Improved procedures for the conjugation of oligosaccharides to protein by reductive amination

1984 ◽  
Vol 62 (5) ◽  
pp. 270-275 ◽  
Author(s):  
René Roy ◽  
Ewa Katzenellenbogen ◽  
Harold J. Jennings
Keyword(s):  
Sialic Acid ◽  
Bovine Serum Albumin ◽  
Bovine Serum ◽  
Reductive Amination ◽  
Reaction Model ◽  
Model Studies ◽  
End Groups ◽  
Coupling Procedure ◽  
Lysine Residues ◽  
Optimized Conditions

The rate of coupling of oligosaccharides having aldose end groups to protein by reductive amination was significantly increased by changing the temperature and pH of the reaction, and even more significantly by the addition of borate ions. Under optimized conditions half of the lysine residues of bovine serum albumin could be derivatized by lactose in 7 h and their complete derivatization was achieved in approximately 24 h. All attempts to carry out similar reductive amination procedures using oligosaccharides having ketose (D-fructose, 3-deoxy-D-manno-octulosonic acid (KDO), and sialic acid) failed owing to the slowness of the reaction. Model studies on the coupling of D-fructose and KDO to glycine indicate that any coupling procedure based on reductive amination of ketose residues would of necessity require the prior introduction of a small functionalized spacer molecule.

scholarly journals Group B Streptococcal Type II and III Conjugate Vaccines: Physicochemical Properties That Influence Immunogenicity

2006 ◽  
Vol 13 (8) ◽  
pp. 936-943 ◽  
Author(s):  
Francis Michon ◽  
Catherine Uitz ◽  
Arun Sarkar ◽  
Anello J. D'Ambra ◽  
Maryline Laude-Sharp ◽  
...  
Keyword(s):  
Sialic Acid ◽  
Reductive Amination ◽  
High Titer ◽  
Type Ii ◽  
Group B Streptococci ◽  
End Groups ◽  
Group B ◽  
Aldehyde Groups ◽  
Opsonophagocytic Killing ◽  
Acid Labile

ABSTRACT Recent efforts toward developing vaccines against group B streptococci (GBS) have focused on increasing the immunogenicity of GBS polysaccharides by conjugation to carrier proteins. However, partial depolymerization of GBS polysaccharides for the production of vaccines is a difficult task because of their acid-labile, antigenically critical sialic acids. Here we report a method for the partial depolymerization of type II and III polysaccharides by mild deaminative cleavage to antigenic fragments with reducing-terminal 2,5-anhydro-d-mannose residues. Through the free aldehydes of their newly formed end groups, the fragments were conjugated to tetanus toxoid by reductive amination. The resulting conjugates stimulated the production in animals of high-titer type II- and III-specific antibodies which induced opsonophagocytic killing of type II and III strains of group B streptococci. For the type II conjugates, immunogenicity increased as oligosaccharide size decreased, whereas for type III conjugates, the size of the oligosaccharides did not significantly influence immunogenicity. When oligosaccharides of defined size were conjugated through sialic acid residues, the resulting cross-linkages were shown to affect immunogenicity. When oligosaccharides were conjugated through terminal aldehyde groups generated by deamination, modification of the exocyclic chain of sialic acid did not influence immunogenicity.

scholarly journals Interaction of dinitrophenyl groups bound to bovine serum albumin with univalent fragments of anti-dinitrophenyl antibody

1979 ◽  
Vol 177 (1) ◽  
pp. 225-236 ◽  
Author(s):  
C. Graham Knight ◽  
N. Michael Green
Keyword(s):  
Bovine Serum Albumin ◽  
Fluorescence Quenching ◽  
Serum Albumin ◽  
Bovine Serum ◽  
Deae Cellulose ◽  
Serum Albumins ◽  
Single Polypeptide Chain ◽  
Quenching Efficiency ◽  
Lysine Residues ◽  
Single Polypeptide

