A protein microarray biosensor based on imaging ellipsometry has been developed as a high-throughput and fast technique for protein analysis. As an automatic technique, it has advanced properties such as label-free, multi-protein simultaneous detection, static or kinetic analysis for protein interaction, and qualitative or quantitative analysis. It has been used for the biomedical applications including tumor markers detection, hepatitis B test, protein competitive adsorption and kinetic visualization for protein interactions. It have demonstrated promising potential for further applications in biomedicine.
Biosensing Techniques ; methods ; trends ; Humans ; Protein Array Analysis ; methods ; trends

Drug Design ; Genomics ; methods ; Oligonucleotide Array Sequence Analysis ; Protein Array Analysis ; Proteomics ; Technology, Pharmaceutical

As one of the proteomic technologies, antibody microarrays can provide a powerful tool for the high-throughput parallel analysis of thousands of proteins in a single experiment. Because antibodies are chemically and structurally complex; immobilizing antibody on solid phase surface of substrates has been a key step during the development of antibody microarrays. This review will focus on the current strategies for antibody immobilization and discuss the advantages and disadvantages of these methods.
Adsorption ; Antibodies, Monoclonal ; chemistry ; metabolism ; Protein Array Analysis ; methods ; Protein Binding

The major bottle-neck in the way of constructing high density protein microarray is the availability and stability of proteins. The traditional methods of generating protein arrays require the in-vivo expression, purification and immobilization of hundreds or thousands of proteins. The cell-free protein array technology employs cell-free expression systems to produce proteins directly onto surface from co-distributed or pre-arrayed DNA or RNA, thus avoiding the laborious and often costly processes of protein preparation in the traditional approach. Here we provide an overview of recently developed novel technology in cell free based protein microarray and their applications in protein interaction analysis, in antibody specificity and vaccine screening, and in biomarker assay.
Cell-Free System ; DNA ; genetics ; Humans ; Protein Array Analysis ; methods ; Protein Biosynthesis ; Protein Interaction Mapping ; Proteins ; analysis ; genetics ; metabolism

OBJECTIVETo establish and evaluate a protein microarray method for combined measurement of serum ferritin (SF) and soluble transferrin receptor (sTfR).

METHODSMicroarrayer was used to print both anti-SF antibodies I and anti-sTfR antibodies I on each protein microarray. Anti-SF antibodies II and anti-sTfR antibodies II were used as detection antibodies and goat antibodies coupled to Cy3 were used as antibodies III. The detection conditions of the quantitative analysis method for simultaneous measurement of SF and sTfR with protein microarray were optimized and evaluated. The protein microarray was compared with commercially available traditional tests with 26 serum samples.

RESULTSBy comparison experiment, mouse monoclonal antibodies were chosen as the probes and contact printing was chosen as the printing method. The concentrations of SF and sTfR probes were 0.5 mg/mL and 0.5 mg/mL respectively, while those of SF and sTfR detection antibodies were 5 μg/mL and 0.36 μg/mL respectively. Intra- and inter-assay variability was between 3.26% and 18.38% for all tests. The regression coefficients comparing protein microarray with traditional test assays were better than 0.81 for SF and sTfR.

CONCLUSIONThe present study has established a protein microarray method for combined measurement of SF and sTfR.

Animals ; Antibodies, Monoclonal ; analysis ; Ferritins ; blood ; Mice ; Protein Array Analysis ; methods ; Rabbits ; Receptors, Transferrin ; blood

In this experiment, a novel biotin-avidin conjugation probe was synthesized and employed in the detection of reverse-phase protein microarray. Firstly, the proportion of the biotin-avidin conjugation probe was optimized. Then the rat IgG and goat anti-rat IgG system was served as a model to optimize the fabrication conditions of reverse-phase protein microarray, including the non-specific absorption of streptavidin-Cy3 molecules, spotting buffer as well as protein activities. At last, the biotin-avidin conjugation probe was applied to the detection of the reverse-phase protein microarray. The results show that the protein microarray prepared by using BSA spotting buffer could prevent non-specific absorptions of fluorescent molecules and improve the sensitivity, effectively. In addition, compared with traditional biotin-avidin system, the detection limit could be improved four times using the biotin-avidin conjugation probe. In conclusion, the biotin-avidin conjugation probe has its merits of easy synthesis, low price and could be further conjugated with other signal amplification techniques, which is promising to be used in the detection of protein microarray.
Avidin ; chemistry ; Biotin ; chemistry ; DNA Probes ; Immunoglobulin G ; analysis ; immunology ; Protein Array Analysis ; methods

