Selective Chemical Inhibition of PGC-1α Gluconeogenic Activity Ameliorates Type 2 Diabetes.

Type 2 diabetes (T2D) is a worldwide epidemic with a medical need for additional targeted therapies. Suppression of hepatic glucose production (HGP) effectively ameliorates diabetes and can be exploited for its treatment. We hypothesized that targeting PGC-1α acetylation in the liver, a chemical modification known to inhibit hepatic gluconeogenesis, could be potentially used for treatment of T2D. Thus, we designed a high-throughput chemical screen platform to quantify PGC-1α acetylation in cells and identified small molecules that increase PGC-1α acetylation, suppress gluconeogenic gene expression, and reduce glucose production in hepatocytes. On the basis of potency and bioavailability, we selected a small molecule, SR-18292, that reduces blood glucose, strongly increases hepatic insulin sensitivity, and improves glucose homeostasis in dietary and genetic mouse models of T2D. These studies have important implications for understanding the regulatory mechanisms of glucose metabolism and treatment of T2D.

AlphaLISA-Based Immunoassay for Detection of Troponin T in Serum of Patients with Acute Myocardial Infarction.

Survival and prognosis of patients with acute myocardial infarction (AMI) are highly dependent on rapid and accurate diagnosis of myocardial damage. Troponin T is the primary diagnostic biomarker and is widely used in clinical practice. Amplified luminescent proximity homogeneous assay (AlphaLISA) may provide a solution to rapidly detect a small amount of analyte through molecular interactions between special luminescent donor beads and acceptor bead. Here, a double-antibody sandwich assay was introduced into AlphaLISA for rapid detection for early diagnosis of AMI and disease staging evaluation. The performance of the assay was evaluated. The study found that the cTnT assay has a linear range of 48.66 to 20,000 ng/L with a limit of detection of 48.66 ng/L. In addition, the assay showed no cross-reactivity with other classic biomarkers of myocardial infarction and was highly reproducible with intra- and inter-batch coefficients of variation of less than 10%, notably, only 3 min was taken, which is particularly suitable for clinical diagnosis. These results suggest that our method can be conveniently applied in the clinic to determine the severity of the patient's condition.

Monitoring hormone and small molecule secretion dynamics from islets-on-chip.

Tissue functions such as hormone secretion involve the interplay of multiple chemical signals and metabolic processes over time. Measuring the different components involved is useful in unraveling the interactions, but often requires use of multiple analytical techniques. The challenge of measuring the necessary components with temporal resolution is greater when tissue samples are limited. Here, an accessible microfluidic platform compatible with multiple measurement techniques to monitor cell secretions has been developed. The platform is applied to islets of Langerhans, micro-organs involved in glucose homeostasis and diabetes. The device houses 1 to 8 islets and the perfusion fluid can be controlled to change conditions, e.g., glucose concentration, in seconds. Samples are collected in fractions and split for offline analysis. The device is paired with a scaled-down immunoassay, AlphaLISA, for hormone quantification and liquid chromatography-mass spectrometry for small molecule quantification to study secretion dynamics. The combined system allows the first simultaneous measurement of insulin, glucagon, biogenic amines, and amino acids from islet secretions. The combined measurements revealed correlation in secretion events and differences in timing of release between hormones and biogenic amines and amino acids. These efforts decreased the number of islets required compared to standard approaches, thus decreasing necessary animal use, reagent use, and cost, while increasing information content achievable from one sample. The microfluidic device is a suitable platform for in-depth characterization of secretion from small tissue samples.

Bis(benzonitrile) dichloroplatinum (II) interrupts PD-1/PD-L1 interaction by binding to PD-1.

