CA125-Tn ELISA assay improves specificity of pre-operative diagnosis of ovarian cancer among patients with elevated serum CA125 levels.

BACKGROUND: CA125 is the most widely used serum marker for preoperative diagnosis of ovarian cancer. However, CA125 elevation is not specific to ovarian cancer. More than 60% of patients who have elevated CA125 levels do not have ovarian cancer. To overcome the low specificity of CA125, we identified a CA125 glycoform that was specifically elevated in ovarian cancer and that may help in the further triage of patients with elevated serum CA125 levels. METHODS: We used antibody-lectin ELISA to detect various CA125 glycoforms. Among 21 lectins tested, VVA, a plant lectin that preferentially binds Tn antigen, showed significantly stronger binding with ovarian cancer-derived CA125 than benign condition-derived CA125. CA125-Tn levels were tested among patients with elevated CA125 levels (n=328, including 68 ovarian cancer, 15 ovarian borderline tumors, and 245 benign conditions). The efficacy of CA125-Tn in diagnosing ovarian cancer was evaluated using ROC analysis. RESULTS: Medians and 25th to 75th quartiles of CA125-Tn levels were 0.31 (0.18-0.65) in ovarian cancer, 0.07 (0.02-0.12) in ovarian borderline tumor, and 0.07 (0.01-0.12) in benign conditions. AUC of the ROC curve was 0.890 (95% CI: 0.845, 0.935) for CA125-Tn to discriminate ovarian cancer cases from nonmalignant cases (borderline tumors and benign conditions). Its performance was even better among patients older than 45 y (AUC: 0.905, 95% CI: 0.841, 0.969). Specificity was improved from 35.1% for CA125 to 75.7% for CA125-Tn among patients older than 45 y when sensitivity was fixed at 90%. CONCLUSIONS: CA125-Tn ELISA assay can improve specificity of the preoperative diagnosis of ovarian cancer and serve as a further triage strategy for patients with elevated CA125 levels.

Response to immune complex vaccine in chronic hepatitis B patients is associated with lower baseline level of serum IgG galactosylation.

The composition of glycan in immunoglobulin G (IgG) has shown to affect various diseases and can be regulated by drugs and preventive vaccination. A hepatitis B surface antigen (HBsAg)-hepatitis B immunoglobulin (HBIG) immune complex (YIC) therapeutic vaccine for chronic hepatitis B (CHB) patients has undergone clinical trials. To explore for markers of CHB, which could be associated with responsiveness to YIC therapeutic vaccine, serum IgG glycosylation in CHB patients was analyzed.Kinetic changes of serum galactosylated IgG in 53 hepatitis Be antigen (HBeAg)-positive CHB patients treated with YIC were monitored by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) analysis. Whole blood cytokines were assayed by cytokine binding assay kits. All samples were back assayed before treatment, during therapy and follow-up for 6 months from a previous completed clinical trial.During YIC treatment, 26 patients with lower IgG galactosylation level at baseline [galactosylation level (Gal-ratio) = -0.29, 0.18 (mean, SD)] showed sustained increase of serum galactosylated IgG, and responded to YIC treatment by HBeAg seroconversion. While those who did not respond to YIC treatment [Gal-ratio = -0.40, 0.15 (mean, SD)] failed to show similar changes. Furthermore, this kinetic increase of galactosylated IgG correlated with marked up-regulated IL-2 level, confirming that effective cellular immune responses have participated in responsiveness.For HBeAg-positive CHB patients lower serum IgG galactosylation level may serve as an indicator for selecting a suitable subpopulation of candidates for YIC therapeutic vaccination.

Carbon nanotubes (2,5-dihydroxybenzoyl hydrazine) derivative as pH adjustable enriching reagent and matrix for MALDI analysis of trace peptides.

