Case-controlled identification of colorectal cancer based on proteomic profiles and the potential for screening.

AIM: Colorectal cancer (CRC) screening programmes detect early cancers but unfortunately have limited sensitivity and specificity. Mass spectrometry-based determination of serum peptide and protein profiles provides a new approach for improved screening. METHOD: Serum samples were obtained from 126 CRC patients before treatment and 277 control individuals. An additional group of samples from 50 CRC patients and 82 controls was used for validation. Peptide and protein enrichments were carried out using reverse-phase C18 and weak-cation exchange magnetic beads in an automated solid-phase extraction and spotting procedure. Profiles were acquired on a matrix-assisted laser desorption/ionization time-of-flight system. Discriminant rules using logistic regression were calibrated for the peptide and protein signatures separately, followed by combining the classifications to obtain double cross-validated predicted class probabilities. Results were validated on an identical patient set. RESULTS: A discriminative power was found for patients with CRC representative for all histopathological stages compared with controls with an area under the curve of 0.95 in the test set (0.93 for the validation set) and with a high specificity (94-95%). CONCLUSION: The study has shown that a serum peptide and protein biomarker signature can be used to distinguish CRC patients from healthy controls with high discriminative power. This relatively simple and cheap test is promising for CRC screening.

High NaCl concentrations induce the nod genes of Rhizobium tropici CIAT899 in the absence of flavonoid inducers.

The nodulation (nod) genes of Rhizobium tropici CIAT899 can be induced by very low concentrations (micromolar to nanomolar range) of several flavonoid molecules secreted by the roots of leguminous plants under a number of different conditions. Some of these conditions have been investigated and appear to have a great influence on the concentration and the number of different Nod factors, which can induce root nodule primordia and pseudonodules in several leguminous plant roots. In one such condition, we added up to 300 mM NaCl to the induction medium of R. tropici CIAT899 containing the nod gene inducer apigenin. At the higher concentrations of NaCl, larger amounts and more different Nod factors were produced than in the absence of extra NaCl. To our surprise, under control conditions (300 mM NaCl without apigenin), some Nod-factor-like spots were also observed on the thin-layer plates used to detect incorporation of radiolabeled glucosamine into newly synthesized Nod factors. This phenomenon was further investigated with thin-layer plates, fusions of nod genes to the lacZ gene, high-performance liquid chromatography, mass spectrometry, and the formation of pseudonodules on bean roots. Here, we report that, in the absence of flavonoid inducers, high concentrations of NaCl induced nod genes and the production of Nod factors.

Glycan labeling strategies and their use in identification and quantification.

Most methods for the analysis of oligosaccharides from biological sources require a glycan derivatization step: glycans may be derivatized to introduce a chromophore or fluorophore, facilitating detection after chromatographic or electrophoretic separation. Derivatization can also be applied to link charged or hydrophobic groups at the reducing end to enhance glycan separation and mass-spectrometric detection. Moreover, derivatization steps such as permethylation aim at stabilizing sialic acid residues, enhancing mass-spectrometric sensitivity, and supporting detailed structural characterization by (tandem) mass spectrometry. Finally, many glycan labels serve as a linker for oligosaccharide attachment to surfaces or carrier proteins, thereby allowing interaction studies with carbohydrate-binding proteins. In this review, various aspects of glycan labeling, separation, and detection strategies are discussed.

Mass spectrometry proteomic diagnosis: enacting the double cross-validatory paradigm.

This paper presents an approach to the evaluation and validation of the diagnostic potential of mass spectrometry data in an application on the construction of an "early warning" diagnostic procedure. Our approach is based on a full implementation and application of double cross-validatory calibration and evaluation. It is a key feature of this methodology that we can jointly optimize the classifiers for prediction while simultaneously calculating validated error rates. The methodology leaves the size of the training data nearly intact. We present application to data from a designed experiment in a colon-cancer study. Subsequent to presentation of results from the double cross-validatory analysis, we explore a post-hoc analysis of the calibrated classifiers to identify the markers that drive the classification.

Current status and prospects of clinical proteomics studies on detection of colorectal cancer: hopes and fears.

Colorectal adenocarcinoma (CRC) is the third most common type of cancer and the fourth most frequent cause of death due to cancer worldwide. Given the natural history of CRC, early diagnosis appears to be the most appropriate tool to reduce disease-related mortality. A field of recent interest is clinical proteomics, which was reported to lead to high sensitivity and specificities for early detection of several solid tumors. This emerging field uses mass spectrometry-based protein profiles/patterns of easy accessible body fluids to distinguish cancer from non-cancer patients. These discrepancies may be a result of: (1) proteins being abnormally produced or shed and added to the serum proteome, (2) proteins clipped or modified as a consequence of the disease process, or (3) proteins subtracted from the proteome owing to disease-related proteolytic degradation pathways. Therefore, protein pattern diagnostics would provide easy and reliable tools for detection of cancer. This paper focuses on the current status of clinical proteomics research in oncology and in colorectal cancer especially, and will reflect on pitfalls and fears in this relatively new area of clinical medicine, which are reproducibility issues and pre-analytical factors, statistical issues, and identification and nature of discriminating proteins/peptides.

