Affinity modulation of platelet integrin alphaIIbbeta3 by beta3-endonexin, a selective binding partner of the beta3 integrin cytoplasmic tail.
AuthorsKashiwagi H, Schwartz MA, Eigenthaler M, Davis KA, Ginsberg MH, Shattil SJ
JournalJ Cell Biol
PubMed ID9182673
'Platelet agonists increase the affinity state of integrin alphaIIbbeta3, a prerequisite for fibrinogen binding and platelet aggregation. This process may be triggered by a regulatory molecule(s) that binds to the integrin cytoplasmic tails, causing a structural change in the receptor. beta3-Endonexin is a novel 111-amino acid protein that binds selectively ... More
Activation of methotrexate-alpha-alanine by carboxypeptidase A-monoclonal antibody conjugate.
AuthorsHaenseler E, Esswein A, Vitols KS, Montejano Y, Mueller BM, Reisfeld RA, Huennekens FM
JournalBiochemistry
PubMed ID1731945
'Carboxypeptidase A (CP-A) and monoclonal antibody KS1/4 directed against an antigen on human lung adenocarcinoma cells (UCLA-P3) were derivatized by treatment with succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate and N-succinimidyl 3-(2-pyridyldithio)propionate, respectively. The derivatized proteins were reacted to produce thioether-linked enzyme-antibody conjugates. Sequential HPLC size-exclusion and DEAE chromatography separated the conjugate preparation from unreacted ... More
Cloning of rat aorta lysyl oxidase cDNA: complete codons and predicted amino acid sequence.
'Lysyl oxidase cDNA clones were identified by their reactivity with anti-bovine lysyl oxidase in a neonatal rat aorta cDNA lambda gt11 expression library. A 500-bp cDNA sequence encoding four of six peptides derived from proteolytic digests of bovine aorta lysyl oxidase was found from the overlapping cDNA sequences of two ... More
Calmodulin and calmodulin-binding proteins in hair bundles.
AuthorsWalker RG, Hudspeth AJ, Gillespie PG
JournalProc Natl Acad Sci U S A
PubMed ID8385344
'Calcium ion plays an important role in the hair cell''s mechanoelectrical transduction process; in particular, Ca2+ controls adaptation to protracted mechanical stimuli. Because calmodulin is a ubiquitous intracellular receptor for Ca2+ and has been shown to accumulate at the tips of stereocilia, we determined its concentration and identified the proteins ... More
The agonist binding site on the bovine bradykinin B2 receptor is adjacent to a sulfhydryl and is differentiated from the antagonist binding site by chemical cross-linking.
AuthorsHerzig MC, Leeb-Lundberg LM
JournalJ Biol Chem
PubMed ID7657637
'Chemical cross-linking was used to analyze the binding sites for the agonist bradykinin (BK) and the antagonists NPC17731 and HOE140 on the bovine B2 bradykinin receptor. [3H]BK and [3H]NPC17731 bound with high affinity to the same B2 receptor in bovine myometrial membranes as determined by the total number of specific ... More
Preparation of monomeric Fab'-horseradish peroxidase conjugate using thiol groups in the hinge and its evaluation in enzyme immunoassay and immunohistochemical staining.
AuthorsIshikawa E, Yoshitake S, Imagawa M, Sumiyoshi A
JournalAnn N Y Acad Sci
PubMed ID6372612
'Horseradish peroxidase and Fab'' were conjugated by using thiol groups in the hinge of Fab''. Maleimide or pyridyl disulfide groups were introduced into peroxidase by treatment with N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate or N-succinimidyl 3-(2-pyridyldithio)propionate and were allowed to react with thiol groups in the hinge of Fab''. The conjugates were obtained in ... More
Development and testing of radio and enzyme immunoassays for acidic fibroblast growth factor (aFGF).
'Acidic fibroblast growth factor (aFGF) and basic fibroblast growth factor from bovine brain stimulate growth in a variety of tissues in several species. Despite the 55% amino acid sequence homology of the two forms of FGF, a specific immunoassay of aFGF has been developed using a polyclonal antibody raised in ... More
Rapid covalent coupling of proteins to cell surfaces: immunological characterization of viable protein-cell conjugates.
AuthorsChristiaansen JE, Gallardo D, Burnside SS, Nelson AA, Sears DW
JournalJ Immunol Methods
PubMed ID6094668
'A rapid and efficient 2-step procedure is described for covalently attaching proteins to cell surfaces by using a heterobifunctional cross-linking agent, succinimidyl-4-(N-maleimidomethylcyclohexane)-1-carboxylate (SMCC). In the first step, protein is derivatized for about 30 min with a 1:1 (mole:mole) stoichiometric ratio of SMCC which creates 4-(N-maleimidomethylcyclohexane)-1-amidyl-protein (MCA-protein) covalent linkages with the ... More
Comparison of four bifunctional reagents for coupling peptides to proteins and the effect of the three moieties on the immunogenicity of the conjugates.
AuthorsPeeters JM, Hazendonk TG, Beuvery EC, Tesser GI
JournalJ Immunol Methods
PubMed ID2499636
'Peptide-carrier conjugates are widely used to raise antipeptide antibodies. In a model system using angiotensin and tetanus toxoid as the peptide and the carrier protein respectively, four cross-linking reagents were employed to study their effect on the immunogenicity of the conjugates. Optimization of the conjugation method for these heterobifunctional reagents, ... More
Tat-conjugated synthetic macromolecules facilitate cytoplasmic drug delivery to human ovarian carcinoma cells.
