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• W2108185963 abstract "Aberrant glycosylation-targeted disease biomarker development is based on cumulative evidence that certain glycoforms are mass-produced in a disease-specific manner. However, the development process has been hampered by the absence of an efficient validation method based on a sensitive and multiplexed platform. In particular, ELISA-based analytical tools are not adequate for this purpose, mainly because of the presence of a pair of N-glycans of IgG-type antibodies. To overcome the associated hurdles in this study, antibodies were tagged with oligonucleotides with T7 promoter and then allowed to form a complex with corresponding antigens. An antibody-bound specific glycoform was isolated by lectin chromatography and quantitatively measured on a DNA microarray chip following production of fluorescent RNA by T7-trascription. This tool ensured measurement of targeted glycoforms of multiple biomarkers with high sensitivity and multiplexity. This analytical method was applied to an in vitro diagnostic multivariate index assay where a panel of hepatocellular carcinoma (HCC) biomarkers comprising alpha-fetoprotein, hemopexin, and alpha-2-macroglobulin (A2M) was examined in terms of the serum level and their fuco-fractions. The results indicated that the tests using the multiplexed fuco-biomarkers provided improved discriminatory power between non- hepatocellular carcinoma and hepatocellular carcinoma subjects compared with the alpha-fetoprotein level or fuco-alpha-fetoprotein test alone. The developed method is expected to facilitate the validation of disease-specific glycan biomarker candidates. Aberrant glycosylation-targeted disease biomarker development is based on cumulative evidence that certain glycoforms are mass-produced in a disease-specific manner. However, the development process has been hampered by the absence of an efficient validation method based on a sensitive and multiplexed platform. In particular, ELISA-based analytical tools are not adequate for this purpose, mainly because of the presence of a pair of N-glycans of IgG-type antibodies. To overcome the associated hurdles in this study, antibodies were tagged with oligonucleotides with T7 promoter and then allowed to form a complex with corresponding antigens. An antibody-bound specific glycoform was isolated by lectin chromatography and quantitatively measured on a DNA microarray chip following production of fluorescent RNA by T7-trascription. This tool ensured measurement of targeted glycoforms of multiple biomarkers with high sensitivity and multiplexity. This analytical method was applied to an in vitro diagnostic multivariate index assay where a panel of hepatocellular carcinoma (HCC) biomarkers comprising alpha-fetoprotein, hemopexin, and alpha-2-macroglobulin (A2M) was examined in terms of the serum level and their fuco-fractions. The results indicated that the tests using the multiplexed fuco-biomarkers provided improved discriminatory power between non- hepatocellular carcinoma and hepatocellular carcinoma subjects compared with the alpha-fetoprotein level or fuco-alpha-fetoprotein test alone. The developed method is expected to facilitate the validation of disease-specific glycan biomarker candidates. Protein-attached glycans are bio-synthesized by a subset of glycosyltransferases mostly located in the endoplasmic reticulum and the Golgi apparatus, and play various functional roles at molecular and cellular levels including molecular interactions, stability, immune function, adhesion, etc. However, cumulative lines of evidence indicate that aberrant glycosylation is associated with various diseases including cancer (1Christiansen M.N. Chik J. Lee L. Anugraham M. Abrahams J.L. Packer N.H. Cell surface protein glycosylation in cancer.Proteomics. 2014; 14: 525-546Crossref PubMed Scopus (370) Google Scholar), either by influencing the functionality of proteins and cells or as nonfunctional participants (2Kim Y.S. Hwang S.Y. Kang H.Y. Sohn H. Oh S. Kim J.Y. Kim C.H. Jeon J.H. Lee J.M. Kang H.A. Miyoshi E. Taniguchi N. Yoo H.S. Ko J.H. Functional proteomics study reveals that N-acetylglucosaminyltransferase V reinforces the invasive/metastatic potential of colon cancer through aberrant glycosylation on tissue inhibitor of metalloproteinase-1.Mol. Cell. Proteomics. 