Identification of meat products by shotgun spectral matching.

A new method, based on shotgun spectral matching of peptide tandem mass spectra, was successfully applied to the identification of different food species. The method was demonstrated to work on raw as well as processed samples from 16 mammalian and 10 bird species by counting spectral matches to spectral libraries in a reference database with one spectral library per species. A phylogenetic tree could also be constructed directly from the spectra. Nearly all samples could be correctly identified at the species level, and 100% at the genus level. The method does not use any genomic information and unlike targeted methods, no prior knowledge of genetic variation within a genus or species is necessary.

Serodiagnostic application of immunohistoperoxidase reactions on antigen-coupled agarose beads.

Agarose beads to which antigens were covalently bound were subjected to indirect immunohistoperoxidase procedures. For detection and titration of serum antibodies against bovine gamma globulin (BGG) this method appeared to be specific and sensitive. One advantage is that no special instruments are needed. As an example of diagnostic applicability the system was successfully used for demonstration of antibodies in human serum containing antibodies against the trematode Schistosoma mansoni. At the microscopical level the technique is suitable for study of basic problems. Microspectrophotometric absorbance scanning of the beads revealed that the method can provide quantitative information, and is probably capable of quantifying stoichiometric relations in immunological reactions.

Antigen-coupled beads adherent to slides: a simplified method for immunological studies.

Small amounts of antigen-coupled beads adherent to object slides provide a simple, quick, economical and sensitive immunohistochemical means of detecting antibodies in serum by both immunofluorescence and immunohistoperoxidase procedures. Sensitivity increases with decreasing quantities of antigen-coupled beads, as was demonstrated in the fluorescence procedure.

A simple and rapid treatment (trichloroacetic acid precipitation) of serum samples to prevent non-specific reactions in the immunoassay of a proteoglycan.

In our laboratory serum components interfering in the immunoassay of the schistosome proteoglycan circulating anodic antigen (CAA) in serum necessitated us to develop a simple technique by which the non-specific reaction of negative control sera could be prevented. Trichloroacetic acid was added to serum samples to precipitate interfering (glyco-)proteins. After centrifugation, the supernatant was neutralized and used directly either in the ELISA or in the indirect haemagglutination. This method gave satisfactory results, i.e., negative control sera did no longer give false positive reactions, while the titre of the positive controls remained unaffected. This method could also be successfully applied for the pretreatment of urine samples.

Automated measurement of immunogalactosidase reactions with a fluorogenic substrate by the aperture defined microvolume measurement method and its potential application to Schistosoma mansoni immunodiagnosis.

For automated read-out of immunogalactosidase assays with a fluorogenic substrate, performed in microtitration trays, an inverted fluorescence microscope equipped with a photometer and a scanning stage and operated by a microprocessor has been used. Trays (60 and 96 wells) were scanned in 1 min and corrected measurements and a histogram of the results printed out in a few minutes. A lower level of 4 ng of the reaction product, 4-methylumbelliferone, was detected. By making the measuring system flexible we were also able to use it for fluorescence measurements of individual agarose beads or cells and for extinction measurements in ELISA reactions with a chromogenic substrate. On comparing FITC and galactosidase conjugates in a DASS-test, and peroxidase and galactosidase conjugates in an ELISA, with Schistosoma mansoni infection as test system, it appeared that the galactosidase modification of both assays was 30-50 times more sensitive than the FITC or peroxidase modification. For S. mansoni egg antigen, a lower detectable level of 0.1 ng antigen/ml was achieved.

Magnetic bead antigen capture enzyme-linked immunoassay in microtitre trays for rapid detection of schistosomal circulating anodic antigen.

We have developed a new magnetic bead antigen capture enzyme-linked immunoassay for the detection of schistosomal circulating anodic antigen. The assay utilizes IgG1 monoclonal antibody coated monodisperse magnetic beads in microtitre trays fitted to a special magnet. The total test time was found to be 1-2 h, using 0.05 mg beads per well. The lower detection level was 0.7 ng AWA-TCA per ml (approximately 0.07 ng CAA per ml). Validation by sera from uninfected and Schistosoma mansoni infected Africans and Norwegians resulted in an assay specificity of 100% and sensitivity was close to 90% for cases excreting more than 100 eggs per gram faeces. At such clinically relevant levels the inter-assay CV was below 10% and photometric absorbance correlated to antigen levels was nearly linear. There was a significant correlation between the magnetic bead EIA absorbance values and the titres obtained using the previously established ELISA. The new bead assay, however, was easier and less laborious because TCA pretreatment and the titration of positive results were unnecessary.

