Detection of H pylori antibody profile in serum by protein array

AIM： To detect multiple H pylori antibodies in serum samples of individuals who carryHpylori by protein array. METHODS： Recombinant H pylori antigens, urease B subunit （UreB）, vacuolating toxin A （VacA） and cytotoxin associated gene A protein （CagA）, were prepared and immobilized in matrixes on nitrocellulose membrane by robotics to bind the specific immunoglobulin G （IgG） antibodies in serum. Staphylococcus protein A （SPA） labeled by colloid gold was used to integrate the immuno-complex and gave red color signal. The scanner based on charge-coupled device （CCD） could collect the image signal and convert it into digital signal. RESULTS： When human IgG was printed on the membrane in increasing concentrations and incubated with immunogold, a linear dose response curve was obtained and the detection limit for IgG was about 0.025 ng. The cutoff values, which were defined as the mean grey level plus 3 times of standard deviation, were 27.183, 28.546 and 27.402, for anti-UreB IgG, anti- CagA IgG and anti-VacA IgG, respectively, as 400 human serum samples with negative H pylori antibodies were detected by the protein array. When 180 serum samples from patients in hospital were employed for detection of IgG against UreB, CagA and VacA, the sensitivity of the protein array was 93.4%, 95.4%, 96.0%, and the specificity was 94.8%, 94.4% and 97.5%, respectively, as compared with the results obtained by ELISA. The assay also showed high reproducibility, uniformity and stability, and the results were available within 30 min. CONCLUSION： The protein array is a very practical method for rapid detection of multiple antibodies in serum samples. It is especially useful for large scale epidemiological investigation of the infection of Hpylori.

Phosphorylated protein chip combined with artificial intelligence tools for precise drug screening

We have developed a protein array system,named"Phospho-Totum",which reproduces the phosphorylation state of a sample on the array.The protein array contains 1471 proteins from 273 known signaling pathways.According to the activation degrees of tyrosine kinases in the sample,the corresponding groups of substrate proteins on the array are phosphorylated under the same conditions.In addition to measuring the phosphorylation levels of the 1471 substrates,we have developed and performed the artificial intelligence-assisted tools to further characterize the phosphorylation state and estimate pathway activation,tyrosine kinase activation,and a list of kinase inhibitors that produce phosphorylation states similar to that of the sample.The Phospho-Totum system,which seamlessly links and interrogates the measurements and analyses,has the potential to not only elucidate pathophysiological mechanisms in diseases by reproducing the phosphorylation state of samples,but also be useful for drug discovery,particularly for screening targeted kinases for potential drug kinase inhibitors.

Protein Array-based Approaches for Biomarker Discovery in Cancer

Biomarkers are deemed to be potential tools in early diagnosis, therapeutic monitoring,and prognosis evaluation for cancer, with simplicity as well as economic advantages compared with computed tomography and biopsy. However, most of the current cancer biomarkers present insufficient sensitivity as well as specificity. Therefore, there is urgent requirement for the discovery of biomarkers for cancer. As one of the most exciting emerging technologies, protein array provides a versatile and robust platform in cancer proteomics research because it shows tremendous advantages of miniaturized features, high throughput, and sensitive detections in last decades. Here, we will present a relatively complete picture on the characteristics and advance of different types of protein arrays in application for biomarker discovery in cancer, and give the future perspectives in this area of research.

Protein array of Coxiella burnetii probed with Q fever sera

Coxiella burnetii is the etiological agent of Q fever.To identify its major seroreactive proteins,a subgenomic protein array was developed.A total of 101 assumed virulence-associated recombinant proteins of C.burnetii were probed with sera from mice experimentally infected with C.burnetii and sera from Q fever patients.Sixteen proteins were recognized as major seroreactive antigens by the mouse sera.Seven of these 16 proteins reacted positively with at least 45% of Q fever patient sera.Notably,HspB had the highest fluorescence intensity value and positive frequency of all the proteins on the array when probed with both Q fever patient sera and mouse sera.These results suggest that these seven major seroreactive proteins,particularly HspB,are potential serodiagnostic and subunit vaccine antigens of Q fever.

