Application of iPDMS protein microarray in screening of tumor-associated antigen autoantibodies / 生物工程学报

The rapid screening of tumor markers is a challenging task for early diagnosis of cancer. This study aims to use highly sensitive chemiluminescent protein microarray technology to efficiently screen a variety of low abundance tumor related markers. A new material, termed integrated polydimethylsiloxane modified silica gel (iPDMS), was obtained by adding a surface polymerization initiator with olefin end to the conventional polydimethylsiloxane, and fixing into the three-dimensional structure of polydimethylsiloxane by thermal crosslinking through silicon hydrogen bonding. In order to make the iPDMS material resistant to non-specific protein adsorption, a poly(OEGMA) polymer brush was synthesized by surface-initiated atom transfer radical polymerization at the active initiation site. Finally, 20 tumor-related antigens were printed into the specific areas of the microarray by high-throughput spray printing technology, and assembled into 48-well detection microtiterplates of the iPDMS microarray. It was found the VEGFR and VEGF121 autoantibodies that obtained from 8 common tumors (breast cancer, lung cancer, colon cancer, gastric cancer, liver cancer, leukemia, lymphoma and ovarian cancer) can be used as potential tumor markers. The chemiluminescence labeled iPDMS protein microarray can be used for the screening of tumor autoantibodies at early stage.

The Immunome of Colon Cancer: Functional In Silico Analysis of Antigenic Proteins Deduced from IgG Microarray Profiling / 基因组蛋白质组与生物信息学报·英文版

Characterization of the colon cancer immunome and its autoantibody signature from differentially-reactive antigens (DIRAGs) could provide insights into aberrant cellular mechanisms or enriched networks associated with diseases. The purpose of this study was to characterize the antibody profile of plasma samples from 32 colorectal cancer (CRC) patients and 32 controls using proteins isolated from 15,417 human cDNA expression clones on microarrays. 671 unique DIRAGs were identified and 632 were more highly reactive in CRC samples. Bioinformatics analyses reveal that compared to control samples, the immunoproteomic IgG profiling of CRC samples is mainly associated with cell death, survival, and proliferation pathways, especially proteins involved in EIF2 and mTOR signaling. Ribosomal proteins (e.g., RPL7, RPL22, and RPL27A) and CRC-related genes such as APC, AXIN1, E2F4, MSH2, PMS2, and TP53 were highly enriched. In addition, differential pathways were observed between the CRC and control samples. Furthermore, 103 DIRAGs were reported in the SEREX antigen database, demonstrating our ability to identify known and new reactive antigens. We also found an overlap of 7 antigens with 48 "CRC genes." These data indicate that immunomics profiling on protein microarrays is able to reveal the complexity of immune responses in cancerous diseases and faithfully reflects the underlying pathology.

Detection of Serum CA19-9 Levels in Patients with Primary Hepatic Carcinoma by Using Chemiluminescent Protein Microarray / 现代生物医学进展

Objective:To develop a simple,effective,low-cost,time saving chemiluminescent protein microarray to detect the serum CA19-9 level of patients with primary hepatic carcinoma.Methods:A protein microarray was developed for detecting CA19-9 levels in the serum samples after spotting mouse-derived CA19-9 monoclonal antibody on an aldehyde-based chip.Serum from primary hepatic carcinoma (n=46) and healthy controls (n=32) were tested by using this assay.Results:The results showed that 24 out of 46 patients with primary hepatic carcinoma had serum CA19-9 levels above 37 U/mL,and 22 out of 46 patients with primary hepatic carcinoma had serum CA19-9 levels under 37 U/mL.In healthy control,30 out of 32 total cases were under 37 U/ml.Only two healthy controls were 37 U/mL.The sensitivity,specificity and AUC of protein microarray were 52.17％,93.75％,0.688 [95％ CI:0.566,0.811].Conclusion:A chemiluminescent protein microarray method was established for detection of CA 19-9 in serum.

Evaluation of mycobacterium tuberculosis IgG antibody protein chip in patients with active and inactive tuberculosis / 中国生化药物杂志

Objective To explore protein microarray chip diagnostic value for patients with active and inactive tuberculosis.Methods 178 cases of active patients tuberculosis and 79 cases of inactive tuberculosis patients and 92 cases of healthy control using protein microarray chip detection.Results Tuberculosis protein chip had a diagnostic value for tuberculosis and the positive rate is 58.4%; combined the diagnostic value of three kinds of proteins is higher than the diagnostic value of a single protein;16 kD protein of inactive tuberculosis positive rate was 16.4%, better than the positive rate of 3.4% for active tuberculosis (P<0.05).Conclusion Tuberculosis protein chip has a diagnostic value for active tuberculosis and inactive tuberculosis.16 kD protein positive rate more than 38 kD protein in patients with inactive tuberculosis (P<0.05).

