John G Bruno

California State University, San Marcos, San Marcos, California, United States

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Publications (71)108.23 Total impact

  • John G. Bruno · Alicia M. Richarte
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    ABSTRACT: DNA aptamer sequences were selected and screened for affinity against recombinant human insulin-like growth factor-I (rhIGF-I) using an ELISA-like aptamer-based assay (ELASA). When the top four aptamers were paired against one another in all possible sandwich assay combinations using capture aptamers on magnetic beads (MBs), one sandwich combination appeared superior. This combination produced a reliably sensitive assay with a probable limit of detection. <16. ng/mL even in human serum. The assay did not require separation of rhIGF-I from IGF binding proteins (IGFBP) in human serum, suggesting that the capture and reporter aptamers may bind an exposed IGF-I epitope in the IGF-I-IGFBP complex.
    No preview · Article · Aug 2015 · Microchemical Journal
  • John G. Bruno · Taylor Phillips · Tiffany Montez
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    ABSTRACT: Two hundred base long DNA aptamers were developed against crude black cobra (Naja melanoleuca) venom and purified honey bee phospholipase A2 (PLA2). These aptamers were screened for the ability to inhibit PLA2 via a colorimetric assay and for the ability to protect murine NIH 3T3 fibroblasts from cobra venom in culture as assessed by trypan blue dye exclusion. Several aptamers were identified which could inhibit cobra venom up to 44% and bee PLA2 over 56%, while an unrelated aptamer and randomly chosen oligonucleotide could not inhibit the enzymatic activity of bee PLA2. Some aptamers were also identified which protected 3T3 cells from cobra venom in vitro (i.e., enhanced viability from 68% to 86%–89% in the presence of cobra venom). Although, snake venoms are complex mixtures of various tissue degradative enzymes such as PLA2, neurotoxins and other toxins, the existence of these aptamers provides proof-of-principle that aptamer-based antidotes for snake, insect and other venomous bites are feasible. DNA aptamerbased antivenoms could also be mass produced at lower cost than traditional antisera with longer shelf-lives at ambient temperatures to aid envenomated victims in underdeveloped countries.
    No preview · Article · Aug 2015 · Journal of Bionanoscience
  • John G Bruno
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    ABSTRACT: Despite the great promise of nucleic acid aptamers in the areas of diagnostics and therapeutics for their facile in vitro development, lack of immunogenicity and other desirable properties, few truly successful aptamer-based products exist in the clinical or other markets. Core reasons for these commercial deficiencies probably stem from industrial commitment to antibodies including a huge financial investment in humanized monoclonal antibodies and a general ignorance about aptamers and their performance among the research and development community. Given the early failures of some strong commercial efforts to gain government approval and bring aptamer-based products to market, it may seem that aptamers are doomed to take a backseat to antibodies forever. However, the key advantages of aptamers over antibodies coupled with niche market needs that only aptamers can fill and more recent published data still point to a bright commercial future for aptamers in areas such as infectious disease and cancer diagnostics and therapeutics. As more researchers and entrepreneurs become familiar with aptamers, it seems inevitable that aptamers will at least be considered for expanded roles in diagnostics and therapeutics. This review also examines new aptamer modifications and attempts to predict new aptamer applications that could revolutionize biomedical technology in the future and lead to marketed products.
    No preview · Article · Apr 2015 · Molecules
  • John G Bruno
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    ABSTRACT: The metachromatic fluorophore acridine orange (AO) has demonstrated green fluorescent staining of dormant Bacillus spores and orange to red staining of transcriptionally active vegetative cells when used in the mid-micoMolar range. Despite the microscopic observation of numerous bright orange to red fluorescent vegetative cells following germination induction, no clear spectral emission peaks > 590 nm have ever been reported for spectrofluorometric analysis involving AO in conjunction with spore germination. This microscopy versus spectrofluorometry paradox is documented in the present report and hypotheses are put forth to explain the very weak spectral changes in the red region which do not appear to correlate with the abundant orange-red fluorescence of nascent vegetative cells seen through the fluorescence microscope.