Changes in proteins related to early nerve repair in a rat model of sciatic nerve injury

Peripheral nerves have a limited capacity for self-repair and those that are severely damaged or have significant defects are challenging to repair. Investigating the pathophysiology of peripheral nerve repair is important for the clinical treatment of peripheral nerve repair and regeneration. In this study, rat models of right sciatic nerve injury were established by a clamping method. Protein chip assay was performed to quantify the levels of neurotrophic, inflammation-related, chemotaxis-related and cell generation-related factors in the sciatic nerve within 7 days after injury. The results revealed that the expression levels of neurotrophic factors(ciliary neurotrophic factor) and inflammationrelated factors(intercellular cell adhesion molecule-1, interferon γ, interleukin-1α, interleukin-2, interleukin-4, interleukin-6, monocyte chemoattractant protein-1, prolactin R, receptor of advanced glycation end products and tumor necrosis factor-α), chemotaxis-related factors(cytokine-induced neutrophil chemoattractant-1, L-selectin and platelet-derived growth factor-AA) and cell generation-related factors(granulocyte-macrophage colony-stimulating factor) followed different trajectories. These findings will help clarify the pathophysiology of sciatic nerve injury repair and develop clinical treatments of peripheral nerve injury. This study was approved by the Ethics Committee of Peking University People's Hospital of China(approval No. 2015-50) on December 9, 2015.

Topical application of glycyrrhizin preparation ameliorates experimentally induced colitis in rats

AIM:To examine the efficacy of glycyrrhizin preparation(GL-p) in the treatment of a rat model of ulcerative colitis(UC).METHODS:Experimental colitis was induced by oral administration of dextran sodium sulfate.Rats with colitis were intrarectally administered GL-p or saline.The extent of colitis was evaluated based on body weight gain,colon wet weight,and macroscopic damage score.The expression levels of pro-inflammatory cytokines and chemokines in the inflamed mucosa were measured by cytokine antibody array analysis.The effect of GL-p on myeloperoxidase(MPO) activity in the inflamed mucosa and purified enzyme was assayed.RESULTS:GL-p treatment significantly ameliorated the extent of colitis compared to sham treatment with saline.Cytokine antibody array analysis showed that GL-p treatment significantly decreased the expression levels of pro-inflammatory cytokines and chemokines,including interleukin(IL)-1β,IL-6,tumor necrosis factor-α,cytokine-induced neutrophil chemoattractant-2,and monocyte chemoattractant protein-1 in the inflamed mucosa.Furthermore,GL-p inhibited the oxidative activity of mucosal and purified MPO.CONCLUSION:GL-p enema has a therapeutic effect on experimental colitis in rats and may be useful in the treatment of UC.

Effects of acupuncture on the number of associated protein phosphorylation in brain tissues of MCAO rats based on protein microarray technique

Objective： To investigate the effects of acupuncture on the number of associated phosphorylated proteins in brain tissues of middle cerebral artery occlusion （MCAO） rats, based on the protein microarray technique. Methods： The MCAO model was prepared according to the modified occlusion method using occlusion lines. Forty healthy Sprague-Dawley （SD） rats were randomly divided into 4 groups using the lottery method： a sham operation group, a model group, a control point group and an acupoint group, with 10 rats in each group. Rats in the sham operation group and the model group only received binding without acupuncture. Rats in the acupoint group received acupuncture at Dazhui （GV 14）, Baihui （GV 20） and Shuigou （GV 25）; rats in the control point group received acupuncture at non-acupoint control points. The needle was twisted once for 1 min after insertion and another time in the middle of the 30 min needle retaining. Acupuncture was conducted once every 12 h for 6 consecutive times. At the end of the experiment, the neurological impairment score was collected, and cells of the ischemic brain tissues were extracted. The protein phosphorylation of the related signaling was detected using the 720 phosphorylated antibody microarray technique, and the differentially expressed proteins between groups were screened. Results： The neurological impairment scores after 72 h of treatment： compared with the sham operation group, the scores of the model group, the control point group and the acupoint group were significantly increased （P〈0.01）; compared with the model group, the scores of the acupoint group and the control point group were significantly decreased （P〈0.01,P〈0.05）; the score of the acupoint group was better than that of the control point group （P〈0.05）. The results of the protein microarray： compared with the sham operation group, 48 proteins showed up-regulated phosphorylation （≥1.5 times） in the model group and the down-regulated was 28; compared with the model group, 35 proteins showed up-regulated phosphorylation in the control point group, and the down-regulated was 24. There were 29 proteins showing up-regulated phosphorylation in the acupoint group and the down-regulated was 51. The numbers of proteins involved in the function and signal transduction pathways were also different. Conclusion： Acupuncture at Dazhui （GV 14）, Baihui （GV 20） and Shuigou （GV 25） can effectively repair brain injury. The ischemic injury of brain tissue may be caused by imbalance of a variety of proteins, and acupuncture can promote brain tissue repair by multi-functional and multi-channel regulation of the protein disorders.