2008; 7: 1-14Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 3Häuselmann I. Borsig L. Altered tumor-cell glycosylation promotes metastasis.Front. Oncol. 2014; 4: 28Crossref PubMed Scopus (257) Google Scholar, 4Meany D.L. Chan D.W. Aberrant glycosylation associated with enzymes as cancer biomarkers.Clin. Proteomics. 2011; 8: 7Crossref PubMed Scopus (184) Google Scholar). Causal roles of aberrant glycosylation have been widely investigated in the development and progression of diseases, and significant progress has been made in the realm of cancer research (5Kim Y.S. Yoo H.S. Ko J.H. Implication of aberrant glycosylation in cancer and use of lectin for cancer biomarker discovery.Protein Pept. Lett. 2009; 16: 499-507Crossref PubMed Scopus (34) Google Scholar). For these reasons, glycoproteins carrying aberrant glycans have been targets for the development of in vitro diagnostics (6Adamczyk B. Tharmalingam T. Rudd P.M. Glycans as cancer biomarkers.Biochim. Biophys. Acta. 2012; 1820: 1347-1353Crossref PubMed Scopus (382) Google Scholar). Alpha feto protein (AFP) -L3, a fucoform of AFP that is retained by the Lens culinaris lectin (LCA), is an extensively proven disease biomarker (7Cheng J. Wang W. Zhang Y. Liu X. Li M. Wu Z. Liu Z. Lv Y. Wang B. Prognostic role of pretreatment serum AFP-L3% in hepatocellular carcinoma: systematic review and meta-analysis.PLOS One. 2014; 9: e87011Crossref PubMed Scopus (48) Google Scholar). AFP is frequently overexpressed in hepatic carcinoma cells and thus exists at high concentrations in blood of patients with hepatocellular carcinoma (HCC) (8Xia Y. Yan Z.L. Xi T. Wang K. Li J. Shi L.H. Wu M.C. Shen F. A case-control study of correlation between preoperative serum AFP and recurrence of hepatocellular carcinoma after curative hepatectomy.Hepatogastroenterol. 2012; 59: 2248-2254PubMed Google Scholar). However, the onco-fetal protein is reported to surge even under non-tumor disease conditions such as inflammation or abnormal pregnancy (9Potapovich A.I. Pastore S. Kostyuk V.A. Lulli D. Mariani V. De Luca C. Dudich E.I. Korkina L.G. alpha-Fetoprotein as a modulator of the pro-inflammatory response of human keratinocytes.Br. J. Pharmacol. 2009; 158: 1236-1247Crossref PubMed Scopus (14) Google Scholar, 10Androutsopoulos G. Gkogkos P. Decavalas G. Mid-trimester maternal serum HCG and alpha fetal protein levels: clinical significance and prediction of adverse pregnancy outcome.Int. J. Endocrinol. Metab. 2013; 11: 102-106Crossref PubMed Scopus (27) Google Scholar). This indicates a limited utility of AFP because of low specificity for prediction or diagnosis of HCC. Because it has been reported that the ratio of AFP-L3 to total AFP could be highly specific for HCC, AFP-L3 has been a preferred HCC biomarker to AFP levels and extensive investigations culminated in FDA-approval of an AFP-L3 lab test to determine the risk of developing liver cancer in patients with chronic liver disease (11Shiraki K. Takase K. Tameda Y. Hamada M. Kosaka Y. Nakano T. A clinical study of lectin-reactive alpha-fetoprotein as an early indicator of hepatocellular carcinoma in the follow-up of cirrhotic patients.Hepatology. 1995; 22: 802-807Crossref PubMed Google Scholar). Besides AFP-L3, several glycan indicators of a relationship with cancer states including CA15-3 and CA19-9, have been reported in terms of relationship with cancer states (12Duffy M.J. Evoy D. McDermott E.W. CA 15–3: uses and limitation as a biomarker for breast cancer.Clin. Chim. Acta. 2010; 411: 1869-1874Crossref PubMed Scopus (232) Google Scholar, 13Galli C. Basso D. Plebani M. CA 19–9: handle with care.Clin. Chem. Lab. Med. 2013; 51: 1369-1383Crossref PubMed Scopus (63) Google Scholar). Because of the potential pitfalls in the clinical use of the glycan biomarkers, the need to analyze cancer-specific changes in glycan structures and to use them as cancer biomarkers is thus increasing and this has to be met to ultimately treat cancer timely and efficiently (14Kang J.G. Ko J.H. Kim Y.S. Pros and cons of using aberrant glycosylation as companion biomarkers for therapeutics in cancer.BMB Rep. 2011; 44: 765-771Crossref PubMed Scopus (17) Google Scholar). However, the development of an aberrant glycosylation-based cancer biomarker has been hampered by the absence of an analytical tool to trace the protein glycan alterations in a sensitive and quantitative manner. Blood is the most preferred source for biomarker-based diagnostic tests, but it is often difficult to measure proteins of medium- or low-abundance levels at which most interesting biomarkers are believed to exist (15Rifai N. Gillette M.A. Carr S.A. Protein biomarker discovery and validation: the long and uncertain path to clinical utility.Nat. Biotechnol. 