A search for novel cancer/testis antigens in lung cancer identifies VCX/Y genes, expanding the repertoire of potential immunotherapeutic targets.

Cancer/testis (CT) antigens are potential immunotherapeutic targets in cancer. However, the expression of particular antigens is limited to a subset of tumors of a given type. Thus, there is a need to identify antigens with complementary expression patterns for effective therapeutic intervention. In this study, we searched for genes that were distinctly expressed at a higher level in lung tumor tissue and the testes compared with other nontumor tissues and identified members of the VCX/Y gene family as novel CT antigens. VCX3A, a member of the VCX/Y gene family, was expressed at the protein level in approximately 20% of lung adenocarcinomas and 35% of squamous cell carcinomas, but not expressed in normal lung tissues. Among CT antigens with concordant mRNA and protein expression levels, four CT antigens, XAGE1, VCX, IL13RA2, and SYCE1, were expressed, alone or in combination, in about 80% of lung adenocarcinoma tumors. The CT antigen VCX/Y gene family broadens the spectrum of CT antigens expressed in lung adenocarcinomas for clinical applications.

Identification of potential glycan cancer markers with sialic acid attached to sialic acid and up-regulated fucosylated galactose structures in epidermal growth factor receptor secreted from A431 cell line.

We have used powerful HPLC-mass spectrometric approaches to characterize the secreted form of epidermal growth factor receptor (sEGFR). We demonstrated that the amino acid sequence lacked the cytoplasmic domain and was consistent with the primary sequence reported for EGFR purified from a human plasma pool. One of the sEGFR forms, attributed to the alternative RNA splicing, was also confirmed by transcriptional analysis (RNA sequencing). Two unusual types of glycan structures were observed in sEGFR as compared with membrane-bound EGFR from the A431 cell line. The unusual glycan structures were di-sialylated glycans (sialic acid attached to sialic acid) at Asn-151 and N-acetylhexosamine attached to a branched fucosylated galactose with N-acetylglucosamine moieties (HexNAc-(Fuc)Gal-GlcNAc) at Asn-420. These unusual glycans at specific sites were either present at a much lower level or were not observable in membrane-bound EGFR present in the A431 cell lysate. The observation of these di-sialylated glycan structures was consistent with the observed expression of the corresponding α-N-acetylneuraminide α-2,8-sialyltransferase 2 (ST8SiA2) and α-N-acetylneuraminide α-2,8-sialyltransferase 4 (ST8SiA4), by quantitative real time RT-PCR. The connectivity present at the branched fucosylated galactose was also confirmed by methylation of the glycans followed by analysis with sequential fragmentation in mass spectrometry. We hypothesize that the presence of such glycan structures could promote secretion via anionic or steric repulsion mechanisms and thus facilitate the observation of these glycan forms in the secreted fractions. We plan to use this model system to facilitate the search for novel glycan structures present at specific sites in sEGFR as well as other secreted oncoproteins such as Erbb2 as markers of disease progression in blood samples from cancer patients.

Directly functionalizable surface platform for protein arrays in undiluted human blood plasma.

Protein arrays are a high-throughput approach for proteomic profiling, vital for achieving a greater understanding of biological systems, in addition to disease diagnostics and monitoring therapeutic treatments. In this work, zwitterionic carboxybetaine polymer (pCB) coated substrates were investigated as an array surface platform to enable convenient amino-coupling chemistry on a single directly functionalizable and unblocked film for the sensitive detection of target analytes from undiluted human blood plasma. Using a surface plasmon resonance (SPR) imaging sensor, the antibody immobilization conditions which provided excellent spot morphology and the largest antigen response were determined. It was found that pCB functionalization and the corresponding antigen detection both increased with pH and antibody concentration. Additionally, immobilization only required an aqueous buffer without the need for additives to improve spot quality. The nonspecific protein adsorption to undiluted human plasma on both the antibody immobilized pCB spots and the background were found to be about 9 and 6 ng/cm(2), respectively. A subsequent array consisting of three antibodies spotted onto pCB revealed little cross-reactivity for antigens spiked into the undiluted plasma. The low postfunctionalized nonfouling properties combined with antibody amplification showed similar sensitivities achievable with conventional spectroscopic SPR sensors and the same pCB films, but now with high-throughput capabilities. This represents the first demonstration of low fouling properties following antibody functionalization on protein arrays from undiluted human plasma and indicates the great potential of the pCB platform for high-throughput protein analysis.