    No preview · Article · Dec 2014 · Journal of Fluorescence
  • John G. Bruno · Alicia M. Richarte · Taylor Phillips
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    ABSTRACT: Fifty-two candidate DNA aptamer sequences were selected for binding to the cardiovascular biomarker B-type or brain natriuretic peptide (BNP). Candidate aptamers were screened to rank their relative affinities against BNP by an aptamer-based ELISA-like microplate assay (ELASA). The highest affinity aptamers from ELASA screening were also paired in all possible combinations and screened for electrochemiluminescence (ECL) assay potential in capture aptamer-magnetic bead and ruthenium trisbipyridine (Ru(bpy)32 +)-reporter aptamer sandwich formats. The top ECL sandwich combinations utilized the same aptamer pair in either capture or reporting roles with nanogram to low picogram per mL levels of detection even in 50% human serum. ECL assay sensitivity and linearity even in 50% human serum suggest that the aptamer-based assay is at least comparable to other reported immunoassays for BNP.
    No preview · Article · Jul 2014 · Microchemical Journal
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    John G Bruno
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    ABSTRACT: Preliminary studies aimed at improving the sensitivity of foodborne pathogen detection via lateral flow (LF) test strips by use of high affinity DNA aptamers for capture and reporter functions when coupled to red-emitting quantum dots (Qdot 655) are reported. A variety of DNA aptamers developed against Escherichia coli, Listeria monocytogenes, and Salmonella enterica were paired in capture and reporter combinations to determine which yielded the strongest detection of their cognate bacteria using a colloidal gold screening system. Several promising sandwich combinations were identified for each of the three bacterial LF strip systems. The best E. coli aptamer-LF system was further studied and yielded a visible limit of detection (LOD) of ~3,000 E. coli 8739 and ~6,000 E. coli O157:H7 in buffer. These LODs were reduced to ~300-600 bacterial cells per test respectively by switching to a Qdot 655 aptamer-LF system. Novel aspects of these assays such as the use of high levels of detergents to avoid quantum dot agglutination and enhance migration in analytical membranes, identification of optimal analytical membrane types, UV-immobilization of capture aptamers, and novel dual biotin/digoxigenin-end labeled aptamer streptavidin-colloidal gold or -Qdot 655 conjugates plus anti-digoxigenin antibody control lines are also discussed. In general, this work provides proof-of-principle for highly sensitive aptamer-Qdot LF strip assays for rapid foodborne pathogen detection.
    Preview · Article · Jun 2014 · Pathogens
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    ABSTRACT: Leishmaniasis is a disease caused by protozoan parasites of the genus Leishmania which affects an estimated 12 million people worldwide. The work presented herein describes characterization of several DNA aptamers which were developed against two different 39-amino acid synthetic peptides derived from the highly conserved repetitive regions of a 230 kD flagellar Leishmania kinesin for their potential use in Leishmania diagnostics. Following ten rounds of aptamer affinity selection against the kinesin peptides and PCR amplification (i.e., SELEX), several candidate aptamers demonstrated strong binding to the kinesin peptides in an enzyme-linked aptamer sorbent assay (ELASA). Confocal fluorescence microscopy using several aptamers with the highest affinities and the microtubule marker paclitaxel revealed localization patterns characterized by diffuse, but often punctate, staining within the cytoplasm and in some cases along the flagella of L. major promastigotes. Aptamer association with microtubules was further evidenced by aptamer-colloidal gold staining and electron microscopy supporting the binding and specificity of these aptamers for Leishmania kinesins. In addition, Southwestern blots confirmed that some of the top aptamers were specific for their respective ∼4 kD peptides. One aptamer in particular (K2-13R) also demonstrated clear detection of a 100 kD protein band and >250 kD bands in L. major promastigote lysate. Given that the presence of antibodies recognizing the 39 kD kinesin (K39) or repetitive region of the 230 kD kinesin in patient serum is useful for the serodiagnosis of visceral leishmaniasis, but that few commercial antibodies exist for detection of K39 itself, these kinesin aptamers may have value as novel cellular and molecular probes for Leishmania diagnosis.