2006; 24: 971-983Crossref PubMed Scopus (1367) Google Scholar, 16Anderson N.L. Anderson N.G. The human plasma proteome: history, character, and diagnostic prospects.Mol. Cell. Proteomics. 2002; 1: 845-867Abstract Full Text Full Text PDF PubMed Scopus (3551) Google Scholar). Given the high complexity and high dynamic range of proteins in blood, it is far more difficult to simultaneously measure a glycoform of multiple glycoproteins with significantly different levels in blood. A possible modality combining immunoprecipitation and a lectin blot analysis is far from meeting an analytical sensitivity required for blood tests. Moreover, antibody-based analyses, for example, the lectin-based enzyme-linked immunosorbent assay (lectin-ELISA) is not feasible for such purposes because of the presence of a pair of N-glycans on immunoglobulin G (17Hong Q. Lebrilla C.B. Miyamoto S. Ruhaak L.R. Absolute quantitation of immunoglobulin G and its glycoforms using multiple reaction monitoring.Anal. Chem. 2013; 85: 8585-8593Crossref PubMed Scopus (98) Google Scholar). A methodological breakthrough is thus needed to advance aberrant glycosylation-based cancer biomarker development in a clinical setting as well as to delineate the glycan structure-function relationship in basic research. Herein, we report a novel quantitative method by which a specific glycoform can be quantitatively measured by using a DNA-tagged antibody and lectin chromatography. With this approach, we validated fucoform biomarkers through a case-control study in a sensitive and multiplexing manner. With the validation results as a basis, we suggested a fuco-index (If) obtained from triple biomarkers that can differentiate non-HCC and HCC more clearly than when AFP or AFP-L3 is used alone. To our best knowledge, it is the first report that aberrant glycan codes are utilized as an in vitro diagnostic multivariate index assay (IVDMIA) in the development of cancer biomarkers. Hep3B cells were transfected with plasmid vector constructs carrying the human FUT8 gene or shRNA for the gene using an electroporator (Neon™, Invitrogen) according to the manufacturer's instructions. The DNA sequences for shRNA are listed in supplemental Table S1. The stable clone was screened and established by RT-PCR and lectin blot analyses. Each clone was maintained at 37 °C in an RPMI 1640 medium containing 10% fetal bovine serum and 1% antibiotic solution (penicillin/streptomycin, WelGene Inc, Daegu, South Korea), supplied with 5% CO2. Mouse embryonic fibroblast (MEF) and the Fut8−/− mutant cell line were kindly donated by the Taniguchi group at the RIKEN. The human AFP gene was stably transfected into both the parental and knock-out cells as aforementioned. Serum samples were obtained from various participants at the Pusan National University Yangsan Hospital, Korea with agreement to participate. The acquisition of samples was reviewed and approved by the Institutional Review Board (KRIBB-IRB-20110808-04). Subjects with HCV, alcohol abuse, or nonalcoholic steatohepatitis were excluded to minimize the associated confounding factors. Blood specimens of HCC patients were obtained pre-operatively and before administration of any drug. Blood samples were taken into Clot-activator-treated polypropylene vacuum capillary tubes (Vacuplus®, Medigene, Seoul, Korea) and then incubated at room temperature for 30 min. After centrifugation at 1100 × g for 10 min to remove fibrinogen aggregates and other cellular components, supernatants were collected, aliquoted, and frozen at −80 °C until use. Once thawed, blood samples were consumed, and no remnants were used for subsequent experiments. Proteins were resolved on 10-12% SDS-PAGE gels and electrically transferred onto PVDF membranes (Immobilon-P, Millipore). The