Checkpoint inhibitors such as PD-1/PD-L1 antibody therapeutics are a promising option for the treatment of multiple cancers. Due to the inherent limitations of antibodies, great efforts have been devoted to developing small-molecule PD-1/PD-L1 signaling pathway inhibitors. In this study we established a high-throughput AlphaLISA assay to discover small molecules with new skeletons that could block PD-1/PD-L1 interaction. We screened a small-molecule library of 4169 compounds including natural products, FDA approved drugs and other synthetic compounds. Among the 8 potential hits, we found that cisplatin, a first-line chemotherapeutic drug, reduced AlphaLISA signal with an EC50 of 8.3 ± 2.2 µM. Furthermore, we showed that cisplatin-DMSO adduct, but not semplice cisplatin, inhibited PD-1/PD-L1 interaction. Thus, we assessed several commercial platinum (II) compounds, and found that bis(benzonitrile) dichloroplatinum (II) disturbed PD-1/PD-L1 interaction (EC50 = 13.2 ± 3.5 µM). Its inhibitory activity on PD-1/PD-L1 interaction was confirmed in co-immunoprecipitation and PD-1/PD-L1 signaling pathway blockade bioassays. Surface plasmon resonance assay revealed that bis(benzonitrile) dichloroplatinum (II) bound to PD-1 (KD = 2.08 µM) but not PD-L1. In immune-competent wild-type mice but not in immunodeficient nude mice, bis(benzonitrile) dichloroplatinum (II) (7.5 mg/kg, i.p., every 3 days) significantly suppressed the growth of MC38 colorectal cancer xenografts with increasing tumor-infiltrating T cells. These data highlight that platinum compounds are potential immune checkpoint inhibitors for the treatment of cancers.

Combinational antibody detection approach increases the clinical validity of colorectal cancer screening.

BACKGROUND: At different stages of the disease, biomarkers can help to determine disease progression and recurrence and provide a personalized indicator of therapeutic effectiveness. The serological identification of antigens by recombinant cDNA expression cloning (SEREX) has identified five SEREX antigens. RESULTS: Compared with healthy donors, anti-FIRΔexon2 and anti-SOHLH antibodies (Abs) in the sera of patients with colorectal cancer (CRC) were markedly higher. Furthermore, no correlation was noted between five SEREX antigens and the three tumor markers (CEA, CA19-9, and anti-p53 Abs), indicating that anti-FIRΔexon2 Abs are an independent candidate marker for patients with CRC. Generally, the levels of anti-FIRΔexon2 Abs combined with clinically available tumor markers were determined to be significantly higher compared with CEA, CA19-9. Moreover, in early-stage CRC, the levels of anti-FIRΔexon2 Abs combined with existing tumor markers were higher than those of CEA, CA19-9. CONCLUSION: Due to the highly heterogeneous nature of CRC, a single tumor marker is unlikely to become a standalone diagnostic test due to its commonly insufficient sensitivity and/or specificity. Using a combination antibody detection approach of tumor markers for CRC diagnosis has the potential to be an effective approach. Therefore, the use of serum protein biomarker candidates holds promise for the development of inexpensive, noninvasive, and inexpensive tests for the detection of CRC.

A Causal Treatment for X-Linked Hypohidrotic Ectodermal Dysplasia: Long-Term Results of Short-Term Perinatal Ectodysplasin A1 Replacement.

X-linked hypohidrotic ectodermal dysplasia (XLHED), caused by a genetic deficiency of ectodysplasin A1 (EDA1), is a rare developmental disorder of ectodermal derivatives such as hair, sweat glands, and teeth. The absence of sweat glands and perspiration can evoke life-threatening hyperthermia. As molecular genetic findings are not always conclusive, the concentrations of circulating EDA1 may help to distinguish between total and partial EDA1 deficiencies. We previously treated nine male patients with obvious signs of XLHED with a recombinant EDA1 replacement protein, Fc-EDA, either shortly after birth (n = 3) or by prenatal administration in gestational week 26 and beyond (n = 6). Here, we present the long-term follow-up for up to six years. In patients who had received Fc-EDA after birth, neither sweat glands nor sweating ability were detected at the age of 12-60 months. In contrast, prenatal EDA1 replacement resulted in ample sweat gland development and pilocarpine-inducible sweating in all treated subjects, who also attained more permanent teeth than their untreated affected relatives. Normal perspiration has persisted for six years in the two oldest boys treated repeatedly with Fc-EDA in utero. When they had a sauna, adequate thermoregulation was evidenced. Lower sweat production after single prenatal dosing may indicate a dose-response relationship. The absence of circulating EDA1 in five prenatally treated subjects proved that these children would have been unable to perspire if they had been left untreated. The sixth infant was shown to produce an EDA1 molecule that, albeit interacting with its cognate receptor, cannot activate EDA1 signaling. In conclusion, a causal treatment of XLHED before birth is feasible.

Structure-Activity relationship of 1-(Furan-2ylmethyl)Pyrrolidine-Based Stimulation-2 (ST2) inhibitors for treating graft versus host disease.