    No preview · Article · Jun 2014 · Journal of Bionanoscience
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    ABSTRACT: A fluorescent peroxidase-linked DNA aptamer-magnetic bead sandwich assay is described which detects as little as 100 ng of soluble protein extracted from Leishmania major promastigotes with a high molarity chaotropic salt. Lessons learned during development of the assay are described and elucidate the pros and cons of using fluorescent dyes or nanoparticles and quantum dots versus a more consistent peroxidase-linked Amplex Ultra Red (AUR; similar to resazurin) fluorescence version of the assay. While all versions of the assays were highly sensitive, the AUR-based version exhibited lower variability between tests. We hypothesize that the AUR version of this assay is more consistent, especially at low analyte levels, because the fluorescent product of AUR is liberated into bulk solution and readily detectable while fluorophores attached to the reporter aptamer might occasionally be hidden behind magnetic beads near the detection limit. Conversely, fluorophores could be quenched by nearby beads or other proximal fluorophores on the high end of analyte concentration, if packed into a small area after magnetic collection when an enzyme-linked system is not used. A highly portable and rechargeable battery-operated fluorometer with on board computer and color touchscreen is also described which can be used for rapid (<1 h) and sensitive detection of Leishmania promastigote protein extracts (∼100 ng per sample) in buffer or sandfly homogenates for mapping of L. major parasite geographic distributions in wild sandfly populations.
    No preview · Article · Nov 2013 · Journal of Fluorescence
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    John G Bruno
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    ABSTRACT: The potential to emulate or enhance antibodies with nucleic acid aptamers while lowering costs has prompted development of new aptamer-protein, siRNA, drug, and nanoparticle conjugates. Specific focal points of this review discuss DNA aptamers covalently bound at their 3' ends to various proteins for enhanced stability and greater pharmacokinetic lifetimes in vivo. The proteins can include Fc tails of IgG for opsonization, and the first component of complement (C1q) to trigger complement-mediated lysis of antibiotic-resistant Gram negative bacteria, cancer cells and possibly some parasites during vulnerable stages. In addition, the 3' protein adduct may be a biotoxin, enzyme, or may simply be human serum albumin (HSA) or a drug known to bind HSA, thereby retarding kidney and other organ clearance and inhibiting serum exonucleases. In this review, the author summarizes existing therapeutic aptamer conjugate categories and describes his patented concept for PCR-based amplification of double-stranded aptamers followed by covalent attachment of proteins or other agents to the chemically vulnerable overhanging 3' adenine added by Taq polymerase. PCR amplification of aptamers could dramatically lower the current $2,000/gram cost of parallel chemical oligonucleotide synthesis, thereby enabling mass production of aptamer-3'-protein or drug conjugates to better compete against expensive humanized monoclonal antibodies.
    Preview · Article · Mar 2013 · Pharmaceuticals
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    ABSTRACT: Hybrid aptamer-C1q and Fc conjugates have recently been used to kill Gram negative bacteria and cancer cells. In this chapter we summarize our past work and present new evidence for how aptamers can couple to the immune system to kill invading cells and endogenous cancer cells. Examples of new aptamer internalization by receptormediated endocytosis for drug delivery and stoichiometric aptamer-toxin neutralization studies are also reported.
    No preview · Article · Jan 2013
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    ABSTRACT: Novel aptamer beacon, competitive and intrachain fluorescence resonance energy transfer (FRET)-aptamer approaches and data are documented for rapid (15-20 minute) detection of chemical and biological (CB) threat agents. Aptamers to detect these potential CB terrorist weapons include aptamers that bind organophosphorus (OP) nerve agents, botulinum neurotoxins (BoNTs) A, B, and E, and Bacillus anthracis (anthrax) spores. We demonstrate here that FRET-aptamers can detect CB threat agents within minutes at very low levels even in turbid soil suspensions in portable and lyophilized formats for long assay shelf life.
    No preview · Article · Jan 2013
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    ABSTRACT: Aptamers have been employed in a variety of traditional immunoassay formats, but because aptamers are composed of nucleic acids, their implementation in some traditional platforms can present unique challenges and opportunities. In this chapter we relay our experiences and advice with regard to aptamer implementation in ELISA-like microplate assays, lateral flow chromatographic test strip, electrochemiluminescent (ECL) and fluorescent magnetic bead sandwich assays for a variety of infectious diseases and clinical analytes of contemporary interest.