membranes were blocked in TBS buffer containing 0.05% (v/v) Tween 20 plus 5% (w/v) skim milk for Western blot or 3% (w/v) BSA for lectin blot. Anti-A2M (Santa Cruz Biotechnology, Santa Cruz, CA) and anti-AFP (Abcam, Cambridge, UK) antibodies were used as primary antibody, and biotin-conjugated Aleuria aurantia lectin (AAL) and Lens culinaris lectin (LCA) were used to probe glycan moieties. After incubation with HRP-labeled secondary antibodies (Cell Signaling Technology Inc., Danvers, MA) or HRP-avidin conjugates (Vector Laboratories, Inc., Burlingame, CA), membranes were allowed to react with ECLTM Western blotting detection reagents (GE Healthcare, Hertfordshire, UK) and exposed to an x-ray film for 1–2 min. Total RNA was extracted from cells using an RNeasy Miniprep kit (Qiagen, Hilden, Germany), quantified, and used for synthesis of cDNA, which was used as templates for RT-PCR. cDNA was synthesized from the total RNA using a reverse-transcription kit (Nanohelix, Daejeon, South Korea), and 2 μg of cDNA was used for PCR. A gene-specific primer pair with sequences of 5′-TGATGCCTCTGCAAACTTCC (forward) and 5′-CGTCCTTCCCAATTTCCTGT (reverse) was used to measure the mRNA level of the human FUT8 gene. Double-stranded oligonucleotides were prepared by incubating 10 μl each of biotinylated 70-mer oligonucleotides (100 pm) with 40-mer oligonucleotides (100 pm) with unique sequences and a hydrazine-modified 30-mer oligonucleotide (100 pm) containing T7 promoter sequences in a thermocycler (MyCycler, Bio-Rad). Ten micrograms of antibodies was treated with 1 m NaIO4 in PBS buffer under complete darkness. The oxidized antibodies were incubated with the double-stranded oligonucleotides at room temperature for 12 h and the formed aldehyde-hydrazine linkage was stabilized by treatment with 5 mm NaBH4 at room temperature for 1 h. The DNA-tagged antibody fraction was prepared and preserved following desalting on a Zeba desalting column with a molecular weight cut-off of 5 kDa (Thermo Scientific). Secreted proteome was retrieved from the conditioned medium of Hep3B or HepG2 cells following filter-concentration using a Centricon filter (Millipore) with a molecular weight cut-off of 10kDa. For detection of targets in sera, serum samples were precleared with Protein G-coupled Sepharose beads (Millipore) at 4 °C, and the supernatants were retrieved for immunoprecipitation. One hundred microliters of the conditioned media or the precleared, PBS-diluted serum was incubated with 5 μg of an oligonucleotide-tagged anti-alpha-2-macroglubulin (Santa Cruz Biotechnology), anti-AFP (Abcam), and anti-hemopexin (Abcam) monoclonal antibodies for 2 h at 4 °C. Antibody-antigen complex was separated on various lectin columns. Beads were extensively washed with 1 ml of PBS at 4 °C. The bound antibody-antigen complexes for lectin chromatography were either eluted or assayed as a bound form on lectin beads. The beads-bound glycoforms were directly used for T7 polymerization reactions and lectin/Western blot analyses following extensive washing. RNA transcripts were synthesized using a T7 RNA polymerase kit (Promega, Madison, WI) according to the manufacturer's instruction. Briefly, DNA-tagged antibodies alone or antigen-bound antibody conjugates were incubated at 37 °C in the presence of 20 units of T7 polymerase in 50 μl of reaction buffer containing 40 mm Tris (pH 7.9), 6 mm MgCl2, 2 mm spermidine, 10 mm DTT, 10 mm NaCl, and 0.25 mm UTP-depleted NTPs plus either 0.25 mm Cyanine3- or Cyanine5-UTP (Enzo Life Sciences, Inc., Farmingdale, NY). Amine-coated slides (Luminano) were incubated in 1% (w/v) glutaraldehyde (Sigma-Aldrich, St. Louis, MO) in PBS for 2 h, washed with distilled water, and air-dried. Oligonucleotide probes as listed in supplemental Table S1 were dissolved at 50 pm in 3×saline sodium citrate (SSC) buffer containing 0.05% SDS and spotted on a slide using a microarray machine (Proteogen, Seoul, Korea). After spotting, the slides were incubated in a humidity chamber (≥ 60% humidity) at room temperature for 12 h, blocked by 2 mg/ml NaBH4 for 5 min, washed by 0.2% SDS for 5 min, and air-dried. The RNA transcripts were diluted with a hybridization solution (5×SSC and 0.5% SDS) and hybridized with probes on a microarray chip with a coverslip at 55 °C for 