Protein microarray for complex apoptosis monitoring of dysplastic oral keratinocytes in experimental photodynamic therapy

BACKGROUND: Photodynamic therapy is an alternative treatment of muco-cutaneous tumors that uses a light source able to photoactivate a chemical compound that acts as a photosensitizer. The phthalocyanines append to a wide chemical class that encompasses a large range of compounds; out of them aluminium-substituted disulphonated phthalocyanine possesses a good photosensitizing potential. RESULTS: The destructive effects of PDT with aluminium-substituted disulphonated phthalocyanine are achieved by induction of apoptosis in tumoral cells as assessed by flow cytometry analysis. Using protein microarray we evaluate the possible molecular pathways by which photodynamic therapy activates apoptosis in dysplastic oral keratinocytes cells, leading to the tumoral cells destruction. Among assessed analytes, Bcl-2, P70S6K kinase, Raf-1 and Bad proteins represent the apoptosis related biomolecules that showed expression variations with the greatest amplitude. CONCLUSIONS: Up to date, the intimate molecular apoptotic mechanisms activated by photodynamic therapy with this type of phthalocyanine in dysplastic human oral keratinocytes are not completely elucidated. With protein microarray as high-throughput proteomic approach a better understanding of the manner in which photodynamic therapy leads to tumoral cell destruction can be obtained, by depicting apoptotic molecules that can be potentially triggered in future anti-tumoral therapies.

Experimental study on infertility related antibody detection in female infertility / 国际检验医学杂志

Objective To study whether the correlation exists between 6 kinds of autoimmune antibodies and female infertility . Methods The protein microarray technique was adopted to detect serum antibodies of anti-sperm antibodies(ASAb) ,anti-endome-trial(AEmAb) ,anti-zona pellucida(AZpAb) ,anti-ovarian(AVoAb) ,anti-hCG(AhCG Ab) and anti-trophoblastic(ATAb) in 140 ca-ses of female infertility and the correlation between these 6 kinds of antibody with female infertility was evaluated .Results Com-pared with the control group ,except AvoAb ,the positive rates of other 5 kinds of antibody had statistically significant differences (P0 .05) .The positive rate of ANA had no statistical difference between the infertility group and the control group (P>0 .05) ,but the titers of ANA in the experimenter were ≥1∶100(6/7) and most of karyotypes were nuclear coarse (5/7) ,which was differed from the low positive titer in the control group .The positive rate of ANA had statistical difference between the infertility related antibody positive and the infertility related antibody negative in the infertility patients .Conclusion ASAb ,AZpAb AEmAb , AhCGAb and ATAb are related with female sterility .The combined detection of these 6 kinds of antibody can significantly increase the detection sensitivity .

Determination of the serum antibody in pneumonic plague patients / 中国地方病学杂志

Objective To analyze the species of the antibody and immune responsibility in pneumonic plague patients in order to pave the way to screen the new sub-unit of the vaccine to provide the experimental basis. Methods Using the virulence-related protein microarray containing 149 proteins of Yersinia pestis (Y.pestis), the species of the antibody and immune responsibility were analyzed in serum of two pneumonic plague patients in six months after onset. Results Eighty-eight gene coded proteins were detected out the related antibodies except YPMT1.23c, YPMT1.86, YPO0406 and YPO1071 in patient 1. Forty-three antibodies from gene coded protein were analyzed, other forty-nine had not been identified in patient 2. Thirty-nine antibodies were detected in both patients. The proteins YPMT1.81c, YPMT1.84, YPCD1.31c, rw10, YPCD1.28, YPCD1.58, YPMT1.62c, YPO3247-related antibodies increased significantly by 109.96,176.4 ;20.64,17.73 ;16.50,7.16 ;23.51,7.65 ;46.00,25.61 ;4.50,8.24 ;5.98,5.08 ;23.98,4.76 folds, respectively. Conclusions The study on the antibody in pneumonic plague patients helps us to select the potential vaccine candidates, which reveals that eight proteins are the immunity diagnosis targets and the research key of sub-unit vaccine.