    No preview · Article · Jan 2013
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    ABSTRACT: Background Nucleic acid aptamers have long demonstrated the capacity to bind viral envelope proteins and to inhibit the progression of pathogenic virus infections. Here we report on initial efforts to develop and screen DNA aptamers against recombinant envelope proteins or synthetic peptides and whole inactivated viruses from several virulent arboviruses including Chikungunya, Crimean-Congo hemorrhagic fever (CCHF), dengue, tickborne encephalitis and West Nile viruses. We also analyzed sequence data and secondary structures for commonalities that might reveal consensus binding sites among the various aptamers. Some of the highest affinity and most specific aptamers in the down-selected libraries were demonstrated to have diagnostic utility in lateral flow chromatographic assays and in a fluorescent aptamer-magnetic bead sandwich assay. Some of the reported aptamers may also be able to bind viral envelope proteins in vivo and therefore may have antiviral potential in passive immunity or prophylactic applications. Results Several arbovirus DNA aptamer sequences emerged multiple times in the various down selected aptamer libraries thereby suggesting some consensus sequences for binding arbovirus envelope proteins. Screening of aptamers by enzyme-linked aptamer sorbent assay (ELASA) was useful for ranking relative aptamer affinities against their cognate viral targets. Additional study of the aptamer sequences and secondary structures of top-ranked anti-arboviral aptamers suggest potential virus binding motifs exist within some of the key aptamers and are highlighted in the supplemental figures for this article. One sequence segment (ACGGGTCCGGACA) emerged 60 times in the anti-CCHF aptamer library, but nowhere else in the anti-arbovirus library and only a few other times in a larger library of aptamers known to bind bacteria and rickettsia or other targets. Diagnostic utility of some of the aptamers for arbovirus detection in lateral flow chromatographic assays and a fluorescent sandwich assay on the surface of magnetic microbeads is also demonstrated. Conclusions This article catalogues numerous DNA aptamer sequences which can bind various important pathogenic arboviruses and have, in some cases, already demonstrated diagnostic potential. These aptamer sequences are proprietary, patent-pending, and partially characterized. Therefore, they are offered to the scientific community for potential research use in diagnostic assays, biosensor applications or for possible passive immunity and prophylaxis against pathogenic viruses.
    Full-text · Article · Nov 2012 · BMC Research Notes
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    ABSTRACT: This study was designed to characterize binding of a DNA aptamer to breast cancer cells and to test whether that aptamer could be used to kill target cells in vitro as part of an aptamer-C1q protein conjugate by coupling to the classic complement cascade. A biotinylated DNA aptamer designated MUC1-5TR-1 was shown to decorate the plasma membranes of human breast adenocarcinoma (MCF7) cells via fluorescence confocal microscopy. Biotinylated aptamer binding successfully initiated the classical complement pathway leading to complement fixation on the target cells via a streptavidin-C1q conjugate as previously reported. Förster Resonance Energy Transfer (FRET) measurements demonstrated membrane depolarization upon aptamer binding, providing indirect evidence of membrane attack complex (MAC) formation as a result of aptamer binding. Transmission electron microscopy (TEM) and immunogold labeling confirmed that aptamer-mediated complement fixation results in MAC formation on the plasma membrane, leading to osmotic swelling and cell death. This approach may provide a much less toxic and more precisely targeted "antibody-like" treatment for cancers by coupling to the patient's innate immune system in much the same way as more expensive humanized monoclonal antibodies.
