PMC:4996395 / 23757-26801 JSONTXT

{"project":"2_test","denotations":[{"id":"27600221-11470888-69478204","span":{"begin":395,"end":397},"obj":"11470888"},{"id":"27600221-16825183-69478205","span":{"begin":918,"end":920},"obj":"16825183"},{"id":"27600221-18204456-69478206","span":{"begin":1275,"end":1277},"obj":"18204456"},{"id":"27600221-20967624-69478207","span":{"begin":1278,"end":1280},"obj":"20967624"},{"id":"27600221-23454659-69478208","span":{"begin":1424,"end":1426},"obj":"23454659"},{"id":"27600221-23616427-69478209","span":{"begin":2367,"end":2369},"obj":"23616427"}],"text":"5. NAPPA Alternative Methods\nBesides NAPPA technology, several in situ expressed microarrays have been developed, such as protein in situ arrays (PISA) and printing protein arrays from DNA (DAPA). The main difference between PISA and NAPPA is that the DNA template is added as a free molecule together with the reaction mixture. Thus, it is not necessary to immobilize the DNA onto the surface [33]. Angenendt and colleagues demonstrated that yield signals for protein expressed with these arrays were comparable to 300 μg/mL directly spotted proteins. Moreover, the volume of required sample was too low (subnanotliter volumes) and the nature of the surfaces determined the protein binding. Thus, the nickel chelate-coated slides generated an unspecific binding. Finally, they refined and miniaturized PISA arrays by using multiple spotting technique to get a high-density protein microarray with up to 13,000 spots [34].\nOn the other hand, the DAPA strategy, developed in 2007, is characterized by assembly face-to-face between a slide containing the DNA templates for the proteins and a second slide pre-coated with a protein-capturing reagent. For the transcription/translation, the cell-free system is placed between the two slides using a cell-extract soaked membrane [35,36]. Although this is the basis of the technology, modifications can be made to improve its functionality. In such a way, Schmidt and colleagues [37] have investigated the influence of different support coatings (Ni-NTA, Epoxy, 3D-Epoxy and Polyethylene glycol methacrylate (PEGMA)) concluding that their optimal combination results in high protein yields and optimized spot morphology. Moreover, using a tag-specific capture antibody on a protein repellent surface coating, they improved the specificity of protein capturing and obtained amounts of expressed proteins comparable to classical protein arrays.\nFinally, ProtoArrays are also a remarkable microarray approach. They are characterized for including thousands of proteins (\u003e9000) in a high-density array allowing a high-throughput screening using low volumes of sample (~10 μL of serum). Several studies have been performed by using these arrays. For instance, they are employed for autoimmune antibody screening studies to discover biomarkers of the Parkinson’s disease (ParkCHIP). In this research, Turewicz and collaborators [38] studied a large cohort of samples and adapted the default workflow for these arrays to their requirements. This constitutes a clear example of the flexibility of this approach. Furthermore, these ProtoArrays have been employed to study systemic erythematosus lupus (SLE), specifically to identify novel autoantibodies associated with the disease. In total, 9500 antigens were screened resulting in 446 IgG and 1218 IgM autoantibodies significantly elevated in SLE patients. In this research, the researchers not only identified previously described autoantibodies (SSA/SSB, Sm/RNP…) but also reported novel antigens associated with the nucleus, cytoplasma or membrane [39]."}