AuthorsNori A, Jensen KD, Tijerina M, Kopecková P, Kopecek J
JournalBioconjug Chem
PubMed ID12526691
'We have synthesized N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-cell penetrating peptide Tat conjugates and evaluated their subcellular distribution in A2780 human ovarian carcinoma cells by confocal fluorescence microscopy and subcellular fractionation. Our data indicate the transport of these conjugates by a single Tat molecule to both the cytoplasm and nucleus via a nonendocytotic ... More
Extended length heterobifunctional coupling agents for protein conjugations.
AuthorsBieniarz C, Husain M, Barnes G, King CA, Welch CJ
JournalBioconjug Chem
PubMed ID8741995
'A series of extended length heterobifunctional coupling agents is described. The successive aminocaproic acid homologation of succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate, a known 9-atom long maleimide active ester linker, yielded 16-, 23-, and 30-atom long maleimide active ester homologues. The performance study of these coupling agents in automated microparticle enzyme immunoassays showed that, ... More
Sensitive non-radioactive dot-blot hybridization using DNA probes labelled with chelate group substituted psoralen and quantitative detection by europium ion fluorescence.
AuthorsOser A, Roth WK, Valet G
JournalNucleic Acids Res
PubMed ID3344204
'A new labelling method for cloned DNA probes used in hybridization assays is described. The DNA insert of recombinant plasmid DNA was made partially single-stranded for the labelling reaction by a restriction enzyme digest, followed by a controlled exonuclease III incubation. A thiol-containing psoralen derivative was covalently bound through irradiation ... More
Cryptomonad algal phycobiliproteins as fluorochromes for extracellular and intracellular antigen detection by flow cytometry.
AuthorsTelford WG, Moss MW, Morseman JP, Allnutt FC
JournalCytometry
PubMed ID11309804
'BACKGROUND: Phycobiliproteins play an important role in fluorescent labeling, particularly for flow cytometry. The spectral properties of R-phycoerythrin (R-PE) and allophycocyanin (APC) have made them the dominant reagents in this class of fluorochromes. In this study, we evaluate a lesser-known but potentially important series of low-molecular weight cryptomonad-derived phycobiliproteins (commercially ... More
Specific and covalent labeling of a membrane protein with organic fluorochromes and quantum dots.
AuthorsBonasio R, Carman CV, Kim E, Sage PT, Love KR, Mempel TR, Springer TA, von Andrian UH
JournalProc Natl Acad Sci U S A
PubMed ID17785425
'The real-time observation of protein dynamics in living cells and organisms is of fundamental importance for understanding biological processes. Most approaches to labeling proteins exploit noncovalent interactions, unsuitable to long-term studies, or genetic fusion to naturally occurring fluorescent proteins that often have unsatisfactory optical properties. Here we used the fungal ... More
A silicon sensor-based filtration immunoassay using biotin-mediated capture.
'A sensitive sandwich immunoassay for human chorionic gonadotropin (hCG) was developed with biotin-mediated filtration capture and silicon sensor detection. A high density of biotin on the membrane assured efficient capture of complexes containing streptavidin and analyte. Capture efficiency was not affected over a wide range of filtration flow rates or ... More
Bactericidal antisense effects of peptide-PNA conjugates.
AuthorsGood L, Awasthi SK, Dryselius R, Larsson O, Nielsen PE
JournalNat Biotechnol
PubMed ID11283595
'Antisense peptide nucleic acids (PNAs) can specifically inhibit Escherichia coli gene expression and growth and hold promise as anti-infective agents and as tools for microbial functional genomics. Here we demonstrate that chemical modification improves the potency of standard PNAs. We show that 9- to 12-mer PNAs, especially when attached to ... More
A highly sensitive immuno-PCR assay for detecting Group A Streptococcus.
AuthorsLiang H, Cordova SE, Kieft TL, Rogelj S
JournalJ Immunol Methods
PubMed ID12969551
'A highly sensitive hybrid assay, based on immuno polymerase chain reaction (immuno-PCR) and enzyme-linked immunosorbent assay (ELISA) techniques, was developed for the detection of pathogenic Group A Streptococcus (Strep A). Cells were disrupted by sonication and then coated onto the walls of Maxisorp microtiter plates. Next, biotinylated anti-Group A monoclonal ... More
A new reagent which may be used to introduce sulfhydryl groups into proteins, and its use in the preparation of conjugates for immunoassay.
AuthorsDuncan RJ, Weston PD, Wrigglesworth R
JournalAnal Biochem
PubMed ID6353995
'A synthesis of the N-hydroxysuccinimide ester of S-acetylthioacetic acid is described. This material is stable when stored dry and has advantages over the currently available reagents used to introduce sulfhydryl groups into a variety of proteins. Proteins modified with this reagent can be used to prepare conjugates for enzyme immunoassay. ... More
A brief survey of methods for preparing protein conjugates with dyes, haptens, and cross-linking reagents.