12 h. The coverslips were removed and then the slides were washed with 2×SSC and 0.1% SDS for 10min and air-dried. Slides were scanned in a fluorescence scanner (GenePix 4200 professional, Axon, Molecular Devices, USA). The fluorescence intensity was measured using GenePix Pro 6.1 software (Axon). ELISA kits for AFP and alpha-2-macroglobulin (A2M) were purchased from Abcam and hemopexin (HPX) from USCN. All assays were performed basically according to the manufacturers' recommendations. Sera were diluted 2-, 50-, and 200- fold prior to assay with PBS for AFP, HPX, and A2M, respectively. All statistics were conducted with MedCalc software (Mariakerke, MedCalc Software bvba, Belgium) including the derivation of the receiver operating characteristic (ROC) curve and the area under the ROC (AUROC) values. The diagnostic performances were compared by constructing the ROC curve, from which the AUROC values and sensitivity and specificity that best discriminate between non-HCC and HCC were derived. p < 0.05 was considered statistically significant. Many lines of evidence indicate that a subtle change in protein–glycan structures is associated with biological functions and various diseases (1Christiansen M.N. Chik J. Lee L. Anugraham M. Abrahams J.L. Packer N.H. Cell surface protein glycosylation in cancer.Proteomics. 2014; 14: 525-546Crossref PubMed Scopus (370) Google Scholar, 14Kang J.G. Ko J.H. Kim Y.S. Pros and cons of using aberrant glycosylation as companion biomarkers for therapeutics in cancer.BMB Rep. 2011; 44: 765-771Crossref PubMed Scopus (17) Google Scholar, 18Kościelak J. Diseases of aberrant glycosylation.Acta Biochim. Pol. 1995; 42: 1-10Crossref PubMed Scopus (15) Google Scholar). Although protein glycan synthesis is a nontemplate process and thus the glycan structures are heterogeneous, it is still necessary to identify and quantify specific glycoforms. Because an aberrant glycoform can be developed as a disease-specific biomarker, a multiplexing technique with high analytical sensitivity and specificity is increasingly demanded in the biomarker development pipeline. These unmet needs prompted us to develop an analytical method to quantify biomarkers of interest with a specific glycan structure. For this, immunoglobulin G-type antibodies were tagged with a double-stranded oligonucleotide with an identifiable DNA sequence at the core mannosyl residues of N-glycan in antibody (Fig. 1A). The IgG-type antibodies have a pair of N-glycans at the CH2 domain in the constant region of the heavy chain. The mannosyl dihydroxyl groups are oxidized by NaIO4 treatment, thereby generating paired aldehyde groups following ring opening (19Zhang H. Li X.J. Martin D.B. Aebersold R. Identification and quantification of N-linked glycoproteins using hydrazide chemistry, stable isotope labeling, and mass spectrometry.Nat. Biotechnol. 2003; 21: 660-666Crossref PubMed Scopus (1275) Google Scholar), and these groups participate in covalent bonding with a hydrazide functional group at the 5′-end of an oligonucleotide (Fig. 2B). The double-stranded oligonucleotides are comprised of a pair of nucleotides, one of which has a T7 promoter and an identifiable unique mRNA coding sequence. The unique coding sequence serves as a barcode and is used to specify an antibody and the bound antigen. The overall procedure for quantification of targets is depicted in Fig. 1C; the DNA-tagged antibody is admixed with biofluidic sources of biomarkers, usually blood or conditioned media, which are pretreated with RNase. A DNA-antibody-antigen complex is trapped on a lectin affinity column through interactions between glycans on an antigen and a lectin. The DNA-tagged antibody is co-eluted with an antigen during the elution step, and the amount of eluted antibody is proportional to that of a lectin-bound glycoform of an antigen. Accordingly, a specific glycoform of an antigen can be quantified from the co-eluted, DNA-bound antibody, which is used as a template for T7 transcription to produce fluorescent transcripts. The transcripts are specifically identified and quantified on a DNA microarray chip.Fig. 2Attachment of a DNA tag to the N-glycan of an antibody and the resultant erasure of lectin-bound glycan moieites. A, N-glycans of an anti-AFP antibody was oxidized with 1 