A fusion protein composed of antibody fragments and β-lactamase was earlier created by Kojima et al. (2011), with antigen specificities against a bone disease marker and a pesticide. The enzyme was circularly permutated and fused to the variable heavy and light chain antibody fragments, thereby ensuring inactivity until binding of the target antigen triggered enzyme activation. Upon activation, the β-lactamase produced a colorimetric signal, which indicated antigen presence. In this work, a similar strategy was used to create two novel fusion proteins composed of circularly permuted β-lactamase and superfolder green fluorescent protein with anti-benzo[a]pyrene variable antibody fragments. The fusion proteins were designed and expressed in E. coli for the development of a single-step visual immunoassay. It was hypothesized that the cp reporter proteins would be activated once the binding of B[a]P to the variable antibody fragments occurred, and this interaction was expected to produce a detectable colorimetric or fluorescent signal. Although positive results were obtained in one instance, substantial supportive evidence in favour of the hypothesis could not be obtained. / SENTINEL Bioactive Paper Network, Natural Sciences and Engineering Research Council of Canada (NSERC), Canada Research Chairs Program.

The aim of this research was the development of mass spectrometry-based methodologies for the high-throughput quantitation of peptides in biological matrices. Glucagon and GLP-1, which are of interest as biomarkers and in the development of therapeutics, were chosen as model peptides. Immunoassays that are traditionally used to quantify these often perform poorly; therefore, necessitating the development of alternative methodologies. Application of mass spectrometry-based methodologies to these analytes has, however, been limited, primarily due to sensitivity challenges, but also due to analytical challenges associated with their endogenous nature and instability in biological matrices. Chapter 2 describes the development and qualification of the first liquid-chromatography coupled tandem mass spectrometry (LC-MS/MS) method for the quantitation of endogenous glucagon from human plasma. A novel 2D extraction procedure was developed to ensure robustness and sensitivity, whilst a novel surrogate matrix quantitation strategy took into account the endogenous nature of the analyte. A lower limit of quantitation (LLOQ) of 25 pg/mL was qualified, which was a considerable improvement over that previously reported in the literature (250 pg/mL) for a LC-MS/MS method. Clinical samples were cross-validated against a conventional radioimmunoassay (RIA), and similar pharmacokinetic (PK) profiles resulted, demonstrating that the methods were complementary. In Chapter 2 glucagon instability in biological matrix was noted. To characterise this further, in Chapter 3 in vitro glucagon metabolites were identified using high-resolution mass spectrometry (HRMS). Metabolites observed by others (glucagon19-29, glucagon3 29 and [pGlu]3glucagon3 29) in alternative matrices were identified, alongside novel metabolites (glucagon20-29 and glucagon21-29). Cross-interference of these metabolites in immunoassays may help to explain their poor performance, whilst knowledge of metabolism may also aid the development of future stabilisation strategies. The method developed in Chapter 2 was refined in Chapter 4 to improve sensitivity, robustness and throughput, and to add GLP-1 as a secondary analyte. The sensitivity achieved (glucagon: 15 pg/mL LLOQ, GLP-1: 25 pg/mL LLOQ) is the highest reported for both peptides for an extraction avoiding immunoenrichment. Specificity of endogenous glucagon quantitation was assured using a novel approach with a supercharging mobile phase additive to access a sensitive qualifier transition. A cross-validation against established immunoassays using physiological study samples demonstrated some similarities between the methods. Differences between the immunoassay results exemplified the need to develop alternative methodologies. The resulting LC-MS/MS method is considered a viable alternative to immunoassays, for the quantitation of endogenous glucagon, dosed glucagon and/or dosed GLP-1 in human plasma.

