uptake by cells, AND OTHER MAJOR EFFECT ON CONTROL AND MANAGEMENT OF DIABETES AR...
Gene Analysis Techniques, 1984
A simple, economical, and efficient procedure for analysis of proteins (Western blotting) and DNA... more A simple, economical, and efficient procedure for analysis of proteins (Western blotting) and DNA (Southern blotting) transferred to nitrocellulose for reaction with antibodies or nucleic acid probes is described. The techniques utilize nonfat dry milk as a protein-nucleic acid source for blocking nonspecific reactions, as an incubation medium, and for subsequent washing to remove unreacted reagents. The incubation cocktail, termed BLOTTO (Bovine Lacto Transfer Technique Optimizer), is superior to bovine serum albumin or gelatin for preventing nonspecific absorption in Western blot analyses and does not require the use of detergents or chaotropic agents to effect efficient reduction of background. BLOTTO, at the proper dilution in NaClNa citrate, is just as efficient in Southern blot analyses as more complicated cocktails typically used in the latter technique. We also found that BLOTTO works well for blocking, incubating, and washing ELISA plate assays relative to the normal BSA carrier, at a considerable savings to the laboratory.
Combinatorial Chemistry & High Throughput Screening, 2003
Fluorescence polarization (FP) has become widely employed for high throughput screening used in p... more Fluorescence polarization (FP) has become widely employed for high throughput screening used in pharmaceutical drug discovery. Assays of important signal transduction targets are now adapted to FP. In this review we examine assays for cyclic adenosine monophosphate, phosphodiesterases, and protein kinases and phosphatases using FP competitive immunoassays and a direct enzymatic method called IMAP.
Analytical Chemistry, 1983
The kinetic and equilibrium characteristics of the interaction of Insulin with a specific antibod... more The kinetic and equilibrium characteristics of the interaction of Insulin with a specific antibody immobilized on siianetreated controlled pore glass beads have been evaluated by using the technique of "high-performance lmmunoaff lnlty chromatography" (HPIC). A previously described equilibrium model was verified and used to establish the equilibrium constants for the reaction of the immobilized antibody with insulln in the mobile phase. Computer simulations and experimental data were used to demonstrate the contribution of nonspectfic interactions with the support to the overall binding process. An estimate Is also made of the rate of the antlbody-antigen complex formation reaction, and its effect on immunochromatographic investlgations is discussed.
Analytical Chemistry, 1980
The authors wish to thank Spectra Physics Inc. for the generous loan of the SP8000 liquid chromat... more The authors wish to thank Spectra Physics Inc. for the generous loan of the SP8000 liquid chromatograph, and Dupont, Inc., for a gift of the Zorbax CN column. Special thanks are also due to Dave Herman for his helpful discussions and ideas on the subject.
Journal of Clinical Investigation, 1986
A monoclonal antibody, selected for reactivity with the Epstein-Barr virus (EBV)-encoded antigen ... more A monoclonal antibody, selected for reactivity with the Epstein-Barr virus (EBV)-encoded antigen EBNA-1, exhibited strong reactivity with the synovial lining cells in joint biopsies from 10 of 12 patients with rheumatoid arthritis (RA) and adherent cells eluted from these tissues. No staining of RA synovial membrane frozen tissue sections or eluted synovial-lining cells was obtained with monoclonal antibodies directed against other EBV-encoded antigens (anti-p160, anti-gp200/350) or with monoclonal antibodies directed against antigens encoded by cytomegalovirus, herpes simplex viruses, or human T cell leukemia virus type I. Among 12 osteoarthritis and normal synovial biopsies only rare reactive cells were noted. Characterization of the antigen(s) in RA synovium by the Western immunoblotting technique revealed a 62,000-molecularweight (mol-wt) protein, in contrast to the 70,000-85,000-molwt EBNA-1 antigen found in EBV-transformed cells. The structural basis for the cross-reactivity of the RA synovial membrane 62,000-mol-wt protein and the EBNA-1 antigen appears to reside in the glycine-alanine rich region of these molecules. A rabbit antibody directed against a synthetic peptide (IR3-VI-2) derived from the glycine-alanine-rich region of EBNA-1 reacted with the 70,000-85,000-mol-wt EBNA-1 antigen in EBV-infected cells and with the 62,000-mol-wt molecule in RA synovial membrane extracts. Since strong antibody responses to EBNA-1 are known to exist in RA patients, these results suggest that immune responses to a cross-reactive antigen may play a role in the pathogenesis of RA.