AuthorsBrinkley M
JournalBioconjug Chem
PubMed ID1616945
Synthesis and purification of horseradish peroxidase-labeled oligonucleotides for tyramide-based fluorescence in situ hybridization.
Authorsvan Gijlswijk RP, van de Corput MP, Bezrookove V, Wiegant J, Tanke HJ, Raap AK
JournalHistochem Cell Biol
PubMed ID10817671
'A method is presented to conjugate horseradish peroxidase (HRP) to oligodeoxynucleotides for fluorescence in situ hybridization assays employing tyramide signal amplification (TSA). HRP is covalently bound to the oligonucleotide by thiol ether linkage and purified by high-performance liquid chromatography. With TSA detection, a single HRP-labeled oligonucleotide probe is sufficient for ... More
Horseradish peroxidase-labeled oligonucleotides and fluorescent tyramides for rapid detection of chromosome-specific repeat sequences.
'We present a sensitive and rapid fluorescence in situ hybridization (FISH) strategy for detecting chromosome-specific repeat sequences. It uses horseradish peroxidase (HRP)-labeled oligonucleotide sequences in combination with fluorescent tyramide-based detection. After in situ hybridization, the HRP conjugated to the oligonucleotide probe is used to deposit fluorescently labeled tyramide molecules at ... More
Time-resolved detection of energy transfer: theory and application to immunoassays.
AuthorsMorrison LE
JournalAnal Biochem
PubMed ID3218725
'Energy-transfer measurements based upon acceptor fluorophore emission are plagued with background fluorescence resulting from absorption of the excitation light by the acceptor fluorophore. The present work examines the use of a long-lifetime donor fluorophore and a short-lifetime acceptor fluorophore, combined with pulsed-laser excitation and electronic gating of detector signals, to ... More
Antibodies that inhibit fusion of human immunodeficiency virus-infected cells bind a 24-amino acid sequence of the viral envelope, gp120.
AuthorsRusche JR, Javaherian K, McDanal C, Petro J, Lynn DL, Grimaila R, Langlois A, Gallo RC, Arthur LO, Fischinger PJ
JournalProc Natl Acad Sci U S A
PubMed ID2452447
'Antisera to recombinant human immunodeficiency virus (HIV) proteins containing the entire envelope, gp160, or the central portion of the envelope, PB1, can inhibit fusion of virally infected cells in culture. This fusion inhibition is HIV-variant specific--that is, anti-gp160-IIIB inhibits fusion of isolate HTLV-IIIB-infected cells but not of isolate HTLV-IIIRF-infected cells. ... More
Conjugation of glucose oxidase from Aspergillus niger and rabbit antibodies using N-hydroxysuccinimide ester of N-(4-carboxycyclohexylmethyl)-maleimide.
AuthorsYoshitake S, Yamada Y, Ishikawa E, Masseyeff R
JournalEur J Biochem
PubMed ID574817
'Glucose oxidase from Aspergillus niger was conjugated with rabbit immunoglobulin G or its monovalent fragments (Fab''). The enzyme was treated with N-hydroxysuccinimide ester of N-(4-carboxycyclohexylmethyl)-maleimide to introduce maleimide groups, which were then allowed to react with thiol groups of reduced IgG or Fab''. More than 40% of immunoglobulin G, Fab'' ... More
Chemical modification of proteins: comments and perspectives.
AuthorsFeeney RE
JournalInt J Pept Protein Res
PubMed ID3570660
'The use of chemical modification of proteins has increased exponentially during the past two decades. Today the many different uses of chemical modification include determination of relative reactivities of side chain groups, the quantitation of individual amino acids, development of affinity reagents, mechanism-based reagents for pharmaceutical uses, cross-linking reagents, special ... More
Quantitative detection of C-reactive protein using phosphocholine-labelled enzyme or microspheres.
AuthorsDeegan O, Walshe K, Kavanagh K, Doyle S
JournalAnal Biochem
PubMed ID12531203
'C-reactive protein (CRP) is a positive, acute-phase protein. Plasma levels rise dramatically in response to tissue injury or inflammation and fall rapidly after recovery or treatment. Antibody-based human CRP test systems do not readily detect CRP from other animals due to the species specificity of antibodies directed against human CRP. ... More
Coupling of monoclonal antibodies with enzymes.
AuthorsHaugland RP
JournalMethods Mol Biol
PubMed ID7550686
Preparation of avidin conjugates.
AuthorsHaugland RP, Bhalgat MK
JournalMethods Mol Biol
PubMed ID9664375
Coupling of monoclonal antibodies with fluorophores.
AuthorsHaugland RP
JournalMethods Mol Biol
PubMed ID7550684
Progress in enzyme immunoassays: production of reagents, experimental design, and interpretation.
AuthorsKurstak E
JournalBull World Health Organ
PubMed ID3910300
The use of phycobiliproteins as fluorescent labels in immunoassay.
AuthorsKronick MN
JournalJ Immunol Methods
PubMed ID3528294
Cross-linking techniques.
AuthorsBäumert HG, Fasold H
JournalMethods Enzymol
PubMed ID2546017
Protein labeling with fluorescent probes.
AuthorsHolmes KL, Lantz LM
JournalMethods Cell Biol
PubMed ID11060842
Dynamic visualization of RGD-quantum dot binding to tumor neovasculature and extravasation in multiple living mouse models using intravital microscopy.