Made available in DSpace on 2011-05-04T12:36:25Z (GMT). No. of bitstreams: 0 Previous issue date: 2010 / Os agrotóxicos são substâncias usadas para exterminar pragas ou doenças que causam danos às plantações. Os organofosforados são agrotóxicos responsáveis por 70 por cento das intoxicações ocupacionais e atuam inibindo a enzima acetilcolinesterase por meio da fosforilação do aminoácido serina do sítio ativo. Existem diversos métodos de diagnóstico para monitorar a exposição a estes compostos, tais como: identificação e análise quantitativa de compostos livres noplasma; identificação e análise quantitativa de metabólitos na urina; determinação da atividade de colinesterases no plasma ou no sangue total. Entretanto, estas metodologias possuem algumas limitações, como por exemplo, a falta de especificidade, tecnologias de análise caras e variações interindividuais (sexo, idade, etc.). Uma tentativa de contornar estas limitações na quantificação da atividade de colinesterases é o desenvolvimento de metodologias baseadas na detecção da enzima fosforilada através de técnicas imunoquímicas. Este estudo teve como objetivo desenvolver um método de imunoensaio para o diagnóstico rápido da exposição humana aos organofosforados pela detecção deacetilcolinesterase inibida no sangue humano por anticorpos aviários produzidos contra uma sequência de aminoácidos ao redor da serina fosforilada do centro catalítico da enzima. Para isto, foram adquiridos dois decapeptídeos sintéticos correspondentes ao sítio catalítico "não fosforilado" (SOH) e outro com o resíduode serina fosforilado (SOP). Estes peptídeos foram conjugados com albumina bovina de soro (BSA), injetados em galinhas poedeiras da raça Isa-Brown e anticorpos IgY foram obtidas das gemas dos ovos das galinhas imunizadas. A especificidade dos anticorpos IgY anti-SOH e anti-SOP foi testada por meio de métodos ELISA constatando-se que estes anticorpos reconhecem os peptídeos livres, os peptídeos conjugados com BSA e a própria BSA. Além disso, ocorreu uma significativa reação cruzada, ou seja, os anticorpos IgY anti-SOH e anti-SOP não conseguiram diferenciar os peptídeo SOH e SOP. Estes resultados não permitem a indicação destes anticorpos para o desenvolvimento de métodos para diagnóstico de intoxicação. / Pesticides are chemical substances used to control plagues and diseases in plantations. Organophosphates are the pesticides involved in 70% of the occupational intoxications and act inhibiting the enzyme acetylcholinesterase by phosphorylation of the serine residue of catalytic centre. There are several methods for monitoring the exposition to these compounds, as so: identification and assay the free compounds into plasma; identification and assay metabolites into urine; cholinesterases assay into plasma or blood. However, these methods have some limitations, as for example lack of specificity, expensive analytical technologies and inter-individual variety (sex, age, etc.). One way to outline these limitations on cholinesterase assay is the development of methods based on phospho-enzyme detection by imunochemical techniques. This study have had the objective to developing an imuno-assay method for rapid diagnosis of human exposition to organophosphorus compounds by imuno-detection of inhibited acetylcholinesterase using aviary antibodies produced against a peptide sequence of the catalytic centre around phospho-serine residue. Two synthetic decapeptides were bought: one with the sequence of "not-phosphorylated" catalytic centre (SOH) and another with a serine-phosphorylated residue. These peptides were conjugated with bovine serum albumin (BSA), injected into Isa-Brown hens and antibodies IgY were isolated from egg yolk of immunized hens. The specificity of IgY anti-SOH and anti-SOP was tested using ELISA methods showing that these antibodies recognize free peptides, conjugated peptides and BSA. In addition, there was a significant cross reaction, or in other words, the antibodies IgY anti- SOH and anti-SOP failed on differentiate the peptide SOH and SOP. These results do not allow the indication of these antibodies for developing methods for intoxication diagnosis.

