Establishment of a 1, 4, 7, 10-tetraazacyclododecane-1,4,7,10-tetraacetic acid mono-N-hydroxysuccinimide ester (DOTA-NHS-ester) based lectin microarray for efficiently detecting serum glycans in gastric cancers.

Development of cancers is involved in changes of a variety of glycans. Lectin microarray is one of the most powerful methodologies for investigation of glycan alterations in biological samples with its advantages of high through-put, selectivity and specificity of the technique. However, utilization of lectin microarrays available commercially keeps of great challenges. In this study, we took use of the molecular self-assembled monolayer technique to modify a gold surface with the reagent 1,4,7,10-tetraazacyclododecane- 1,4,7,10-tetraacetic acid mono-N-hydroxysuccinimide ester (DOTA-NHS-ester) in combination with 16-amino-1-hexadecanethiol hydrochloride. Cross-linking effect of DOTA-NHS-ester is brought about via activating three -OH ends to three terminals of succinylimidines, making selective binding of the terminal amino groups in proteins possible. We immobilized ten commercial lectins on the platform and measured changes of serum lectin-matched glycans in patients with gastric cancer. The results demonstrated that this biochip modification platform conferred impressive chemical surface stabilization, sensitivity and geometric images. We observed that all the serum glycans tested in the patients were significantly higher than those in the controls (P < 0.05). The biochip would provide a versatile platform for investigation of potential glycan biomarkers in making tumor diagnosis decision and analyzing escape of tumors from immunity.

A simplified direct on-chip forward or reverse immunoassay for evaluating protein-protein interactions in the serum.

BACKGROUND: The identification of protein-protein interactions is a great challenge. In this study, we fabricated a gold surface-modified biochip with activated sophorolipids (SLs) in combination with 16-amino-1-hexadecanethiol hydrochloride to detect serum proteins. MAIN METHODS AND MAJOR RESULTS: The on-chip immunoassay reported here included a forward assay, in which a ligand is immobilized on the biochip surface and allowed to interact with its free specific receptor in liquid phase, and a reverse assay, in which a receptor is loaded on the biochip surface and combined with its free specific ligand in solution. The specificity of the molecular interactions on the biochip was evaluated using immunological blocking assays and chemiluminescent immunoassays (CLIA). Hemophagocytic lymphohistiocytosis (HLH) serum was used to test the potential utilization of the biochip. Reverse receptor CD25-based interleukin (IL)-2 and forward ligand IL-2-based CD25 assays revealed that the limit of detection of the target proteins was as low as 156 and 78 pg/ml, respectively. Using receptor- or ligand-based platforms, we found that the positive rates of free IL-2 and soluble CD25 (sCD25) monomers in the sera of HLH patients were 14.3% and 71.4%, respectively. In addition, the biochip showed good compatibility with CLIA for the measurement of sCD25 (r = 0.77, p < 0.01). CONCLUSIONS AND IMPLICATIONS: Biochip platforms, such as on-chip immunoprecipitation (IP), can be used to evaluate the interactions between proteins, ligands, and receptors, or enzymes and substrates in serum.

Do combined assays of serum AFP, AFP-L3, DCP, GP73, and DKK-1 efficiently improve the clinical values of biomarkers in decision-making for hepatocellular carcinoma? A meta-analysis.

Objectives: Serum biomarkers are valuable for clinical decision-making for patients with hepatocellular carcinoma (HCC), among which the most promising are AFP, AFP-L3, DCP, DKK-1, and GP73; however, the efficacy of using combined biomarkers remains controversial. This meta-analysis provides insights regarding this topic.Methods: After systematically surveying the literature available in PubMed, Embase, and Cochrane Library, we identified 28 qualified articles published since January 2015. A random-effects model was used to assess pooled sensitivity, specificity, positive and negative likelihood ratios (PLRs and NLPs), and diagnostic odds ratio (DOR).Results: Values under the summary receiver operating characteristic (SROC) curve varied in different panels of the five biomarkers. Importantly, the sum of sensitivity and specificity of AFP+GP73 was 1.76 (P= 0.0001), which was the best among all the panels. The sum of the triple biomarker panel of AFP, AFP-L3, and DCP was larger (1.64, P= 0.0001) than those of any double biomarker panels of AFP, AFP-L3, and DCP.Conclusions: To the best of our knowledge, this is the first meta-analysis to focus solely on combination assays of multiple biomarkers in HCC. The combined assay of AFP and GP73 conferred the best outcome among all panels. The triple combined panel of AFP, AFP-L3, and DCP showed higher diagnostic potential than individual random double combinations of the three biomarkers. Multiple-biomarker combined assays will be clinically important for decision-making processes for HCC.

A simple lectin-based biochip might display the potential clinical value of glycomics in patients with spontaneous intracerebral hemorrhage.

BACKGROUND: Intracerebral hemorrhage (ICH) is a cerebrovascular disease with extremely high disability and mortality rates. Glycans play critical roles in biological processes. However, whether glycans can serve as potential biomarkers for determining clinical diagnosis and prognosis in ICH remains determined. METHODS: In this study, we established a lectin-biochip to measure serum glycans levels in ICH patients (n=48) and healthy controls (n=16). An enzyme-linked immunosorbent assay (ELISA) was carried out to determine serum levels of IL-10 and TNF-α in the patients. Correlation analyses of the serum glycan and cytokine levels and the clinicopathological parameters of patients were performed. RESULTS: The biochip-based data revealed that the serum levels of α-Man/α-Glc (ConA), Galß3GalNAc (PNA), GalNAc (VVA), Fucα6GlcNAc (AAL), α-Fuc (LTL), and Galß3GalNAc-Ser/Thr (AIL) significantly increased in the super-acute phase of ICH in comparison with healthy controls. Clinicopathological analysis indicated the serum levels of ConA, VVA, and LTL had significant associations with the National Institute of Health Stroke Scale (NIHSS), and serum VVA levels had a significant association with the Mini-Mental State Examination (MMSE) at day 90 after ICH. Correlation coefficient analysis revealed significant correlations between TNF-α and ConA (P<0.001) as well as between IL-10 and ConA (P<0.001), PNA (P=0.02), VVA (P<0.001), and MAL (P=0.04), respectively. CONCLUSIONS: We established a proof-of-concept platform for detecting serum glycomics and highlighted their potential value in diagnosing and predicting ICH patients' outcomes.