Proteinchip profiling is a powerful and innovative proteomic technology for biomarker discovery and diagnostic/prognostic assay development. Based on surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS), Ciphergen's proteinchip system offers a single, unified, high-throughput platform for a multitude of proteomic research applications. Proteins are the major functional components of the cell, the study of proteomics provides mankind with a better understanding of disease and life. The remarkable findings in disease biomarkers have shed light to the early diagnosis, monitoring and predicting prognosis of various diseases, especially for cancer. In this article, the development and technology of SELDI-TOF MS are introduced. Some research progress and encouraging research results in oncoproteomics, infectious diseases, neurological diseases and diabetes mellitus using SELDI-TOF MS are also reviewed. The paper is closed by the appraisals on its pros and cons, as well as the future prospective is also expounded.
Animals ; Computational Biology ; Humans ; Lasers ; Mass Spectrometry ; methods ; Protein Array Analysis ; methods ; Proteomics ; Surface Properties

Animals ; Doping in Sports ; methods ; Drug Design ; Drug Evaluation, Preclinical ; Humans ; Molecular Biology ; methods ; Protein Array Analysis ; methods ; Proteomics ; Toxicology ; methods

We selected 12 antigens corresponding to commonly used autoantibodies in clinical practice to prepare antigen microarray. We chose NBT/BCIP color reaction as the end detection strategy to develop a new autoantibody protein chip detection system. Using this system, we optimized the best spotting solution, spotting concentration of the 12 antigens and the dilution of serum. We prepared a protein chip that could detect 12 autoantibodies simultaneously using the optimized antigen concentration. We established a new method to determine the cutoff of each autoantibodies by evaluation of 678 positive and 120 negative serum of clinical sample. We also evaluated the sensitivity and specificity of our new detection system. The optimal spotting solution was 0.1% TBST, the dilution of serum was 1:4 and the best spotting concentration of the 12 antigens were ANA 520 microg/mL, Ro-60/SSa 465 microg/mL, La/SSb 530 microg/mL, Jo-1 530 microg/mL, Scl-70 525 microg/mL, Sm 520 microg/mL, Ro-52/SSa 615 microg/mL, RF 340 microg/mL, CCP 465 microg/mL, ulRNP 410 microg/mL, CENP-B 490 microg/mL and dsDNA 580 microg/mL respectively. It had higher coincidence rate compared to current clinical used methods. We have developed a 12 antigens protein chip for the detection of autoantibodies based on the NBT/BCIP color reaction system. This system was fast, convenient, efficient, and cost-effective.
Autoantibodies ; blood ; immunology ; Autoimmune Diseases ; blood ; immunology ; Humans ; Protein Array Analysis ; instrumentation ; methods ; Sensitivity and Specificity

Avidin layer was bound on the substrate surface of Silicon wafer modified with aldehyde. The interaction between avidin and biotin was adopted for the immobilization of mouse monoclonal biotin-anti-M13 (antibody GP3)-labeled biotin. The surface was incubated in a solution containing phage M13KO7, which was trapped by the antibody GP3 with the interaction between phage M13KO7 and antibody GP3, resulting in a variation of layer thickness that was detected by imaging ellipsometry. The results showed a saturated layer of antibody GP3 with a thickness about 6.9 nm on the surface of the silicon wafer. The specific interaction between phage M13KO7 and antibody GP3 resulted in a variation of the layer thickness. The layer of phage M13KO7 bound with antibody GP3 was 17.5 nm in the concentration of 1.1 x 10(10) pfu/mL. Each variation of layer thickness corresponded to a concentration of phage M13KO7 in the range of 0.1 x 10(10) approximately 2.5 x 10(10) pfu/mL, with the sensitivity of 10(9) pfu/mL. Compared with other methods, the optical protein-chip requires only short measurement time, is label free, is a quantitative test, and can be visualized. This study could be significant on the investigation of interactions between the antibody and virus, and shows potential in the early diagnosis of virosis.
Animals ; Antibodies, Viral ; immunology ; Bacteriophage M13 ; immunology ; isolation & purification ; Mice ; Protein Array Analysis ; methods