Specific single chain variable fragment (ScFv) antibodies to angiotensin II AT(2) receptor: evaluation of the angiotensin II receptor expression in normal and tumor-bearing mouse lung.

To gain insight into the mechanism by which angiotensin II type 2 receptor (AT(2)) regulates carcinogen-induced lung tumorigenesis, we have newly developed anti-AT(2) single chain variable fragment (ScFv) antibodies using a rodent phage-displayed recombinant antibody library with various peptide fragments of the receptor protein, and investigated the expression of the AT(2) receptor protein. The specificity of the antibodies was verified using AT(2) over-expressing COS-7 cells and AT(2) naturally expressing PC12W cells. In control wild type mouse lung, a stronger immunoreactivity was observed in bronchial epithelial cells. A moderate immunoreactivity was detected in pulmonary vascular walls and vascular endothelial cells. In the lungs possessing tobacco-specific nitrosamine (NNK)-induced tumors, significantly increased AT(2) and AT(1 )immunostaining was observed in adenomatous lesions. These data suggest that the increase in both receptors' expression in the alveolar epithelial cells may be accompanied with the onset of NNK-induced tumorigenesis and hence play important roles in lung tumorigenesis.

Production and characterization of mouse ureteric bud cell-specific rat hybridoma antibodies utilizing subtractive immunization and high-throughput screening.

The highly branched collecting system of the kidney arises developmentally from the ureteric bud (UB) by a process of branching morphogenesis. This process is critical for the normal development of the collecting ducts and pelvis of the kidney, and is tightly controlled by the spatial and temporal expression of numerous proteins. To identify cell proteins that are differentially expressed by the UB relative to those expressed by the highly differentiated collecting ducts of the adult kidney, two mouse cell populations derived from either the early UB or the adult inner medullary collecting duct (IMCD) were used. A subtractive immunization strategy was performed in rats to generate monoclonal antibodies that preferentially reacted with antigens on UB, but not IMCD cells. In addition, the technique of antibody printing, a novel high-throughput antibody screening method for determining the specificities of a large number of monoclonal antibodies, is described. The methodologies outlined in this manuscript have broad applicability as they demonstrate that subtractive immunization can be performed in rats with cells derived from mice. Additionally, the high-throughput screening methods should facilitate the use of subtractive immunization for identifying antibodies that can distinguish differences in proteins expressed in closely related cell types.

Assays for selection of single-chain fragment variable recombinant antibodies to metal nanoclusters.

The protocols herein describe colony-lift and fluorescent immunoassays that were used to identify bacterial colonies that produced single-chain fragment variable (ScFv) recombinant antibodies reactive with zero-state silver. A large (approx 2.9-billion member) phage-displayed antibody library was panned against zero valent silver. Bacterial colonies obtained after two rounds of selection were either lifted onto nitrocellulose filters or picked to individual wells of 384-well microtiter culture plates. Colonies lifted onto filters were placed onto zero valent silver-coated filters and induced to produce soluble ScFv antibodies. ScFv antibodies, expressed by individual colonies, that bound to silver nanocluster-coated filters were detected using an anti-ScFv antibody conjugated to horseradish peroxidase and a chemiluminescent substrate. Colonies picked to 384-well micotiter culture plates were induced to express soluble ScFv antibodies. ScFv antibodies in bacterial periplasmic extract were transferred from 384-well culture plates to 384-well assay plates containing zero-state silver particles and an anti-ScFv antibody conjugated to a fluorescent dye. ScFv antibodies, expressed by individual bacterial clones, that bound to zero valent silver nanoparticles in 384-well assay plates were detected using an FMAT 8100 fluorescent plate reader. The colony-lift and fluorescent immunoassays detected ScFv antibodies reactive with zero valent silver. Similar assay formats should also be useful to detect bacterially expressed recombinant antibodies or proteins to other nanoclusters.

Heterogeneity among Helicobacter pylori strains in expression of the outer membrane protein BabA.

The BabA adhesin of Helicobacter pylori is an outer membrane protein that binds to the fucosylated Lewis b histo-blood group antigen on the surface of gastric epithelial cells. We screened a phage-displayed ScFv (single-chain fragment variable) recombinant antibody library for antibodies reactive with a recombinant BabA fragment and identified two such antibodies. Each antibody recognized an approximately 75-kDa protein present in wild-type H. pylori strain J99 but absent from an isogenic babA mutant strain. An immunoreactive BabA protein was detected by at least one of the antibodies in 18 (46%) of 39 different wild-type H. pylori strains and was detected more commonly in cagA-positive strains than in cagA-negative strains. Numerous amino acid polymorphisms were detected among BabA proteins expressed by different strains, with the greatest diversity occurring in the middle region of the proteins. Among the 18 strains that expressed a detectable BabA protein, there was considerable variation in the level of binding to Lewis b in vitro. Heterogeneity among H. pylori strains in expression of the BabA protein may be a factor that contributes to differing clinical outcomes among H. pylori-infected humans.

Selection of active ScFv to G-protein-coupled receptor CCR5 using surface antigen-mimicking peptides.

This study describes the use of cyclic peptides for use in the selection of single-chain (ScFv) antibodies specific for the HIV-1 coreceptor CCR5, a representative G-protein-coupled receptor (GPCR). A tandem ligation strategy was developed for preparing biotinylated cyclic peptides, first through an orthogonal end-to-end ligation and then a chemoselective ligation with functionalized biotin. Cyclic peptides mimicking the extracellular loops of CCR5 and their unconstrained counterparts were then used for solution-phase selection of ScFv antibodies from a phage display antibody library. Antibodies reactive with CCR5 on cells were detected using a homogeneous high throughput assay. Of 19 isolated ScFv antibodies that bound to CCR5+ cells, three inhibited CCR5-mediated but not CXCR4-mediated HIV infection. Only ScFvs selected by binding to cyclic constrained peptides exhibited inhibitory activity. Our results demonstrate that surface-antigen mimetics of a GPCR are effective tools for selecting active, site-specific ScFv antibodies that hold promise as immunological reagents and therapeutics.