A comparative study on the preparation of immunoglobulin-galactosidase conjugates.

For the application of beta-D-galactosidase-immunoglobulin conjugates in the enzyme-linked immunosorbent assay (ELISA), four techniques for the preparation of such conjugates were compared. Sheep immunoglobulin (Ig) (against soluble egg antigens of the trematode Schistosoma mansoni) was coupled to beta-D-galactosidase by means of 1) glutaraldehyde treatment, 2) the heterobifunctional reagent N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP), and 3,4) two different procedures using the coupling agent m-maleimidobenzoyl-N-hydroxysuccinimide ester(MBS). The prepared conjugates were then fractionated by gel filtration on Sepharose 6B and the resultant molecular weight fractions were tested in an ELISA for the detection of S. mansoni antigen. Optimal results were obtained with a conjugate that was synthesized according to one of the two techniques using MBS. With this conjugate, 10(-9) g antigen/ml could still be detected in an ELISA with a chromogenic substrate, which was at least ten times as sensitive as with the other conjugates. Application of a fluorogenic substrate resulted in a lower detection level of 10(-10) g antigen/ml.

Quantitative and morphological aspects of Unicryl versus Lowicryl K4M embedding in immunoelectron microscopic studies.

In this study we compared the recently commercialized electron microscopy embedding resin Unicryl with the well-known resin Lowicryl K4M with regard to morphological and immunohistochemical preservation properties. The standard embedding procedure recommended by the manufacturer for the use of Unicryl resulted in considerable morphological alterations of the tissue, with the appearance of large gaps in and between the cells of the examined tissue. Morphometric analysis pointed to a swelling of the extracellular matrix as the main cause of these morphological artifacts. A slight modification in the protocol to correct this artifact is proposed and tested. Immunohistochemically, tissue embedded in Unicryl resulted in a significantly stronger immunogold labeling than identical tissue embedded in Lowicryl K4M. From the results of this technical study, it can be concluded that Unicryl embedding is a valuable new tool to supplement the available techniques for immunoelectron microscopic studies.

Application of the FITC-anti-FITC-gold system to ultrastructural localization of antigens.

We report the application of a fluorescein isothiocyanate (FITC)-anti-FITC method to localize antigens at the ultrastructural level. In the systems studied, the anti-FITC-based detection method displays high specificity and sensitivity. These observations, combined with ease of production and with availability of FITC-protein conjugates, suggest that the FITC-anti-FITC method is a good alternative to presently used methods and is widely applicable to immunochemical and immunocytochemical procedures. The same preparation and protocol can be used for light and electron microscopic studies, thereby reducing possible artifacts introduced if different procedures are used. In the present study, two systems were used to test the method. One system used an FITC-labeled monoclonal antibody (MAb) to schistosome circulating cathodic antigen. In this system, the label was detected in the gut of adult Schistosoma mansoni by an anti-FITC MAb conjugated to 10-nm gold particles. The second system used human IgM antibodies pooled from patients infected with Schistosoma mansoni. In this system detection was accomplished using an anti-human IgM-FITC conjugate followed by the anti-FITC-Au antibody conjugate.

Dot immunoassay with biotinylated antigen for determination of antibodies against the circulating cathodic antigen (CCA) in schistosomiasis japonica.

Based on the fact that schistosomiasis patients in both the acute and chronic phase of the infection show a strong humoral immune response against the gut-associated circulating cathodic antigen, a simple and sensitive dot immunobinding assay for schistosomiasis japonica was developed. Circulating cathodic antigen that had been purified by immunoadsorption using monoclonal antibody was biotinylated with biotin aminocaproylhydrazide via the carbohydrate moiety of the antigen. Serum samples dotted onto nitrocellulose strips were then tested in an assay involving a combined incubation step of biotinylated antigen and streptavidin peroxidase, and a subsequent staining; the total assay time was 1.5 hr. Assaying the sera of 105 uninfected controls and 104 Schistosoma japonicum-infected individuals showed a specificity of 99.0%, and sensitivities of 96.2% (acute infections) and 94.1% (chronic infections). The described assay is economic, rapid, and reproducible and lends itself to use under field conditions.