AuthorsSmith BR, Cheng Z, De A, Rosenberg J, Gambhir SS,
JournalSmall
PubMed ID20862677
In this work, we exploited intravital microscopy to develop in vivo nanoparticle binding and extravasation assays, comparing the experimental RGD-Qdot condition with controls in various tumor models in living mice. ... More
Enzyme immunoassay for captopril.
AuthorsKinoshita H, Nakamaru R, Tanaka S, Tohira Y, Sawada M
JournalJ Pharm Sci
PubMed ID3531460
A simple enzyme immunoassay for the determination of captopril was developed. A specific antibody for captopril was produced in rabbits that were immunized with a hapten-bovine immunoglobulin G conjugate, which was prepared by using 4-(maleimidomethyl)cyclohexane carboxylic acid as a spacer group. The limit of detection in plasma is 0.5 ng/mL. ... More
Fluorochrome-labeled tyramides: use in immunocytochemistry and fluorescence in situ hybridization.
The peroxidase-mediated deposition of hapten- and fluorochrome-labeled tyramides has recently been shown to increase the sensitivity of immunofluorescence and fluorescence in situ hybridization techniques. We have evaluated a number of red, green, and blue fluorescent tyramides for detection of antigens in tissue sections and cytospin preparations and for the detection ... More
Scp160p, an RNA-binding, polysome-associated protein, localizes to the endoplasmic reticulum of Saccharomyces cerevisiae in a microtubule-dependent manner.
AuthorsFrey S, Pool M, Seedorf M
JournalJ Biol Chem
PubMed ID11278502
Scp160p is an RNA-binding protein containing 14 tandemly repeated heterogenous nuclear ribonucleoprotein K-homology domains, which are implicated in RNA binding. Scp160p interacts with free and membrane-bound polysomes that are dependent upon the presence of mRNA. Despite its presence on cytosolic polysomes, Scp160p is predominantly localized to the endoplasmic reticulum (ER). ... More
Antibody caging of a nuclear-targeting signal.
AuthorsHalleck MS, Rechsteiner M
JournalProc Natl Acad Sci U S A
PubMed ID2170983
We have developed a technique for reversibly masking a peptide-targeting signal. A fluoresceinated derivative of the simian virus 40 large tumor antigen nuclear-targeting signal was synthesized and cross-linked to bovine serum albumin. The conjugated protein was efficiently transported into rat liver nuclei unless the peptide-targeting signal was sterically hindered by ... More
Novel protein transfection of primary rat cortical neurons using an antibody that penetrates living cells.
AuthorsWeisbart RH, Baldwin R, Huh B, Zack DJ, Nishimura R
JournalJ Immunol
PubMed ID10820286
An Ab-based system to deliver functional proteins into neurons was developed using the murine mAb, mAb 3E10. This was achieved by covalently conjugating catalase to the Ab so that the conjugate retained high activity for the degradation of hydrogen peroxide. Three-dimensional fluorescence microscopy was used to demonstrate penetration of the ... More
A simple method for the conjugation of affinity-purified Fab' to horseradish peroxidase and beta-D-galactosidase from Escherichia coli.
AuthorsHashida S, Imagawa M, Ishikawa E, Freytag JW
JournalJ Immunoassay
PubMed ID3926824
A simple method was described for the conjugation of affinity-purified Fab' to horseradish peroxidase and beta-D-galactosidase from Escherichia coli. IgG was subjected to successive processes of pepsin digestion, reduction with 2-mercaptoethylamine, affinity-purification and reaction with maleimide groups introduced into the enzymes. In the present method, gel filtration was required only ... More
A sensitive assay for maleimide groups.
AuthorsSingh R
JournalBioconjug Chem
PubMed ID7948101
A sensitive spectrophotometric assay has been developed for maleimide groups incorporated into proteins. The assay involves the reaction of maleimide with an excess of cysteine and quantitation of the remaining cysteine using an enzymatic assay that is based on the stoichiometric conversion of an inactive disulfide form of papain to ... More
Preparation and characterization of xanthine oxidase-antibody and -hapten conjugates for use in sensitive chemiluminescent immunoassays.
AuthorsFert V, Baret A
JournalJ Immunol Methods
PubMed ID2391429
Methods have been developed to label haptens or antibodies with xanthine oxidase for use in chemiluminescent enzyme immunoassays. We have optimised coupling reactions involving the use of heterobifunctional cross-linkers, the introduction of sulfhydryl groups and the utilization of accessible cysteine residues on the native enzyme. The versatility of xanthine oxidase ... More
Near-infrared fluorescence lifetime assay for serum glucose based on allophycocyanin-labeled concanavalin A.
AuthorsMcCartney LJ, Pickup JC, Rolinski OJ, Birch DJ
JournalAnal Biochem
PubMed ID11355853
We describe an assay scheme for glucose based on fluorescence resonance energy transfer (FRET) between concanavalin A (con A), labeled with the near-infrared fluorescent protein allophycocyanin (APC) as donor, and dextran labeled with malachite green (MG) as acceptor. Glucose competitively displaces dextran-MG and leads to reduction in FRET, assessed by ... More
Liposome immunoassay by long-lived fluorescence detection.