Expression of Narcissus pseudonarcissus lectin and mannose receptor positive macrophages predict progression and prognosis of patients with gastric cancer.

BACKGROUND: Mannose receptor (MR) is an immune adhesion molecule and is mainly expressed in macrophages and nonmature dendritic cells. The ligand mannose, one of the natural ligands of MR, is a monosaccharide, which is localized in the envelope or cytoplasm of macrophages. The aim of this study was to investigate expression of MR and its ligand mannose in tumor tissues of primary advanced gastric cancer and to evaluate the predictive and prognostic value of the positive cells in gastric cancer patients. METHODS: Histochemical staining for Narcissus pseudonarcissus lectin (NPL) and immunohistochemical envision two-step assay for MR were used to detect expression of NPL and MR in primary advanced gastric adenocarcinoma tissues. Adjacent non-cancerous gastric tissues of the patients were used as controls. Relationship of NPL and MR expression in the tumor tissues with clinicopathological features and survival time of the gastric cancer patients were analyzed. RESULTS: Numbers of NPL+ and MR+ macrophages in stromal tissues of gastric cancer were significantly higher than those in the adjacent non-cancerous gastric tissues (P=0.006; P<0.001). NPL expression in the primary tumor tissues was significantly more dominant than that in the adjacent non-cancerous gastric tissues (P=0.003). Expression of both the molecules in macrophages in tumor tissues was negatively correlated (r=-0.363, P=0.009). TNM stage of the patients was closely correlated to number of MR+ macrophages and NPL expression in the stromal tissues of gastric cancer (P=0.009 and P=0.020). Kaplan-Meier survival model data showed that the patients with low counting of NPL+ macrophages and high counting of MR+ macrophages significantly led to worse disease progression and poorer prognosis (P=0.008). Cox regression analysis further demonstrated that high expression of MR+ macrophages was an independent predictor of poor prognosis in patients with gastric cancer (P=0.033). CONCLUSIONS: Occurrence of mannose and MR in tumor tissues of gastric cancer might be prognostic factors for estimating risk of gastric cancer patients.

Development of a dendrimer PAMAM­based gold biochip for rapid and sensitive detection of endogenous IFN­Î³ and anti­IFN­Î³ IgG in patients with hemophagocytic lymphohistiocytosis.

Hemophagocytic lymphohistiocytosis (HLH) is a rare but severe disease characterized by immune hyperactivation and cytokine storm. Given the high mortality rate of HLH, there is a need for more effective diagnostic tools and treatments. The present study developed a dendrimer­based protein biochip for rapid, sensitive and simultaneous detection of serum interferon (IFN)­Î³ and endogenous anti­IFN­Î³ antibody (Ab) in patients with HLH. A gold biochip was modified with 1, 4­phenylene diisothiocyanate (PDITC), polyamidoamine (PAMAM) or PDITC­activated PAMAM. The optimal immobilization concentration for Ab capture and the reaction concentration for detecting Ab on the PDITC­activated PAMAM­modified biochip were 6.25 and 3.12 µg/ml, respectively; the limit of detection of IFN­Î³ protein was 50 pg/ml. The efficiency of the protein­probed biochip in detecting IFN­Î³ and anti­IFN­Î³ Ab in serum samples from 77 patients with HLH was evaluated; the positive rates for IFN­Î³ and anti­IFN­Î³ IgG Ab were 63.6% (49/77) and 61.0% (47/77), respectively. The present results demonstrated that the PDITC­activated PAMAM­modified biochip might be a sensitive tool for the specific detection of IFN­Î³ and anti­IFN­Î³ Ab in serum, and might have clinical applicability for the diagnosis of HLH.

Establishment of a protein biochip to detect serum IgG antibodies against IL-2 and soluble CD25 in hemophagocytic lymphohistiocytosis.

BACKGROUND: Interleukin-2 (IL-2) and soluble CD25 (sCD25) are among the most important cytokines and diagnostic biomarkers in hemophagocytic lymphohistiocytosis (HLH). Detecting serum level of IL-2 and sCD25 is valuable for making clinical diagnosis and treatment decision in HLH. METHODS: Since tests showing serum IgG antibody against IL-2 or sCD25 have never been carried out, a new protein biochip, which was modified with cysteine and activated sophorolipid (Cys-SL), was developed. RESULTS: Limits of detection on the biochip were 78 pg/ml for IL-2 and 39 pg/ml for sCD25, respectively. The data showed that on-chip seroimmunological responses to IL-2 and sCD25 proteins were 20.8% and 83.1% and the seroprevalence of IL-2 and sCD25 IgG antibodies were 45.5% and 57.2%, respectively. Data collection for the seroprevalence of serum antigen-antibody complex of sCD25 was 68.8%. The new biochip model shared similar sensitivity and specificity to chemiluminescent immunoassay (CLIA) in its measuring capacity of serum sCD25. CONCLUSIONS: We addressed and confirmed the involvement of serum IgG antibodies against IL-2 and sCD25 as well as Ag-Ab complex of sCD25 in HLH patients. Therefore, this biochip platform would offer a new technological substitution for clinical serological diagnosis of HLH.