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An anti- peptide antibody, designated alpha-1468, was used to detect the native form of the cystic fibrosis transmembrane conductance regulator (CFTR) in human pancreas and sweat gland. Alpha-1468 detected a 155- to 175-kDa protein, which in T84 cells was localized to the apical region. Further analysis showed that this protein could serve as a protein kinase substrate, thus confirming its identity as a CFTR. These results show that alpha-1468 may be used for studyingother aspects of the regulation and function of CFTR.

Context.--Uroplakin II is a 15-kDa protein component of the urothelial plaques that enhance the permeability barrier and strength of the urothelium. Studies have shown uroplakin II messenger RNA to be expressed in bladder cancer tissues and peripheral blood of patients with urothelial carcinoma. Little is known about the protein expression of uroplakin II in urothelial carcinoma, possibly because of the absence of a commercially available uroplakin II antibody. Pathologists have used the uroplakin III antibody (AU1) to identify tumors of urothelial origin; however, the use of AU1 is limited because of its poor sensitivity. Objectives.--To evaluate a newly developed mouse monoclonal uroplakin II antibody (BC21) in urothelial carcinoma and to compare it with previously developed mouse monoclonal uroplakin III (BC17 and AU1). Design.--Uroplakin II and III antibodies were optimized for staining using a horseradish peroxidase-polymer detection system and were visualized with 3,3 -diaminobenzidine. Results.--BC21, BC17, and AU1 demonstrated sensitivities in urothelial carcinoma of the bladder of 79% (44 of 56), 55% (31 of 56) (P = .002), and 34% (19 of 56) (P < .001), respectively. Subsequently, the increased staining sensitivity and intensity of BC21, compared with BC17, was validated in a larger study (134 of 174; 77% and 94 of 174; 54%, respectively) (P < .001). BC21 was found to be highly specific when evaluated in various normal and neoplastic tissues, including prostatic and renal carcinomas. Conclusions.--The mouse monoclonal uroplakin II antibody (BC21) demonstrated superior sensitivity and specificity in urothelial carcinoma, compared with uroplakin III (BC17 and AU1), suggesting its advantages in the differential diagnosis of urothelial carcinoma and in the detection of tumors of unknown origin. (Arch Pathol Lab Med. 2014; 138:943-949; doi: 10.5858/arpa.2013-0221-0A), Bladder cancer is the sixth most commonly occurring carcinoma in the United States, with 73 510 new cases expected to be diagnosed in 2012. (1) This cancer is particularly associated [...]

A central goal of HIV-1 vaccine research is the elicitation of antibodies capable of neutralizing diverse primary isolates of HIV-1. Here we show that focusing the immune response to exposed N-terminal residues of the fusion peptide, a critical component of the viral entry machinery and the epitope of antibodies elicited by HIV-1 infection, through immunization with fusion peptide-coupled carriers and prefusion stabilized envelope trimers, induces cross-clade neutralizing responses. In mice, these immunogens elicited monoclonal antibodies capable of neutralizing up to 31% of a cross-clade panel of 208 HIV-1 strains. Crystal and cryoelectron microscopy structures of these antibodies revealed fusion peptide conformational diversity as a molecular explanation for the cross-clade neutralization. Immunization of guinea pigs and rhesus macaques induced similarly broad fusion peptide-directed neutralizing responses, suggesting translatability. The N terminus of the HIV-1 fusion peptide is thus a promising target of vaccine efforts aimed at eliciting broadly neutralizing antibodies. An alternative HIV vaccine design facilitates generation of HIV-1-antibodies, with promising neutralization breadth in rodents and nonhuman primates., Author(s): Kai Xu [sup.1] , Priyamvada Acharya [sup.1] [sup.2] , Rui Kong [sup.1] , Cheng Cheng [sup.1] , Gwo-Yu Chuang [sup.1] , Kevin Liu [sup.1] , Mark K. Louder [sup.1] [...]