Two lysine residues of bovine serum albumin reacted with 1-fluoro-2,4-dinitrobenzene with apparent second-order rate constants approx. 500-times greater than those observed in similar reactions with low-molecular-weight lysine derivatives. A series of dinitrophenyl (Dnp)-bovine serum albumins were prepared and their ability to bind univalent fragments of anti-Dnp antibody was measured by fluorescence-quenching titrations. Compared with the Dnp group of the free hapten, 6-N-Dnp-aminohexanoate, the majority of the protein-bound Dnp groups were unavailable to the antibody at pH8.0. When the same Dnp-albumins were titrated at pH3.0 the availability of the Dnp groups increased approx. 3-fold. Dnp-albumins were treated with pepsin at pH3.0 and Dnp-containing fragments isolated by chromatography on DE-52 DEAE-cellulose. Fluorescence-quenching titrations showed that the Dnp groups on the fragments behaved like the free hapten with respect to quenching efficiency, although with an increased dissociation constant. The association between the Dnp-albumins and the antibody was measured also by difference-spectral titrations at high protein concentrations. Antibody binding was increased under these conditions, but the Dnp group of mono-Dnp-albumin remained unavailable to antibody. We propose that the reactive lysine residues are located in clefts between the globular sub-domains of the single polypeptide chain. Dnp groups attached to these lysine residues are fully exposed to the solvent, but binding of the macromolecular probe, anti-Dnp antibody, is sterically hindered by the adjacent surface of the albumin molecule.

Synthesis and Identification of the Ciprofloxacin Hapten-Protein Conjugates

2013 ◽  
Vol 781-784 ◽  
pp. 1244-1247
Author(s):  
Xuan Yun Huang ◽  
Dong Mei Huang ◽  
Yong Fu Shi ◽  
Bing Feng ◽  
Hui Juan Yu ◽  
...  
Keyword(s):  
Bovine Serum Albumin ◽  
Serum Albumin ◽  
Bovine Serum ◽  
Sulfonic Acid ◽  
High Sensitivity ◽  
Animal Tissues ◽  
Benzene Sulfonic Acid ◽  
Protein Conjugates ◽  
Optimized Conditions ◽  
Carbodiimide Coupling

In order to generate the specific antibody of small molecular hapten-ciprofloxacin for detecting the Ciprofloxacin residues in edible animal tissues. A modified Carbodiimide coupling method was developed to obtain the immunogenic conjugates through coupling ciprofloxacin with bovine serum albumin in five different proportions. The conjugates were characterized and verified by electrophoresis,the trinitro-benzene-sulfonic acid (TNBS) method and ultraviolet spectra scanning. A quantitative analysis of the density of ciprofloxacin-bovine serum albumin conjugates suggested that a high conjugate density 30:1 could be obtained in the optimized conditions. By immunizing the mice, the antibody showed high sensitivity toward ciprofloxacin with an IC50of 4.69 ng/ml. This study confirmed a practical approach for the ciprofloxacin-bovine serum albumin conjugates, through which the high potent antisera specific for ciprofloxacin hapten can be produced for immunoassay.

scholarly journals Detection of β-Amyloid by Sialic Acid Coated Bovine Serum Albumin Magnetic Nanoparticles in a Mouse Model of Alzheimer's Disease

Small ◽  
2017 ◽  
Vol 14 (3) ◽  
pp. 1701828 ◽  
Author(s):  
Seyedmehdi Hossaini Nasr ◽  
Hovig Kouyoumdjian ◽  
Christiane Mallett ◽  
Sherif Ramadan ◽  
David C. Zhu ◽  
...  
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Sialic Acid ◽  
Magnetic Nanoparticles ◽  
Bovine Serum Albumin ◽  
Mouse Model ◽  
Serum Albumin ◽  
Bovine Serum ◽  
Model Of Alzheimer’S Disease ◽  
Β Amyloid

Synthesis of protein conjugates and analogues of N-acetylneuraminic acid

1990 ◽  
Vol 68 (11) ◽  
pp. 2045-2054 ◽  
Author(s):  
René Roy ◽  
Craig A. Laferrière
Keyword(s):  
Bovine Serum Albumin ◽  
Serum Albumin ◽  
Tetanus Toxoid ◽  
Bovine Serum ◽  
Reductive Amination ◽  
Reductive Ozonolysis ◽  
Acetylneuraminic Acid ◽  
Protein Conjugates ◽  
Step Procedure ◽  
Acidic Resin