Made available in DSpace on 2016-12-23T13:56:12Z (GMT). No. of bitstreams: 1 Aline Lyra Pereira.pdf: 1742611 bytes, checksum: fac56687c54e0ab6545343dd5ab0a212 (MD5) Previous issue date: 2012-03-30 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A paracoccidioidomicose (PCM) é uma micose sistêmica causada pelo fungo dimórfico Paracoccidioides brasiliensis. Está entre as infecções fúngicas mais frequentes da América Latina, sendo o Brasil o país de maior endemicidade, com maior prevalência na região Sudeste. O diagnóstico considerado por muitos autores padrão-ouro para a PCM é o encontro de células fúngicas sugestivas de Paracoccidioides brasiliensis no Exame Microscópico Direto (EMD) de escarro ou outros espécimes clínicos, como raspado de lesão, aspirado de linfonodos, biopsia, etc. As provas sorológicas têm grande importância, complementando ou substituindo o exame micológico no diagnóstico/prognóstico da doença. O objetivo do presente estudo foi comparar dois desses testes sorológicos para PCM: a imunodifusão dupla (IDD) e o ensaio imunoenzimático ELISA. A metodologia foi estabelecida comparando-se títulos de anticorpos obtidos de três grupos de pacientes: 145 amostras de soros de 83 pacientes com PCM (Grupo I), 49 doadores saudáveis (Grupo II) e 13 pacientes com IDD positiva para outras micoses pulmonares: 11 com histoplasmose e 2 com aspergilose (Grupo III). Nossos resultados mostraram que 8 (9,6%) e 5 (6,0%) dos pacientes com PCM foram falso-negativos ao teste de IDD e ELISA, respectivamente. Dentro do grupo de pacientes saudáveis, não houve casos de falso positivo na IDD, porém, 5 (10,2%) amostras foram positivas com títulos baixos no ELISA. Entre os 13 pacientes com outras micoses, o ELISA mostrou que 10 (77,0%) foram positivos enquanto a IDD não apresentou qualquer caso de reação cruzada. A sensibilidade do ELISA (93,9%) foi um pouco superior ao da IDD (90,3%). A especificidade do ELISA calculada como base o controle saudável e os pacientes com outras micoses foi 89,7% e 23,1%, respectivamente. A IDD mostrou uma especificidade de 100% para ambos os casos. Os valores preditivos positivos e negativos foram de 100% e 83,8% para IDD e de 88,5% e 90,3% para ELISA, respectivamente. A acurácia dos testes também foi calculada: a IDD teve 94,4% e o ELISA 86,2% de acurácia. / Paracoccidioidomycosis (PCM) is a systemic mycosis caused by the dimorphic fungus Paracoccidioides brasiliensis. It is among the most common fungal infections in Latin America, with Brazil being the country of highest endemicity, mainly in the Southeast part. The diagnosis considered by many authors as gold standard for the PCM is the finding of suggestive yeast cells of Paracoccidioides brasiliensis in the Direct Microscopic Examination of sputum or other clinical specimens, such as scraping the lesion, lymph node aspirate, biopsy, etc. The serological tests are of great importance, since they complement or replace the mycological examination in the diagnosis / prognosis of the disease. The aim of this study was to compare two serological tests for PCM: a double immunodiffusion (IDD) and Enzyme-Linked Immunoabsorbent Assay (ELISA). The methodology was established by comparing antibody titers obtained from three groups of patients: 145 serum samples from 83 patients with PCM (Group I), 49 healthy blood donors (Group II) and 13 patients with IDD positive for other pulmonary mycoses: 11 with histoplasmosis and 2 with aspergillosis (Group III). Our results showed that eight (9.6%) and 5 (6.0%) of patients with PCM were false negative to the IDD and ELISA tests, respectively. In the group of healthy patients, no cases of false positive in IDD were observed; however, 5 (10.2%) of samples were positive with low titers in ELISA. Among the 13 patients with other mycoses, the ELISA showed that 10 (77.0%) were positive while the IDD tests did not show any case of cross-reaction. The sensitivity of the ELISA (93.9%) was slightly higher than the IDD (90.3%). The specificity of the ELISA calculated basing on the healthy control and patients with other mycoses was 89.7% and 23.1%, respectively. IDD showed 100% of specificity for both cases. The positive predictive and negative predictive values were 100% and 83.8% for IDD and 88.5% and 90.3% for ELISA, respectively. The accuracy of the tests was also calculated: the IDD was 94.4% while ELISA was 86.2%.