An elevated plasma level of soluble ST2 (sST2) is a risk biomarker for graft-versus-host disease (GVHD) and death in patients receiving hematopoietic cell transplantation (HCT). sST2 functions as a trap for IL-33 and amplifies the pro-inflammatory type 1 and 17 response while suppressing the tolerogenic type 2 and regulatory T cells activation during GVHD development. We previously identified small-molecule ST2 inhibitors particularly iST2-1 that reduces plasma sST2 levels and improved survival in two animal models. Here, we reported the structure-activity relationship of the furanylmethylpyrrolidine-based ST2 inhibitors based on iST2-1. Based on the biochemical AlphaLISA assay, we improved the activity of iST2-1 by 6-fold (∼6 µM in IC50 values) in the inhibition of ST2/IL-33 and confirmed the activities of the compounds in a cellular reporter assay. To determine the inhibition of the alloreactivity in vitro, we used the mixed lymphocyte reaction assay to demonstrate that our ST2 inhibitors decreased CD4+ and CD8+ T cells proliferation and increased Treg population. The data presented in this work are critical to the development of ST2 inhibitors in future.

Measurement of anti SARS-CoV-2 RBD IgG in saliva: validation of a highly sensitive assay and effects of the sampling collection method and correction by protein.

OBJECTIVES: To develop and evaluate a new highly sensitive assay to detect IgG anti-SARS-CoV-2 RBD in saliva samples. METHODS: A two-step sandwich type immunoassay based on the amplified luminescent proximity homogeneous technology was developed and an analytical validation was performed. As a part of this validation, the influence of factors, such as different sampling conditions (stimulated saliva and passive drool) and the correction of values by total protein content, in the ability of saliva to detect increases in antibodies after an immune stimulus and be an alternative to serum, was evaluated. For this purpose, paired samples of saliva and serum at different times after vaccination were used. RESULTS: Saliva concentrations were lower than serum, but both fluids showed similar kinetics, with higher correlations when saliva was obtained by passive flow and the results were not corrected by protein. CONCLUSIONS: The developed method showed a good analytical performance and can properly measure antibody concentrations in saliva of vaccinated individuals. However, saliva could have a lower sensitivity compared to serum at initial stages of the immune response and also when the antibody response decreased after a stimulus.

Anti-FIRΔexon2 autoantibody as a novel indicator for better overall survival in gastric cancer.

There is no clinically available biomarker for efficiently indicating the overall survival or therapy response of gastric cancer (GC). The autoantibodies (Abs) in the sera of anti-far-upstream element-binding protein-interacting repressor-lacking exon2 (FIRΔexon2), anti-sorting nexin 15, and anti-spermatogenesis and oogenesis-specific basic helix-loop-helix 1 were markedly higher in GC patients than in healthy donors (HDs). These Abs were identified by large-scale serological identification of antigens by recombinant cDNA expression cloning screenings and their expression levels were evaluated by amplified luminescence proximity homogeneous assay. In particular, compared with age-matched HDs, the level of anti-FIRΔexon2 Abs in GC patients was significantly higher (P < .001). The Spearman's rank correlation analysis between anti-FIRΔexon2 Abs and clinically available tumor markers such as carcinoembryonic antigen (CEA) was statistically insignificant, indicating that FIRΔexon2 Abs is an independent biomarker. We performed receiver-operating curve analysis to evaluate the anti-FIRΔexon2 Ab as a candidate biomarker with CEA and carbohydrate antigen 19-9 (CA19-9). The overall survival of GC patients with high anti-FIRΔexon2 Abs titer was significantly favorable (P = .04) than that of GC patients who were below detection level of anti-FIRΔexon2 Abs. However, clinical stages were not apparently correlated with the levels of anti-FIRΔexon2 Ab, CEA, and CA19-9. In conclusion, anti-FIRΔexon2 Abs detected in GC patients is a potential biomarker for monitoring a better prognosis. Hence, anti-FIRΔexon2 Abs is a promising biomarker for indicating better overall survival of gastric cancer patients.

Application of an AlphaLISA method for rapid sensitive detection of African swine fever virus in porcine serum.

