Immobilization of Saccharides and Peptides on 96-Well Microtiter Plates Coated with Methyl Vinyl Ether–Maleic Anhydride Copolymer

Satoh, Ayano; Kojima, Kensuke; Koyama, Tamami; Ogawa, Haruko; Matsumoto, Isamu

doi:10.1006/abio.1998.2668

Cited by 36 publications

(23 citation statements)

References 14 publications

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“…6 Although this approach using ABEE was also useful in structural analyses on HPLC and in measurements of lectin-carbohydrate interaction analysis in ELISA, pyrene label surpassed ABEE by about ten times in sensitivity. The immobilization methods used by other groups for the oligosaccharides include covalent coupling to modified surface of plates, 6,8 the binding of biotinylated oligosaccharides to avidin-coated plates, 9 and the production of neoglycolipids. 10 In these methods, additional or entirely different procedures were necessary to modify the oligosaccharides from those used for other detections.…”

Section: Elisamentioning

confidence: 99%

Fluorescence Labeling of Oligosaccharides Useful in the Determination of Molecular Interactions

2003

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Glycoconjugates on cell surfaces play important roles in cellcell interactions in a variety of biological processes. Glycans on the surfaces of glycosylated proteins also regulates intracellular and body-wide trafficking of these molecules. They play important roles in the regulation of the proper folding of the proteins. It is crucial to determine the specificity and affinity of the interaction between carbohydrates and the recognition molecules to evaluate the biological significance of carbohydrate residues. Fluorescence polarization (FP) is a useful method because molecular interactions can be determined without separating the bound and free ligands. We previously determined the interactions between recombinant human macrophage C-type lectin and synthetic glycopeptides by measuring FP. 1 Antigen-antibody interactions can also be determined. 2However, because the fluorescent labeling of oligosaccharides at minute quantity suitable for the detection by FP was not previously available, there were only a few applications in the determination of glycan-lectin interactions. [3][4][5] ELISA is another potentially useful approach in oligosaccharide characterizations, though difficulty in quantitative immobilization of oligosaccharides hampered the use of this procedure. In the present study, we developed multifunctional oligosaccharide probes by introducing pyrene groups through a simple procedure. The oligosaccharide probes were proven to be applicable to FP and ELISA at picomole quantities of oligosaccharides obtained from biological samples, such as those from cells and tissues. Because fluorescence derivatives of oligosaccharides previously used with 2-aminopyridine or aminobenzoethyl ester (ABEE) were not suited for FP and ELISA, as described below, the pyrene labeling of oligosaccharide developed in the present report should be of great value. Experimental Oligosaccharide labelingThe preparation of labeled oligosaccharides was according to procedure used for the amino benzoate ethyl ester (ABEE) labeling, except that a reducing reagent was not used. 6 Pyrene butanoic acid hydrazide (Molecular Probes, Leiden, The Netherlands) in methanol (500 nmol/20 µl) mixed with 2 µl acetic acid diluted with methanol (1:8, v/v) was added to dried oligosaccharides in a glass vial with a screw cap. The mixture was tightly sealed with a cap, mixed well and heated at 80˚C. After incubating for 20 min, the reaction mixture was neutralized by adding a 1 M sodiumhydroxide solution. The greatest amounts of fluorescent oligosaccharides were obtained at 80˚C. Pyrene-labeled oligosaccharides were obtained in the aqueous phase by chloroform/water (1:1) extraction, and unreacted pyrene derivatives were removed by this procedure. The aqueous phase was evaporated to dryness. The residue was dissolved with water and applied onto a Sep-Pak C18 cartridge (Millipore Corporation, Bedford, MA). After washing the cartridge with water, labeled oligosaccharides were eluted with a mixture of water and acetonitorile (6:4, v/v). The fraction was evapo...