Journal of Biomolecular Screening, 2002
Cyclic nucleotide phosphodiesterases (PDEs) catalyze the hydrolysis of the 3′-ester bond of cycli... more Cyclic nucleotide phosphodiesterases (PDEs) catalyze the hydrolysis of the 3′-ester bond of cyclic AMP (cAMP) and cyclic GMP (cGMP), important second messengers in the transduction of a variety of extracellular signals. There is growing interest in the study of PDEs as drug targets for novel therapeutics. We describe the development of a homogeneous fluorescence polarization assay for PDEs based on the strong binding of PDE reaction products (i.e., AMP or GMP) onto modified nanoparticles through interactions with immobilized trivalent metal cations. This assay technology (IMAP) is applicable to both cAMP- and cGMP-specific PDEs. Results of the assay in 384- and 1536-well microplates are presented.
Journal of Biomolecular Screening, 2002
Cyclic nucleotide phosphodiesterases (PDEs) catalyze the hydrolysis of the 3′-ester bond of cycli... more Cyclic nucleotide phosphodiesterases (PDEs) catalyze the hydrolysis of the 3′-ester bond of cyclic AMP (cAMP) and cyclic GMP (cGMP), important second messengers in the transduction of a variety of extracellular signals. There is growing interest in the study of PDEs as drug targets for novel therapeutics. We describe the development of a homogeneous fluorescence polarization assay for PDEs based on the strong binding of PDE reaction products (i.e., AMP or GMP) onto modified nanoparticles through interactions with immobilized trivalent metal cations. This assay technology (IMAP) is applicable to both cAMP- and cGMP-specific PDEs. Results of the assay in 384- and 1536-well microplates are presented.
Methods in Enzymology, 2002
... J. Richard Sportsman. Available online 29 November 2003. ... [231 Fluorescence Anisotropy in ... more ... J. Richard Sportsman. Available online 29 November 2003. ... [231 Fluorescence Anisotropy in Pharmacologic Screening By J. RICHARD SPORTSMAN Introduction The enterprise of drug discovery screening has undergone an industrial revolu-tion in the past decade. ...
To quantify concentrations of anti-growth hormone antibody in >600 serum samples by radioimmunoas... more To quantify concentrations of anti-growth hormone antibody in >600 serum samples by radioimmunoassay, we devised a system ("B0LAc") to process data and to execute the curvefitting program LIGAND-PC automatically with an IBM PCcompatible computer. We fit data from each sample to four binding models comprising different combinations of nonspecific binding and one-or two-antibody binding sites. Total antibody concentration is then calculated from the model that is statisticaHy "best." This process occasionally selects a two-binding-site model that severely overestimates the antibody concentration. Errors of this kind are discarded by constraining the product of the second-site antibody's affinity and its concentration to exceed a minimum value (0.05). We evaluated the performance of the B0L.Acsystem by assaying controls and by using computer simulations to demonstrate the high confidence levels attainable in estimation of antibody concentrations. Between-assay variability (CV) was <25%, and analytical recovery exceeded 90%. These figures are acceptable for an assay based on curve-fining of competitive radioimmunoassay data, allowing clinically relevant assessments of antibody responses in patients' samples. The advantages of the aoic system include high throughput and the reporting of results in absolute units of affinities and concentrations. Additional Keyphrases: natural and modified proteins as antigens somatotropinsomat rem assessing immunogenicity assuming multiple antibody populations; for most purposes two sites are adequate to describe the data (17). Problems arise when nonspecific binding becomes appreciable relative to low-affinity binding, which can lead curve-fitting procedures to indicate erroneously high concentrations of low-affinity sites. The unreliability of curve-fitting techniques in these situations has led some researchers to Lilly Research Laboratories, Lilly CorporateCenter, Indianapolis, IN 46285. 'Nonstandard abbreviations: hGH, human growth hormone; NLLS, nonlinear least squares; BOLAC, batch operationof LIGAND for antibodycapacity;and SBT, serum binding test.
Methods in enzymology, 2003
... J. Richard Sportsman. Available online 29 November 2003. ... [231 Fluorescence Anisotropy in ... more ... J. Richard Sportsman. Available online 29 November 2003. ... [231 Fluorescence Anisotropy in Pharmacologic Screening By J. RICHARD SPORTSMAN Introduction The enterprise of drug discovery screening has undergone an industrial revolu-tion in the past decade. ...