Infection with African swine fever virus (ASFV) causes an acute and highly lethal hemorrhagic disease that has been responsible for huge economic losses in China. To exactly detect the antigen of ASFV, we established a rapid, no-wash, one-step sandwich-type immunoassay based on the amplified luminescent proximity homogenous assay-linked immunosorbent assay (AlphaLISA) using two monoclonal antibodies (mAbs) M-5 and M-6 against ASFV p72. ASFV p72 in samples was captured by biotinylated mAb M-5 connected to the donor bead surface via streptavidin and "sandwiched" by mAb M-6 which was coated onto the acceptor bead. Efficacy and sensitivity trials revealed that the AlphaLISA could detect ≥0.78 ng/ml of purified p72 and with a linear range of 0.78-100 ng/ml. The AlphaLISA was specific for ASFV and did not cross-react with other common pathogenic porcine viruses. Compared with RealPCR ASFV DNA test and ASFV antigen detection kit, the sensitivity of the AlphaLISA evaluated in 60 porcine serum samples was 93% and 100%, respectively. The specificity was 100% and 91.7%, respectively. This study presents a good laboratory diagnostic tool for sensitive and efficient detection of ASFV in porcine serum. KEY POINTS: • MAbs M-5 and M-6 recognized various epitopes of ASFV p72. • The established ASFV p72 AlphaLISA showed well specificity, high sensitivity, and satisfied correlation coefficient.

Development of a novel AlphaLISA ImmunoAssay for Big angiotensin-25.

We previously described the discovery of Big angiotensin-25 (Bang-25), an angiotensin-related peptide isolated from human urine. Bang-25 consists of the first 25 amino acids of the N-terminus of angiotensinogen (Aogen), with N-linked glycosylation on the 14th amino acid and a cysteine conjugated to the 18th amino acid. Bang-25 is rapidly converted into angiotensin II (Ang II) by chymase. Because Bang-25 is widely distributed in human tissues, including islet cells in the pancreas and podocytes in the kidney, we hypothesized that it may participate in the Ang II production system in these tissues. To test this hypothesis, we developed a specific assay for Bang-25 and used it to examine the urinary concentrations of Bang-25 in patients with diabetes mellitus (DM). The assay used the Amplified Luminescent Proximity Homogeneous Assay (Alpha)-based ELISA method (AlphaLISA) of PerkinElmer Japan and included antibodies specific to the N-terminus of Ang II and the C-terminus of Bang-25. The AlphaLISA ImmunoAssay specifically recognized Bang-25 and had no cross-reactivity with Aogen or Ang I. Bang-25 was detected in healthy volunteers' urine samples but not in their plasma samples. In patients with DM, the urinary Bang-25 concentration was significantly higher than in healthy volunteers. Moreover, the results indicated that the Bang-25 concentration in the urine may offer a different perspective on disease status from that provided by urinary albumin. This assay could provide a useful tool for determining urinary Bang-25, which may prove an important biomarker for diabetic kidney disease.

Targeting the Integrated Stress Response Kinase GCN2 to Modulate Retroviral Integration.

Multiple viral targets are now available in the clinic to fight HIV infection. Even if this targeted therapy is highly effective at suppressing viral replication, caregivers are facing growing therapeutic failures in patients due to resistance, with or without treatment-adherence glitches. Accordingly, it is important to better understand how HIV and other retroviruses replicate in order to propose alternative antiviral strategies. Recent studies have shown that multiple cellular factors are implicated during the integration step and, more specifically, that integrase can be regulated through post-translational modifications. We have shown that integrase is phosphorylated by GCN2, a cellular protein kinase of the integrated stress response, leading to a restriction of HIV replication. In addition, we found that this mechanism is conserved among other retroviruses. Accordingly, we developed an in vitro interaction assay, based on the AlphaLISA technology, to monitor the integrase-GCN2 interaction. From an initial library of 133 FDA-approved molecules, we identified nine compounds that either inhibited or stimulated the interaction between GCN2 and HIV integrase. In vitro characterization of these nine hits validated this pilot screen and demonstrated that the GCN2-integrase interaction could be a viable solution for targeting integrase out of its active site.

Mapping Interactions among Cell-Free Expressed Zika Virus Proteins.

