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A new microarray technology is described for rapid, inexpensive, multiplex diagnostics assays. Referred to as 'in-plane parallel scanning' (IPPS), this technology replaces expensive laser scanning with a grid of 100-[micro]m-wide waveguides embedded in the chip's substrate, enabling real-time quantification of molecular complex formation on the chip's surface. Compared to conventional microarray technology, IPPS has advantages of shorter assay time and lower instrument cost and complexity so that the platform can potentially be used in point-of-care (POC) settings. Two different chip formats are described: a low-density microarray with 10 sensing wells (IPPS- 10) and a medium-density one with 100 sensing wells (IPPS100). Performance was evaluated in two different proof-of-principle immunoassays: intedeukin-1[beta] (IL-1[beta]) and Clostridium difficile toxin A. The two assays gave similar limits of detection of 0.67 and 0.94 pM, respectively. A saturation kinetics model described the sensor response with apparent dissociation constants of 511 pM for IL1[beta] and 6.47 nM for C. difficile toxin A toxoid. The multiplexing capabilities of the IPPS technology were also demonstrated in a multiplex assay for both analytes on the same IPPS- 10 chip. Based on these results, the IPPS technology holds promise for translating diagnostic microarrays into near-patient environments. 10.1021/ac101571b

Binding studies with 'head-to-tail' cyclized peptide analogs were performed to investigate the importance of peptide conformation on the overall binding of MN12H2 to conformationally restricted epitope sequences. Results indicated a strong correlation between the binding of MN12H2 to cyclic peptides and the capability of these peptides to elicit bactericidal antibodies.

A study was conducted to analyze the predictions of computer simulations of the molecular dynamics of the anti-fluorescein 4-4-20 antigen-binding fragment. Site-specific conjugation chemistry was used to attach a fluorescent probe to the 4-4-20 Fab at its C-terminus. Two rotational components were observed: a longer correlation time corresponding to global Fab rotation and a shorter correlation time attributed to local or segmental motion of the constant domain dimer.

A study was conducted to model the molecular dynamics of the anti-fluorescein 4-4-20 antigen-binding fragment with and without fluorescein using computer simulations. Time-resolved fluorescence experiments showed that the Fab minus fluorescein possessed more segmental flexibility than the Fab without fluorescein. The three-dimensional structure of the unliganded Fab was predicted using the simulations.

Antifluorescyl IgG antibody and Fab binding to two fluorescein-conjugated lipids was measured using the quartz crystal microbalance methodology. By use of the Langmuir-Blodgett technique, the fluorescein lipids, which were diluted to 5% in a L-Alpha-dipalmitoyl phosphatidylethanolamine (DPPE) matrix, were deposited directly onto one gold electrode of the quartz crystal. Binding to films containing the fluorescein hapten was significantly enhanced compared to films of the pure DPPE matrix lipid, indicating that binding occurred primarily through a specific interaction. Association constants were 40-300 times less than for binding to haptens free in solution. Binding of IgG to the lipid in which the hydrocarbon chains and the fluorescein hapten were linked via a hydrophilic spacer was approximately 7 times as great as to the lipid containing no spacer. IgG binding to the lipid containing the spacer was increased 1.5-4.4 times compared to Fab binding for the same lipid. Equilibrium binding curves and kinetic measurements are analyzed quantitatively and compared.

A novel concept is described for directly coupling fluorescence emission to protein-ligand binding. It is based on shifting the intramolecular monomer in equilibrium dimer equilibrium of two fluorescent dyes linked by a short spacer. A 13-residue peptide, recognized by a monoclonal antibody against human chorionic gonadotrophin (hCG), was labeled with fluorescein (F) and tetramethylrhodamine (T) at its N- and C-terminus, respectively. Spectral evidence suggests that when the conjugate is free in solution, F and T exist as an intramolecular dimer. Fluorescence quenching of fluorescein and rhodamine is approximately 98% and approximately 90%, respectively, due to dimerization. When the double-labeled peptide is bound to anti-hCG, however, the rhodamine fluorescence increases by up to 7.8-fold, depending upon the excitation wavelength. This is attributed to the dissociation of intramolecular dimers brought about by conformational changes of the conjugate upon binding. Fluorescein fluorescence, on the other hand, was still quenched because of excited-state energy transfer and residual ground-state interactions. Antibody binding also resulted in a approximately 3.4-fold increase in fluorescence anisotropy of the peptide. These changes in intensity and anisotropy allow direct measurement of antigen-antibody binding with a fluorescence plate reader or a polarization analyzer, without the need for separation steps and labeling antibodies. Because recent advances in peptide technology have allowed rapid and economical identification of antigen-mimicking peptides, the double-labeled peptide approach offers many opportunities for developing new diagnostic assays and screening new therapeutic drugs. It also has many potential applications to techniques involving recombinant antibodies, biosensors, cell sorting, and DNA probes.

This paper describes a homogeneous immunoassay based on fluorescence polarization that enables subnanomolar detection of high molecular weight analytes. A monoclonal antibody (Mab) to human chorionic gonadotrophin (hCG) was screened against a panel of 221 synthetic peptides using the method of Geysen et al. (Geysen, H. M.; et al. J. Immunol. Methods 1987, 102, 259-274. Geysen, H. M.; et al. J. Mol. Immunol. 1986, 23, 709-715). One of these peptides, which was located near the C-terminus of the hCG Beta chain, bound to the Mab with high affinity. It was labeled with tetramethylrhodamine (TMR) and used as the tracer antigen in a competitive fluorescence polarization immunoassay (FPIA) for hCG. The peptide-TMR conjugate binds specifically to the anti-hCG Mab with an antigen-binding affinity (K(sub a)) of 1.5 X 10 to the seventh power M to the -1 at 6 degrees C. Its fluorescence intensity was enhanced by approximately 20% upon binding as a result of a prolonged excited-state lifetime. In a typical embodiment, hCG was determined at a level of 1 X 10 to the -9 M (95% confidence limit) - a 100-fold improvement over similar systems reported in the literature. This is mainly attributed to the large difference in hydrodynamic volume between the tracer and the antibody, which resulted in large changes in polarization of the peptide tracer upon binding. Issues related to sensitivity, specificity, and reversibility were also investigated. This method is believed to be of significant importance to rapid and economical measurements of high molecular weight antigens of clinical interest.

The enolic group of Arg-L34 and F1 exhibit a salt link, and the phenylcarboxylate group of Tyr-L32 and F1 reveal a hydrogen bond which reacts. The potency of the reaction is examined by fluorescence spectroscopy. F1 hapten has an enolic group with an ionization state. The dissociation constant relies on the pH levels. Time-resolved fluorescence spectroscopy is used to examine the structure of the active site, following the ionization of the phenolic group of Tyr-L32.

A study was conducted on the importance of electrostatics in protein adsorption to preformed negatively-charged and neutral liposomes at low ionic strength and at varying pH values. Results revealed that significant protein adsorption to negatively-charged liposomes occurred only at pH values in which protein positive charge moieties exceeded negative charge moieties by a minimum of three charge units. Between pH 4 and pH 9, negligible adsorption to liposomes consisting of zwitterionic lipids occurred. Results indicate that adsorption was controlled by liposome charge neutralization caused by positively-charged protein adsorption.