Real-time quantification of fatty acid uptake using a novel
Procedures for identifying compounds having at least one characteristic selected from a group con... more Procedures for identifying compounds having at least one characteristic selected from a group consisting of a composition (A) MODULATES kinase activity of the insulin receptor; And / or (B) potentiates insulin activation of the insulin receptor; And / or (C) DIFFERENCE stimulation of glucose uptake by insulin CELLULAR; (D) stimulates glucose uptake in cells INDICATING insulin receptor; (E) REDUCES BLOOD GLUCOSE DIABETIC INDIVIDUALS; (F) stimulates phosphorylation of IRS - 1; (G) stimulates the activity of PL KINASE 3; And / or (H) stimulates translocation of GLUT - 4; ALL which are described ASI. Substances that have these characteristics alter the conformation of the scope of Cytoplasmic QUINASA lobules or preferentially bind the points that have been identified as binding sites Modulators CHAIN INSULIN BE RECEIVING. Furthermore, the modulating the activity of the insulin receptor, increasing glucose uptake by cells, AND OTHER MAJOR EFFECT ON CONTROL AND MANAGEMENT OF DIABETES AR...
Gene Analysis Techniques, 1984
A simple, economical, and efficient procedure for analysis of proteins (Western blotting) and DNA... more A simple, economical, and efficient procedure for analysis of proteins (Western blotting) and DNA (Southern blotting) transferred to nitrocellulose for reaction with antibodies or nucleic acid probes is described. The techniques utilize nonfat dry milk as a protein-nucleic acid source for blocking nonspecific reactions, as an incubation medium, and for subsequent washing to remove unreacted reagents. The incubation cocktail, termed BLOTTO (Bovine Lacto Transfer Technique Optimizer), is superior to bovine serum albumin or gelatin for preventing nonspecific absorption in Western blot analyses and does not require the use of detergents or chaotropic agents to effect efficient reduction of background. BLOTTO, at the proper dilution in NaClNa citrate, is just as efficient in Southern blot analyses as more complicated cocktails typically used in the latter technique. We also found that BLOTTO works well for blocking, incubating, and washing ELISA plate assays relative to the normal BSA carrier, at a considerable savings to the laboratory.
Combinatorial Chemistry & High Throughput Screening, 2003
Fluorescence polarization (FP) has become widely employed for high throughput screening used in p... more Fluorescence polarization (FP) has become widely employed for high throughput screening used in pharmaceutical drug discovery. Assays of important signal transduction targets are now adapted to FP. In this review we examine assays for cyclic adenosine monophosphate, phosphodiesterases, and protein kinases and phosphatases using FP competitive immunoassays and a direct enzymatic method called IMAP.
Analytical Chemistry, 1983
The kinetic and equilibrium characteristics of the interaction of Insulin with a specific antibod... more The kinetic and equilibrium characteristics of the interaction of Insulin with a specific antibody immobilized on siianetreated controlled pore glass beads have been evaluated by using the technique of "high-performance lmmunoaff lnlty chromatography" (HPIC). A previously described equilibrium model was verified and used to establish the equilibrium constants for the reaction of the immobilized antibody with insulln in the mobile phase. Computer simulations and experimental data were used to demonstrate the contribution of nonspectfic interactions with the support to the overall binding process. An estimate Is also made of the rate of the antlbody-antigen complex formation reaction, and its effect on immunochromatographic investlgations is discussed.
Analytical Chemistry, 1980
The authors wish to thank Spectra Physics Inc. for the generous loan of the SP8000 liquid chromat... more The authors wish to thank Spectra Physics Inc. for the generous loan of the SP8000 liquid chromatograph, and Dupont, Inc., for a gift of the Zorbax CN column. Special thanks are also due to Dave Herman for his helpful discussions and ideas on the subject.
Journal of Clinical Investigation, 1986
A monoclonal antibody, selected for reactivity with the Epstein-Barr virus (EBV)-encoded antigen ... more A monoclonal antibody, selected for reactivity with the Epstein-Barr virus (EBV)-encoded antigen EBNA-1, exhibited strong reactivity with the synovial lining cells in joint biopsies from 10 of 12 patients with rheumatoid arthritis (RA) and adherent cells eluted from these tissues. No staining of RA synovial membrane frozen tissue sections or eluted synovial-lining cells was obtained with monoclonal antibodies directed against other EBV-encoded antigens (anti-p160, anti-gp200/350) or with monoclonal antibodies directed against antigens encoded by cytomegalovirus, herpes simplex viruses, or human T cell leukemia virus type I. Among 12 osteoarthritis and normal synovial biopsies only rare reactive cells were noted. Characterization of the antigen(s) in RA synovium by the Western immunoblotting technique revealed a 62,000-molecularweight (mol-wt) protein, in contrast to the 70,000-85,000-molwt EBNA-1 antigen found in EBV-transformed cells. The structural basis for the cross-reactivity of the RA synovial membrane 62,000-mol-wt protein and the EBNA-1 antigen appears to reside in the glycine-alanine rich region of these molecules. A rabbit antibody directed against a synthetic peptide (IR3-VI-2) derived from the glycine-alanine-rich region of EBNA-1 reacted with the 70,000-85,000-mol-wt EBNA-1 antigen in EBV-infected cells and with the 62,000-mol-wt molecule in RA synovial membrane extracts. Since strong antibody responses to EBNA-1 are known to exist in RA patients, these results suggest that immune responses to a cross-reactive antigen may play a role in the pathogenesis of RA.