Comparison of four schistosome excretory-secretory antigens: phenol sulfuric test active peak, cathodic circulating antigen, gut-associated proteoglycan, and circulating anodic antigen.

Several carbohydrate-containing antigens of schistosomes have been characterized and tests for these antigens or for the corresponding antibodies are increasingly being used for the diagnosis and clinical analysis of human schistosomiasis. Phenol sulfuric active peak (PSAP) and cathodic circulating antigen (CCA) are 2 soluble glycoproteins found in adult worms of Schistosoma mansoni. These antigens have some similar characteristics including solubility in trichloroacetic acid and non-binding or weak binding to DEAE cellulose which suggests these 2 compounds to be identical. PSAP and CCA were therefore compared using radioimmunoassays employing monoclonal antibodies to CCA and radiolabeled PSAP as well as inhibitory assays using the original glycoproteins as inhibitors. By these criteria PSAP and CCA were found to be different glycoproteins. Using similar techniques, 2 anodic schistosome compounds, gut-associated proteoglycan (GASP) and circulating anodic antigen (CAA) were found to be identical. We now propose to call this later material GASCAP (gut-associated circulating anodic proteoglycan).

Ultrastructural localization of the circulating anodic antigen in the digestive tract of Schistosoma mansoni using monoclonal antibodies in an immunogold labeling procedure.

The purpose of this study was to determine the ultrastructural localization of a major schistosome circulating antigen-the circulating anodic antigen (CAA)-in the digestive tract of various life cycle stages of Schistosoma mansoni. The presence of CAA was determined by an indirect gold-labeling procedure using CAA-specific monoclonal antibodies. In cercariae, gold label was found in the cytoplasm and in the surface coat of the gut epithelium. A minimal amount of gold particles was also observed in the esophagus epithelium, but this was limited to the luminal surface coat and located proximally to the gut. In 3 1/2-week-old worms and in adult male and female worms CAA was demonstrable in the Golgi apparatus, in cytoplasmic vesicles, and in the luminal surface coat of the gut epithelium. As determined in the adult worms, CAA-positive lysosome-like bodies were only encountered in the most caudal quarter of the gut. In the gut lumen CAA was associated with host white blood cells and with a thick layer of finely granular, moderately electron-dense material covering the gut epithelium. The esophagus of these worms did not show CAA reactivity. These results definitely prove that CAA is a gut-specific antigen produced by various life cycle stages of S. mansoni, from the cercarial stage on.

Antibody response to Schistosoma mansoni adult worm cysteine proteinases in infected individuals.

Antigens of the Schistosoma mansoni digestive tract are recognized early in the infective process. Two immunogenic components of the excretory/secretory products are proteolytic enzymes that degrade host hemoglobin in the lumen of the parasite gut. These enzymes, CP1 and CP2, belong to the class of cysteine proteinases. In this study, a preparation containing both proteinases has been used to detect proteinase antibodies in the sera of individuals living in Burundi. Of 133 individuals tested, 92% were excreting schistosome eggs. All patients with documented infections had positive anti-proteinase IgG titers (mean = 1:614), while 82% had positive IgM titers (mean = 1:267). Six weeks following praziquantel treatment, patients were assessed for egg excretion and antibody titer. Anti-proteinase IgG titers were significantly lower (mean = 1:259) than pre-treatment titers. Patients who were infected with S. japonicum or S. haematobium typically showed a cross-reactive IgG response. Patients from non-endemic regions yielded negative titers, and those with non-trematode parasites were negative (79%) or weakly positive. S. mansoni cysteine proteinases may be used for the detection of schistosome infections.

Day-to-day fluctuation of schistosome circulating antigen levels in serum and urine of humans infected with Schistosoma mansoni in Burundi.

Day-to-day fluctuations of both circulating anodic antigen (CAA) and circulating cathodic antigen (CCA) in serum and urine were examined simultaneously in a group of Schistosoma mansoni-infected individuals from Burundi and compared with each other and with fecal egg count fluctuations. Significant correlations were found between fecal egg counts and circulating antigens (CAA and CCA) and between circulating antigen levels in serum and urine samples. The cumulative percentage of positive results after three samplings was highest for urine CCA detection, followed by fecal egg counts, serum CCA, serum CAA, and urine CAA detection, respectively. It was demonstrated that circulating antigen levels in both serum and urine showed less fluctuation than fecal egg counts, except for urine CAA levels. The serum CAA detection assay in particular, although less sensitive in this low endemic area in Burundi, gave very constant measurements over a period of one week. Our results indicate that detection of circulating antigens in a single serum or urine sample provides a quantitatively more stable diagnosis of S. mansoni infection than fecal egg counts based on a single stool examination.