Transmissive spongiform encephalopathies (TSEs) are neurodegenerative diseases characterized by depositions of abnormally folded prion protein (PrPTSE ) in brain. PrPTSE is at present the only specific biochemical marker of human and animal TSEs. Diagnostic tests are based on the detection of PrPres after proteinase K digestion of brain homogenate using Western blot or on the immunohistochemistry of fixed brain tissue, which are both difficult and time consuming. In this work we focused on development of a new type of tests based on PrP detection without need of proteinase K digestion. As deposits of PrPTSE remain in the body for a long time, there is a substantial chance of them being nonenzymatically modified by glycation. The detection of glycated PrPTSE may have a potential to serve as a diagnostic marker. We prepared monoclonal antibodies specific for carboxymethyl lysine/arginine modified prion protein. Bacterially expressed and purified recombinant human prion protein (rhPrP) was modified by glyoxylic acid that introduces carboxymethyl groups on lysine and arginine residues present within the molecule of the protein. Modified rhPrP (rhPrP-CML) was used for immunization of laboratory mice and hybridoma cells were prepared. Screening of cell supernatants resulted in the selection of 4...

Abstract Cancer progression is a multistep process dependent on tumour-stroma interactions. Various cell types, such as fibroblasts, endothelial, inflammatory and stem cells, as well as extracellular matrix (ECM) proteins, such as collagens, contribute to the tumour outcome. Tumour growth and invasion is accompanied by the proteolysis of ECM components mediated by various enzymes, such as matrix metalloproteases (MMPs). Proteolytic fragments released into the circulation may reflect cancer progression. The aim of this study was to develop novel in vitro methods for investigating cell invasion and for measuring cancer-associated collagen degradation. Human carcinoma cell invasion studies in vitro are often performed in organotypic cell culture models, mainly composed of rat or mouse ECM proteins. To create a human microenvironment for invasion studies, a novel organotypic model based on human uterine myoma (benign tumour) tissue was developed. Compared to the conventional collagen-based organotypic model, in the myoma model the carcinoma cell invasion depth was about eightfold and the invasion resembled, to a greater degree, the invasion pattern of dissected tissue samples of cancer patients. In addition, the invasion was easily quantified with a novel radioimmunoassay measuring type III collagen degradation products from the organotypic culture media. As human mesenchymal stem cells (MSCs) are one of the stromal cell types that may affect tumour progression, the mechanisms of stem cell invasion were also studied. On the surface of MSCs, Toll-like receptor 9 (TLR9) functions in immune defence against microbes. The activation of TLR9 with microbial DNA-resembling molecules induced human MSC invasion into the myoma tissue in a MMP-13-mediated fashion. To analyse cancer-associated soft tissue degradation, a novel enzyme immunoassay was developed. This novel assay enabled, for the first time, the measurement of type III collagen degradation products from human serum samples. In head and neck cancer patient sera, high levels of type III and type I collagen degradation products were shown to predict poor survival. In conclusion, the novel myoma model showed that the tumour microenvironment crucially affects carcinoma cell invasion. In addition, cancer-associated type III collagen degradation was successfully measured in cell cultures and in human sera by novel immunoassays. / Tiivistelmä Syövän eteneminen on monivaiheinen tapahtuma, jossa syöpäsolut ovat vuorovaikutuksessa lähiympäristönsä kanssa. Ympäristön eri solutyypit, kuten kantasolut