N-Acetylneuraminic acid (1) was prepared from the salivary gland mucins of the Chinese swiftlet by an improved procedure using acidic resin hydrolysis. Compound 1 was transformed into the known acetochloroneuraminic acid (3) by a new two-step procedure. Koenigs–Knorr glycosylation of 3 followed by subsequent reductive ozonolysis of the 2-propenyl α-glycoside afforded the key aldehyde precursors 7 and 8, which were coupled to bovine serum albumin or tetanus toxoid by reductive amination. The factors influencing the extent of incorporation were investigated. A series of N-acetylneuraminic acid analogues modified at strategic functionalities were also synthesized. Keywords: sialic acid, N-acetylneuraminic acid, neoglycoproteins, bovine serum albumin, tetanus toxoid.

scholarly journals An Optimised Di-Boronate-ChemMatrix Affinity Chromatography to Trap Deoxyfructosylated Peptides as Biomarkers of Glycation

Molecules ◽  
2020 ◽  
Vol 25 (3) ◽  
pp. 755 ◽  
Author(s):  
Monika Kijewska ◽  
Francesca Nuti ◽  
Magdalena Wierzbicka ◽  
Mateusz Waliczek ◽  
Patrycja Ledwoń ◽  
...  
Keyword(s):  
Bovine Serum Albumin ◽  
Serum Albumin ◽  
Bovine Serum ◽  
Diabetes Complications ◽  
Ad Hoc ◽  
Complex Mixtures ◽  
The Novel ◽  
Peptide Mixtures ◽  
Lysine Residues ◽  
Modified Peptides

We report herein a novel ChemMatrix® Rink resin functionalised with two phenylboronate (PhB) moieties linked on the N-α and N-ε amino functions of a lysine residue to specifically capture deoxyfructosylated peptides, compared to differently glycosylated peptides in complex mixtures. The new PhB-Lys(PhB)-ChemMatrix® Rink resin allows for exploitation of the previously demonstrated ability of cis diols to form phenylboronic esters. The optimised capturing and cleavage procedure from the novel functionalised resin showed that only the peptides containing deoxyfructosyl-lysine moieties can be efficiently and specifically detected by HR-MS and MS/MS experiments. We also investigated the high-selective affinity to deoxyfructosylated peptides in an ad hoc mixture containing unique synthetic non-modified peptides and in the hydrolysates of human and bovine serum albumin as complex peptide mixtures. We demonstrated that the deoxyfructopyranosyl moiety on lysine residues is crucial in the capturing reaction. Therefore, the novel specifically-designed PhB-Lys(PhB)-ChemMatrix® Rink resin, which has the highest affinity to deoxyfructosylated peptides, is a candidate to quantitatively separate early glycation peptides from complex mixtures to investigate their role in diabetes complications in the clinics.

Localization of Intracellular Antigens in thin Sections with Ferritin Labeled Antibody

1970 ◽  
Vol 28 ◽  
pp. 174-175
Author(s):  
M.S. Shahrabadi ◽  
T. Yamamoto
Keyword(s):  
Bovine Serum Albumin ◽  
Bovine Serum ◽  
Antigenic Determinants ◽  
Conjugate Prior ◽  
Thin Sections ◽  
Specific Attachment ◽  
Intracellular Antigen ◽  
Antigen Antibody ◽  
Ideal Method ◽  
The Ideal

The technique of labeling of macromolecules with ferritin conjugated antibody has been successfully used for extracellular antigen by means of staining the specimen with conjugate prior to fixation and embedding. However, the ideal method to determine the location of intracellular antigen would be to do the antigen-antibody reaction in thin sections. This technique contains inherent problems such as the destruction of antigenic determinants during fixation or embedding and the non-specific attachment of conjugate to the embedding media. Certain embedding media such as polyampholytes (2) or cross-linked bovine serum albumin (3) have been introduced to overcome some of these problems.

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