In nanopore analysis, peptides and proteins can be detected by the change in current when single molecules interact with an α-hemolysin pore embedded in a lipid membrane. Studies into the effects of fluorenylmethoxycarbonyl (Fmoc), acetylation or proline modification to negatively charged α-helical peptides showed that Fmoc peptides give more translocations than acetylated peptides. The addition of a proline in the middle of an acetylated peptide further reduces the number of translocations compared to Fmoc. The effect of peptide conformation on translocation or intercalation was studied with small α-helical and β-sheet hairpins. The capped β-hairpin increased translocations compared to the uncapped. The Fmoc-α-helical hairpin, containing a disulfide link, displayed both bumping and translocations whereas in the unlinked peptide the proportion of translocations was greater. Prion diseases arise from the misfolding and aggregation of the normal cellular prion protein. Nanopore analysis of prion peptides with α-helical and β-strand sequences show changes to the event parameters that help distinguish them. The interaction of bovine prion protein (bPrP), with α-hemolysin showed both bumping (type-I) and intercalation/translocation (type-II) events. There are several lines of evidence that indicate these type-II events with a blockade current of -65 pA for bPrP, represent translocations. Nanopore analysis showed that about 37% events were translocations. The interaction of metal ions with bPrP showed that Cu(II) or Zn(II) reduced translocations. Surprisingly, Mn(II) caused an increase in translocation events to about 64%. Complex formation between antibodies and prion peptides and proteins can be detected by nanopore analysis. The PrP/antibody complex is too large to translocate whereas the event parameters for unbound molecules are unchanged. In principle, a nanopore can detect a single molecule; thus, this work represents the first step towards the development of a prion detector. The nanopore will provide the sensitivity and the antibodies will provide the specificity to distinguish between PrPC and PrPSc. Also, the prion N- and C-terminal signal peptides interact with bPrP changing the event parameters, relating to a new mechanism. Finally, the folding intermediates of bPrP at 0.86 M Gdn-HCl suggests that the protein unfolds and then refolds into a different conformation with event parameters similar to those of bPrP.

In nanopore analysis, peptides and proteins can be detected by the change in current when single molecules interact with an α-hemolysin pore embedded in a lipid membrane. Studies into the effects of fluorenylmethoxycarbonyl (Fmoc), acetylation or proline modification to negatively charged α-helical peptides showed that Fmoc peptides give more translocations than acetylated peptides. The addition of a proline in the middle of an acetylated peptide further reduces the number of translocations compared to Fmoc. The effect of peptide conformation on translocation or intercalation was studied with small α-helical and β-sheet hairpins. The capped β-hairpin increased translocations compared to the uncapped. The Fmoc-α-helical hairpin, containing a disulfide link, displayed both bumping and translocations whereas in the unlinked peptide the proportion of translocations was greater. Prion diseases arise from the misfolding and aggregation of the normal cellular prion protein. Nanopore analysis of prion peptides with α-helical and β-strand sequences show changes to the event parameters that help distinguish them. The interaction of bovine prion protein (bPrP), with α-hemolysin showed both bumping (type-I) and intercalation/translocation (type-II) events. There are several lines of evidence that indicate these type-II events with a blockade current of -65 pA for bPrP, represent translocations. Nanopore analysis showed that about 37% events were translocations. The interaction of metal ions with bPrP showed that Cu(II) or Zn(II) reduced translocations. Surprisingly, Mn(II) caused an increase in translocation events to about 64%. Complex formation between antibodies and prion peptides and proteins can be detected by nanopore analysis. The PrP/antibody complex is too large to translocate whereas the event parameters for unbound molecules are unchanged. In principle, a nanopore can detect a single molecule; thus, this work represents the first step towards the development of a prion detector. The nanopore will provide the sensitivity and the antibodies will provide the specificity to distinguish between PrPC and PrPSc. Also, the prion N- and C-terminal signal peptides interact with bPrP changing the event parameters, relating to a new mechanism. Finally, the folding intermediates of bPrP at 0.86 M Gdn-HCl suggests that the protein unfolds and then refolds into a different conformation with event parameters similar to those of bPrP.

A method for extraction of therapeutic proteins from dried blood spots (DBS) followed by quantification on Gyrolab(TM) has been developed. The method makes it possible to measure the concentration of the analyte in the range 100-6000 ng/mL. The procedure can generate full analytical information from 15 μL blood originally sampled from a subject. The modest sample requirements allows for sampling a full pre-clinical pharmacokinetic profile from a single mouse. This may allow for reduced usage of animals during preclinical development of new therapeutic proteins in accordance with the 3R’s, replace, refine and reduce.