A functionalized carbon nanotube (CNT), CNT 2,5-dihydroxybenzoyl hydrazine derivative, was synthesized and used as both pH adjustable enriching reagent and matrix in matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis of trace peptides. The derivative reagent, 2,5-dihydroxybenzoyl hydrazine, introduced phenolic hydroxyl and phenyl groups to the surface of the CNT. The former group can provide adjustable surface charge and a source of protons for chemical ionization, and the latter helps to keep strong ultraviolet absorption for enhancing pulsed laser desorption and ionization. It was found that the functionalized CNT was less twisted in a basic condition (pH 10.5), which afforded an increased surface area to volume ratio for adsorption towards trace peptides. However, functionalized CNT becomes deposited in an acidic condition (pH 5) and can be isolated readily from the sample solutions once the nanoparticles have trapped the target analytes, thus providing a novel and convenient alternative method for quick isolation. Compared with the previously reported method on enriching analytes using the pristine CNT, it is observed that the detection limit for analytes can be greatly improved due to enhancing adsorption capacity of the functionalized CNT. Moreover, peptide mixture at concentration as low as 0.01 pg/microL still can be detected after enrichment mediated by the functionalized CNT, while it is difficult to be detected without enrichment at concentration 0.1 pg/microL using alpha-cyano-4-hydroxycinnamic acid (CHCA) as matrix. Therefore, high efficiency of adsorption and enrichment towards trace peptides can be achieved by adjusting pH value of the functionalized CNT dispersion.

Immobilized carbon nanotubes as matrix for MALDI-TOF-MS analysis: applications to neutral small carbohydrates.

In this work, we reported on the advantages of immobilized carbon nanotubes as a novel MALDI-matrix. Recently, carbon nanotubes have been reported to be an effective MALDI matrix for small molecules (Anal. Chem.2003, 75, 6191), as it can eliminate the interfering matrix peaks as well as form a web morphology to fully disperse the analyte and allow strong ultraviolet absorption for enhanced pulsed laser desorption and ionization. In our study, to overcome the problem that the carbon nanotube matrix may fly off from the target, a type of polyurethane adhesive, NIPPOLAN-DC-205, is introduced to immobilize carbon nanotubes on the target, which enables widespread application of carbon nanotubes as matrix for MALDI-MS analysis. At the same time, the properties of the carbon nanotubes as an efficient matrix remained after immobilization. The presence of NIPPOLAN-DC-205 increases the time for analysis at a particular desorption spot by minimizing the time-consuming search for "hot spots" and facilitating experiments such as post source decay (PSD) which need longer-lasting signals. Moreover, NIPPOLAN-DC-205 produces no interference peaks and can easily be cleaned with acetone. Fast evaporation technology may be used to enhance signal reproducibility in MALDI analysis using carbon nanotubes as matrix. Consequently, the applicability of the carbon nanotube as matrix for matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis of low molecular mass analytes is highly improved. The feasibility of the method employing polyurethane is demonstrated by comparison of the results produced from the carbon nanotube matrix with and without immobilization. In addition, neutral small carbohydrates, which are difficult to be ionized normally, can be cationized with high efficiency by MALDI-TOF-MS using the immobilized carbon nanotube matrix. The method was further applied to analyze peptides and detect urine glucose successfully.

Oxidized carbon nanotubes as matrix for matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysis of biomolecules.

Oxidized carbon nanotubes (CNTs), which can form a stable homogeneous suspension in water close to a solution phase, were synthesized and used for matrix-assisted desorption/ionization mass spectrometric (MALDI-MS) analysis of biomolecules. Infrared (IR) spectra, transmission electron microscopy (TEM) and particle size analysis were used for the characterization of the oxidized CNTs. The results indicate that the physical structure of the CNTs was not damaged, but carboxylate groups were introduced onto the surface of the CNTs. In addition, impurities including amorphous carbon, which is one of the main reasons for ion source contamination, were destroyed by the oxidization. The carboxyl groups on the oxidized surface of the CNTs can not only provide an additional proton source, but can also increase the surface polarity and solubility of the CNTs, making it easier to manipulate which is important for MALDI analysis of some biomolecules, especially larger peptides and proteins. The oxidized CNTs were successfully applied to the analysis of neutral oligosaccharides, peptides, and insulin, and thus promise to be an efficient matrix for MALDI-MS analysis of biomolecules.