AuthorsOkabayashi Y, Ikeuchi I
JournalAnalyst
PubMed ID9764512
Immunoassay by fluorescence energy transfer from a europium chelate in liposome to allophycocyanin (APC) was demonstrated. Streptavidin or antibody to biotin was bonded to the liposome containing the europium chelate of 2-naphthoyltrifluoroacetone in the bilayer. When the biotin bonded to APC (APC-BT) was added to the prepared liposomes and the ... More
Improved sensitivity in homogeneous enzyme immunoassays using a fluorogenic macromolecular substrate: an assay for serum ferritin.
AuthorsArmenta R, Tarnowski T, Gibbons I, Ullman EF
JournalAnal Biochem
PubMed ID3922243
A new highly sensitive nonseparation enzyme immunoassay for human serum ferritin is described. Reagents include a beta-galactosidase-ferritin conjugate, sheep anti-ferritin, anti-sheep IgG, and dextran-linked beta-galactosylumbelliferone as enzyme substrate. The method is based on inhibition of enzyme activity when anti-ferritin binds to the enzyme-ferritin conjugate. Ferritin in the sample and enzyme-labeled ... More
The voltage-clamp fluorometry technique.
AuthorsGandhi CS, Olcese R,
JournalMethods Mol Biol
PubMed ID18998096
Ion channels are the cell's gatekeepers. These proteins selectively allow ionic current to flow down its electrochemical gradient. In some cases, specialized chemical or voltage sensing domains respond to environmental changes and signal the cell to adjust its internal chemistry in response to its surroundings. Because of their importance in ... More
Imaging epidermal growth factor receptor expression in vivo: pharmacokinetic and biodistribution characterization of a bioconjugated quantum dot nanoprobe.
AuthorsDiagaradjane P, Orenstein-Cardona JM, E Colón-Casasnovas N, Deorukhkar A, Shentu S, Kuno N, Schwartz DL, Gelovani JG, Krishnan S,
JournalClin Cancer Res
PubMed ID18245533
PURPOSE: To develop and validate an optical imaging nanoprobe for the discrimination of epidermal growth factor (EGF) receptor (EGFR)-overexpressing tumors from surrounding normal tissues that also expresses EGFR. EXPERIMENTAL DESIGN: Near-infrared (NIR) quantum dots (QD) were coupled to EGF using thiol-maleimide conjugation to create EGF-QD nanoprobes. In vitro binding affinity ... More
Reversibility of covalent electrophile-protein adducts and chemical toxicity.
AuthorsLin D, Saleh S, Liebler DC,
JournalChem Res Toxicol
PubMed ID19548357
The biotin-tagged electrophiles 1-biotinamido-4-(4'-[maleimidoethylcyclohexane]-carboxamido)butane (BMCC) and N-iodoacetyl-N-biotinylhexylenediamine (IAB) have been used as model electrophile probes in complex proteomes to identify protein targets associated with chemical toxicity. Whereas IAB activates stress signaling and apoptosis in HEK293 cells, BMCC does not. Cysteine Michael adducts formed from BMCC and nonbiotinylated analogues rapidly disappeared ... More
Bioluminescent assays using glucose-6-phosphate dehydrogenase: application to biotin and streptavidin detection.
AuthorsTérouanne B, Bencheick M, Balaguer P, Boussioux AM, Nicolas JC
JournalAnal Biochem
PubMed ID2479285
A streptavidin-glucose-6-phosphate dehydrogenase (G6PDH) conjugate was synthesized and its properties were studied, along with those of biotin-G6PDH conjugates. Two bioluminescent assays were used. Streptavidin was assayed in two steps: streptavidin samples were first incubated with a small amount of biotin-G6PDH and then with biotinylated rabbit gamma-globulins. The complex was immobilized ... More
Chemically optimized antimyosin Fab conjugates with chelating polymers: importance of the nature of the protein-polymer single site covalent bond for biodistribution and infarction localization.
Murine antimyosin Fab fragment was conjugated with 111In-labeled N-terminal-modified DTPA-polylysine using three bifunctional reagents: N-hydroxysuccinimide esters of 3-(2-pyridyldithio)propionic acid (SPDP conjugate), 4-(maleimidomethyl)cyclohexanecarboxylic acid (SMCC conjugate) and bromoacetic acid (BrAc conjugate) for potential localization of experimental myocardial infarction. Using various antibody preparations and a rabbit acute myocardial infarction model the following ... More
The development and application of protein-liposome conjugation techniques.
AuthorsHeath TD, Martin FJ
JournalChem Phys Lipids
PubMed ID3742677
Numerous techniques have been developed over the past 10 years for the conjugation of proteins to liposomes. Early procedures involved coupling with reagents such as glutaraldehyde or EDCI. Subsequently, more sophisticated approaches involving selective bifunctional coupling agents have been developed. These later procedures are also much more efficient for coupling ... More
Endocytosis of beta 2 integrins by stimulated human neutrophils analyzed by flow cytometry.
AuthorsChambers JD, Simon SI, Berger EM, Sklar LA, Arfors KE
JournalJ Leukoc Biol
PubMed ID8097762
Flow cytometry and fluorescently labeled monoclonal antibodies were used to investigate endocytosis of human neutrophil beta 2 integrins following cellular activation. CD18 initially present on the cell surface cycled in two phases after exposure to formyl peptide or platelet-activating factor. The first phase lasted 3 min at 37 degrees C; ... More
Bioconjugated quantum dots for multiplexed and quantitative immunohistochemistry.
