Computational analysis of non-covalent polymer-protein interactions governing antibody orientation.

The ALYGNSA is an affinity-based antibody orientation system produced through the interaction of the polymer poly(methyl methacrylate) (PMMA) and recombinant protein G (rProG), a streptococcal protein. This improved orientation suggests a specific non-covalent attachment of the rProG to PMMA that leaves the IgG binding region of the rProG more readily available. In this study, a full tertiary structure model of the rProG molecule of 198 amino acid residues containing a signal region, two IgG binding domains, and an anchor region, was computationally generated using the iterative threading assembly refinement (I-Tasser) server. The rProG model having the highest confidence score was subject to docking experiments with varied-length short chains of PMMA polymer via the graphic processing units-based Hex server. A five-residue section of the rProG anchor region, with the sequence TPATP, was identified as a potential interaction site. A complete ternary model (rProG, PMMA, and IgG) was assembled and provides insight into a plausible mechanism for non-covalent antibody orientation by the ALYGNSA system.

AFM imaging of ALYGNSA polymer-protein surfaces: evidence of antibody orientation.

Previous investigations found the combination of recombinant bacterial protein G (rProG) and poly(methyl methacrylate) (PMMA) to produce a greater proportion of oriented antibodies. PMMA-rProG yielded a sixfold greater availability of antibody Fab regions compared with other bacterial affinity linker protein and polymer pairings, including commercially available polystyrene (PS) high-binding 96-well microplates. Given the name ALYGNSA, the PMMA-rProG combination was developed into a fluorescence assay and evaluated in conjunction with commercially available cancer biomarker enzyme-linked immunosorbent assays (ELISAs). In each study, a lower limit of detection was seen with the ALYGNSA assay. The purpose of this investigation was to examine the ALYGNSA substrate in contrast with a commonly used ELISA substrate and analyze the affinity-immobilized antibodies for additional evidence of orientation. Non-contact atomic force microscopy is a logical method as it operates in ambient conditions, can be used directly on biological samples without modification, and offers the resolution necessary to identify the position of the antibody on the surface. Dynamic contact angle studies were employed to examine untreated PMMA and PS samples and revealed important differences in their surface characters. Comparative height threshold grain analysis of the prepared ALYGNSA surface, a similarly treated mica surface, and a gold colloid sizing standard evaluated and confirmed the antibody orientation of the ALYGNSA system.

Immuno-interferometric sensor for the detection of influenza A nucleoprotein.

Influenza A virus, both seasonal and pandemic, has the potential to cause rampant devastating disease around the world. The most relied upon methods of viral detection require days, skilled workers, and laboratory settings to complete properly. Here, we report two methods for the detection of the nucleoprotein from inactivated influenza A (IFA-NP), a patented polymer-protein antibody orientation immuno-method, termed ALYNGSA, and a newly fabricated optical label-free Fabry-Perot interferometric immunosensor. The ALYGNSA assay for IFA-NP had a level of detection below 5 microg/mL of inactivated virus sample. The label-free detection through Fabry-Perot interferometry with the ALYGNSA orientation yielded an improved sensitivity to 1 microg/mL over the fluorescence sandwich assay alone. Characterization of the detection surface by fluorescence microscopy and non-contact AFM corroborated interferometry results. The resulting label-free detection method has the prospective for adaptation into a portable multi-chip sensor capable of real-time in situ detection of influenza A virus.

Polymer-protein-enhanced fluoroimmunoassay for prostate-specific antigen.

Prostate-specific antigen (PSA) is a serum glycoprotein overproduced in prostate cancer, the total of which is comprised of two major forms, free and complexed. The common method for measuring of PSA is an Enzyme-linked immunosorbent assay (ELISA). Limits of detection using commercial PSA ELISA kits for free and total PSA were determined in our laboratory to be 1 and 10 ng/mL, respectively. A value of 0.10 was obtained for the free to total PSA ratio, a ratio of 0.25 being indicative of prostate cancer. A possible improvement in the sensitivity and detection limits of free and total PSA may be achieved by the ALYGNSA system (Clarizia et al., Anal Bioanal Chem 393:1531-1538, 2009). This fluorescent assay system employed a selective polymer biolinker system proven to enhance primary antibody orientation. Using this system, free and total PSA detection limits of 0.13 and 0.63 ng/mL, respectively, were realized. This amounted to an 8- and 15-fold improvement in detection limits for free and total PSA, respectively. A free to total PSA ratio of 0.20 was maintained in this study and may be useful for definitive diagnosis of prostate, as well as, other cancers.

Novel fluoroimmunoassay for ovarian cancer biomarker CA-125.

Cancer antigen 125 (CA-125) is a glycoprotein biomarker that denotes the presence of ovarian and reproductive cancers in women, with serum concentrations of CA-125 greater than 35 U/ml considered indicative of potential malignancies. A fluorescent immunoassay recently developed in our laboratory employing the ALYGNSA antibody-orientation system has been used to measure CA-125 levels. This system displayed significantly increased sensitivity with a detection limit of 1.5 U/ml compared to that of a commercial CA-125 enzyme-linked immunosorbent assay (15 U/ml) This tenfold lower level of detection of the ALYGNSA CA-125 assay should permit better identification and monitoring of ovarian cancer.