An alternate method utilizing small quantities of ligand for affinity purification of monospecific antibodies

Hn, Aithal; Km, Knigge; Kartha, Sreedharan; Ea, Czyzewski; Toback, F. Gary

doi:10.1016/0022-1759(88)90034-8

Cited by 21 publications

(7 citation statements)

References 12 publications

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“…Antibodies to the C terminus of CCR5 were produced using the sequence Cys-Nle-344-352. All peptides were coupled to thyroglobulin through their cysteine residues using the sulfo-m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester method (1). Polyclonal receptor antibodies were produced by Pocono Rabbit Farm (Canadensis, Pa.) and were purified by protein A-Sepharose chromatography.…”

Section: Methodsmentioning

confidence: 99%

CCR5, CXCR4, and CD4 Are Clustered and Closely Apposed on Microvilli of Human Macrophages and T Cells

Singer

Scott

Kawka

et al. 2001

J Virol

151

129

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The chemokine receptors CCR5 and CXCR4 act synergistically with CD4 in an ordered multistep mechanism to allow the binding and entry of human immunodeficiency virus type 1 (HIV-1). The efficiency of such a coordinated mechanism depends on the spatial distribution of the participating molecules on the cell surface. Immunoelectron microscopy was performed to address the subcellular localization of the chemokine receptors and CD4 at high resolution. Cells were fixed, cryoprocessed, and frozen; 80-nm cryosections were double labeled with combinations of CCR5, CXCR4, and CD4 antibodies and then stained with immunogold. Surprisingly, CCR5, CXCR4, and CD4 were found predominantly on microvilli and appeared to form homogeneous microclusters in all cell types examined, including macrophages and T cells. Further, while mixed microclusters were not observed, homogeneous microclusters of CD4 and the chemokine receptors were frequently separated by distances less than the diameter of an HIV-1 virion. Such distributions are likely to facilitate cooperative interactions with HIV-1 during virus adsorption to and penetration of human leukocytes and have significant implications for development of therapeutically useful inhibitors of the entry process. Although the mechanism underlying clustering is not understood, clusters were observed in small trans-Golgi vesicles, implying that they were organized shortly after synthesis and well before insertion into the cellular membrane. Chemokine receptors normally act as sensors, detecting concentration gradients of their ligands and thus providing directional information for cellular migration during both normal homeostasis and inflammatory responses. Localization of these sensors on the microvilli should enable more precise monitoring of their environment, improving efficiency of the chemotactic process. Moreover, since selectins, some integrins, and actin are also located on or in the microvillus, this organelle has many of the major elements required for chemotaxis.Human immunodeficiency virus (HIV) therapies have been highly successful in slowing disease progression, increasing health and well-being, and prolonging life. However, viral resistance is now becoming common, and since most existing drugs target only two viral proteins, reverse transcriptase and protease, cross-resistance is a significant problem. One solution to the issue of resistance is development of new complementary therapies based on novel mechanisms of action. The discovery that the chemokine receptors CCR5 and CXCR4, in addition to CD4, are required for viral entry not only furthered understanding of the fusion and infection process but provided two new targets for therapeutic intervention (3,12,14,17,18,22,44).The entry mechanism as currently understood is an ordered process in which the viral envelope protein, gp120, following interaction with CD4, undergoes a conformational change allowing binding to the appropriate chemokine receptor, CCR5 for macrophagetropic or R5 strains, and CXCR4 for T-celltropic or X4 s...

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Section: Methodsmentioning

confidence: 99%

CCR5, CXCR4, and CD4 Are Clustered and Closely Apposed on Microvilli of Human Macrophages and T Cells

Singer

Scott

Kawka

et al. 2001

J Virol

151

129

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“…m-Maleimidobenzoyl-N-hydoxysuccinimide ester (Sulfo-MBS) (Pierce), an NHS ester-maleimide heterobifunctional crosslinker, was reacted with aminopentamidine so as to form a sulfhydryl reactive intermediate ( Fig. 2, Step II) (30). A three-fold molar excess of Sulfo-MBS to the calculated number of available sulfhydryl groups on the ovalbumin (see below) was dissolved in borate buffer (generally 32 mM in 200 µl).…”

Section: Hapten Activation and Conjugationmentioning

confidence: 99%

Immunoassays for pentamidine and related compounds: Development of a facile inhibitory ELISA suitable for clinical use

Reisner

Gray

Jones

et al. 2000

J. Clin. Lab. Anal.

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Aromatic dicationic drugs have a broad spectrum of activity against protozoal and fungal pathogens including Pneumocystis carinii, Leishmania mexicana amazonensis, Cryptosporidium parvum and Cryptococcus neoformans. Pentamidine serves as the exemplar for an extensive collection of newly synthesized related compounds, which have reduced toxicity and a wider range of target organisms. Assays of pentamidine and related compounds have depended on HPLC‐tandem mass spectrometry (HPLC‐TMS) for the quantitation and identification of drug and metabolites. Immunoassays for pentamidine would have many advantages over the HPLC methods including relative simplicity of assay format and required equipment, convenience in sample preparation and reduction in time and cost of assays. In this report we describe a simple ELISA based immunoassay for pentamidine and pentamidine‐like drugs with requisite sensitivity and specificity for use as a clinical assay (EC50 value of about 50 nanomolar). Immunogen was synthesized by coupling the hapten aminopentamidine to ovalbumin (chemically modified to provide an optimal number of –SH groups) using sulfo‐MBS. Maleic‐anhydride activated ELISA plates were covalently sensitized using the aminopentamidine hapten and used in an inhibitory ELISA assay format whereby the ability of analyte to suppress antibody binding to sensitized plate was measured. The assay detects primarily the phenolic amidine of pentamidine when in a para position and hence can also detect structurally related derivatives of pentamidine of potential interest as new therapeutic agents. J. Clin. Lab. Anal. 14:73–82, 2000. © 2000 Wiley‐Liss, Inc.

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“…Polyacrylamide gels (10%) were run and either stained with Coomassie Brilliant Blue R-250 and then autoradiographed or Western blotted. Blotted membranes were probed with polyclonal antibodies against annexins I, III, VI, and VII (obtained from Santa Cruz Biotechnology Inc.) and against annexin XI, the latter raised against the synthetic peptide SPAQFGNRGTITD (amino acids 182-194) [19] conjugated to thyroglobulin [20]. The anti-annexin antibodies were first individually used to probe Western blots of THP-1 cytosolic fractions in order to establish the exact position of the bands of the different annexins.…”

Section: Electrophoresis and Immunoblottingmentioning

confidence: 99%

Annexins VII and XI are present in a human macrophage-like cell line. Differential translocation on FcR-mediated phagocytosis

Pittis

García

1999

Journal of Leukocyte Biology

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An alternate method utilizing small quantities of ligand for affinity purification of monospecific antibodies

Cited by 21 publications

References 12 publications

CCR5, CXCR4, and CD4 Are Clustered and Closely Apposed on Microvilli of Human Macrophages and T Cells

CCR5, CXCR4, and CD4 Are Clustered and Closely Apposed on Microvilli of Human Macrophages and T Cells

Immunoassays for pentamidine and related compounds: Development of a facile inhibitory ELISA suitable for clinical use

Annexins VII and XI are present in a human macrophage-like cell line. Differential translocation on FcR-mediated phagocytosis

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