AuthorsXing Y, Chaudry Q, Shen C, Kong KY, Zhau HE, Chung LW, Petros JA, O'Regan RM, Yezhelyev MV, Simons JW, Wang MD, Nie S
JournalNat Protoc
PubMed ID17546006
Bioconjugated quantum dots (QDs) provide a new class of biological labels for evaluating biomolecular signatures (biomarkers) on intact cells and tissue specimens. In particular, the use of multicolor QD probes in immunohistochemistry is considered one of the most important and clinically relevant applications. At present, however, clinical applications of QD-based ... More
More useful maleimide compounds for the conjugation of Fab' to horseradish peroxidase through thiol groups in the hinge.
AuthorsHashida S, Imagawa M, Inoue S, Ruan KH, Ishikawa E
JournalJ Appl Biochem
PubMed ID6490581
Nine different maleimide compounds were evaluated for the conjugation of Fab' to horseradish peroxidase through thiol groups in the hinge. The compounds evaluated were succinimidyl maleimidoacetate (I), succinimidyl 4-maleimidobutyrate (II), succinimidyl 6-maleimidohexanoate (III), succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (IV), succinimidyl m-maleimidobenzoate (V), succinimidyl 4-(p-maleimidophenyl)butyrate (VI), sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (VII), sulfosuccinimidyl m-maleimidobenzoate (VIII), and sulfosuccinimidyl ... More
Optimized procedures for the coupling of proteins to liposomes.
AuthorsLoughrey HC, Choi LS, Cullis PR, Bally MB
JournalJ Immunol Methods
PubMed ID2391438
A general, optimized method for coupling proteins to liposomes is presented. This procedure utilizes streptavidin covalently coupled to liposomes to allow the subsequent attachment of a variety of biotinated proteins of interest. In the first part of this study, covalent methods for coupling proteins to liposomes which contain the lipid ... More
Mild and efficient conjugation of rabbit Fab' and horseradish peroxidase using a maleimide compound and its use for enzyme immunoassay.
AuthorsYoshitake S, Imagawa M, Ishikawa E, Niitsu Y, Urushizaki I, Nishiura M, Kanazawa R, Kurosaki H, Tachibana S, Nakazawa N, Ogawa H
JournalJ Biochem (Tokyo)
PubMed ID6759501
A mild and efficient procedure for conjugating rabbit Fab' and horseradish peroxidase using a maleimide compound was developed. The enzyme was treated with N-hydroxysuccinimide ester of N-(4-carboxycyclohexylmethyl) maleimide to introduce maleimide groups. Then, the maleimide-enzyme was allowed to react with thiol groups of Fab', and the conjugate formed was separated ... More
Novel roles of CpG oligodeoxynucleotides as a leader for the sampling and presentation of CpG-tagged antigen by dendritic cells.
AuthorsShirota H, Sano K, Hirasawa N, Terui T, Ohuchi K, Hattori T, Shirato K, Tamura G
JournalJ Immunol
PubMed ID11418633
Oligodeoxynucleotides containing CpG motifs have been highlighted as potent Th1 activators. We previously reported that Ag and CpG, when conjugated together, synergistically promoted the Ag-specific Th1 development and inhibited the Th2-mediated airway eosinophilia. In this study, we examined the mechanisms underlying the synergism of the covalent conjugation. The CpG-OVA conjugate ... More
Imparting bone affinity to glycoproteins through the conjugation of bisphosphonates.
AuthorsGittens SA, Matyas JR, Zernicke RF, Uludag H
JournalPharm Res
PubMed ID12880282
PURPOSE: To develop a novel means of conjugating bisphosphonates onto the carbohydrate moieties of glycoproteins to enhance protein affinity to bone. METHODS: 1-Amino-1,1-diphosphonate methane (aminoBP) was conjugated onto the carbohydrate moietites of oxidized fetuin by using 4-(maleimidomethyl)cyclohexane-1-carboxyl-hydrazide (MMCCH). Bone affinity of the resulting conjugates was compared to proteins obtained from ... More
Pseudomonas exotoxin A mutants. Replacement of surface-exposed residues in domain III with cysteine residues that can be modified with polyethylene glycol in a site-specific manner.
AuthorsBenhar I, Wang QC, FitzGerald D, Pastan I
JournalJ Biol Chem
PubMed ID8175770
Pseudomonas exotoxin A (PE) is composed of three structural and functional domains. Domain Ia is responsible for cell recognition, domain II for translocation of PE across the cell membrane, and domain III for ADP-ribosylation of elongation factor 2. To investigate the role of the amino acids exposed on the surface ... More
Double-enzyme conjugates, producing an intermediate color, for simultaneous and direct detection of three different intracellular immunoglobulin determinants with only two enzymes.