* Context.--Anti-promyelocytic leukemia (PML) immunofluorescence staining is a known diagnostic tool for rapid diagnosis of acute promyelocytic leukemia (APL). Objective.--We describe our methods using the recently developed, commercially available, tetramethylrhodamine-5-isothiocyanate-labeled PG-M3 anti-PML antibody for APL testing. Design.--Immunofluorescence staining with the tetramethylrhodamine-5-isothiocyanate-labeled PG-M3 antibody was used to detect PML-RARA in bone marrow aspirate and/or peripheral blood smears from 30 patients with acute leukemia. The results were compared with those of concurrent testing with our in-house polyclonal anti-PML antibody and with established tests. Results.--All APL cases showed a positive (fine/micro granular) immunofluorescence staining pattern, whereas non-APL cases showed a negative (chunky/macrogranular) pattern. These results, which were available within 2 hours, were validated by testing with the polyclonal anti-PML antibody and with established cytogenetic and molecular testing methods. Conclusions.--We validated the utility of the tetramethylrhodamine-5-isothiocyanate-labeled anti-PML antibody PG-M3 for the diagnosis of APL. Our results indicate that immunofluorescence staining with this antibody is a rapid and reliable method for the diagnosis of APL. (Arch Pathol Lab Med. 2013;137:1669-1673; doi: 10.5858/arpa.2012-0565-OA), Acute promyelocytic leukemia (APL) is characterized by fusion of the promyelocytic leukemia (PML) and retinoic acid receptor alpha (RARA) genes, leading to expression of the aberrant PML-RARA protein. This abnormality [...]

Molecular imprinting on polymers to make them mimic antibodies can be used in drug assays, is a cheaper process and the polymers are less delicate and so do not require special handling. A comparison of the accuracy between molecularly imprinted sorbent assay and the traditional enzyme-multiplied immunoassay techniques shows excellent correlation. One disadvantage is that the imprinting technique requires extra steps: extraction, incubation and handling of radioactive materials.

Several methods are described for assessing viral infectivity in environmental samples by using PCR methods. The combination of direct PCR method with viral capture systems, such as use of antibodies or cell receptors to separate virions from environmental matrix, has helped in reducing the effect of PCR-inhibitory compounds and has provided a more feasible method for analyzing the viral genomes in environmental samples.

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.

This investigation was pursued to test the use of intracellular antibodies (intrabodies) as a means of blocking the pathogenesis of Huntington's disease (HD). HD is characterized by abnormally elongated polyglutamine near the N terminus of the huntingtin protein, which induces pathological protein--protein interactions and aggregate formation by huntingtin or its exon 1-containing fragments. Selection from a large human phage display library yielded a single-chain Fv (sFv) antibody specific for the 17 N-terminal residues of huntingtin, adjacent to the polyglutamine in HD exon 1. This anti-huntingtin sFv intrabody was tested in a cellular model of the disease in which huntingtin exon 1 had been fused to green fluorescent protein (GFP). Expression of expanded repeat HD-polyQ-GFP in transfected cells shows perinuclear aggregation similar to human HD pathology, which worsens with increasing polyglutamine length; the number of aggregates in these transfected cells provided a quantifiable model of HD for this study. Coexpression of anti-huntingtin sFv intrabodies with the abnormal huntingtin-GFP fusion protein dramatically reduced the number of aggregates, compared with controls lacking the intrabody. Anti-huntingtin sFv fused with a nuclear localization signal retargeted huntingtin analogues to cell nuclei, providing further evidence of the anti-huntingtin sFv specificity and of its capacity to redirect the subcellular localization of exon 1. This study suggests that intrabody-mediated modulation of abnormal neuronal proteins may contribute to the treatment of neurodegenerative diseases such as HD, Alzheimer's, Parkinson's, prion disease, and the spinocerebellar ataxias.

