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.

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.

HER-2/neu oncogene is over-expressed and amplified in patients associated with metastatic breast cancer. An increased level (>15 ng/mL) in the shed extracellular domain (sECD-HER 2/neu) is indicative of the potential presence and associated progression of this disease. A fluorescent ELISA incorporating the newly developed ALYGNSA antibody-orientation system revealed a 10-fold increase in sensitivity (≤0.63 ng/mL) of sECD-HER 2/neu when compared to a control standard ELISA kit (≤7.5 ng/mL). This enhanced mode of detection has the potential to not only address breast and other cancers per se but also permit an in depth evaluation of "shed extracellular domains", in general, and the role of these "proteolytic derived factors" in physiological signalling at normal levels.

Breast cancer is the second leading cause of cancerrelated deaths in women worldwide; a prime cancer biomarker to aid in the diagnosis, directed treatment, clinical management, and reoccurrence of this cancer is a MUC1 peptide fragment: cancer antigen 15-3 (CA 15-3). Herein, an immuno-fluorescence assay for CA 15-3 was developed; this ALYGNSA system consists of a protein biolinker (Protein G’) adsorbed onto Poly (methyl methacrylate) (PMMA). The unique interaction of Protein G’ with PMMA, a thermo-plastic polymer has been demonstrated to improve human IgG capture antibody alignment/ orientation and result in greater assay sensitivity. Indeed a previous report (HEALTH 1 325 - 329, 2009) on the shed extracellular domain of HER-2/neu revealed a 10-fold increase in sensitivity of the ALYGSNA assay over a control ELISA assay. Results from this ALYGNSA assay study revealed that a 16-fold increase in detection (≤0.94 U/mL) of CA 15-3 was found in comparison to a commercial control ELISA kit (≤15 U/mL). In conclusion, this enhanced sensitivity of the ALYGNSA assay for CA 15-3, may provide insights into the role/function of this biomarker in normal, as well as, breast cancer and other epithelial cancers

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.