A study of APin-protein interactions using protein microarray / 대한치과보존학회지

Protein microarray or protein chips is potentially powerful tools for analysis of protein-protein interactions. APin cDNA was previously identified and cloned from a rat odontoblast cDNA library. The purpose of this study was to investigate the APin-protein interactions during ameloblast differentiation. Protein microarray was carried with recombinant APin protein and MEF2, Aurora kinase A, BMPR-IB and EF-hand calcium binding protein were selected among 74 interacting proteins. Immortalized ameloblast cells (ALCs) were transfected with pCMV-APin construct and U6-APin siRNA construct. After transfection, the expression of the mRNAs for four proteins selected by protein micoarrays were assessed by RT-PCR. The results were as follows: 1. APin expression was increased and decreased markedly after its over-expression and inactivation, respectively. 2. Over-expression of the APin in the ALCs markedly down-regulated the expression of MEF2 and Aurora kinase A, whereas their expression remained unchanged by its inactivation. 3. Expression of BMPR-IB and EF-hand calcium binding protein were markedly increased by the overexpression of the APin in the ALCs, whereas expression of BMPR-IB remained unchanged and expression of EF-hand calcium binding protein was markedly decreased by its inactivation. These results suggest that APin plays an important role in ameloblast differentiation and mineralization by regulating the expression of MEF2, Aurora kinase A, BMPR-IB and EF-hand calcium binding protein.

In situ Fabrication and Application of Protein Microarray With Cell-free System / 生物化学与生物物理进展

Protein is one of major bio-functional performers. As one of several crucial proteomic research approaches, protein microarray has these following advantages: high-throughput, high sensitivity, quick detection and so on. Meanwhile, there are some critical factors that are important to the further development of protein microarray technology, for example, how to express and purify proteins for the research of protein microarray, how to immobilize proteins onto the substrate and keep the bio-function of proteins immobilized. Nano-biotechnology and cell-free expression system have been used to fabricate protein microarray by the way of immobilizing target genes onto the substrate and directly expressing corresponding proteins, which provides a new strategy to fabricate more complicated microarray. The stragegy and its progress were summarized———fabrication of protein microarray based on DNA, including immobilization of target genes, cell-free expression to proteins, immobilization of renascence proteins, advantages and drawbacks of the methods of protein chip fabrication etc.

Investigation of Viral Hepatitis Seroepidemiology by Using Protein Microarray / 医学研究杂志

Objective The anti-HCV positive results obtained from ELISA assay was confirmed by using protein microarray, and then the difference of detection rate of different antibody function domains was studied through observing low risk population. Methods The HCV mixing antigen, core area antigen, NS3 domain antigen, NS4 domain antigen, NS5 domain antigen were spotted on the slide. The 48 serum samples, which was positive HCV detected by using ELISA assay, were chosen from the specimens applying to investigation of viral hepatitis seroepidemiology in 2001, and furthermore the antibodies containing different function domain were detected by using protein microarray. Results The negative results were obtained from different domains of 15 samples. Then 1-4 antibodies from 33 samples were positive. The total detection rate of anti-C, anti-NS3, anti-NS4, anti-NS5 was 51.5%, 51.5%, 48.5% and 57.6% respectively. In these above statistics, the detection rate of anti-C from Zhoushan, Quzhou, Wenzhou, Shaoxing was 53.3%, 33.3%, 44.4%, 66.7% respectively; the detection rate of anti-NS3 was 33.3%, 100%, 55.6%, 66.7% respetively; the detection rate of anti-NS4 was 60.0%, 0%, 33.3%, 66.7% respectively; 抗the detection rate of anti-NS5 was 66.7%, 0%, 44.4%, 83.3% respectively. Conclusion The difference of distribution of different antibody function domains existed through studying low risk population. Moreover, the protein microarray assay was highly specific for detecting and identifying HCV antibody in low risk population.

Application and analysis of protein microarray in different drug resistant cell lines of ovarian cancer / 中国病理生理杂志

AIM: To identify the key factors responsible for drug resistance in different ovarian cancer cell lines using protein microarray system. METHODS: Six ovarian cancer cell lines were employed. The sensitivity of ovarian cancer cell line to common chemotherapeutic drugs was determined by using MTT assays. The expression of 78 cytokines and other factors was examined by using cytokine antibody array technology. RESULTS: Different ovarian cancer cell line responded to chemotherapeutic agents differently. The drug resistance was correlated with certain cytokine expression. Cell line SKOV3 was less sensitive to first line chemotherapeutic drug (ADM, CBPDA) and accumulated high amounts of GRO and TIMP-2 compared with other 5 cell lines. OVCAR4 cells were more resistant to second line chemotherapeutic drug (TAXOL, VP16) and had higher levers of IL-6 and IL-8 than IGROV1, OVCAR3 and OVCAR5. CONCLUSIONS: Among the most common excretive cytokines, increasing of GRO, IL-6, IL-8 and TIMP-2 might be related to drug-resistance of ADM and CBPDA in ovarian cancer cell, while IL-6 and IL-8 might also be related with drug resistance of TAXOL and VP16. The different types of ovarian cancer cell might have roughly similar excretive cytokines-induced mechanism of drug resistance.