Adenovirus E1A mediates its effects on cellular transformation and transcription by interacting with critical cellular proteins involved in cell growth and differentiation. The amino terminus of E1A binds to CBP/p300 and associated histone acetyltransferases such as P/CAF. The carboxyl terminus binds to the carboxyl-terminal binding protein (CtBP), which associates with histone deacetylases. We show that 12S E1A can be acetylated by p300 and P/CAF and map one of the acetylation sites to Lys-239. This Lys residue is adjacent to the consensus CtBP binding motif, PXDLS. Mutation of Lys-239 to Gin or Ala blocks CtBP binding in vitro and disrupts the E1A-CtBP interaction in vivo. Peptide competition assays demonstrated that the interaction of E1A with CtBP is also blocked by Lys-239 acetylation. Supporting a functional role for Lys-239 in CtBP binding, mutation of this residue to Ala decreases the ability of E1A to block cAMP-regulated enhancer (CRE)-binding protein (CREB)-stimulated gene expression. Finally, we demonstrate that Lys-239 is acetylated in cells by using an antibody directed against an acetyl-Lys-239 E1A peptide. CtBP interacts with a wide variety of other transcriptional repressors through the PXDLS motif, and, in many instances, this motif is followed by a Lys residue. We suggest that acetylation of this residue by histone acetyltransferases, and the consequent disruption of repressor complexes, might be a general mechanism for gene activation.

In crustaceans, the synthesis of steroid molting hormones, ecdysteroids, is negatively regulated by a neuropeptide molt-inhibiting hormone (MIH). Amino acid sequence data place MIH in the crustacean hyperglycemic hormone (CHH) family. As part of our studies of the control of growth and molting in blue crabs (Callinectes sapidus), we have generated antipeptide antibodies for detection of MIH. Using GCG (University of Wisconsin) sequence analysis software programs, two regions of the blue crab MIH peptide were selected for antibody production. These fragments were selected using multiple alignment and peptide structure programs that predicted regions of MIH having high surface probability, flexibility and antigenicity, but which were not highly conserved between MIH and other members of the CHH family. The two 10-mer peptides were commercially synthesized and used to generate polyclonal antisera in rabbits. The binding properties of the antisera were characterized by immunocytochemistry (ICC) and Western blot. Thin section ICC showed specific immunoreactivity in the X-organ/sinus gland complex of eyestalk neural ganglia with both antisera. Western blot analysis revealed that both antisera also bound to proteins of the predicted size in blue crab sinus gland extracts and cell lysates containing recombinant MIH protein. We anticipate that these antipeptide antibodies will be useful for developing an immunoassay for quantification of MIH in hemolymph. Supported by MS/AL Sea Grant and NSF.

Mice infected with vesicular stomatitis virus (VSV), a cytopathic virus closely related to rabies virus, mount a virus-neutralizing antibody response protecting against lethal disease. VSV-neutralizing monoclonal IgGs isolated from primary immune responses were devoid of somatic mutations, whereas most secondary and all hyperimmune response IgGs tested were hypermutated. A comparative analysis of recombinant single-chain antibody fragments (scFv-C[Kappa]) revealed that even the germ-line precursor of one hypermutated antibody bound and neutralized VSV. Four somatic amino acid substitutions in [V.sub.H] increased by 300-fold the binding strength of monovalent scFv-C[Kappa]. The multivalent binding avidity of germ-line scFv-C[Kappa] was increased by more than 10-fold compared with the monovalent binding strength. In contrast, hypermutated scFv-C[Kappa] did not show such avidity effects. Thus the overall binding difference between the germ-line and the hypermutated VSV-neutralizing antibody was only 10- to 15-fold. This may explain why primary germ-line antibodies and secondary hypermutated antibodies directed against pathogens such as viruses and bacteria expressing repetitive antibody determinants show rather similar binding qualities, whereas monovalently binding hapten-specific antibodies can show 'affinity maturation' effects of up to 1000-fold.

Efforts to make a prophylactic HIV vaccine have identified monoclonal antibodies that potently suppress viral replication. studies in monkeys show that these reagents effectively treat HIV infection. A major breakthrough [...]

We are systematically developing a biosensor platform that is fast in response time, versatile that can be used to detect a variety of chemicals species and metabolites, and ultra-sensitive that [...]