Surface plasmon resonance biosensor for determination of tetrodotoxin: prevalidation study.

A label-free surface plasmon resonance biosensor method was applied to determine tetrodotoxin (TTX) in pufferfish matrixes using an antibody inhibition assay format. A prevalidation study was conducted to demonstrate the assay performance characteristics, such as selectivity, LOD, LOQ, repeatability, reproducibility, and accuracy. Three participating laboratories reported standard curves in buffer and pufferfish matrix. A set of blind samples with TTX spiked into buffer as well as in 10% pufferfish extract were analyzed. Additionally, three blind naturally contaminated samples were analyzed, and the results were compared to those obtained using a reference method (HPLC/electrospray ionization-selected reaction monitoring-MS). The developed method was demonstrated to be capable of detecting TTX in pufferfish matrix standard samples in a broad concentration range (2-9000 ng/mL) with an LOD of 1.5 ng/mL. Between-laboratory recovery values were in the range of 51-190% with a mean of 107%, and 64-180% with a mean of 103% for TTX-spiked samples in buffer and pufferfish matrix, respectively. Between-laboratory recoveries were in the satisfactory range of 101-119% for naturally contaminated samples. This robust, rapid, and noninvasive method may serve as an attractive alternative to established methods for detection of TTX in pufferfish extracts.

Towards an integrated proteomic and glycomic approach to finding cancer biomarkers.

Advances in mass spectrometry have had a great impact on the field of proteomics. A major challenge of proteomic analysis has been the elucidation of glycan modifications of proteins in complex proteomes. Glycosylation is the most structurally elaborate and diverse type of protein post-translational modification and, because of this, proteomics and glycomics have largely developed independently. However, given that such a large proportion of proteins contain glycan modifications, and that these may be important for their function or may produce biologically relevant protein variation, a convergence of the fields of glycomics and proteomics would be highly desirable. Here we review the current status of glycoproteomic efforts, focusing on the identification of glycoproteins as cancer biomarkers.

Direct detection of carcinoembryonic antigen autoantibodies in clinical human serum samples using a surface plasmon resonance sensor.

The ability to detect biomarkers in human serum is important for cancer diagnostics. The work presented focuses on the establishment of a surface plasmon resonance (SPR) biosensor as a means for detecting varying levels of autoantibody biomarkers in human serum samples. Carcinoembryonic antigen (CEA) is a biomarker that is present in human serum. It is thought that CEA levels become elevated in patients with colon and ovarian cancer, causing a corresponding increase in the autoantibody level in human serum. Detection of this CEA autoantibody increase could be used to diagnose cancer in patients. Using a SPR biosensor, human serum samples were screened directly for CEA antibody levels. Results using a sandwich assay with a SPR sensor demonstrated the same linear trend as seen from an established enzyme-linked immunosorbent assay (ELISA) method. Serum samples from five healthy individuals were used to establish a threshold value for differentiating a cancerous serum sample from a negative sample with a 95% confidence. Three serum samples from cancer patients with positive CEA antibody levels as evaluated by ELISA were used to test the criterion.

Comparative study of SPR and ELISA methods based on analysis of CD166/ALCAM levels in cancer and control human sera.

Surface plasmon resonance (SPR), as a label free method for analysis of various analytes, has significantly advanced in recent years. However, assessment of the performance of SPR compared to label-based immunoassays such as the commonly used multiplexed enzyme-linked immunosorbent assay (ELISA) is limited, particularly for applications involving complex media. In this work, an optimized SPR assay was implemented and its performance compared with an ELISA assay for CD166/activated cell leukocyte adhesion molecule (ALCAM), as candidate pancreatic cancer marker, based on direct and amplified detection in buffer and in human serum samples from healthy individuals and subjects with cancer. ALCAM antibody was immobilized on the surface of a four-channel SPR sensor via physical adsorption onto charged amine-terminated alkanethiolates to mimic the ELISA plate surface. Excellent correlations between SPR and ELISA results were achieved in buffer and in human serum. SPR detected the target protein with a similar sensitivity to sandwich ELISA, with a detection limit below ng/mL. The detection time, sample consumption, throughput, signal referencing, and surface blocking and washing for detection in human serum were evaluated. It is demonstrated that SPR can distinguish between ALCAM levels in cancer and control sera using direct detection without the need for additional amplification steps.

Label-free detection of cancer biomarker candidates using surface plasmon resonance imaging.

