Objectives“PAULA’s” test (Protein Assays Utilizing Lung cancer Analytes) is a novel multiplex immunoassay blood test that incorporates both tumor antigens and autoantibodies to determine the risk that lung cancer (LC) is present in individuals from a high-risk population. The test’s performance characteristics were evaluated in a study using 380 retrospective clinical serum samples.MethodsPAULA’s test is performed on the Luminex xMAP technology platform, and detects a panel of 3 tumor antigens (CEA, CA-125, and CYFRA 21–1) and 1 autoantibody marker (NY-ESO-1). A training set (n = 230) consisting of 115 confirmed diagnoses of non-small cell lung carcinoma (NSCLC) cases and 115 age- and smoking history-matched controls was used to develop the LC predictive model. Data from an independent matched validation set (n = 150) was then used to evaluate the model developed, and determine the ability of the test to distinguish NSCLC cases from controls.ResultsThe 4-biomarker panel was able to discriminate NSCLC cases from controls with 74% sensitivity, 80% specificity, and 0.81 AUC in the training set and with 77% sensitivity, 80% specificity, and 0.85 AUC in the independent validation set. The use of NY-ESO-1 autoantibodies substantially increased the overall sensitivity of NSCLC detection as compared to the 3 tumor markers alone. Overall, the multiplexed 4-biomarker panel assay demonstrated comparable performance to a previously employed 8-biomarker non-multiplexed assay.ConclusionsThese studies confirm the value of using a mixed panel of tumor antigens and autoantibodies in the early detection of NSCLC in high-risk individuals. The results demonstrate that the performance of PAULA’s test makes it suitable for use as an aid to determine which high-risk patients need to be directed to appropriate noninvasive diagnostic follow-up testing, especially low-dose CT (LDCT).
The mammaglobin gene has been shown to be preferentially expressed in breast tissue. Few genes match its specificity. Mammaglobin has generated much interest, and studies are ongoing to develop diagnostic tests for breast cancer based on the detection of mammaglobin. While searching the Incyte Genomics Lifeseq database for tissue-specific markers, we observed a second secretoglobin, BU101, also known as lipophilin B. We report here that mammaglobin, in breast tissue, is found as a complex with BU101. The complex was isolated from breast cancer tissue and was characterized as the biologically relevant form of mammaglobin.
In this report we describe a cDNA sequence, BS106, identified from Incyte Genomics LifeSeq® Expressed Sequence Tag database. A multi-tissue mRNA expression array, northern blots, and RT-PCR assays demonstrate the expression of BS106 in mammary, salivary and prostate glands, but not in other tissue types. BS106 mRNA was detected in 90% of the breast tissues examined. The cDNA encodes a 90-amino acid protein characterized as a small, mucin-like protein based on amino acid composition, extensive O-linked glycosylation, and expression profile. BS106 protein was recombinantly expressed in human embryonic kidney 293 cells and the secreted product was purified from the culture media. Monoclonal antibodies were prepared and used for immunohistochemical analysis of early stage breast cancer. BS106 protein was detected in the vast majority of carcinomas (70–100%) and overexpressed in approximately 30% of the 22 specimens analyzed. BS106 protein was not detected in other solid tumor types including bladder carcinoma, colon carcinoma, endometrial carcinoma, gastric carcinoma, squamous cell lung carcinoma, adenocarcinoma of the lung, ovarian carcinoma, pancreatic and prostatic carcinoma.
