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Quantification of a PEGylated peptide in human plasma using LC--MS/MS to support clinical studies presented challenges in terms of assay sensitivity, selectivity, and ruggedness. To ensure specific recognition of PEGylated species, an immunoaffinity purification method (IAP) using anti-PEG antibody followed by two-dimensional (2D) LC--MS/MS was developed for MK-2662, an investigational peptide containing 38 amino acids with a 40 kDa branched PEG [poly(ethylene glycol)] at C-terminus. Biotinylated anti-PEG antibody, bound to streptavidin-coated magnetic beads, was used to capture MK-2662 and its stable-isotope-labeled internal standard from human plasma. After on-bead digestion with trypsin, the supernatant was injected on a 2D high-performance liquid chromatography (HPLC) system constructed with strong cation-exchange and reversed-phase columns, followed by MS/MS detection of the surrogate [N.sub.1-12]-mer of MK-2662 on an API5000. The assay ruggedness was improved by optimizing the trypsin digestion and sample storage conditions. The intraday validation, conducted in parallel with protein precipitation (PPT) assay, demonstrated 94.8-105.8% accuracy with 10.1021/ac1009832

Low abundant (< 100 cells [mL.sup.-1]) E. coli O157:H7 cells were isolated and enriched from environmental water samples using a microfluidic chip. The poly(methylmethacrylate), PMMA, chip contained 8 devices, each equipped with 16 curvilinear high aspect ratio channels that were covalently decorated with polyclonal anti-O157 antibodies (pAb) and could search for rare ceils through a pAb mediated process. The chip could process independently 8 different samples or one sample using 8 different parallel inputs to increase volume processing throughput. After cell enrichment, cells were released and enumerated using benchtop real-time quantitative polymerase chain reaction (PCR), targeting genes which effectively discriminated the O157:H7 serotype from other nonpathogenic bacteria. The recovery of target cells from water samples was determined to be ~72%, and the limit-of-detection was found to be 6 colony forming units (cfu) using the slt1 gene as a reporter. We subsequently performed analysis of lake and wastewater samples. The simplicity in manufacturing and ease of operation makes this device attractive for the selection of pathogenic species from a variety of water supplies suspected of containing bacterial pathogens at extremely low frequencies. 10.1021/ac100323k

Magnetic nanoparticles functionalized with anti-Escherichia coli O157:H7 or anti-Salmonella typhimurium anti-bodies that can specifically bind to their target organisms were used to isolate E. coil O157:H7 and S. typhimurium separately from a cocktail of bacteria and from food matrixes. The pathogens were then detected using label-free IR fingerprinting. The binding and detection protocol was first validated using a benchtop FT-IR spectrometer and then applied to a portable mid-IR spectrometer to enable this approach as a point-of-detection technology. Highly selective detection was achieved in less than 30 rain at both species (E. coli O157:H7 vs S. typhimurium) and strain (E. coli O157:H7 vs E. coli K12) levels in complex food matrixes (2% milk, spinach extract) with a detection limit of [10.sup.4]-[10.sup.5] CFU/mL. The combined approach of functionalized magnetic nanoparticles and IR spectroscopy imparts specificity through spectroscopic fingerprinting and selectivity through species-specific antibodies with an in-built sample extraction step and could be applied in the field for on-site food-borne pathogen monitoring.

A highly sensitive (pM), efficient (t < 20 min) detection assay was developed by designing surfaces with grafted antibodies. Through this approach, a short half-life antigen, glucagon, was rapidly detected in a biologically complex plasma/blood environment. Tailoring of graft composition eliminates the need for time-consuming blocking steps, significantly reducing antigen-antibody incubation times, while maintaining antibody specificity and activity toward target antigen. Grafted antibodies were bound through solvated, mobile polymer chains, thereby circumventing problems associated with antibody accessibility, analyte diffusion, and steric limitations. The efficiency of this assay is provided through grafting synthesized, acrylated antibodies in the presence of PEG monoacrylate. This procedure eliminates the need for blocking steps, due to a decrease in nonspecific protein interactions. These polymerizable antibodies were tethered with a range of densities while retaining biological activity. Moreover, biological activity of acrylated antibodies was compared to that of unmodified antibodies and remained comparable. The acrylated antibodies were grafted from substrate surfaces using controlled radical photopolymerization, maintaining the advantages of classical antibody immobilization techniques while providing improved detection. Through integrating this antibody conjugation chemistry and immunoassay approach with photolithographic techniques, construction of spatial patterns on a microfluidic device was demonstrated for efficient, parallel screening of multiple antigens.