In this work, we present an antibody array for the detection of cancer biomarker candidates by a surface plasmon resonance (SPR) imaging sensor with polarization contrast. Responses from the SPR imaging sensor are shown to be similar to those from a conventional spectroscopy-based SPR sensor. Antibodies are spotted onto a self-assembled monolayer (SAM) composed of oligo(ethylene glycol) (OEG)-containing alkanethiol chains. Detection of two cancer biomarker candidates, activated leukocyte cell adhesion molecule/CD 166 (ALCAM) and transgelin-2 (TAGLN2), is demonstrated. Limits of detection for ALCAM and TAGLN2 are established at 6 ng/mL and 3 ng/mL, respectively, in buffer. No cross-reactivity is observed between immobilized antibodies and nonspecific antigen. Biomarker candidates are also detected in a 10% human serum solution.

Functionalizable surface platform with reduced nonspecific protein adsorption from full blood plasma--material selection and protein immobilization optimization.

In this work, zwitterionic polymers are investigated as ultra-low fouling and functionalizable coatings for biosensors, nanoparticle-based diagnostics, and microarrays to enable detections in real-world complex media. The effect of the spacer length between the two charged groups on the nonfouling properties of zwitterionic poly(carboxybetaine acrylamide) (polyCBAA) was studied in blood plasma and serum. The polyCBAA polymer with an ethylene spacer was selected for protein immobilization studies. A polyCBAA-coated surface was functionalized with antibodies using a simple and fast amino coupling chemistry for direct protein immobilization in two simple steps: surface activation and protein immobilization/background deactivation. The effect of pH was found to be very important for both steps and it was optimized. The functionalized polyCBAA surface exhibited very low fouling properties even when exposed to undiluted blood plasma for more than 6h with <7ng/cm(2) of adsorbed proteins. The biological activity of the immobilized proteins was demonstrated with the detection of a model protein in undiluted blood plasma. A recently developed highly sensitive four-channel surface plasmon resonance (SPR) sensor was used for the evaluation of specific and nonspecific protein adsorption to these surfaces.

Hybrid surface platform for the simultaneous detection of proteins and DNAs using a surface plasmon resonance imaging sensor.

In this work, we present a novel surface and assay for the simultaneous detection of DNA and protein analytes on a surface plasmon resonance (SPR) imaging sensor. A mixed DNA/oligo (ethylene glycol) (OEG) self-assembled monolayer (SAM) is created using a microarrayer. Thiol-modified single-stranded DNA sequences are spotted onto a gold-coated glass substrate. Backfilling with an OEG-modified alkanethiol creates a protein-resistant surface background. Antibodies conjugated to complementary single-stranded DNA sequences are immobilized on the surface through DNA hybridization. By converting only part of the DNA array into a protein array, simultaneous detections of DNA and protein analytes are possible. A model system of two cDNA sequences and two human pregnancy hormones are used to demonstrate the assay. No cross-reactivity was observed between DNA or protein analytes and nontargeted immobilized cDNA sequence or antibodies. A response from a detection of a single analyte in a mixture of protein and DNA analytes corresponds well with that of a single-analyte solution.

Quantitative and simultaneous detection of four foodborne bacterial pathogens with a multi-channel SPR sensor.

We report the quantitative and simultaneous detection of four species of bacteria, Escherichia coli O157:H7, Salmonella choleraesuis serotype typhimurium, Listeria monocytogenes, and Campylobacter jejuni, using an eight-channel surface plasmon resonance (SPR) sensor based on wavelength division multiplexing. Detection curves showing SPR response versus analyte concentration were established for each species of bacteria in buffer at pH 7.4, apple juice at native pH 3.7, and apple juice at an adjusted pH of 7.4, as well as for a mixture containing all four species of bacteria in buffer. Control experiments were performed to show the non-fouling characteristics of the sensor surface as well as the specificity of the amplification antibodies used in this study. The limit of detection (LOD) for each of the four species of bacteria in the tested matrices ranges from 3.4 x 10(3) to 1.2 x 10(5) cfu/ml. Detection curves in buffer of an individual species of bacteria in a mixture of all four species of bacteria correlated well with detection curves of the individual species of bacteria alone. SPR responses were higher for bacteria in apple juice at pH 7.4 than in apple juice at pH 3.7. This difference in sensor response could be partly attributed to the pH dependence of antibody-antigen binding.