Development of a rapid, immunochromatographic strip test for serum asialo alpha1-acid glycoprotein in patients with hepatic disease.
Serum asialoglycoprotein (desialylated glycoproteins) concentrations have been reported to be elevated in patients with hepatic disease as compared with that of normal subjects. We recently developed a solid-phase sandwich assay for asialo alpha1-acid glycoprotein (AsAGP) as a representative of the serum asialoglycoproteins and evaluated the utility of this AsAGP as a diagnostic marker for liver cirrhosis (LC) and/or hepatocellular carcinoma (HCC). In this study, we developed a rapid, one-step immunochromatographic strip capable of specifically detecting AsAGP in serum specimens. We have produced a monoclonal antibody (mAb) to AGP, and based on ELISA and Western blot analysis, we have selected four hybridoma clones which generated mAbs to recognize AsAGP. In the immunochromatographic strip test, one mAb was used for conjugation with colloidal gold microparticles. Ricinus communis agglutinin (RCA) was immobilized onto a nitrocellulose membrane strip to form a result line in the path of chromatographic migration. Likewise, a control line was created above the result line by the immobilization of anti-mouse IgG. A serum specimen was then applied to the sample pad. The AsAGP in the sample specifically bound to the microparticles via mAb (As16.89) and co-migrated upward until the AsAGP was sandwiched with the immobilized lectin (RCA), revealing a visible result line. The colloidal gold microparticles without bound AsAGP continued to migrate, forming a visible control line. Thus, an AsAGP-positive specimen (>1.5 microg/mL) yielded a result line and a control line, whereas an AsAGP-negative specimen (<1.5 microg/mL) produced only a single control line. The entire test procedure was completed in less than 5 min. In order to examine the reliability of the testing procedures, we carried out the immunochromatographic strip test with 102 serum samples and compared the results of these tests with those obtained by ELISA. The two methods showed excellent correlation, with 83-100% above/below the cut-off value (1.5 microg/mL). Therefore, we concluded that the results of the immunochromatographic test are in excellent accordance with those of the sandwich ELISA.
Lee
Eun Young
EY
The Laboratory of Cell Biology, Korea Research Institute of Bioscience and Biotechnology, P. O. Box 115, Yusong, Daejeon 305-600, Republic of Korea.
Kang
Ji Hyun
JH
Kim
Kyoung A
KA
Chung
Tai Wha
TW
Kim
Hee Jung
HJ
Yoon
Do Young
DY
Lee
Hee Gu
HG
Kwon
Dur Han
DH
Kim
Jae Wha
JW
Kim
Cheorl Ho
CH
Song
Eun Young
EY
D000818:Animals; D000911:Antibodies, Monoclonal; D001212:Asialoglycoproteins / Q000097:blood* / Q000276:immunology; D015415:Biomarkers / Q000097:blood; D006528:Carcinoma, Hepatocellular / Q000097:blood; D002845:Chromatography / Q000379:methods*; D003101:Collodion; D004797:Enzyme-Linked Immunosorbent Assay; D017979:Gold Colloid; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D008103:Liver Cirrhosis / Q000097:blood; D008107:Liver Diseases / Q000097:blood* / Q000175:diagnosis; D008113:Liver Neoplasms / Q000097:blood; D051379:Mice; D009961:Orosomucoid / Q000031:analogs & derivatives* / Q000276:immunology; D037121:Plant Lectins; D011817:Rabbits
D000911:Antibodies, Monoclonal; D001212:Asialoglycoproteins; D015415:Biomarkers; D017979:Gold Colloid; D009961:Orosomucoid; D037121:Plant Lectins; C017707:Ricinus communis agglutinin-1; C017030:asialoorosomucoid; D003101:Collodion