Silicon microchips with immobilized antibodies were used to develop microfluidic enzyme immunoassays using chemiluminescence detection and horseradish peroxidase (HRP) as the enzyme label. Polyclonal anti-atrazine antibodies were coupled to the silicon microchip surface with an overall dimension of 13.1 x 3.2 mm, comprising 42 porous flow channels of 235-[micro]m depth and 25-[micro]m width. Different immobilization protocols based on covalent or noncovalent modification of the silica surface with 3-aminopropyltriethoxysilane (APTES) or 3-glycidoxypropyltrimethoxysilane (GOPS), linear polyethylenimine (LPEI, MW 750 000), or branched polyethylenimine (BPEI, MW 25 000), followed by adsorption or covalent attachment of the antibody, were evaluated to reach the best reusability, stability, and sensitivity of the microfluidic enzyme immunoassay ([mu]FEIA). Adsorption of antibodies on a LPEI-modified silica surface and covalent attachment to physically adsorbed BPEI lead to unstable antibody coatings. Covalent coupling of antibodies via glutaraldehyde (GA) to three different functionalized silica surfaces (APTES--GA, LPEI--GA, and GOPS-BPEI--GA) resulted in antibody coatings that could be completely regenerated using 0.4 M glycine/HCl, pH 2.2. The buffer composition was shown to have a dramatic effect on the assay stability, where the commonly used phosphate buffer saline was proved to be the least suitable choice. The best long-term stability was obtained for the LPEI--GA surface with no loss of antibody activity during one month. The detection limits in the [micro]FEIA for the three different immuno surfaces were 45, 3.8, and 0.80 ng/L (209, 17.7, and 3.7 pM) for APTES--GA, LPEI--GA, and GOPS--BPEI--GA, respectively.

A new, rapid method for selective extraction of hydroxylated polycyclic aromatic hydrocarbons metabolites (OH-PAHs) in human urine was developed using an immunosorbent of anti-pyrene antibodies which were encapsulated in a sol-gel glass (SGG) matrix. Resulting chromatograms after immunoextraction of urine samples and HPLC analysis of the extracts were free from matrix interferences. The LODs for the determination of OH-PAHs in these difficult samples were in the low-ppt range (1-16 ng/L). In addition to its high selectivity, the immunosorbent proved to be robust and reusable. Obtained recoveries in spiked urine samples ranged from 83 to 107% for the hydroxyphenanthrene and hydroxypyrene compounds under investigation, while recovery for 3-hydroxybenzo[a]pyrene was only 45-62%. In a biomonitoring study, the SGG immunosorbent was successfully used for trace-level analysis of OH-PAHs in 20 human urine samples. Results were compared to data obtained by an independent reference analysis method and revealed good correlation between both methods.

Research discusses the design and synthesis of haptens of naturally occurring (S)-nicotine, (S)- and (R)-nornicotine, and the (S)-cotinine in optically pure form. Antibodies generated using haptens are tested for their efficacy as immunopharmacologic agents to combat nicotine dependence.

A conformation-dependent neoepitope in activated protein C-protein C inhibitor complex and the cleaved, loop-inserted protein C inhibitor complex is recognized by a mouse monoclonal antibody. Data indicate that part of the epitope represents the inserted reactive center loop and involves residues ArgP13-AlaP9.

