Quantitation of glycoproteins on electroblots using the biotin-streptavidin complex

O’Shannessy, Daniel J.; Voorstad, P. J.; Quarles, Richard H.

doi:10.1016/0003-2697(87)90114-x

Cited by 106 publications

(36 citation statements)

References 12 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Direct labeling of glycoproteins by low-molecularmass compounds and their detection with their respective binding partners (e.g. the biotidstreptavidin system) has proved to be successful [ 18,261. Tagging reducing oligosaccharides with biotinylated diaminopyridine was recently described [27], allowing their characterization and their biological study.…”

Section: Discussionmentioning

confidence: 99%

“…0.5 mg/ml of ManLAM in 100 mM sodium acetate pH 5.5 was oxidized at 0°C (in an ice bath) by incubation in 1 mM sodium periodate for 10 min in the dark (from experimental procedures used for glycoproteins in [18]). The oxidation time was determined so that the structural and functional integrities of ManLAM were minimally altered and was fixed at 10 min.…”

Section: Preparation Of Manlammentioning

confidence: 99%

“…the sodium periodate concentration, the temperature and the reaction time, controlled the extent of ManLAM oxidation. 1 mg ManLAM (60 nmol) was oxidized with 1 mM sodium periodate at O"C, mild conditions based upon previous reports for the glycoprotein labeling [18]. This amount of periodate would be able to cleave nearly 50% of the monosaccharide units since 60nmol Man-LAM approximatively contained 6 pmol monosaccharide.…”

Section: Labeling Of Manlam With a Biotin Hydrazide Derivativementioning

confidence: 99%

See 2 more Smart Citations

Mannosylated Lipoarabinomannan Interacts with Phagocytes

Venisse

Fournié

Puzo

1995

European Journal of Biochemistry

View full text Add to dashboard Cite

Infection by Mycobacterium tuberculosis first involves its adhesion to mononuclear host phagocytes. Various macrophage opsonic and non-opsonic receptors are known to mediate this adhesion, with some specificity of mannosyl receptors for the more virulent strains. Mannosylated lipoarabinomannan, a major component of cell walls from M. tuberculosis and Mycobacterium bovis BCG, is endowed with mannooligosaccharide units that could mediate its binding to these latter receptors. To explore its interaction with murine immune cells by flow cytometry, we report a new procedure to fluorescently tag the polysaccharide molecules. We covalently labeled mannosylated lipoarabinomannan from M. bovis BCG with biotin, allowing formation of stable complexes with streptavidin coupled to a fluorochrome. In this work, we demonstrated that this major carbohydrate antigen interacts selectively with murine phagocytes, i.e. granulocytes and macrophages. This binding was affected by temperature and was serum-and divalent-cationdependent. It also appears to involve a metabolically recycling protein receptor on the phagocyte surface and mannosyl aggretopes on the mannosylated lipoarabinomannan molecule. Thus, the latter may provide a means for mycobacteria to bind to and invade their host phagocytes. This molecule could constitute one of the early factors of mycobacterial virulence. all the known ones share a basic structure composed of a mannan core linked to an arabinan chain to which are attached arabinosy1 side chains and a phosphatidylinositol anchor located at the mannan core reducing end [ 13 -151. Moreover, the majority of the terminal oligoarabinosyl motifs in lipoarabinomannan from virulent strains of M. tuberculosis and from the vaccine strain M. bovis BCG are themselves extensively capped with either mono-, di-, or trimannosyl residues [16, 171, leading to the terms mannosylated or mannose-capped lipoarabinomannan (ManLAM). KeywordsIn an attempt to study its binding to murine immune cells by flow cytofluorimetry, the ManLAM from M. bovis BCG was covalently tagged after mild oxidation by sodium periodate. We describe the experimental procedure for the covalent coupling of biotinamidohexanoyl hydrazide to ManLAM whose functional and structural integrity was not significantly altered by this procedure. Then, the biotinylated ManLAM was recognized with high affinity by streptavidin coupled to a fluorochrome which allowed its detection at the cell surface. By this approach, we showed that ManLAM selectively interacts with granulocytes and macrophages. This interaction appears to be dependent upon the presence of serum and divalent cations, and to be sensitive to temperature. It was also found to involve a proteasesensitive membrane receptor. The binding was competitively inhibited by mannan, suggesting a mannosyl-defined interaction. Therefore, the implication of ManLAM in the first step of infection will be discussed with regard to its role in the mediation of adhesion and phagocytosis of mycobacteria. MATERIALS AND METHODSPreparat...

show abstract

Section: Discussionmentioning

confidence: 99%

Section: Preparation Of Manlammentioning

confidence: 99%

Section: Labeling Of Manlam With a Biotin Hydrazide Derivativementioning

confidence: 99%

See 1 more Smart Citation

Mannosylated Lipoarabinomannan Interacts with Phagocytes

Venisse

Fournié

Puzo

1995

European Journal of Biochemistry

View full text Add to dashboard Cite

show abstract

“…AT was also tested for carbohydrate using an enzyme immunoassay system to detect digoxigenin-labeled glycans (11). Transferrin was used as a positive control.…”

Section: Protein Analysesmentioning

confidence: 99%

Characterization of rat hepatic acetyltransferase.

