Biochemical and immunological studies indicate that rod outer segments (ROS) of bovine photoreceptor cells contain a Mr 240,000 polypeptide related to the ∝-subunit of red blood cell (RBC) spectrin. With the use of sodium dodecyl sulfate gel electrophoresis in conjunction with the immunoblotting technique, monoclonal antibody 4B2 was found to bind to a Mr 240,000 polypeptide in ROS that is distinct from the prominent Mr 220,000 concanavalin A binding glycoprotein. The Mr 240,000 polypeptide is highly susceptible to degradation by endogenous proteases. It does not appear to be an integral membrane protein but is tightly membrane associated since it can be partially extracted from ROS membranes with urea in the absence of detergent. The 4B2 antibody cross-reacted with RBC ghost membranes and bovine brain microsomal membranes. Radioimmune assays and immunoblotting analysis of purified bovine RBC spectrin further revealed that the 4B2 antibody predominantly labelled the ∝-chain of RBC spectrin having an apparent Mr of 240,000. Monoclonal antibody 3A6 was found to bind to a polypeptide with a slightly lower Mr than the 4B2-specific polypeptide. It is also highly susceptible to degradation by endogenous proteases, but unlike the 4B2 antibody, it predominantly labelled the β-chain of RBC spectrin having an apparent M of 220,000. Polyclonal anti-spectrin antibodies that bound to both the ∝ - and β-chain of RBC spectrin predominantly labelled a Mr 240,000 polypeptide of ROS membranes. Two faintly labelled bands in the Mr range of 210,000-220,000 were also observed. These components may represent variants of the β -chain of spectrin that are weakly cross-reacting or present in smaller quantities than the ∝-chain. Immunocytochemical labelling studies using the 4B2 antibody and immunogold-dextran markers indicated that the ROS spectrin-like protein is preferentially localized in the region where the discs come in close contact to the plasma membrane of ROS. Immunoblotting analysis indicated that rhodopsin and peripherin which constitute over 90% of total disc membrane proteins were selectively solubilized in Triton X-100, whereas a set of polypeptides including the 4B2-specific polypeptide and the Mr 220,000 concanavalin A-binding glycoprotein was only partially soluble. Electron microscopy of a negatively stained Triton-extracted ROS pellet revealed a filamentous network. These studies indicate that ROS contain a protein related to RBC spectrin, which may constitute a major component of a filamentous network lining the inner surface of the ROS plasma membrane as previously seen by electron microscopy. This membrane skeletal system may serve to stabilize the ordered ROS structure and maintain a constant distance between the rim region of the discs and the plasma membrane. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

Two hybridoma clones, labelled 4D10 C1 and 2H4 H12, produced monoclonal antibodies which recognized the outer membrane of an enteropathogenic Escherichia coli (EPEC) 0142:K86:H6 in an enzyme-linked immunosorbent assay (ELISA) and the whole cell in an immunofluorescence assay. Large scale production of the monoclonal antibodies was accomplished through ascites production in balb/c mice. Purification of the ascites fluid was achieved by gel filtration and ion exchange chromatography. Isotyping of the purified fractions showed 4D10 C1 to be an IgG2 and 2H4 H12 an IgM. These monoclonal antibodies were screened by immunofluorescence assay against several pathogenic and nonpathogenic strains of E.coli in addition to other Enterobacteriaciae. Results of the screening showed these antibodies to be specific for the E.coli serotype to which they were raised. Minimal cross-reactivity with other Enterobacteriaceae was observed. In a separate and concurrent project, the use of an ELISA capable of detecting ß-N-acetylglucosaminidase (NAGase) was examined. White Leghorn hens were injected with commercially prepared bovine NAGase. Eggs were collected and the immunoglobulin fraction separated from the egg yolk by polyethylene glycol precipitation followed by ion exchange on a DEAE-Sephacel column. The use of the purified immunoglobulins was examined in a sandwich, double-sandwich and a competitive ELISA. A statistically significant standard curve for the detection of NAGase was successfully derived using a double-sandwich ELISA when rabbit immunoglobulin was used to coat the microwell plates. This assay was used to measure the NAGase concentration in press juice and fish extract of fresh and frozen salmon muscle samples. The ratio of the NAGase concentration in the press juice to the total NAGase concentration was compared. No significant difference was found between the calculated concentration ratios of the fresh muscle samples and samples frozen for 1 week at -20°C. / Land and Food Systems, Faculty of / Graduate

