A simple and reliable method for the detection of sialylation in complex/hybrid-type carbohydrate chains of glycoproteins by mixed lectins.
A practical and convenient method for discriminating between the presence and the absence of sialic acid in carbohydrate chains of glycoproteins was devised using paramagnetic beads and two lectins, Sambucus sieboldiana lectin (SSA) and Ricinus communis agglutinin (RCA120). The glycoproteins of transferrin or thyroglobulin were firstly captured to paramagnetic beads that were previously coated with corresponding antibody, and then the lectins of RCA120-biotin and SSA-FITC were bound to the glycoproteins on the paramagnetic beads. Finally, the fluorescence intensity of the beads was measured to determine the ratios of lectins RCA120-biotin/Cy5-streptavidin to SSA-FITC. The mixed lectins method showed bigger difference of the ratios between the presence and the absence of sialic acid, indicating higher discrimination efficiency than the ordinary non-mixed lectins method. Furthermore, statistical analysis by two-side t-test indicated that the mixed lectins method was more highly reliable than the ordinary non-mixed lectins method in discriminating between the presence and the absence of sialic acid. The reaction with the two lectins can be performed in a single tube and readily automated taking advantage of the use of paramagnetic beads.
Natsuki
Jun
J
National Institute of Advanced Industrial Science and Technology (AIST), Central 6, Higashi 1-1-1, Tsukuba, Ibaraki 305-8566, Japan.
Machida
Masayuki
M
D000906:Antibodies / Q000737:chemistry; D002241:Carbohydrates / Q000737:chemistry*; D006023:Glycoproteins / Q000737:chemistry* / Q000276:immunology; D006801:Humans; D008280:Magnetics; D008840:Microfilament Proteins / Q000737:chemistry / Q000276:immunology; D008863:Microspheres; D009124:Muscle Proteins / Q000737:chemistry / Q000276:immunology; D019158:N-Acetylneuraminic Acid / Q000032:analysis*; D037121:Plant Lectins / Q000737:chemistry*; D054788:Ribosome Inactivating Proteins; D013050:Spectrometry, Fluorescence / Q000379:methods*; D014168:Transferrin / Q000737:chemistry / Q000276:immunology
D000906:Antibodies; D002241:Carbohydrates; D006023:Glycoproteins; D008840:Microfilament Proteins; D009124:Muscle Proteins; D037121:Plant Lectins; C017707:Ricinus communis agglutinin-1; C050600:Sambucus nigra lectins; D014168:Transferrin; C051615:transgelin; D054788:Ribosome Inactivating Proteins; D019158:N-Acetylneuraminic Acid