ObjectiveThe race for finding effective treatments for nonalcoholic fatty liver disease (NAFLD) has been slowed down by the high screen-failure rate for including patients in trials due to the lack of a noninvasive biomarker that can identify patients with significant disease. Recently, Wisteria floribunda agglutinin-positive Mac-2-binding protein (WFA+ -M2BP) has shown promise in predicting liver fibrosis. The aims of this study were to evaluate the utility of WFA+ -M2BP as a biomarker to sub-classify patients with NAFLD according to their disease severity and to assess its correlation with histologic features of NAFLD.MethodsPatients undergoing biopsy for clinical suspicion of NAFLD and healthy controls were included. Patients with NAFLD were classified into: NAFL, early NASH (F0-F1), fibrotic NASH (F2-F3), and NASH cirrhosis (F4). Levels of WFA+ -M2BP in sera was measured by a HISCL™ M2BPGi™ assay kit using an automated immunoanalyzer (HISCL™-800; Sysmex, Kobe, Japan). Analysis of covariance was used to assess difference in WFA+ -M2BP between the groups and Spearman’s correlation coefficients were used to assess correlation with histological features.ResultsOur cohort consisted of 20 healthy controls and 198 patients with biopsy-proven NAFLD divided as follows: 52 with NAFL, 62 with early NASH, 52 with fibrotic NASH, and 32 with NASH cirrhosis. WFA+ -M2BP level was found to be significantly increased in the fibrotic NASH and NASH cirrhosis groups compared to healthy controls and those with early NAFLD after adjusting for age, gender and BMI. Furthermore, patients with NASH cirrhosis had significantly higher WFA+ -M2BP levels (2.4[1.5, 4.2] C.O.I (Cut-off Index)) than those with fibrotic NASH (1.2[0.79, 1.9]), p < 0.001. WFA+ -M2BP level had moderate correlation with inflammation, ballooning and NAFLD activity score and strong correlation with fibrosis stage. Additionally, ROC curve analysis demonstrated that WFA+ -M2BP accurately differentiated F2-4 from F0-F1.ConclusionIn a large cohort of patients with the full spectrum of NAFLD, WFA+ -M2BP levels predicted the presence of advanced disease and correlated strongly with fibrosis stage.
Selectively labeled polypeptides comprising the gamma-carboxyglutamic acid (Gla) domain (GD) and helical stack (HS) regions of human protein C (PC), and consisting of amino acid residues 1-47, have been chemically synthesized and their Ca2+ binding properties assessed by [13C]-NMR methods. A total of nine such polypeptides have been studied, each containing one of the Gla residues fully enriched with [13C] at its two gamma-carboxylate carbon atoms. Additions of Ca2+ resulted in readily measurable [13C] chemical shifts, titrations of which were used to obtain apparent dissociation constants for each Gla residue in the presence of all other such residues. The Ca2+ titration data obtained on each of the nine polypeptides showed that Gla residues 6, 16, 25, and 26 were involved in the higher affinity Ca2+ binding sites, whereas the remaining Gla residues, viz., 7, 14, 19, 20, and 29, coordinated Ca2+ more weakly. The results are consistent with conclusions drawn from functional studies obtained with site-directed mutations of individual Gla residues and with the structural model of the GD/HS of human PC. In these cases, Gla residues 6, 16, and 26 served as coordination loci for internally located Ca2+ ions, and GD-related Ca(2+)- and PL-dependent properties of PC and activated PC were dependent on the integrity of these Gla residues.
We have developed a solid-phase procedure for protein-protein conjugation that gives greater control over product size and composition than previous methods. Conjugates are assembled by sequential addition of activated proteins to the support under conditions suitable for maintaining the activity of the proteins. The total number of conjugate units to be prepared is fixed in the first step by the quantity of the first protein absorbed by the support. In each following step, the added protein links only to previously bound protein. The final conjugate is released to solution by cleaving the linker holding the first protein to the support. This stepwise assembly provides uniformly sized conjugates of the desired size and composition with placement of components at the desired positions within the structure. Using this approach, we have prepared a series of conjugates containing R-phycoerythrin as the central protein, with varying quantities of alkaline phosphatase and IgG with expected molecular masses ranging from 1.6 to 11.5 MDa. Size-exclusion chromatography and atomic force microscopy demonstrate homogeneity and control of the conjugate size. In an immunoassay for human thyroid stimulating hormone, the conjugates show signals consistent with their compositions.
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