    No preview · Article · Aug 2012
  • John G Bruno · Maria P Carrillo
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    ABSTRACT: A library of 92 DNA aptamer sequences was developed against Bacillus anthracis (nonpathogenic Sterne strain) spores and anthrose sugar immobilized on magnetic beads. The selected DNA sequences were studied for similarities and potential binding pockets between the B. anthracis spore and anthrose aptamers. Several recurring loop structures were identified and tested for their potential to act as aptamer beacons when labeled with TYE 665 dye on their 5' ends and Iowa Black quencher on their 3' ends. Of these candidate sequences, two beacons designated BAS-6F and BAS-6R emerged which gave strong fluorescence responses at high spore concentrations (greater than 30,000 spores/ml). These aptamer beacons also detect B. cereus and B. thuringiensis spores with greater fluorescence intensity, but do not strongly detect vegetative cells from an array of other bacterial species. BAS-6F and 6R are also not capable of detecting pure anthrose, thereby probably ruling that epitope out as a spore surface target for these particular beacons. While not extremely sensitive, the BAS-6F and 6R aptamer beacons are potentially valuable for rapid presumptive detection of anthrax or Bacillus spores in suspect powders or bioterrorist activity where spore concentrations are anticipated to be high. The sequence similarities of these beacons to other published Bacillus spore aptamers are also discussed.
    No preview · Article · Jan 2012 · Journal of Fluorescence
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    ABSTRACT: A dominant aptamer loop structure from a library of nearly 100 candidate aptamer sequences developed against immobilized 25-hydroxyvitamin D(3) (calcidiol) was converted into a 5'-TYE 665 and 3'-Iowa black-labelled aptamer beacon. The aptamer beacon exhibited a mild 'lights on' reaction in buffer as a function of increasing concentrations of several vitamin D analogues and metabolites, with a limit of detection of approximately 200 ng/mL, and was not specific for any particular congener. In 10% or 50% human serum, the same aptamer beacon inverted its fluorescence behaviour to become a more intense 'lights off' reaction with an improved limit of detection in the range 4-16 ng/mL. We hypothesized that this drastic change in fluorescence behaviour was due to the presence of creatinine and urea in serum, which might destabilize the quenched beacon, causing an increase in fluorescence followed by decreasing fluorescence as a function of vitamin D concentrations that may bind and quench increasingly greater fractions of the denatured beacons. However, the results of several control experiments in the presence of physiological or greater concentrations of creatinine and urea, alone or combined in buffer, failed to produce the beacon fluorescence inversion. Other possible mechanistic hypotheses are also discussed.
    No preview · Article · Jan 2012 · Luminescence
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    ABSTRACT: Sixty candidate DNA aptamers were developed against botulinum neurotoxin (BoNT) type A light chain (LC) from ten rounds of selection, resulting in several identical sequences. Secondary structures of the identical aptamers were compared to structures of previously reported BoNT A DNA aptamers. A series of ten candidate loop structures were selected from this comparison as potential binding pockets and aptamer beacons. These candidate beacons were synthesized with 5'-TYE 665 and 3'-Iowa Black quencher labels for comparison of fluorescence levels as a function of BoNT A LC concentration. Only three of the ten candidates exhibited any fluorescence response to increasing levels of BoNT A LC. However, of the two most responsive candidates, one represented a subset loop of the larger more intensely fluorescent double-looped structure, designated Beacon 10. This beacon yielded a lower limit of detection of 1 ng/mL in buffer using a spectrofluorometer and a portable handheld fluorometer, but also responded substantially to BoNT A, B, E holotoxins and heavy or light chain components even in a dilute soil suspension, but not in 50% human serum. Beacon 10 did not respond strongly to a variety of other divergent peptides, suggesting that it is relatively specific to the level of botulinum toxins and is only useful for environmental testing. Beacon 10 also shared short sequence segments with other published BoNT aptamer DNA sequences, suggesting that these may be points of physical contact between the aptamers and BoNTs.