The rapid spread of arthropod-borne Zika virus poses a serious public health threat that calls for effective ways of controlling and treating viral infection. This in turn necessitates better understanding of the mechanisms of virus assembly and its interaction with the host cells. In order to facilitate such efforts, we developed a new multihost expression vector pmCellFree that allows rapid and multiplexed production of ZIKV proteins in any in vitro translation system as well as in mammalian cells. Using a combination of in vitro expression in Leishmania cell-free system and AlphaLISA interaction assay, pairwise protein interactions of all ZIKV proteins were systematically tested. We identified thirty-three intraviral binary protein interactions, of which 13 interactions are novel. These findings were further validated by expressing selected protein pairs in mammalian HEK293T cell line and assessing their interactions in the cellular lysate. The results of these interaction assays were identical to those obtained with in vitro expressed proteins. The observed novel protein-protein interactions were further validated using a pulldown assay. The unrevealed novel protein interactions may point to the previously unappreciated complexity of the ZIKV assembly process and may play an important role in the infection process. These interactions may represent new targets for antiviral drug development.

Anti-FIRΔexon2, a splicing variant form of PUF60, autoantibody is detected in the sera of esophageal squamous cell carcinoma.

Anti-PUF60 autoantibodies are reportedly detected in the sera of patients with dermatomyositis and Sjögren's syndrome; however, little is known regarding its existence in the sera of cancer patients. FIR, a splicing variant of the PUF60 gene, is a transcriptional repressor of c-myc. In colorectal cancer, there is an overexpression of the dominant negative form of FIR, in which exon 2 is lacking (FIRΔexon2). Previously, large-scale SEREX (serological identification of antigens by recombinant cDNA expression cloning) screenings have identified anti-FIR autoantibodies in the sera of cancer patients. In the present study, we revealed the presence and significance of anti-FIR (FIR/FIRΔexon2) Abs in the sera of patients with esophageal squamous cell carcinoma (ESCC). Our results were validated by an amplified luminescence proximity homogeneous assay using sera of patients with various cancer types. We revealed that anti-FIRΔexon2 Ab had higher sensitivity than anti-FIR Ab. Receiver operating characteristic (ROC) analysis was applied for evaluating the use of anti-FIRΔexon2 Ab as candidate markers such as anti-p53 Ab and carcinoembryonic antigen, and the highest area under the ROC curve was observed in the combination of anti-FIRΔexon2 Ab and anti-p53 Ab. In summary, our results suggest the use of anti-FIRΔexon2 Ab in combination with the anti-p53 Ab as a predictive marker for ESCC. The area under the ROC curve was further increased in the advanced stage of ESCC. The value of anti-FIRΔexon2 autoantibody as novel clinical indicator against ESCC and as a companion diagnostic tool is discussed.

Discovery of potent DOT1L inhibitors by AlphaLISA based High Throughput Screening assay.

DOT1L (the disruptor of telomeric silencing 1-like), through its methyltransferase activity of H3K79, plays essential roles in transcriptional regulation, cell cycle regulation, and DNA damage response. In addition, DOT1L is believed to be involved in the development of MLL-rearranged leukemia driven by the MLL (mixed-lineage leukemia) fusion proteins, which thus to be a crucial target for leukemia therapy. Hence, discovering of novel DOT1L inhibitors has been in a great demand. In this study, we initiated the discovering process from setting up the AlphaLISA based High Throughput Screening (HTS) assay of DOT1L. Combining with radioactive inhibition assay and Surface Plasmon Resonance (SPR) binding assay, we identified compound 3 and its active analogues as novel DOT1L inhibitors with IC50 values range from 7 µM to 20 µM in vitro. Together with the analysis of structure activity relationships (SAR) and binding modes of these compounds, we provided clues to assist in the future development of more potent DOT1L inhibitors. Moreover, compounds 3 and 9 effectively inhibited the proliferation of MLL-rearranged leukemia cells MV4-11, which could induce cell cycle arrest and apoptosis. In conclusion, we developed a HTS platform based on AlphaLISA method for screening and discovery of DOT1L novel inhibitor, through which we discovered compound 3 and its analogues as potent DOT1L inhibitors with promising MLL-rearranged leukemia therapeutic application.

Serologic assay for diagnosis of celiac disease based on a patient-derived monoclonal antigliadin antibody.