Paraferritin is involved in the mucosal absorption and transport of inorganic iron in intestinal cells in rats. Antibody studies indicate that the protein is a complex of integrin, mobilferrin and flavin monooxygenase and the hydrodynamic volume is similar to that of ferritin. The protein has flavin monooxygenase ferrireductase activity that is NADPH-dependent. Antibodies against integrin and mobiliferrin suppress the monooxygenase activity that results in a decrease in radioiron uptake in intestinal cells in culture.

Polyclonal site-directed antibodies and proteinase K are used to analyze the peptide chain of the oxoglutarate carrier situated inside proteoliposomes in a direction opposite to mitochondria. Anti-C and anti-N antibody sequences are seen. Four cleavages site external to the liposomal membrane and three sites internal to the membrane are seen. The peptide chain of the carrier contains seven transmembrane segments with the N- and C-terminal sequences growing into the cytosol.

Use of antibodies which are specific to acetylated H4 facilitates the immunoprecipitation of newly duplicated or newly formed nucleosomes. When duplication occurs along with histone preparation, majority of new H3 and at least 60-65% of new H2A/H2B become complexes with H4 which is acetylated. Duplication of chromatin in the absence of butyrate leads to decreased immunoprecipitation of old and new chromatin.

Low temperature processed, porous sol-gel glasses represent a new class of materials for immobilization of biomolecules. The ability to form these materials into films, monoliths, and fibers to produce high-purity, porous glasses and the fact that they are optically transparent and chemically inert make them an intriguing platform for the development of chemical biosensors. In this paper, we report on the first attempt to encapsulate an intact antibody in a sol-gel glass matrix. Specifically, we present results on the affinity of sol-gel-encapsulated polyclonal antifluorescein. The results demonstrate that the sol-gel-encapsulated antibody retains an affinity for fluorescein; the affinity constant (K(sub f)) for antibody-hapten complex is on the order of 10 to the seventh power M to the -1. The encapsulation process decreases K(sub f) by about 2 orders of magnitude compared to the native system in buffer solution. The effect of aging and drying on the K(sub f) for sol-gel-encapsulated antifluorescein is reported. Finally, we demonstrate that the intact antibody affinity can be maintained using simple storage protocols.

The combination of limited proteolysis and MALDI-TOF mass spectrometry has become an important tool for the determination of epitopes but works best with highly purified antibodies. Here we report the use of capture antibodies to reduce the need for purification of the antibody in the mass spectrometric determination of the epitope. In this new method, a secondary Fc-specific antibody, covalently bound to Sepharose beads, is used to capture the primary antibody (the antibody of interest). After capture, the two antibodies are cross-linked. The antigen is then bound to the immobilized antibodies and subjected to proteolysis using several successive proteinases. In this study, this strategy is demonstrated with a crude mouse anti-ACTH IgG solution and adrenocorticotropin (ACTH). Comparing this strategy with previous methods where the antibody is bound directly to activated beads, the new method (1) results in a higher binding capacity of the bound antibody to ACTH, (2) does not require purification of the antibody of interest, and (3) dramatically reduces the chemical background in the MALDI mass spectra.

The feasibility of antibody catalysis during the cyclodehydration of beta-diketone hapten was analyzed during enantioselective Robinson annulation. Analysis of the reaction of 2-methyl-1,3-cyclohexadienone with nethyl vinyl ketone in aqueous medium indicated the ability of the natural class of aldolase enzymes in catalyzing the dehydration step of Robinson annulation reactions. Furthermore, the antibodies exhibited high rates of control over prochiral discrimination.

Ab initio computations were performed to identify transition structures for specific processes, to compute the energy difference between 5-exo and 6-endo epoxide openings in investigations of an antibody that catalyzes the Baldwin's Rules-disfavored cyclization of oxirane 1 to form product 3, and overwhelms the normal preference for fine-membered ring formation (2) evident in the acid-catalyzed cyclization of the hydroxy epoxide. Computations were performed with hydrogen instead of the benzyl group in 1. Harmonic frequency analysis was used to characterize the transition states.