ja sidekudoksen fibroblastit sekä soluväliaineen proteiinit kuten kollageenit, vaikuttavat syöpäsolujen invaasioon eli tunkeutumiseen ympäröivään kudokseen. Syövän invaasiossa useat entsyymit, mm. matriksin metalloproteaasit (MMP:t), hajottavat soluväliainetta. Kasvaimen kehittymisen aikana verenkiertoon vapautuu soluväliaineen hajoamistuotteita, joiden määrä voi kuvastaa sairauden etenemistä. Väitöstutkimuksen tarkoituksena oli kehittää uusia menetelmiä solujen invaasion ja syöpään liittyvän kollageenin hajoamisen tutkimiseen. Ihmisen karsinoomasolujen invaasion tutkimuksessa on perinteisesti käytetty kolmiulotteisia soluviljelymalleja, jotka koostuvat pääasiassa rotan tai hiiren soluväliaineproteiineista. Työssä kehitettiin uusi viljelymalli, jossa soluja kasvatettiin ihmisen hyvälaatuisen kohtukasvainkudospalan eli myooman päällä. Perinteiseen kollageenimalliin verrattuna myoomamallissa karsinoomasolut tunkeutuivat noin kahdeksan kertaa syvemmälle, ja solujen kasvu muistutti enemmän potilaiden syöpäkudosnäytteissä havaittua kasvutapaa. Invaasion voimakkuuden määrittämiseen kehitettiin vasta-aineisiin perustuva menetelmä, jolla mitattiin soluviljelmän kasvatusliuokseen myoomakudoksesta vapautuneiden tyypin III kollageenin hajoamistuotteiden määrää. Koska kantasolujen tiedetään voivan vaikuttaa syöpäkasvaimen leviämiseen, tutkimme myös ihmisen luuytimen kantasolujen invaasiota. Kantasolujen pinnalla TLR9-reseptori osallistuu immuunipuolustukseen mikrobeja vastaan. Kun reseptoria aktivoitiin mikrobi-DNA:ta muistuttavilla molekyyleillä, kantasolut alkoivat invasoitua myoomakudokseen ja MMP-13:n aktiivisuus soluissa lisääntyi. Syöpään liittyvän pehmytkudoksen hajoamisen tutkimiseksi kehitettiin vasta-ainemenetelmä, jolla onnistuttiin ensi kertaa mittaamaan potilaiden seeruminäytteistä tyypin III kollageenin hajoamistuotteita. Pään ja kaulan alueen syöpäpotilailla korkean tyypin III kollageenin hajoamistuotepitoisuuden todettiin liittyvän huonoon ennusteeseen. Tutkimus osoitti, että kasvaimen ympäristö vaikuttaa olennaisesti syöpäsolujen leviämiseen. Syöpään liittyvää tyypin III kollageenin hajoamista pystyttiin työssä kehitetyillä menetelmillä mittaamaan sekä soluviljelmistä että potilaiden seeruminäytteistä.

Abstract Integrated optical (IO) sensor allowing sensitive, label-free, real-time and multi-parameter monitoring of bio-molecular interactions are conventionally fabricated with inorganic dielectrics inherited from CMOS manufacturing technology. Polymers as complement materials to inorganic dielectrics are becoming to have an increasing market share for IO circuits in optical communications networks owing to its good optical properties, versatile processibility and low cost. This work aims at developing disposable low-cost biosensors based mainly on polymeric materials, with a performance comparable to inorganic-dielectric based IO biosensors. This thesis describes the development of polymer IO biosensors based on the Young interferometer (YI) transducer platform for ambient noise compensation and a complete periodic intensity fringe pattern. Three different waveguide configurations were utilized, taking into consideration operational simplicity, fabrication simplicity and enhanced sensitivity. Among the developed polymer biosensors, an unconventional interferometer structure: a vertically placed dual-slab waveguide interferometer and an inverted rib waveguide configuration were employed. To enhance the sensitivity of the waveguides, deposition of Ta2O5 high index coating was performed on the rib waveguide configuration. Along with the development of polymer biosensors based on the inverted-rib waveguide configuration, a fabrication process was also developed featuring UV-imprinting and spin coating. The simple two-step fabrication process demonstrated using a polymer mold is potentially transferable to the roll-to-roll