Land

King

1994

Environ Health Perspect

View full text Add to dashboard Cite

Rat liver cytosol is capable of N-acetylation (NAT) of arylamines, O-acetylation (OAT) of arylhydroxylamines, and NO-acetyltransfer (AHAT) of arylhydroxamic acids. Physical, enzymatic, and immunochemical techniques now support the conclusion that a single 32 kDa protein accounts for all of these activities. Of the five immunoglobulin (lgG1) mouse monoclonal antibodies (mAb) produced against this protein, each affected one or more of these acetylation activities. When mixed with rat hepatic cytosol and then chromatographed on a gel filtration column, mAbs 1 F2 and 5F8 increased the apparent size of all enzymes capable of acetylation from 32 kDa to the exclusion volume. Each of the mAbs reacted with only a single 32 kDa protein on SDS-PAGE/VVestern blots, regardless of the state of purity of the enzyme. This enzyme is unstable in low salt solutions, as reflected by a relative loss in NAT versus AHAT activity, but it does not result in changes in either molecular weight or isoelectric point (pl). A second form of instability is shown by the formation of more basic peptides with pls as high as 6, again without change in molecular weight. Although NAT activity is retained in acetyltransferase (AT) that has a minimally modified pl, further increases in pl result in total loss of enzyme activity. The differential effects of the mAbs on AT suggest that the ratios of NAT, OAT, and AHAT may be highly dependent on the conformation of the enzyme and, consequently, provide insight as to why the abilities of ATs from different species exhibit such dissimilar potentials for the activation of aromatic amines by OAT and AHAT. -Environ Health Perspect 102(Suppl 6): 91-93 (1994)

show abstract

“…The utilization of the carbohydrate moiety as the binding site has an advantage since these regions are generally not involved with their biological activities [27]. Therefore, many glycoproteins, including hormones [28], antibodies [29] and enzymes such as horseradish peroxidase, lactamases, hexokinase, glucose-6-phosphate dehydrogenase [30][31][32], retain most of their biological activities even when their carbohydrate regions are modified or altered. We speculated that APBA-agarose presaturated with HRPO could purify the BsMAb as HRPO-labelled BsMAb complex.…”

Section: Introductionmentioning

confidence: 99%

Sequential affinity purification of peroxidase tagged bispecific anti-SARS-CoV antibodies on phenylboronic acid agarose

Bhatnagar

Das

Suresh

2008

Journal of Chromatography B

View full text Add to dashboard Cite

Hybrid hybridomas (quadromas) are derived by fusing at least two hybridomas, each producing a different antibody of predefined specificity. The resulting cell secretes not only the immunoglobulins of both parents but also hybrid molecules manifesting the binding characteristics of the individual fusion partners. Purification of the desired bispecific immunoprobe with high specific activity from a mixture of bispecific and monospecific monoclonal antibodies requires special strategies. Using a dual, sequential affinity chromatography (Protein-G chromatography followed by maminophenyleboronic acid agarose column), we have purified bispecific monoclonal antibodies (BsMAb) as a preformed HRPO (Horseradish Peroxidase) complex (BsMAb-HRPO). The quadroma culture supernatant was initially processed on a Protein-G column to isolate all the species of immunoglobulins. This pre-enriched fraction was subsequently passed through the aminophenyleboronic acid column super saturated with HRPO. The column matrix has the ability to bind to proteins such as HRPO with vicinal diols. The enzyme loaded column captures the desired bispecific anti-SARS-CoV × anti-HRPO species with the elimination of the monospecific anti-SARS-CoV MAb to result in a high specific activity diagnostic probe. The presence of anti-HRPO MAb is an acceptable impurity as it will not bind to the target SARS-CoV NP antigen and will get washed out during the ELISA procedure.

show abstract

Quantitation of glycoproteins on electroblots using the biotin-streptavidin complex

Cited by 106 publications

References 12 publications

Mannosylated Lipoarabinomannan Interacts with Phagocytes

Mannosylated Lipoarabinomannan Interacts with Phagocytes

Characterization of rat hepatic acetyltransferase.

Sequential affinity purification of peroxidase tagged bispecific anti-SARS-CoV antibodies on phenylboronic acid agarose

Contact Info

Product

Resources

About