Site-specific photocoupling of pBpa mutated scFv antibodies for use in affinity proteomics.

Recombinant antibody libraries can provide a source of renewable and high-performing binders tailored for use in affinity proteomics. In this context, the process of generating site-specific 1:1 tagging/functionalization and/or orientated surface immobilization of antibodies has, however, proved to be challenging. Hence, novel ways of generating such engineered antibodies for use in affinity proteomics could have a major impact on array performance. In this study, we have further tailored the design of human recombinant scFv antibodies for site-specific photocoupling through the use of an unnatural amino acid (UAA) and the Dock'n'Flash technology. In more detail, we have generated the 2nd generation of scFvs carrying the photoreactive UAA p-benzoyl-l-phenylalanine (pBpa). Based on key properties, such as expression levels, activity, and affinity, a preferred choice of site for pBpa, located in the beginning of the C-terminal affinity-tag, was for the first time pin-pointed. Further, the results showed that pBpa mutated antibody could be site-specifically photocoupled to free and surface immobilized ß-cyclodextrin (an affinity ligand to pBpa). This paves the way for use of scFv antibodies, engineered for site-specific photochemical-based tagging, functionalization, and orientated surface immobilization, in affinity proteomics.

Urinary levels of triclosan and parabens are associated with aeroallergen and food sensitization.

BACKGROUND: Endocrine-disrupting compounds (EDCs) have immune-modulating effects. We were interested in determining their association with allergic sensitization. OBJECTIVE: We sought to determine the association between EDCs and allergic sensitization and whether this relationship depends on the antimicrobial properties of the EDCs, sex, or both. METHODS: Data were obtained from the 2005-2006 National Health and Nutrition Examination Survey in which urinary bisphenol A; triclosan; benzophenone-3; propyl, methyl, butyl, and ethyl parabens; and specific IgE levels were available for 860 children. Aeroallergen and food sensitizations were defined as having at least 1 positive (≥ 0.35 kU/L) specific IgE level to an aeroallergen or a food. Logistic regression was used to determine the association of EDCs and sensitization. Analyses were adjusted for urinary creatinine level, age, sex, ethnicity, and poverty index ratio. RESULTS: The odds of aeroallergen sensitization significantly increased with the level of the antimicrobial EDCs triclosan and propyl and butyl parabens (P ≤ .04). The odds of food sensitization significantly increased with the level of urinary triclosan among male subjects (odds ratio for third vs first tertiles, 3.9; P= .02 for trend). There was a significant interaction between sex and triclosan level, with male subjects being more likely to be food sensitized with exposure (P= .03). Similar associations were not identified for the nonantimicrobial EDCs bisphenol A and benzophenone-3 (P > .2). CONCLUSIONS: As a group, EDCs are not associated with allergen sensitization. However, levels of the antimicrobial EDCs triclosan and parabens were significantly associated with allergic sensitization. The potential role of antimicrobial EDCs in allergic disease warrants further study because they are commonly used in Western society.

An improved method for the production of antibodies to lipophilic carboxylic hapten using small amount of hapten-carrier conjugate.

A rapid, simple and low cost procedure for preparing minute amount of hapten-protein conjugates was developed using 4-acetyl benzoic acid (ABA) and two other closely related small chromophoric haptens. The amide bond-generating mixed anhydride method of Erlanger was modified to promote conjugation to various proteins (bovine serum albumin, ovalbumin, casein and hemocyanin) or to a synthetic homopolymer (Poly-DL-lysine). The key process in this synthesis is the use of a reversed micellar medium allowing strong carrier haptenization as determined by spectrophotometric measurement at characteristic hapten absorption peaks. This coupling procedure is applicable to as little hapten material as 0.2 micromol and is disclosed to be most valuable for other rare lipid haptens which pose analytical problem in biological fluids and matrices. Specific mice polyclonal antibodies were produced following multiple intraperitoneal injections of (ABA)23-BSA conjugate as revealed by indirect and competitive ELISA. Calculated KD for the interaction of the antibodies with free ABA was found to be 5 X 10(-5)M.