Šio darbo tikslas buvo gauti monokloninius antikūnus prieš Hendra ir Nipah virusų nukleokapsidės baltymus. Dėl didelės Henipavirus genties virusų nukleokapsidės baltym�� homologijos sunku gauti monokloninius antikūnus, specifiškus konkretaus viruso nukleokapsidės baltymui. Siekiant išspręsti šią problemą, buvo panaudoti chimeriniai rekombinantiniai baltymai, sukonstruoti pelės poliomos viruso pagrindinio kapsidės baltymo VP1 pagrindu, į kurį buvo įterptos nehomologiškos Nipah ir Hendra virusų nukleokapsidės baltymų sekos. Imunizacijoms panaudojus tokius chimerinius baltymus, buvo nustatyta, kad jie sukelia stiprų imuninį atsaką. Buvo sukurti nauji monokloniniai antikūnai, specifiški tik Nipah viruso nukleokapsidės baltymui ir nereaguojantys su Hendra viruso nukleokapsidės baltymu. Taip pat buvo sukurti monokloniniai antikūnai prieš baltymą-nešiklį – pelės poliomos viruso pagrindinį kapsidės baltymą VP1. Naujai sukurtų antikūnų specifiškumas buvo patvirtintas imunofermentinės analizės ir imunoblotingo metodais. Monokloninių antikūnų prieš Hendra viruso nukleokapsidės baltymą gauti nepavyko. / The aim of this study was to generate monoclonal antibodies against Hendra and Nipah virus nucleocapsid proteins. There is high homology between nucleocapsid proteins of Henipavirus genus members, therefore it is difficult to generate monoclonal antibodies that do not show any cross-reactivity with both antigens. This problem was solved by using recombinant chimeric proteins designed by insertion of non-homological segments of Hendra and Nipah virus nucleocapsid proteins into the mouse polyomavirus capsid protein VP1. Mice were immunized with these chimeric proteins and it was determined that they induce a strong immune response. Monoclonal antibodies against Nipah virus nucleocapsid protein as well as carrier protein – mouse polyomavirus capsid protein VP1 – were generated. The specificities of newly developed monoclonal antibodies were confirmed by ELISA and immunoblot. The generation of specific monoclonal antibodies against Hendra virus nucleocapsid protein failed.

Tyrimo tikslas buvo įvertinti sensitizaciją ŽLA antigenais, inksto transplantatų recipientams, kurie greta standartinės imunosupresijos vartojo monokloninius antikūnus prieš IL-2 receptorių ir monokloninių antikūnų nevartojusiems recipientams. Tyrime dalyvauja VULSK pacientai, kuriems 2000-2005 metais imtinai buvo atliktos inkstų transplantacijos (Tx), bei kurie prieš ir po Tx buvo tirti dėl teigiamai su limfocitų panele reaguojančių antikūnų skaičiaus, išreikšto procentais (PRA). Iš viso tyrime dalyvauja 189 recipientai. Dalis jų (n=83) greta standartinės imunosupresijos vartojo monokloninius antikūnus prieš IL-2 receptorių (basiliksimabą ar daklizumabą), kiti (n=106) gavo tik standartinę imunosupresiją. Pagrindiniai sensitizaciją ŽLA antigenais lemiantys veiksniai abiejose grupėse pasiskirstė nevienodai. Didesnė monokloninius antikūnus vartojusių dalis gavo kraujo perpylimus (72% vs. 57,3%), šioje grupėje taip pat daugiau buvo pakartotinų Tx (9,6% vs. 7,5%), tik gimdžiusių moterų skaičius didesnis buvo monokloninių antikūnų nevartojusioje grupėje (47,7% vs. 30,8%). Tirtos ligonių grupės palygintos taikant &#967;² kriterijų, skirtumas laikytas statistiškai reikšmingas, kai p<0,05. Išanalizavus recipientų sensitizaciją prieš Tx paaiškėjo, kad dauguma (58%; 110/189) buvo nesensitizuoti (PRA 0-10%), likę 42% (79/189) - sensitizuoti, iš kurių 14% (11/189) – labai sensitizuoti (PRA 50-100%). Po Tx monokloninius antikūnus vartojusių recipientų grupėje (n=83) 2% padaugėjo... [toliau žr. visą tekstą] / The aim of this study was to evaluate the sensitization with HLA antigens in kidney transplant recipients, who received induction therapy with monoclonal antibodies against IL-2R and in the group of patients, who were only under the triple drug therapy. This study comprises recipients, who received kidney transplant in the year 2000-2005, and who were tested for panel reactive antibody test before and after transplantation (Tx). The total number of 189 kidney transplant recipients takes part in this study. 83 received monoclonal antibodies against IL-2R (basiliximab or daclizumab), others (n=106) – did not. These groups were unequal in comparison to the main factors causing sensitization with HLA antigens. The group of patients, who received induction therapy with monoclonal antibodies had more blood transfuzions (72% vs. 57,3%), and previous transplantations (9,6% vs. 7,5%), in comparison with the other group. Only the number of pregnancies was higher in the group of patients who were only under the triple drug therapy (47,7% vs. 30,8%). Statistical analyses were caried out using chi-square test, differences were considered significant at p<0,05. 58% (110/189) of kidney transplant recipients were unsensitized (PRA 0-10%) before Tx, the rest 42% (79/189) were sensitized, from which 14% (11/189) were highly sensitized (PRA 50-100%). After Tx the number of medium sensitized (PRA 11-50%) kidney transplant recipients, who received induction therapy by monoclonal antibodies... [to full text]