Journal of Biomolecular Screening, 2002
Cyclic nucleotide phosphodiesterases (PDEs) catalyze the hydrolysis of the 3′-ester bond of cycli... more Cyclic nucleotide phosphodiesterases (PDEs) catalyze the hydrolysis of the 3′-ester bond of cyclic AMP (cAMP) and cyclic GMP (cGMP), important second messengers in the transduction of a variety of extracellular signals. There is growing interest in the study of PDEs as drug targets for novel therapeutics. We describe the development of a homogeneous fluorescence polarization assay for PDEs based on the strong binding of PDE reaction products (i.e., AMP or GMP) onto modified nanoparticles through interactions with immobilized trivalent metal cations. This assay technology (IMAP) is applicable to both cAMP- and cGMP-specific PDEs. Results of the assay in 384- and 1536-well microplates are presented.
Journal of Biomolecular Screening, 2002
Cyclic nucleotide phosphodiesterases (PDEs) catalyze the hydrolysis of the 3′-ester bond of cycli... more Cyclic nucleotide phosphodiesterases (PDEs) catalyze the hydrolysis of the 3′-ester bond of cyclic AMP (cAMP) and cyclic GMP (cGMP), important second messengers in the transduction of a variety of extracellular signals. There is growing interest in the study of PDEs as drug targets for novel therapeutics. We describe the development of a homogeneous fluorescence polarization assay for PDEs based on the strong binding of PDE reaction products (i.e., AMP or GMP) onto modified nanoparticles through interactions with immobilized trivalent metal cations. This assay technology (IMAP) is applicable to both cAMP- and cGMP-specific PDEs. Results of the assay in 384- and 1536-well microplates are presented.
Methods in Enzymology, 2002
... J. Richard Sportsman. Available online 29 November 2003. ... [231 Fluorescence Anisotropy in ... more ... J. Richard Sportsman. Available online 29 November 2003. ... [231 Fluorescence Anisotropy in Pharmacologic Screening By J. RICHARD SPORTSMAN Introduction The enterprise of drug discovery screening has undergone an industrial revolu-tion in the past decade. ...
To quantify concentrations of anti-growth hormone antibody in >600 serum samples by radioimmunoas... more To quantify concentrations of anti-growth hormone antibody in >600 serum samples by radioimmunoassay, we devised a system ("B0LAc") to process data and to execute the curvefitting program LIGAND-PC automatically with an IBM PCcompatible computer. We fit data from each sample to four binding models comprising different combinations of nonspecific binding and one-or two-antibody binding sites. Total antibody concentration is then calculated from the model that is statisticaHy "best." This process occasionally selects a two-binding-site model that severely overestimates the antibody concentration. Errors of this kind are discarded by constraining the product of the second-site antibody's affinity and its concentration to exceed a minimum value (0.05). We evaluated the performance of the B0L.Acsystem by assaying controls and by using computer simulations to demonstrate the high confidence levels attainable in estimation of antibody concentrations. Between-assay variability (CV) was <25%, and analytical recovery exceeded 90%. These figures are acceptable for an assay based on curve-fining of competitive radioimmunoassay data, allowing clinically relevant assessments of antibody responses in patients' samples. The advantages of the aoic system include high throughput and the reporting of results in absolute units of affinities and concentrations. Additional Keyphrases: natural and modified proteins as antigens somatotropinsomat rem assessing immunogenicity assuming multiple antibody populations; for most purposes two sites are adequate to describe the data (17). Problems arise when nonspecific binding becomes appreciable relative to low-affinity binding, which can lead curve-fitting procedures to indicate erroneously high concentrations of low-affinity sites. The unreliability of curve-fitting techniques in these situations has led some researchers to Lilly Research Laboratories, Lilly CorporateCenter, Indianapolis, IN 46285. 'Nonstandard abbreviations: hGH, human growth hormone; NLLS, nonlinear least squares; BOLAC, batch operationof LIGAND for antibodycapacity;and SBT, serum binding test.
Methods in enzymology, 2003
... J. Richard Sportsman. Available online 29 November 2003. ... [231 Fluorescence Anisotropy in ... more ... J. Richard Sportsman. Available online 29 November 2003. ... [231 Fluorescence Anisotropy in Pharmacologic Screening By J. RICHARD SPORTSMAN Introduction The enterprise of drug discovery screening has undergone an industrial revolu-tion in the past decade. ...