m NaIO4 to yield aldehyde functional groups, which were reacted with either hydrazine biotin or 5′-hydrazine double-stranded oligonucleotide labeled with biotin. The formed bond was stabilized in the presence of 5 mm NaBH4. Tagging was confirmed by an immunoblot analysis using HRP-conjugated streptavidin. B The erasure of lectin-bound glycan moieties was tested by lectin-affinity chromatography. The DNA-tagged antibody was run on a Con-A column with a minimal flow rate and the presence of the DNA-tagged antibody was tested by ab immunoblot analysis. The DNA-tagged antibody was found only in the pass-through (P) and washing (W) fractions, but not in the elution (E) fractions. C, The DNA-tagged anti-AFP antibody was incubated with AFP to form an antigen-antibody complex, and the complex was again run on a Con-A column under the same protocol as that in B. The lectin blot analysis indicates the binding of the complex on a Con-A column. D, The binding of either intact or DNA-tagged antibody with various lectins was tested including Con-A, LCA, AAL, DSA, l-PHA, E-PHA, SSA, and E-selectin. The lectin beads were precipitated by centrifugation and the lectin-bound antibodies were assessed by an immunoblot analysis. Relative binding in the y axis indicates the ratio of lectin-bound antibodies to the starting amounts after three rounds of washing with PBS. Values are means of three independent experiments with standard error.View Large Image Figure ViewerDownload Hi-res image Download (PPT) It was assumed that this method would be compatible with multiplexed and highly sensitive quantification of targeted glycoforms. Multiplexity becomes possible by using probe cocktails containing a mixture of DNA-tagged antibodies. As described in Fig. 1, the DNA-antibody-antigen complex can be captured through antigen-attached glycan-lectin interactions. An analytical validation of our method began with tagging an anti-AFP monoclonal antibody with oligonucleotides labeled with biotin. The DNA-tagging procedure consists of a consecutive step comprising oxidation with NaIO4, covalent bonding between aldehyde-hydrazine groups, and stabilization with NaBH4. The aldehyde-hydrazide reaction was found to occur spontaneously by simple incubation as assessed by incubation with biotin hydrazide. The Western blot analysis indicates that, despite the marginal presence of a heavy chain with two molar nucleotides bound, the heavy chain of an antibody was linked with oligonucleotides with a 1:1 stoichiometry (Fig. 2A), which may be explained by competition between mannose-oligonucleotide binding. It appears that the attachment of one oligonucleotide molecule confers significant hindrance to the attachment of another. For a targeted glycoform of a biomarker to be quantified by RNA transcripts that are produced from the antibody-tagged DNA as a template, it is critical that any free DNA-antibody conjugates, that is, an antigen-unbound form, be separated from the DNA-antibody-antigen complex. To this end, a binding test was performed where the DNA-tagged antibody was run on a concanavalin-A (Con-A) column and the presence of the DNA-tagged antibody was investigated in all fractions. As seen in Fig. 2B, antibody was not detected in the elution fractions, indicating that the DNA-tagged antibody can be removed from Con-A beads by simple washing. It is assumed that the lectin-glycan interactions were disturbed by alterations in the N-glycan structure of the antibody during the oxidation and attachment of bulky oligonucleotides. In contrast, the AFP-antibody complex was captured on a Con-A column (Fig. 2C). These results indicate that glycans of AFP, but not of the antibody, participated in the lectin-affinity chromatography, thereby supporting the notion that a specific glycoprotein can be quantified without the interference of free DNA-tagged antibody. Next, we examined which lectin-glycan interactions were disturbed by the DNA-tagging procedure, because it is necessary to define the types of lectins that are applicable without background during assays. To this end, five lots of commercially available monoclonal antibodies were tagged with oligonucleotides and their lectin-binding properties were monitored (Fig. 2D). It was found that lectins recognizing the glycan moiety including