Detection of serum thermolabile beta-2 macroglycoprotein (Hakata antigen) by enzyme-linked immunosorbent assay using polysaccharide produced by Aerococcus viridans.
Although a serum thermolabile beta-2 macroglycoprotein (TMG) may play a role in host defense as a lectin, little is known of its related physiological functions, mainly due to a lack of appropriate methods for tracing the functions of TMG. We identified a polysaccharide from Aerococcus viridans, PSA, which reacts with TMG, and based on this finding, we developed an enzyme-linked immunosorbent assay to trace the functions of TMG. Using ethanol precipitation and DEAE-Sepharose and Sephacryl S-400 column chromatographies, we isolated PSA from cultured medium of A. viridans, and it exhibited specific binding against TMG in blood samples. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the isolated PSA showed ladder bands that implied the existence of repeating units composed of D-glucose, N-acetyl-D-glucosamine, D-mannose, and D-xylose, as confirmed by gas chromatography-mass spectrometry. SDS-PAGE and immunochemical analysis, using rabbit anti-TMG antibody, showed that PSA specifically binds solely to intact serum TMG but not to TMG heated at 56 degrees C for 30 min, a condition under which antigenicity is lost. TMG in serum samples bound to PSA in a dose-dependent manner, and this binding was clearly suppressed by addition of PSA. These observations indicate that PSA is a useful adsorbent to TMG and can be used to develop appropriate methods for tracing the functions of TMG.
Fukuoka Red Cross Blood Center, 1-2-1 Kamikoga, Chikushino, Fukuoka 818-8588, Japan.
M
M
Tsujimura
C
C
Ishida
Y
Y
Sagara
T
T
Miyazaki
K
K
Murakami
H
H
Shiraki
K
K
Okochi
Y
Y
Maeda
D000907:Antibodies, Bacterial / Q000276:immunology; D001323:Autoantibodies / Q000097:blood / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006023:Glycoproteins / Q000032:analysis* / Q000276:immunology / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D037102:Lectins; D008180:Lupus Erythematosus, Systemic / Q000276:immunology; D011135:Polysaccharides, Bacterial / Q000276:immunology* / Q000302:isolation & purification / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding / Q000276:immunology; D013289:Streptococcaceae / Q000276:immunology*
D000907:Antibodies, Bacterial; D001323:Autoantibodies; C109662:FCN3 protein, human; D006023:Glycoproteins; D037102:Lectins; D011135:Polysaccharides, Bacterial