Improvement of the Lectin-Antibody Enzyme Immunoassay of the Alphafetoprotein Carbohydrate Chain for Automation with the Enzyme Immunoassay Robot

Tamano, Koichi; Sugiura, Mika; Natsuki, Jun; Sawakami-Kobayashi, Kazumi; Tajima, Hideji; Machida, Masayuki

doi:10.1271/bbb.69.1616

Cited by 2 publications

(2 citation statements)

References 5 publications

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“…The molecular mass determined by electrospray mass spectrometry (ES-MS) was approximately 4% less than the 68,803 Da determined for native human AFP derived from a hepatoblastoma cell line (Ferranti et al, 1995). This difference was ascribed to the absence in the E. coli rAFP of the carbohydrate structures that are present on mammalian-derived AFP; thus, rAFP is not helpful for enzyme immunoassay used to detect many kinds of disease biomarkers in medical laboratories and hospitals (Tamano et al, 2005). However, the whole purification usually requires a few days to be completed and the recovery of purified monomer rAFP was not particularly high (0.5-1 mg/l of bacterial culture).…”

Section: Introductionmentioning

confidence: 95%

Purification and characterization of Alpha-Fetoprotein from the human hepatoblastoma HepG2 cell line in serum-free medium

et al. 2007

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Alpha-fetoprotein (AFP) is a tumor-associated embryonic molecule whose precise biological function remains unclear. A complete definition of the physiological activities of this oncofetal protein has been severely limited, until now, by the lack of a purification procedure appropriate to obtain pure AFP in appreciable amount. The present report describes a purification procedure extremely rapid and simple and takes advantage of the well-known fact that AFP contains copper. We have developed a single-step purification procedure by immobilized copper-chelate affinity chromatography using the culture medium from human hepatoblastoma cell line HepG2 grown in the absence of serum. This method yields AFP at high purity and high yield. Purified AFP amino acid sequence, molecular mass, carbohydrate structure, and copper content were found to be in line with previous studies. Moreover, we found that the purified AFP has superoxide dismutase activity with efficiency similar to that of the native Cu, Zn SODs at physiological pH. This result may provide further support to the idea that AFP is a bifunctional protein, acting in cellular defence against oxidative stress both as a copper buffer and as a superoxide radical scavenger.

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Section: Introductionmentioning

confidence: 95%

Purification and characterization of Alpha-Fetoprotein from the human hepatoblastoma HepG2 cell line in serum-free medium

et al. 2007

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“…Much research has focused on HCC-specific heterogeneity of the glycosylation structures of AFP. − Recently, several methods for measuring AFP-L3 have been developed, increasing its value in the diagnosis of HCC. − For example, Tamano et al improved a lectin-antibody enzyme immunoassay for AFP-L3, and Wu et al developed a protein microarray for detecting AFP and AFP-L3 . Notably, we developed a sensitive mass spectrometry (MS)-based assay, combining immunoprecipitation (IP) and LCA-binding AFP fractionation, to simultaneously quantify AFP and AFP-L3 in a previous study .…”

Section: Introductionmentioning

confidence: 99%

Comparison of Fucose-Specific Lectins to Improve Quantitative AFP-L3 Assay for Diagnosing Hepatocellular Carcinoma Using Mass Spectrometry

et al. 2022

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Glycoproteins have many important biological functions. In particular, aberrant glycosylation has been observed in various cancers, such as liver cancer. A well-known glycoprotein biomarker is α-fetoprotein (AFP), a surveillance biomarker for hepatocellular carcinoma (HCC) that contains a glycosylation site at asparagine 251. The low diagnostic sensitivity of AFP led researchers to focus on AFP-L3, which has the same sequence as conventional AFP but contains a fucosylated glycan. AFP-L3 has high affinity for Lens culinaris agglutinin (LCA) lectin, prompting many groups to use it for detecting AFP-L3. However, a few studies have identified more effective lectins for fractionating AFP-L3. In this study, we compared the amounts of enriched AFP-L3 with five fucose-specific lectinsLCA, Lotus tetragonolobus lectin (LTL), Ulex europaeus agglutinin I (UEA I), Aleuria aurantia lectin (AAL), and Aspergillus oryzae lectin (AOL)to identify better lectins and improve HCC diagnostic assays using mass spectrometry (MS). Our results indicate that LTL was the most effective lectin for capturing AFP-L3 species, yielding approximately 3-fold more AFP-L3 than LCA from the same pool of HCC serum samples. Thus, we recommend the use of LTL for AFP-L3 assays, given its potential to improve the diagnostic sensitivity in patients having limited results by conventional LCA assay. The MS data have been deposited to the PeptideAtlas (PASS01752).

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Improvement of the Lectin-Antibody Enzyme Immunoassay of the Alphafetoprotein Carbohydrate Chain for Automation with the Enzyme Immunoassay Robot

Cited by 2 publications

References 5 publications

Purification and characterization of Alpha-Fetoprotein from the human hepatoblastoma HepG2 cell line in serum-free medium

Purification and characterization of Alpha-Fetoprotein from the human hepatoblastoma HepG2 cell line in serum-free medium

Comparison of Fucose-Specific Lectins to Improve Quantitative AFP-L3 Assay for Diagnosing Hepatocellular Carcinoma Using Mass Spectrometry

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