the core N-glycan structure were applicable to our strategy, including Con-A, LCA, L4-PHA, and E4-PHA. Some lots of antibodies still carry affinity with AAL and DSA. None of the antibodies were affected in the interaction with SSA. It was interpreted that there exists a fraction of antibodies that carry a certain glycoform that is not affected by DNA attachment. However, when precleared with the corresponding lectins, the undesirable glycoforms of the antibody could be completely removed, and thus a few rounds of a preclearing step allowed most antibodies to be applicable to our purposes (supplemental Fig. S1), except for sialic acid-binding lectins. Because most commercially available and home-made antibodies are not quality-controlled for glycan structures, the preclearing step should be accompanied for analytical robustness and reproducibility. Double-stranded oligonucleotide attached to an antibody has a T7-promoter sequence that enables production of transcripts by a RNA-polymerization reaction. When incubated with a DNA-tagged antibody, T7 RNA-polymerase synthesized transcripts in the presence of NTPs (Fig. 3A). A detectable amount of molecules were observed in the absence of NTPs; this turned out to be a fraction of oligonucleotides that had been attached to antibodies and leaked during incubation. Treatment with DNase could remove the DNA molecules and thus any possible interference in the analysis on a DNA microarray chip (Fig. 3B). The produced molecule was confirmed to be RNA transcripts by RNase treatment. The production of transcripts was assessed with reaction time. It increased with time and became saturated after 30 min of incubation (Fig. 3C). The T7 polymerase reaction time was fixed at 30 min for the ensuing experiments. To test the feasibility of our method, stable cell lines of Hep3B with overexpression or down-regulation of fucosyltransferase 8 (hFUT8) gene were generated by either forcible expression of hFUT8 gene or a short hairpin RNA for hFUT8. Data obtained from the real-time PCR indicated the establishment of two stable cell lines (Fig. 4A). For a more quantitative analysis, quantitative real-time PCR was performed. From the analysis, the relative expression ratios for up- and down-regulation cells were 8.39 ± 1.70 and 0.47 ± 0.12, respectively, when compared with the expression level of parental cells (Fig. 4B). Total secreted proteins were prepared from conditioned media of stably transfected and parental cells, and each protein preparation was differently diluted so that the input of A2M was equal as assessed by an immunoblot analysis (Fig. 4C). An equal amount of A2M was subjected to immunoprecipitation and the immunoprecipitated proteins were blotted against anti-A2M and AAL. Even though A2M was detectable by an immunoblot analysis, the fucoform of A2M could not be detected in these conditions. In contrast, our method using DNA-tagged anti-A2M antibody could differentiate the fucosylation status under the same conditions as those for the immunoblot, and the measured levels were proportional to the values obtained from the quantitative RT-PCR (Fig. 4D). In addition to high sensitivity, specificity was also obtained in the assay, where the RNA transcript of interest was detected on an intended spot only. Despite the evidence presented in Fig. 4, we reasoned that the feasibility of our method should be tested in a more demonstrative setting. Fig. 4 shows that the fucosylation level of A2M could be differentiated between the parental and shFUT8 cells. A characteristic of protein glycosylation is that the level of a specific glycoform may differ depending on the availability of substrate, cell conditions, etc. As shown in Fig. 5A, the glycan level is not likely to be uniformly altered by shFUT8, and there appears to be a subset of proteins that show an equivalent or even higher level of fucosylation in the FUT8 knock-down cells, compared with the parental cells. Because these nonanalytical variations need to" @default.
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• W2108185963 title "Semi-quantitative Measurement of a Specific Glycoform Using a DNA-tagged Antibody and Lectin Affinity Chromatography for Glyco-biomarker Development *" @default.
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