Evidências epidemiológica e imunológica sugerem que o desenvolvimento da imunidade natural contra doença meningocócica pode está associado com a reação cruzada de antígenos em comuns com Neisseria meningitidis e outras bactérias comensais, como Neisseria lactamica. O Objetivo deste trabalho foi de investigar a imunogenicidade de antígenos de vesículas de membrana externa (OMV) de N. lactamica, com ou sem a presença de Bordetella pertussis (BP), utilizada como adjuvante. Grupos de camundongos neonatos da linhagem BALB/c foram imunizados com antígenos de N. lactamica. Os resultados de nossos estudos mostraram o predomínio de altos títulos de anticorpos dos isótipos IgG e IgM com alta e intermediária avidez, depois das imunizações pela via (i.n) com N. lactamica. A análise do soro por immunoblot mostrou proteínas com reatividade cruzada entre as espécies do gênero Neisseria e os anticorpos monoclonais utilizados neste trabalho. Estes resultados sugerem que antígenos de N. lactamica e N. meningititdis em comum, possam ser importantes na imunidade natural contra doença meningocócica, e no desenvolvimento de vacina. / Immunological and epidemiological evidences suggest that the development of natural immunity to meningogoccal disease may be associated with crossreactive antigens together with Neisseria meningitidis and other commensal bacteria, like Neisseria lactamica. The present study aimed to investigate the immunogenicity of antigens of outer membrane vesicles (OMV) of N. lactamica with or without the presence of Bordetella pertussis (BP) used as an adjuvant. Groups of neonate BALB/c mice were immunized intranasally antigens of N. lactamica. The results of our studies showed the predominance of high titers of antibodies of IgG and IgM isotipes with high and intermediate avidity after intranasal immunization with N. lactamica. Immunoblot analysis of serum showed cross-reactivity proteins between the species of the gender Neisseria and the monoclonal antibodies used in this study. These results suggest that antigens of N. lactamica and N. meningitidis in common may be important in natural immunity against meningogoccal disease and in the development of vaccine.

Diisocyanates are very reactive low molecular weight chemicals that are widely used in the manufacture of polyurethane products. Diisocyanate exposure is one of the most commonly reported causes of occupational asthma. Although diisocyanates have been identified as causative agents of respiratory diseases, the specific mechanisms by which these diseases occur remain largely unknown. Tandem mass spectrometry was used to unambiguously identify the binding site of isocyanates within four model peptides (Leu-enkephalin (Leu-enk, YGGFL), Angiotensin I (DRVYIHPFHL), Substance P-amide (RPKPQQFFGLM-NH2), and Fibronectin-adhesion promoting peptide (FAPP, WQPPRARI)). In each case, isocyanates were observed to react to the N-terminus of the peptide. No evidence of side chain/isocyanate adduct formation exclusive of the N-terminus was observed. However, significant intra-molecular diisocyanate crosslinking between the N-terminal amine and a side chain amine group was observed for arginine, when located within two residues of the N-terminus. Addition of multiple isocyanates to the peptide occurs via polymerization at the N-terminus, rather than addition of multiple isocyanate molecules to varied residues within the peptide. Toluene diisocyanate (TDI)-specific monoclonal antibodies (mAbs) with potential use in immunoassays for exposure and biomarker assessments were produced. A total of 59 unique mAbs were produced (29 IgG1, 14 IgG2a, 4 IgG2b, 2 IgG3 and 10 IgM) against 2,4 and 2,6 TDI bound protein. The reactivities of these mAbs were characterized by a solid phase indirect enzyme-linked immunosorbent assay (ELISA), Dot ELISA and Western immunoblot against various monoisocyanate, diisocyanate and dithioisocyanate protein conjugates. A subset of the mAbs were specific for 2,4 or 2,6 TDI-conjugated proteins only while others reacted to multiple dNCO conjugates including methylene diphenyl diisocyanate- and hexamethelene diisocyanate- human serum albumin . Western blot analyses demonstrated that some TDI conjugates form inter- and intra-molecular links resulting in multimers and a change in the electrophoretic mobility of the conjugate. In general, 2,4/2,6 TDI reactive mAbs displayed (1) stronger recognition of monoisocyanate haptenated proteins when the isocyanate was in the ortho position relative to the tolyl group, and were able to discriminate between (2) isocyanate and isothiocyanate conjugates (i.e. between the urea and thiourea linkage); and (3) between aromatic and aliphatic diisocyanates. The mAbs produced were not carrier protein specific with estimated affinity constants toward toluene diisocyanate conjugated human serum albumin ranging from 2.21 x 107 to 1.07 x 1010 M-1 for IgG mAbs. Studies using TDI vapor exposed lung and epithelial cell lines suggest potential utility of these mAbs for both research and biomonitoring of isocyanate exposure.

