1985;85:409C419. in the non-specific binding and a 6-fold increase in specific binding of SEB. fluorescence signals, the differences between the PEG-derivatized hydrogels became more apparent. Physique 4 shows a comparison of the fluorescence signal response in sandwich immunoassays using the three PEG-derivatized hydrogels, as well as control hydrogels. A clear dose-response curve was observed with the three hydrogels, although signals saturated at SEB concentrations of 0.1 g/mL and above in hydrogels modified with PEG-methacrylate. Both PEG-diacrylate and PEG-methacrylate gave approximately 3-fold higher net signals than the PEG-dimethacrylate-modified hydrogels at all concentrations of SEB tested. However, the PEG-methacrylate also showed significantly higher net signals in negative controls without SEB (i.e., non-specific binding of tracer antibody in the absence of SEB; P 0.005). PEG-diacrylate-modified hydrogels, on the other hand, demonstrate superior performance in terms MK-0517 (Fosaprepitant) of both specific and non-specific binding (i.e., lower nonspecific binding, higher specific binding) and therefore may prove to be a more appropriate candidate for further improvement of this and other (see supplemental material) array-based assays incorporating hydrogel matrices. Open in a separate window Physique 4. Comparison of net fluorescence signal responses obtained in sandwich immunoassays using control (no PEG) and three different PEG-incorporated hydrogels. The three PEG candidates ([img]), PEG-diacrylate ([img]), PEG-methacrylate and ([img]) PEG-dimethacrylate were incorporated into galactose-based polyacrylate hydrogels. Rabbit anti-SEB (capture antibody) was bound within the hydrogel and fluorescence was obtained with Cy3-labeled sheep anti-SEB (tracer molecule). Values reported represent replicates of six ( SD). 3.?Experimental Section 3.1. Antibodies, antigens and Rabbit Polyclonal to CDH11 reagents Poly(ethylene glycol) (n) methacrylate n = 526; Poly(ethylene glycol) (n) diacrylate n = 400; Poly(ethylene glycol) (n) dimethacrylate n = 400 and = 3; SD). Of the three PEG complexes investigated (PEG-diacrylate, -methacrylate and -dimethacrylate) PEG-diacrylate was chosen as the optimum candidate for subsequent immunoassays MK-0517 (Fosaprepitant) based on the following factors: 1) significantly lower nonspecific background signal by the tracer molecule measured at the control concentration where no SEB (0 g/mL) is usually applied, 2) clearer and more discrete patterned fluorescence arrays around the hydrogel and 3) higher overall fluorescence signal response in the SEB sandwich immunoassays. Results clearly showed that this addition of PEG-diacrylate reduced the non-specific binding by a factor of 10. In addition to the reduction in non-specific binding from the antigen and/or Cy3-labeled anti-SEB, a 6-fold increase in the fluorescence signal for specific binding of the SEB antigen to the immobilized antibody was observed with a detection level of 1 ng/mL, which is comparable to most antibody-based immunoassay systems. In all, PEG-modified hydrogels provide a conducive, hydrophilic micro-environment for the antibody that can dramatically reduce non-specific protein binding, enhance antibodyCantigen interactions, and improve immunoassay sensitivity. Further optimization of the PEG monomer and antibody concentrations will hopefully provide a means of achieving even lower detection limits. Acknowledgments This research was supported by the Office of Naval Research (NRL 6.2 WU# 69-6008). The views expressed herein are those of the authors and do not represent those of the US Navy, the MK-0517 (Fosaprepitant) US Department of Defense, or the US government. References and Notes 1. Rubina A.Y., Dementieva E.I., Stomakhin A.A., Darii E.L., Pan’kov S.V., Barsky W.E., Ivanov S.M., Konovalova E.V., Mirzabekov A.D. Hydrogel-based protein microchips: manufacturing, properties, and applications. Biotechniques. 2003;34:1008C1022. [PubMed] [Google Scholar] 2. Revzin A., Russell R.J., Yadavalli V.K., Koh W.G., Deister C., Hile D.D., Mellot M.B., Pishko M.V. Fabrication of poly(ethylene glycol) hydrogel microstructure using photolithography. Langmuir. 2001;17:5440C5447. [PubMed] [Google Scholar] 3. Barsky V.E., Kolchinsky A.M., Lysov Y.P., Mirzabekov A.D. Biological.