AuthorsClaassen E, Boorsma DM, Kors N, Van Rooijen N
JournalJ Histochem Cytochem
PubMed ID2419394
A new double-enzyme conjugate was synthesized by coupling alkaline phosphatase (AP) to horseradish peroxidase (HRP). After AP (blue) and subsequent HRP (red) cytochemistry, this new conjugate produced a stable intermediate-colored (violet) product. By coupling this double-enzyme conjugate to an antigen (trinitrophenyl, TNP) or an antibody (anti-mouse immunoglobulin G2a), anti-TNP or ... More
Characterization of RFB4-Pseudomonas exotoxin A immunotoxins targeted to CD22 on B-cell malignancies.
AuthorsMansfield E, Pastan I, FitzGerald DJ
JournalBioconjug Chem
PubMed ID8889017
To develop an immunotoxin for the treatment of B-cell malignancies, we constructed several candidate conjugates with RFB4, a B-cell specific anti-CD22 IgG1, and truncated forms of Pseudomonas exotoxin (PE). The four versions of PE included PE35 and PE35KDEL, which were linked to RFB4 via a disulfide bond, and PE38 and ... More
Purified immunotoxins that are reactive with human lymphoid cells. Monoclonal antibodies conjugated to the ribosome-inactivating proteins gelonin and the pokeweed antiviral proteins.
AuthorsLambert JM, Senter PD, Yau-Young A, Blättler WA, Goldmacher VS
JournalJ Biol Chem
PubMed ID4044586
Seven different monoclonal antibodies of the IgG class that are reactive with four different antigens on human lymphoid cells were utilized to form immunotoxins with the ribosome-inactivating proteins gelonin and the three known pokeweed antiviral proteins. Thirteen different immunotoxin combinations were prepared. The ribosome-inactivating proteins were modified with 2-iminothiolane. The ... More
Development of sulfhydryl-reactive silica for protein immobilization in high-performance affinity chromatography.
AuthorsMallik R, Wa C, Hage DS
JournalAnal Chem
PubMed ID17297940
Two techniques were developed for the immobilization of proteins and other ligands to silica through sulfhydryl groups. These methods made use of maleimide-activated silica (the SMCC method) or iodoacetyl-activated silica (the SIA method). The resulting supports were tested for use in high-performance affinity chromatography by employing human serum albumin (HSA) ... More
Cell-ELISA using beta-galactosidase conjugated antibodies.
AuthorsLiu Z, Gurlo T, von Grafenstein H
JournalJ Immunol Methods
PubMed ID10669780
Cell-enzyme-linked immunosorbent assay (cell-ELISA) is a technique for the rapid, convenient, and quantitative detection of molecules expressed on the cell surface. Here we present an evaluation of beta-galactosidase as an antibody-tag for cell-ELISA. In contrast to substrates for horseradish peroxidase (HRP) and alkaline phosphatase, murine splenocytes do not hydrolyze the ... More
Anomalous diffusion of major histocompatibility complex class I molecules on HeLa cells determined by single particle tracking.
AuthorsSmith PR, Morrison IE, Wilson KM, Fernández N, Cherry RJ
JournalBiophys J
PubMed ID10354459
Single-particle tracking (SPT) was used to determine the mobility characteristics of MHC (major histocompatibility complex) class I molecules at the surface of HeLa cells at 22 degrees C and on different time scales. MHC class I was labeled using the Fab fragment of a monoclonal antibody (W6/32), covalently bound to ... More
Preparation and characterization of anti-tenascin monoclonal antibody-streptavidin conjugates for pretargeting applications.
AuthorsFoulon CF, Bigner DD, Zalutsky MR
JournalBioconjug Chem
PubMed ID10502355
Radioimmunopretargeting is based on the separate injection of a modified mAb and the radionuclide and most frequently exploits the very high avidity of biotin for streptavidin (SA). Currently, we are evaluating the therapeutic potential of directly labeled monoclonal antibody (mAb) 81C6, reactive with the extracellular matrix protein tenascin, in surgically ... More
Sterically stabilized anti-HER2 immunoliposomes: design and targeting to human breast cancer cells in vitro.
AuthorsKirpotin D, Park JW, Hong K, Zalipsky S, Li WL, Carter P, Benz CC, Papahadjopoulos D
JournalBiochemistry
PubMed ID8993319
Liposomes (70-100 nm) of 1-palmitoyl-2-oleoylphosphatidylcholine, cholesterol, and poly(ethylene glycol) (PEG)-modified phosphatidylethanolamine (PEG-DSPE) were conjugated to Fab' fragments of a humanized recombinant MAb against the extracellular domain of HER2/neu to create sterically stabilized immunoliposomes (anti-HER2 SL) as a drug carrier targeting HER2-overexpressing cancers. Conjugation employed maleimide-terminated membrane-anchored spacers of two kinds: ... More
Construction and characterization of a Pseudomonas aeruginosa mucoid exopolysaccharide-alginate conjugate vaccine.
AuthorsTheilacker C, Coleman FT, Mueschenborn S, Llosa N, Grout M, Pier GB
JournalInfect Immun
PubMed ID12819072
Deterioration of lung function in patients with cystic fibrosis (CF) is closely associated with chronic pulmonary infection with mucoid Pseudomonas aeruginosa. The mucoid exopolysaccharide (MEP) from P. aeruginosa has been shown to induce opsonic antibodies in mice that are protective against this chronic infection. MEP-specific opsonic antibodies are also commonly ... More
The carboxyl terminus of interferon-gamma contains a functional polybasic nuclear localization sequence.