The dissertation describes development and characterization of monoclonal antibodies against recombinant yeast-expressed antigens: nucleocapsid (N) proteins of human parainfluenza virus type 3, Menangle virus, hantavirus and rabies virus. The newly developed antibodies were investigated by different immunochemical assays for their specificity, affinity and ability to recognize native viruses in infected cells. It was determined that the antibodies raised against recombinant yeast-expressed viral proteins are suitable to identify virusinfected cells. These data confirmed that recombinant yeast-expressed viral N proteins possess antigenic properties similar to that of native viral nucleocapsids. The monoclonal antibodies were also used to study the antigenic structure of viral N proteins and localize their immunodominant regions. The obtained results may have impact on the development of new immunodiagnostic test systems for the detection of viral infections. The dissertation consists of the introduction, three sections, references, and the list of author’s publications. In the Introductory Chapter, the research topic, the actuality, the aim and tasks, scientific novelty and practical value of the dissertation are discussed. Author’s publications and conference reports are also presented. The first Chapter of the dissertation provides literature overview on the genome organization, structural proteins, pathogenesis and epidemiology of parainfluenza viruses, Menangle virus... [to full text] / Disertacijoje aprašomi monokloniniai antikūnai, sukurti prieš rekombinantinius mielėse susintetintus antigenus: žmogaus paragripo treciojo tipo viruso, Menangle viruso, hantavirusų bei pasiutligės viruso nukleokapsidės (N) baltymus. Sukurtieji antikūnai buvo visapusiškai charakterizuoti įvairiais imunocheminės analizės metodais, įvertintas jų specifiškumas, afiniškumas, sugebėjimas atpažinti natyvius virusus infekuotų ląstelių kultūrose. Buvo nustatyta, kad antikūnai, sukurti prieš rekombinantinius mielėse susintetintus virusų baltymus, tinka virusų nustatymui infekuotose ląstelėse. Šie tyrimai patvirtino, kad rekombinantiniai mielėse susintetinti virusu N baltymai turi panašias antigenines savybes, kaip natyvūs virusų N baltymai, formuojantys nukleokapsides. Sukurtieji monokloniniai antikūnai taip pat buvo panaudoti išsamiems minėtų virusų N baltymų antigeninės struktūros tyrimams bei imunodominuojančių sekų nustatymui. Disertaciniame darbe gauti duomenys svarbūs, kuriant naujas imunodiagnostikos sistemas, skirtas virusų infekcijoms nustatyti. Disertacija sudaro įvadas, trys skyriai, naudotos literatūros sąrašas ir autorės publikacijų sąrašas. Įvadiniame skyriuje aptariama tiriamoji problema, darbo aktualumas, formuluojamas darbo tikslas bei uždaviniai, darbo mokslinis naujumas ir praktinė reikšmė, pristatomos paskelbtos publikacijos ir pranešimai konferencijose. Pirmasis disertacijos skyrius skirtas literatūros apžvalgai: jame apibūdinamos paragripo virusų, Menangle viruso... [toliau žr. visą tekstą]

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Access to abstract permanently restricted to Ball State community only / Access to thesis permanently restricted to Ball State community only / Department of Physiology and Health Science

Autoantibodies generated against M proteins can cause post-streptococcal disorders such as Rheumatic Fever. A severe complication of rheumatic fever is rheumatic heart disease which may involve both cardiomyopathy and valvulitis. Rheumatic fever has been associated with the class I M protein epitope of Group A streptococcus (GAS). This epitope can be recognized by monoclonal antibodies (mAbs) 10B6 and 10F5. Previously, we demonstrated binding of streptococcal mAb10F5 in the heart tissue (apex, atria, and valves) of Lewis rats as compared to anti-myosin binding. To determine if mAb10F5 binding in the heart is due to virulence of the antibody or antibody subtype, rats were injected with control IgG2 antibodies and euthanized after 24, 48, or 72 hrs. Hearts were harvested and immunofluorescence was used to analyze the hearts. The immunofluorescence intensities for IgG2b were compared to mAb10F5 using previously acquired data. Control IgG2b rats showed significantly less immunofluorescence intensities in the heart regions than mAb10F5 injected rats at the 48 and 72 hr time points. These findings reaffirm mAb10F5 as an anti-cardiac antibody thatbinds heart tissue due its own virulence. To differentiate between the two IgG subtypes, binding intensities of IgG2a were compared to the binding intensities of IgG2b. The binding intensities of IgG2a increased with time. This finding was supported by previous work in our laboratory suggesting IgG2a remained in the bloodstream longer than the IgG2b. / Access to thesis permanently restricted to Ball State community only. / Department of Physiology and Health Science