BACKGROUND & AIMS: Patients with celiac disease can be identified based on the detection of serum antibodies to deamidated gliadin peptides (DGPs). Recombinant human monoclonal antibodies (hmAb) against gliadin are produced by cloning antibody genes from single IgA-producing plasma cells isolated from lesions of patients with celiac disease. We developed an assay to identify patients with celiac disease based on the ability of antibodies from their serum to inhibit the binding of a gliadin-specific hmAb (1002-1E03) to a specific peptide antigen (inhibition assay). METHODS: We selected 2 peptides (a 34-mer and a 26-mer) found in ω-gliadins and low-molecular-weight glutenins that had been identified as specific targets of the hmAb 1002-1E03 from a digest of gliadin treated by transglutaminase 2. These peptides contained repeat sequence motifs; their interaction with hmAb 1002-1E03 was assessed in an amplified luminescent proximity homogeneous inhibition assay. We also tested peptides we created that included 3 repeated sequence motifs. Serum samples from untreated patients diagnosed with celiac disease (n = 106) and 2 control groups (198 blood donors, 151 patients with Crohn's disease) were analyzed using the assay, as well as in conventional commercial assays that measure IgA against transglutaminase 2 (TG2) or IgG against DGP. RESULTS: In our inhibition assays, the 34-mer peptide showed the best results, and identified patients with celiac disease with 86.8% sensitivity and 98.6% specificity. Its diagnostic accuracy was comparable with that of commercial anti-DGP IgG (sensitivity, 87.9%; specificity, 98.0) and anti-TG2 IgA (sensitivity, 81.1%; specificity, 98.9) assays, and it detected most of the patients with anti-TG2 IgA-negative celiac disease without a significant decrease in specificity. Combined use of the anti-ω34 and the anti-TG2 assays produced specificity and sensitivity values of 95.3% and 98.0%, respectively. CONCLUSIONS: We developed an antigliadin inhibition assay that identifies patients with celiac disease with high levels of specificity and sensitivity. It may prove useful as an adjunct to the current assay for anti-TG2 IgA.

Development of a sensitive amplified luminescent proximity homogeneous assay to monitor the interactions between pTEFb and Tat.

The viral transactivator protein (Tat) plays an essential role in the replication of human immunodeficiency type 1 virus (HIV-1) by recruiting the host positive transcription elongation factor (pTEFb) to the RNA polymerase II transcription machinery to enable an efficient HIV-1 RNA elongation process. Blockade of the interaction between Tat and pTEFb represents a novel strategy for developing a new class of antiviral agents. In this study, we developed a homogeneous assay in AlphaLISA (amplified luminescent proximity homogeneous assay) format using His-tagged pTEFb and biotinylated Tat to monitor the interaction between Tat and pTEFb. On optimizing the assay conditions, the signal-to-background ratio was found to be greater than 10-fold. The assay was validated with untagged Tat and peptides known to compete with Tat for pTEFb binding. The Z' of the assay is greater than 0.5, indicating that the assay is robust and can be easily adapted to a high-throughput screening format. Furthermore, the affinity between Tat and pTEFb was determined to be approximately 20 pM, and only 7% of purified Tat was found to be active in forming tertiary complex with pTEFb. Development of this assay should facilitate the discovery of a new class of antiviral agents providing HIV-1 patients with broader treatment choices.

A novel immunoassay for the quantization of CYFRA 21-1 in human serum.

BACKGROUND: Cytokeratin 19 fragment antigen (CYFRA 21-1) is used to diagnose and monitor neoplasms. However, the main disadvantages of the currently available CYFRA 21-1 assays include heterogenous technology, being time-consuming, and having low through-put with low insensitivity. This study investigated the use of amplified luminescent proximity homogeneous immunoassay (AlphaLISA) for the quantization of CYFRA 21-1 in human serum. METHODS: The AlphaLISA kit was developed based on AlphaScreen detection technology with two different anti-CYFRA 21-1 monoclonal antibodies. One was coated on AlphaLISA acceptor beads and the other was biotinylated. Donor beads were coated with streptavidin. The test conditions were optimized and analytical performance was studied. RESULTS: The measurement range of AlphaLISA CYFRA 21-1 kit was 0.08-500 ng/ml. Assay detection limit was 0.08 ng/ml. The intra- and interassay coefficients of variation were 3.00-9.00% and 4.00-10.00%, respectively. There was no cross-reaction to alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), neuron-specific enolase (NSE), cancer antigen 19-9 (CA19-9), cytokeratins 8 (CK8), and cytokeratins 18 (CK18). The correlation coefficient of blood samples involved was 0.974 between CYFRA 21-1-AlphaLISA assay and a commercial electrochemiluminescence immunoassay (ECLIA) CYFRA 21-1 kit (Roche). CONCLUSIONS: The AlphaLISA CYFRA 21-1 kit developed in this study had favorable performance characteristics for clinical application with acceptable analytical sensitivity, specificity, and accuracy.

