[Establishment of the optimal conditions for detecting anti-M2 by western blotting in primary biliary cirrhosis].
Anti-mitochondrial antibodies(AMA) are serodiagnostic markers for primary biliary cirrhosis(PBC) but heterogenous in antigen molecules which they recognize. A disease-specific AMA for PBC is anti-M2. The conventional examination methods are indirect immunofluorescence and ELISA. However, there are some problems in specificity, because the antigen preparations used are crude. Thus, analysis with Western-blotting(W-B) is needed, because it allows the identification of a molecule which the antibody reacts with. In this report, we established the optimal conditions for detecting anti-M2 with W-B in PBC. As antigen, we used mitochondrial fractions derived from beef hearts. Because a positive band at 74 kDa became negative after absorbing sera with PDH purified from porcine hearts, this band corresponded to major antigeneity of anti-M2. Titration experiments with SDS-PAGE showed that the optimal concentration of this antigen preparation for loading is 0.04 mg/ml. We also performed titration experiments to determine the optimal dilutions for second antibodies and serum samples. The results showed that the optimal dilution for second antibodies, anti-IgG and anti-IgM, were 1:3000 and 1:1000, respectively. The optimal dilution for serum samples was shown to be 1:10(2). Moreover, the W-B technique gave a positive result even for sera from AMA-negative PBC patients which had tested negative with conventional methods, if undiluted sera were examined or if anti-IgG or anti-IgM was used as a second antibody. Thus, the W-B technique is more sensitive than conventional methods of analysis. Based on these results, we will be able to detect anti-M2 with maximum efficiency, thus improving our ability to study the relationship between anti-M2 and the pathological conditions in PBC.
Kawaguchi
N
N
Central Laboratory, Teikyo University Hospital, Kawasaki.
Miyakawa
H
H
Abe
K
K
Kitazawa
E
E
Kako
M
M
D001323:Autoantibodies / Q000097:blood*; D015415:Biomarkers / Q000097:blood; D015153:Blotting, Western / Q000379:methods*; D006801:Humans; D007074:Immunoglobulin G / Q000097:blood; D007075:Immunoglobulin M / Q000097:blood; D008105:Liver Cirrhosis, Biliary / Q000175:diagnosis*; D008928:Mitochondria / Q000276:immunology; D013347:Subcellular Fractions
D001323:Autoantibodies; D015415:Biomarkers; D007074:Immunoglobulin G; D007075:Immunoglobulin M