AuthorsSubramaniam PS, Mujtaba MG, Paddy MR, Johnson HM
JournalJ Biol Chem
PubMed ID9867857
Cytokines such as interferon-gamma (IFN-gamma), which utilize the well studied JAK/STAT pathway for nuclear signal transduction, are themselves translocated to the nucleus. The exact mechanism for the nuclear import of IFN-gamma or the functional role of the nuclear translocation of ligand in signal transduction is unknown. We show in this ... More
Synthesis and evaluation of luminescent tracers and hapten-protein conjugates for use in luminescence immunoassays with immobilised antibodies and antigens. A critical study of macro solid phases for use in immunoassay systems, Part II.
AuthorsGadow A, Fricke H, Strasburger CJ, Wood WG
JournalJ Clin Chem Clin Biochem
PubMed ID6470624
This article describes the synthesis of labels and hapten-protein conjugates for use in bio- and chemiluminescent immunoassay systems, together with the problems encountered. The effects of maleimide upon acetate-, adenylate- and pyruvate kinase activity have been studied, as well as upon the luciferin-luciferase monitoring system. Maleimide inhibited both acetate and ... More
Modification of the binding site(s) of lectins by an affinity column carrying an activated galactose-terminated ligand.
AuthorsMoroney SE, D'Alarcao LJ, Goldmacher VS, Lambert JM, Blättler WA
JournalBiochemistry
PubMed ID3442663
An affinity column approach is described, aimed at the modification of the galactose binding site(s) of ricin in an effort to block the binding of ricin to cells. The affinity column was prepared by linking N-(2'-mercaptoethyl)lactamine to pyridyldithio-activated polyacrylamide heads. The linker between the ligand and the solid support thus ... More
Phycoerythrin-allophycocyanin: a resonance energy transfer fluorochrome for immunofluorescence.
AuthorsTjioe I, Legerton T, Wegstein J, Herzenberg LA, Roederer M
JournalCytometry
PubMed ID11309805
BACKGROUND: As immunofluorescence experiments become more complex, the demand for new dyes with different properties increases. Fluorescent dyes with large Stoke's shifts that are very bright and have low background binding to cells are especially desirable. We report on the properties of the resonance energy tandems of phycoerythrin and allophycocyanin ... More
Molecular immunolabeling with recombinant single-chain variable fragment (scFv) antibodies designed with metal-binding domains.
AuthorsMalecki M, Hsu A, Truong L, Sanchez S
JournalProc Natl Acad Sci U S A
PubMed ID11756693
To study the molecular structure and function of gene products in situ, we developed a molecular immunolabeling technology. Starting with cDNA from hybridomas producing monoclonal antibodies against biotin, catalase, and superoxide dismutase, we bioengineered recombinant single-chain variable fragment antibodies (scFv) and their derivatives containing metal-binding domains (scFv:MBD). As tested with ... More
Insulin binding to its receptor induces a conformational change in the receptor C-terminus.
AuthorsBaron V, Gautier N, Komoriya A, Hainaut P, Scimeca JC, Mervic M, Lavielle S, Dolais-Kitabgi J, Van Obberghen E
JournalBiochemistry
PubMed ID2196938
Antibodies against peptides corresponding to sequences in the C-terminus of the insulin receptor beta-subunit were used to approach the putative role of this receptor domain in signal generation. Two sequences were chosen and correspond to peptide C1, comprising amino acids 1309-1326, and peptide C2, comprising amino acids 1294-1317. The two ... More
Application of a new method of antibody-enzyme conjugation with maleimide derivative for immunohistochemistry: hepatocellular production, interstitial tissue distribution, and renal cell reabsorption of plasma albumin in guinea pig.
AuthorsTsuruta J, Yamamoto T, Kozono K, Kambara T
JournalJ Histochem Cytochem
PubMed ID3926865
A new sophisticated method for enzyme-antibody conjugation developed in quantitative solid-phase enzyme immunoassay was revealed to be an applicable method for immunohistochemistry; one that offered several advantages over present methods. Using a new maleimide derivative as a coupling reagent, monomeric conjugate of horseradish peroxidase and Fab' antibody was easily prepared ... More
Synthesis of a stable and specific surface plasmon resonance biosensor surface employing covalently immobilized peptide nucleic acids.
AuthorsBurgener M, Sänger M, Candrian U
JournalBioconjug Chem
PubMed ID11087321
Biosensors allow the real-time and label-free observation of biochemical reactions between various ligands including antigen-antibody reactions and nucleic acids hybridizations. In our studies, we used a surface plasmon resonance biosensor to elucidate the hybridization characteristics of a peptide nucleic acid (PNA) ligand immobilized on sensor surfaces either through covalent or ... More
Pseudomonas exotoxin A mutants. Replacement of surface exposed residues in domain II with cysteine residues that can be modified with polyethylene glycol in a site-specific manner.
AuthorsKuan CT, Wang QC, Pastan I
JournalJ Biol Chem
PubMed ID8125985
Pseudomonas exotoxin A (PE) is a three-domain protein in which domain Ia is involved in recognition of receptors on eukaryotic target cells, domain II promotes translocation of PE into the cytosol, and domain III enzymatically ADP-ribosylates elongation factor 2. Modification of proteins with polyethylene glycol (PEG) has been shown to ... More