Application of Nanogold Probe Coupled with Silver Enhancement in Rapid cTnI Colorimetric Immunoassay

A rapid, inexpensive, reliable, and flexible quantitative immunoassay for cardiac troponin I （cTnI） based on the concepts of one-step dual monoclonal antibody “sandwich” principle. The low density protein array, the nanogold probe, and the silver enhancement on the gold particle were provided. The whole detection procedure of the assay could be fulfilled within 40 min with the pretreated colloidal gold-labeled detection antibody and supporting substrate. The assay showed good specific response to cTnI with very low cross-reactivity ratio to the skeletal isoforms of troponin I （sTnI）, cardiac troponin T （cTnT）, and myoglobin. 588 serum samples were assayed simultaneously by enzyme-linked immuno sorbent assay （ELISA） and this colloidal gold method to test the validity of the method and the data were analyzed using the statistical package SPSS version 11.0 （SPSS Inc.）. There was no significant difference between these two assays （P=0.66〉0.05）. The agreement between this method （〉 or 〈0.3 ng/mL） and ELISA was 86%.

Serum cytokine levels in chronic hepatitis B patients receiving peginterferon alpha-2a therapy

BACKGROUND: The relationship between cytokines and responses to peginterferon α-2a treatment in chronic hepatitis B patients has not yet been fully elucidated. We analyzed the serum levels of interleukin (IL)-1α, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, vascular endothelial growth factor, interferon-γ, tumor necrosis factor-α, monocyte chemotactic protein-1 (MCP1) and epidermal growth factor during the treatment with peginterferon α-2a. METHODS: Ninety-three serum samples from 20 chronic hepatitis B patients were collected before, during and after 48 weeks of peginterferon therapy and were assayed for 12 cytokines. The patients were categorized as either virologic responders (VRs) or non-responders (NRs) according to their HBV DNA levels taken at 6th month during treatment. The Evidence Investigator (Randox, Antrim, UK), a protein chip analyzer, was used to quantify cytokines. RESULTS: Among the 12 cytokines, the levels of MCP1 were increased and the levels of IL-4 were decreased during the treatment in VRs. However these cytokines were not significantly changed in NRs in the treatment phases. Area under the receiver operating characteristic curve (AUROC) value of HBV DNA measured before the treatment was 0.81 in predicting VRs, and that of the baseline MCP1 was 0.76. IL-6 levels at 3rd and 6th months during the treatment also showed AUROC values 0.85 and 0.78 respectively in predicting sustained VRs. CONCLUSION: Serum cytokine levels reflect the pathological differences of individual treatment phases and could also be useful in monitoring responses to peginterferon treatment in chronic hepatitis B patients.

Applied research on serum protein fingerprints for prediction of Qi deficiency syndrome and phlegm and blood stasis in patients with non-small cell lung cancer

OBJECTIVE:This study screened serum tumor biomarkers by surface enhanced laser desorption/ionization time-of-flight mass spectrometry(SELDI-TOF-MS) to establish a subset which could be used for the prediction of Qi deficiency syndrome and phlegm and blood stasis in patients with non-small cell lung cancer;and as diagnostic model of Chinese medicine.METHODS:Serum samples from 63 lung cancer patients with Qi deficiency syndrome and phlegm and blood stasis,and 28 lung cancer patients with non-Qi deficiency syndrome and phlegm and blood stasis were analyzed using SELDI-TOF-MS with a PBS II-C protein chip reader.Protein profiles were generated using immobilized metal affinity capture(IMAC3) protein chips.Differentially-expressed proteins were screened.Protein peak clustering and classification analyses were performed using Biomarker Wizard and Biomarker Pattern software packages,respectively.RESULTS:A total of 268 effective protein peaks were detected in the 1,000-10,000 Da molecular range for the 15 serum proteins screened(P<0.05).The decision tree model was M 2284.97,with a sensitivity of 96.2% and a specificity of 66.7%.CONCLUSION:SELDI-TOF-MS techniques,combined with a decision tree model,can help identify serum proteomic biomarkers related to Qi deficiency syndrome and phlegm and blood stasis in lung cancer patients;and the predictive model can be used to discriminate between Chinese medicine diagnostic models of disease.