    No preview · Article · Oct 2011 · Biosensors & Bioelectronics
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    ABSTRACT: A novel DNA aptamer beacon is described for quantification of a 26-amino acid C-telopeptide (CTx) of human type I bone collagen. One aptamer sequence and its reverse complement dominated the aptamer pool (31.6% of sequenced clones). Secondary structures of these aptamers were examined for potential binding pockets. Three-dimensional computer models which analyzed docking topologies and binding energies were in agreement with empirical fluorescence experiments used to select one candidate loop for beacon assay development. All loop structures from the aptamer finalists were end-labeled with TYE 665 and Iowa Black quencher for comparison of beacon fluorescence levels as a function of CTx concentration. The optimal beacon, designated CTx 2R-2h yielded a low ng/ml limit of detection using a commercially available handheld fluorometer. The CTx aptamer beacon bound full-length 26-amino acid CTx peptide, but not a shorter 8-amino acid segment of CTx peptide which is a common target for commercial CTx ELISA kits. The prototype assay was shown to detect CTx peptide from human urine after creatinine and urea were removed by size-exclusion chromatography to prevent nonspecific denaturing of the aptamer beacon. This work demonstrates the potential of aptamer beacons to be utilized for rapid and sensitive bone health monitoring in a handheld or point-of-care format.
    No preview · Article · Jun 2011 · Journal of Fluorescence
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    ABSTRACT: Several different approaches have been taken to development of homogeneous fluorescent aptamer assays including end-labeled beacons and signaling aptamers which are intrinsically quenched by nucleotides. Two new strategies dubbed "intrachain" and "competitive" FRET-aptamer assays are summarized in this review. Intrachain and competitive FRET-aptamers can be engineered on the molecular level through a series exploratory experiments involving prior knowledge of aptamer secondary or tertiary structures and hypotheses about aptamer conformational changes. However, there is an intrinsic risk of altering aptamer affinity or specificity associated with chemical modifications of an aptamer. Natural selection methods for FRET-aptamers have also been devised to potentially obviate the chemical modification problem. The naturally selected aptamers are subjected to fluorophore (F)- and or quencher (Q)-conjugated nucleotide triphosphate (NTP) incorporation by polymerase chain reaction (PCR) with permissive polymerases such as Deep Vent exo-, but still demonstrate sensitive and specific assay performance despite modified bases, because they are ultimately selected after decoration with F and Q. This paper summarizes work in this area and presents some new examples of the engineered and naturally selected FRET-aptamers for detection of vitamin D.
    No preview · Article · May 2011 · Combinatorial chemistry & high throughput screening
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    ABSTRACT: Detection of athletes who use synthetic human growth hormone (hGH; or somatotropin) to enhance physical strength and obtain an advantage in competitive sports is a formidable problem, as rhGH is virtually identical to the natural pituitary hormone. However, some post-translational and other modifications have been documented by chromatographic separation and mass spectrometry (MS) in a small percentage of rhGH. In the present work, development of DNA aptamers against research-grade rhGH and natural hGH with adsorption of the rhGH aptamers against natural hGH was shown to produce a small family of aptamer sequences that bound consistently with greater affinity to rhGH over a low nanogram-to-microgram range in ELISA-like microplate assays. This collection of rhGH discriminatory aptamer sequences shared some short sequence segments and secondary structural features. The top rhGH discriminatory aptamers also appeared to cross-react with human myoglobin and BSA but not with bone collagen peptides and an unrelated viral envelope peptide. The cross-reactivity results suggested several strings of up to five consecutive amino acids that might serve as common epitopes for aptamer binding. SDS-PAGE revealed that the rhGH existed largely as a 45-kDa dimer, and the natural hGH was almost exclusively monomeric. The existence of the rhGH dimer suggests that a discontinuous "bridge" epitope may exist on the rhGH, which spans the subunits, thereby accounting somewhat for the difference in detection. Overall, these results suggest that aptamers might be useful for routine, presumptive laboratory screening to identify athletes who are potentially cheating by administration of rhGH.
    Preview · Article · Apr 2011 · Journal of biomolecular techniques: JBT

Publication Stats

1k Citations
108.23 Total Impact Points

Institutions

  • 2007
    • California State University, San Marcos
      • Department of Chemistry and Biochemistry
      San Marcos, California, United States
  • 2000
    • Trinity University
      • Department of Biology
      San Antonio, Texas, United States
  • 1998-2000
    • University of Texas Health Science Center at San Antonio
      • Department of Radiology
      San Antonio, Texas, United States
    • Wright-Patterson Air Force Base
      Dayton, Ohio, United States
  • 1997
    • Tyndall Air Force Research Laboratory
      Tyndall Air Force Base, Florida, United States