manufacture process. Calibration of the developed sensors was performed by homogeneous refractive index (RI) sensing with glucose de-ionized water solutions. By investigating an antibody – antigen binding interaction involving C-reactive protein and its conjugates, this thesis confirmed the applicability of the developed sensors to specific molecule detection. Moreover, to establish the influence of water molecular absorption on measurement stability, an evaluation was carried out on the polymeric waveguide. Finally, the thesis presented a comparison between the developed sensors, exploring their sensitivities, stabilities, limits of detection (LODs) and other aspects related to operation and fabrication. The results indicated that the Ta2O5-coated polymer waveguide sensor had a high sensing capability. In homogeneous RI sensing, the achieved detection limits were 9×10-7 RIU (refractive index unit), i.e., three times the noise level, and 270 fg/mm2 for surface mass density. / Tiivistelmä Integroidulla optiikalla toteutetut anturit mahdollistavat biomolekulaarisen vuorovaikutuksen tutkimisen käyttäen herkkiä moniparametrisia ja merkkiaineettomia menetelmiä. Näiden bioantureiden valmistukseen käytetään tavallisesti CMOS-teknologian piiristä tuttuja epäorgaanisia puolijohteita ja eristemateriaaleja. Viime aikoina on kuitenkin polymeeristen materiaalien käyttöä integroidussa optiikassa tutkittu merkittävästi johtuen polymeerien hyvistä optisista ominaisuuksista, monipuolisesta työstettävyydestä ja edullisista kustannuksista. Tämän työn tarkoituksena on kehittää edullisia, kertakäyttöisiä, pääasiallisesti polymeerisistä materiaaleista valmistettuja bioantureita, jotka vastaavat suorituskyvyltään epäorgaanisista materiaaleista valmistettuja integroidun optiikan antureita. Tässä työssä kehitetyt polymeeriset integroidun optiikan bioanturit perustuvat Youngin interferometriin mahdollistaen ympäristökohinan kompensoinnin ja ne tuottavat pintavuorovaikutusten tutkimiseen jaksoittaisen interferenssikuvion. Työssä hyödynnettiin kolmea erilaista valokanavarakennetta huomioiden niiden käytön helppous, valmistuksen yksinkertaisuus ja mittausherkkyys. Yksi kehitetyistä polymeerisistä bioantureista koostui päällekkäisistä kerrostetuista polymeerikerroksista. Toisen tutkitun rakenteen toiminta puolestaan perustui käänteiseen harjannevalokanavaan. Mittausherkkyyttä parannettiin pinnoittamalla polymeerirakenne Ta2O5-pinnoitteella. Näin muodostui kerrostettu komposiittivalokanava, joka oli tässä työssä tutkittu kolmas sensorirakenne. Itse bioanturien lisäksi kehitettiin myös valmistusprosessi, jossa hyödynnettiin UV-painatusta ja nestefaasipinnoitusta. Tässä työssä havaittiin lisäksi, että kehitetty yksinkertainen valmistusmenetelmä on paitsi toimiva, myös mahdollisesti siirrettävissä rullalta rullalle valmistus- ja tuotantoteknologiaan. Kehitettyjen anturien kalibrointi suoritettiin homogeenisella taitekerroinmittauksella käyttäen liuoksia, jotka valmistettiin glukoosista ja deionisoidusta vedestä. Kehitettyjen anturien soveltuvuus spesifien molekyylien tunnistamista varten todennettiin tutkimalla vasta-aineiden ja antigeenien sitoutumisreaktioita ja vuorovaikutusta C-reaktiivisella proteiinilla ja sen konjugaateilla. Lisäksi työssä tutkittiin veden absorption vaikutusta mittauksen stabiilisuuteen. Tutkimuksessa suoritettiin vertailu kehitettyjen anturien ja niiden ominaisuuksien välillä kiinnittäen huomiota mittausherkkyyteen, stabiilisuuteen, määritys- ja toteamisrajoihin ja muihin anturien valmistukseen sekä käyttöön liittyviin keskeisiin piirteisiin. Tulokset osoittavat, että Ta2O5-pinnoitetun polymeerivalokanavan mittausherkkyys oli suurin vertailluista rakenteista. Homogeenisessä taitekerroinmittauksessa saavutettu määritys- ja toteamisraja oli 9×10-7 taitekerroinyksikköä (RIU). Pintamassatiheysmittauksessa saavuttu tulos oli 270 fg/mm2.