Influence of irbesartan on the urinary excretion of cytokines in patients with chronic kidney disease

Background The non-hemodynamic effects of angiotensin receptor blocker （ARB） in the delay of progression of chronic kidney disease （CKD） remain unclear. In this study, we investigated the influence of irbesartan on the urinary excretion of cytokines in patients with CKD. Methods In this randomized perspective clinical trial, different doses of irbesartan （150 mg/d and 300 mg/d） were given to two groups of patients in a cross-over design. Blood pressure （BP）, creatinine clearance （Ccr） and 24-hour proteinuria were examined. Urinary excretion of cytokines was determined by human inflammatory cytokine antibody array. A two-fold change in spot intensity was considered significant. Results Urinary excretion of cytokines （granulocyte colony stimulating factor （GCSF）, intercellular cell adhesion molecule-1 （ICAM-1）, interferon y （IFN-y）, interleukin 1β （IL-1β）, IL-2, IL-6, IL-8, IL-11, IL-15 and macrophage inflammatory protein 1δ （MIP-Iδ） in group B （irbesartan 300 mg/d） was significantly decreased in comparison to group A （irbesartan 150 mg/d） after 8-week treatment. In group A, 8 weeks of treatment induced a two- to nine-fold reduction in urinary cytokine levels （GCSF, GM-CSF, IFN-γ, IL-1α, IL-11, IL-12p40, MCP-2, MIP-1α）, while increasing the dosage to 300 mg/d further decreased the excretion of GCSF, GM-CSF, IL-12p40, MCP-2 and MIP-1α by week 18. There was no significant difference in BP or Ccr between the two groups. However, 24-hour proteinuria was significantly reduced in both groups, and in group A the reduction was dose dependent.Conclusion Irbesartan offers additional renoprotection in a dose-dependent manner by reducing pro-inflammatory cvtokines excretion in the urine of CKD patients.

Apoptosis of non-tumor cells contributes to increased serum cytochrome c level in a neuroblastoma xenograft model

Background Neuroblastoma （NB） is one of the most common malignant solid tumors of childhood.It is still not clear whether the apoptosis of tumor cells or the non-tumor cells contributes to the increase of concentration of cytochrome c （Cyt c） in the serum of the cancer patients.The aim of this research was to identify the source of the Cyt c in the serum when the tumor grows up by subcutaneous inoculation of human NB cells into nude mice.Methods We subcutaneously inoculated human NB cells （KP-N-NS） into nude mice and collected the sera of tumor-bearing mice （n=14） and control mice （n=25） 4 weeks later in order to screen for and identify differentially expressed proteins in the serum.Differentially expressed proteins in the serum were screened by surface-enhanced laser desorption/ionization-time-of-fiight （SELDI-TOF） mass spectrometry.Results The relative intensity of a protein having a mass-to-charge ratio （m/z） of 11 609 was 3338.37±3410.85 in the tumor group and 59.84±40.74 in the control group,indicating that the expression level of this protein in the tumor group was 55.8 times higher than that in the control group.Serum proteins were separated and purified by high-performance liquid chromatography （HPLC）.Liquid chromatography coupled with tandem mass spectrometry （LC-MS/MS） was performed to produce peptide mass fingerprints （PMFs）.Spectrum analysis and a database search revealed that the highly expressed protein （m/z=11605.4） from the serum of tumor-bearing mice was the mouse Cyt c.Conclusions Increased concentration of Cyt c in the serum of tumor-bearing nude mice might be partially attributed to the secretion of this protein by non-tumor cells.