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Cells process a wide range of signals by means of multi-component receptors that span the plasma membrane. Our knowledge about the individual proteins involved in these signaling cascades has grown considerably over recent years. However, critical information about the detailed mechanisms of receptor activation, and the quantitative relationships between stimulus and biological response, is still missing. Here, I used the RET receptor tyrosine kinase (RTK), together with its glycosylphosphatidylinositol-coupled co-receptor GFRα3 and their activating growth factor artemin (ART), as a model system to investigate the quantitative and mechanistic features of receptor activation and signaling. I used a set of anti-RET agonist antibodies to induce different extents of receptor clustering on the cell surface, and studied how this factor affects the amplitude and kinetics of membrane-proximal and downstream signaling events, as well as the biological response of neurite outgrowth. Using simulations of the RET-GFRα3-ART system, I studied the effect of co-receptor involvement in the activation mechanism, as well as the importance of the specific activation pathway for the RET system’s response to variations in the expression levels of different components. The principal findings of my work include the following: 1) Higher order receptor clustering is required for full RET activation, as well as for the biological response of neurite outgrowth. 2) The activated forms of the receptor brought about by the agonist antibodies and by ART plus GFRα3 are identical with respect to the ability to activate the transient extracellular signal-regulated kinase (ERK) and Akt responses, but the antibodies show a reduced ability to induce sustained activation of ERK, Akt or c-Jun N-terminal kinase (JNK). 3) The involvement of GFRα3 co-receptor in the activation mechanism of RET provides cells with the ability to regulate their sensitivity to ligand without affecting the maximum amplitude of the pRET response. 4) This effect is limited if the co-receptor GFRα3 is pre-dimerized. Overall, my work aims to elucidate broad principles that underlie the quantitative relationships between RET activation, signaling, and the resulting cellular functional response, that can be applied to other receptor systems.

The development of safer and more accessible contraceptive options is necessary to reduce the high number of unintended pregnancies worldwide. As monoclonal antibody engineering continues to revolutionize drug development, a variety of strategies are being harnessed to establish antibody-based contraceptives. Human Contraception Antibody (HCA), an immunoglobulin G1 (IgG1) monoclonal antibody that potently agglutinates human sperm, is a promising candidate for nonhormonal immunocontraception in women. Our group recently established the safety and efficacy of a topical IgG1 HCA-formulated dissolvable vaginal film. Though successful, we are currently working to further optimize and improve the HCA product. In this study, we characterized engineered variants of HCA. Bioactivities, specifically agglutination and effector functions, of multimeric and fragment crystallizable (Fc)-mutated variants were compared and inform further engineering of an optimal clinical profile. We then established an atomized mRNA mechanism for delivery of HCA to the female reproductive tract (FRT). The use of mRNA could provide several advantages including: efficiency, reversibility, safety, durability, and cost-effectiveness. mRNA-encoded HCAs were expressed in several models of the FRT and were functional, sperm-specific, and safe. We also analyzed Fc N-glycans at the conserved glycosylation site on IgGs that regulate effector functions and compared the site-specific glycosylation on antibodies generated by two HCA expression platforms of interest, namely Nicotiana benthamiana and mRNA-transfected vaginal cells. Disparities in glycan site occupancy and glycoform populations between the two platforms were observed. Platform-specific HCA glycans resulted in differing levels of sperm phagocytosis, an Fc function. In summary, these studies provide a clearer understanding of engineered variants and delivery platforms to further advance the development of HCA as a novel, antibody-based female contraceptive.

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