In der vorliegenden Arbeit wird eine integrierbare Pumpentechnologie für polymerbasierte mikrofluidische Systeme entwickelt. Ausgehend von den Anforderungen für die Durchführung molekulardiagnostischer Nachweise kommen dabei Fertigungsverfahren zum Einsatz, die sich auch für Einweg-Anwendungen eignen. Das genutzte Aktorprinzip für die integrierten Mikropumpen basiert auf der Elektrolyse von Wasser. Zur besseren technologischen Integrierbarkeit wird das Wasser in Form eines Hydrogels appliziert. Der Elektrolyt wird dabei mit einer Polymermembran mit geringer Wasserdampfdurchlässigkeit verschlossen. Die Membran wird in ihrem plastischen Verformbereich genutzt. Zur Dimensionierung der Mikropumpen und des mikrofluidischen Systems werden analytische und numerische Modelle entwickelt, die eine gute Übereinstimmung mit den Messwerten zeigen. Die Funktionsfähigkeit wird anhand zweier vollständig integriert ablaufender Immunoassays demonstriert. Dabei kommt ein polymerbasierter, optischer Biosensor zum Einsatz.

Transmissive spongiform encephalopathies (TSEs) are neurodegenerative diseases characterized by depositions of abnormally folded prion protein (PrPTSE ) in brain. PrPTSE is at present the only specific biochemical marker of human and animal TSEs. Diagnostic tests are based on the detection of PrPres after proteinase K digestion of brain homogenate using Western blot or on the immunohistochemistry of fixed brain tissue, which are both difficult and time consuming. In this work we focused on development of a new type of tests based on PrP detection without need of proteinase K digestion. As deposits of PrPTSE remain in the body for a long time, there is a substantial chance of them being nonenzymatically modified by glycation. The detection of glycated PrPTSE may have a potential to serve as a diagnostic marker. We prepared monoclonal antibodies specific for carboxymethyl lysine/arginine modified prion protein. Bacterially expressed and purified recombinant human prion protein (rhPrP) was modified by glyoxylic acid that introduces carboxymethyl groups on lysine and arginine residues present within the molecule of the protein. Modified rhPrP (rhPrP-CML) was used for immunization of laboratory mice and hybridoma cells were prepared. Screening of cell supernatants resulted in the selection of 4...

Surface-enhanced Raman scattering (SERS) microscopy is a novel method of optical imaging for the localization and quantification of target molecules in cells and tissue specimens. The major advantages of SERS over fluorescence are quantification and spectral multiplexing due to the small line width of vibrational Raman bands. The position of the plasmon band of both hollow gold/silver nanoshells and silica-encapsulated gold nanoclusters can be tuned for maximum SERS enhancement upon red laser excitation, which is optimal for minimizing the disturbing autofluorescence of tissue. In this work, silica-encapsulated and non-encapsulated SERS particles were used for the localization of target proteins in prostate tissue specimens. Two different biofunctionalization methods were established for each type of SERS particles. The cross-linking method based on s-NHS/EDC chemistry was modified for covalently conjugating proteins to hollow gold/silver nanoshells and gold nanostars in order to minimize the aggregation of SERS nanoparticles during and after cross-linking. As an alternative to covalent conjugation chemistry, the noncovalent binding of antibodies to the SERS particles via an adapter protein (protein A/G) was established. The influence of several factors that determine the quality of results obtained by SERS imaging, such as the number of immuno-SERS conjugates, incubation time, antigen retrieval and blocking buffer, were investigated. Rapid SERS microscopy with 30 msec acquisition time per pixel was enabled by using silica-encapsulated gold nanoclusters for the localization of p63 proteins on prostate tissue specimens from healthy donors. Two-color SERS experiments for the parallel localization of PSA and p63 were performed with silica-encapsulated and non-encapsulated nanoshells. The quality of the results depends less on the nature of the surface chemistry of the nanoparticles (with or without silica encapsulation), but more on the blocking buffer and the antigen retrieval method. Silica-encapsulated gold nanoclusters were also used for the simultaneous quantification of three cytokines (IL1, IL8 and TNF- α) in a SERS-based sandwich immunoassay with a detection limit of ca. 0.3 pM. Keywords: Raman, SERS microscopy, biocompatibility of nanoparticles, cross-linking, antigen unmasking methods, antigen detection, immunohistochemistry, immunoassay.