Identification of antibody against wingless­type MMTV integration site family member 7B as a biliary cancer tumor marker.

Biliary cancer has a poor prognosis due to a lack of specific biomarkers and difficulty in diagnosis. The present study aimed to identify serum tumor markers for the diagnosis of biliary cancer via serological identification of antigens by recombinant cDNA expression cloning. Wingless­type MMTV integration site family, member 7 (WNT7B) was identified as a target antigen, suggesting the presence of serum antibodies against this antigen. Deletion mutants were then prepared to evaluate the response to serum antibodies. When serum antibody levels against WNT7B deletion mutants (WNT7B-92­2, -92­260, 2-260 and 184-260) were examined using amplified luminescence proximity homogeneous assay­linked immunosorbent assay, the levels of the antibody against WNT7B with amino acids 184­260 were higher in patients with biliary cancer than in healthy donors. Therefore, the region covering residues 184­260 of WNT7B was decomposed to generate seven peptides, and the levels of antibodies against these peptides were measured. Among them, the levels of antibodies against WNT7B234­253 and WNT7B244­260 were higher in patients with biliary cancers than in healthy donors (WNT7B234­253, P=0.0009; WNT7B244­260, P=0.0005). The levels of the antibody against the former were specifically high in patients with biliary cancer but not in those with esophageal, gastric, colorectal, pancreatic, or breast cancer. Furthermore, analysis by the cutoff value of WNT7B234­253 defined by ROC showed a high sensitivity of 70% in patients with biliary cancer. Therefore, the serum levels of the antibody against WNT7B234­253 may be useful as a marker for biliary cancer diagnosis.

Diagnosis of septic shock by serum measurement of human neutrophil lipocalin by a rapid homogeneous assay.

BACKGROUND: Human neutrophil lipocalin (HNL) is a marker of neutrophil activation and has a high efficacy in diagnosing bacterial infections. In this study, we applied the AlphaLISA technique to measure the serum level of HNL, evaluate HNL's efficacy in diagnosing septic shock, and identify any association between HNL level and septic patients' prognosis. METHODS: We collected 146 serum samples from the Fifth Medical Center of Chinese PLA General Hospital. HNL was measured by AlphaLISA and results were compared with commercial ELISA kits. We studied 78 patients admitted to the ICU with sepsis and data on their clinical and physiological characteristics were recorded. Blood levels of HNL, procalcitonin (PCT), high-sensitivity C-reactive protein (hs-CRP), and lactate were measured. A receiver operating characteristic (ROC) curve was used to evaluate the performance of each marker. RESULTS: The AlphaLISA assay for serum HNL had a detection range from 1.5 ng/mL to 1000 ng/mL, with a detection limit of 1 ng/mL and a detection time of approximately 25 min. The AlphaLISA assay's results were in high agreement with ELISA results (R2 = 0.9413). HNL levels were analyzed in sepsis patients, and HNL was significantly higher in sepsis patients with shock compared to sepsis patients without shock (median 356.47 ng/mL vs 158.93 ng/mL, P < 0.0001) and in the 28-day non-survivor group compared to the 28-day survivor group (median 331.83 ng/mL vs 175.17 ng/mL, P < 0.0001). ROC curve analysis was performed for the biomarkers. In differentiating the diagnosis of septic shock from sepsis patients, HNL was the most effective marker (AUC = 0.857), followed by PCT (AUC = 0.754) and hs-CRP (AUC = 0.627). In predicting the prognosis of septic patients, lactate had the best effect (AUC = 0.805), followed by HNL (AUC = 0.784), PCT (AUC = 0.721), and hs-CRP (AUC = 0.583). CONCLUSIONS: As an assessment tool, we found that our AlphaLISA had good consistency with an ELISA and had several other advantages, including requiring a shorter processing time and detecting a wider range of serum HNL concentrations. Monitoring serum HNL levels of patients admitted to the ICU might be useful in distinguishing sepsis patients who have septic shock from other sepsis patients, indicating its value in the prediction of sepsis patient prognosis.