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Section: Elisamentioning

confidence: 99%

Fluorescence Labeling of Oligosaccharides Useful in the Determination of Molecular Interactions

2003

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“…Then, a 30 mg/ml ADHZ solution (almost saturated solution) was injected for 21 min. Fifty milligrams per milliliter of CS or 0.1 M lactose in 10 mM 1 To whom correspondence should be addressed. Fax: ϩ81-3-5978-5344.…”

Section: Resultsmentioning

confidence: 99%

“…To elucidate these phenomena, it is essential to identify the biological molecules that recognize these carbohydrates and characterize quantitatively the interaction between the carbohydrates and their recognition molecules. To do so requires immobilization of carbohydrates on microtiter plates; we recently reported one method (1). In ELISA, 2 biological substances are generally immobilized in the wells of microtiter plates by nonspecific hydrophobic interactions between the substances and the plastic surface of the well.…”

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confidence: 99%

Analysis of Interaction between Lectin and Carbohydrate by Surface Plasmon Resonance

Satoh

Matsumoto

1999

Analytical Biochemistry

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“…In all cases, bound peptide was detected with alkaline phosphatase-conjugated streptavidin (016-050-084; Jackson ImmunoResearch Labs Inc., West Grove, PA) in an ELISA. Heparin (H93990; Sigma, St. Louis, MO) was immobilized on methyl vinyl ether-maleic anhydride-coated Costar 9102 strip-well microtiter plates (Corning Inc., Corning, NY) treated with adipic acid dihydrazide (37).…”

Section: Methodsmentioning

confidence: 99%

The Virucidal EB Peptide Protects Host Cells from Herpes Simplex Virus Type 1 Infection in the Presence of Serum Albumin and Aggregates Proteins in a Detergent-Like Manner

Bultmann

Girdaukas²,

Kwon³

et al. 2010

Antimicrob Agents Chemother

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The linear cationic amphiphilic EB peptide, derived from the FGF4 signal sequence, was previously shown to be virucidal and to block herpes simplex type I (HSV-1) entry (H. Bultmann, J. S. Busse, and C. R. Brandt, J. Virol. 75:2634؊2645, 2001). Here we show that cells treated with EB (RRKKAAVALLPAVLLALLAP) for less than 5 min are also protected from infection with HSV-1. Though protection was lost over a period of 5 to 8 h, it was reinduced as rapidly as during the initial treatment. Below a 20 M concentration of EB, cells gained protection in a serum-dependent manner, requiring bovine serum albumin (BSA) as a cofactor. Above 40 M, EB coprecipitated with BSA under hypotonic conditions. Coprecipitates retained antiviral activity and released active peptide. NaCl (>0.3 M) blocked coprecipitation without interfering with antiviral activity. As shown for ␤-galactosidase, EB below 20 M acted as an enzyme inhibitor, whereas above 40 to 100 M EB, ␤-galactosidase was precipitated as was BSA or other unrelated proteins. Pyrene fluorescence spectroscopy revealed that in the course of protein aggregation, EB acted like a cationic surfactant and self associated in a process resembling micelle formation. Both antiviral activity and protein aggregation did not depend on stereospecific EB interactions but depended strongly on the sequence of the peptide's hydrophobic tail. EB resembles natural antimicrobial peptides, such as melittin, but when acting in a nonspecific detergent-like manner, it primarily seems to target proteins.

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Immobilization of Saccharides and Peptides on 96-Well Microtiter Plates Coated with Methyl Vinyl Ether–Maleic Anhydride Copolymer

Cited by 36 publications

References 14 publications

Fluorescence Labeling of Oligosaccharides Useful in the Determination of Molecular Interactions

Fluorescence Labeling of Oligosaccharides Useful in the Determination of Molecular Interactions

Analysis of Interaction between Lectin and Carbohydrate by Surface Plasmon Resonance

The Virucidal EB Peptide Protects Host Cells from Herpes Simplex Virus Type 1 Infection in the Presence of Serum Albumin and Aggregates Proteins in a Detergent-Like Manner

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