Enhanced Detection of Host Response Antibodies to Borrelia burgdorferi Using Immuno-PCR

Lyme disease is the fastest-growing zoonotic disease in North America. Current methods for detection of Borrelia burgdorferi infection are challenged by analysis subjectivity and standardization of antigen source. In the present study, we developed an immuno-PCR (iPCR)-based approach employing recombinant in vivo-expressed B. burgdorferi antigens for objective detection of a host immune response to B. burgdorferi infection. iPCR is a liquid-phase protein detection method that combines the sensitivity of PCR with the specificity and versatility of immunoassay-based protocols. Use of magnetic beads coated with intact spirochetes provided effective antigen presentation and allowed detection of host-generated antibodies in experimentally infected mice at day 11 postinoculation, whereas host-generated antibodies were detected at day 14 by enzyme-linked immunosorbent assay (ELISA) and day 21 by immunoblotting. Furthermore, magnetic beads coated with recombinant B. burgdorferi in vivo-expressed antigen OspC or BmpA demonstrated positive detection of host-generated antibodies in mice at day 7 postinoculation with markedly increased iPCR signals above the background, with the quantification cycle (C-q) value for each sample minus the mean background C-q plus 3 standard deviations (Delta C-q) being 4 to 10, whereas Delta C-q was 2.5 for intact spirochete-coated beads. iPCR demonstrated a strong correlation (Spearman rank correlation = 0.895, P \u3c 0.0001) with a commercial ELISA for detection of host antibodies in human Lyme disease patient sera using the B. burgdorferi VlsE C6 peptide. In addition, iPCR showed potential applicability for direct detection of spirochetes in blood. The results presented here indicate that our iPCR assay has the potential to provide an objective format that can be used for sensitive detection of multiple host response antibodies and isotypes to B. burgdorferi infection

Avaliação de um dispositivo microfluídico baseado em fios têxteis para a determinação amperométrica de estriol

Orientador: Prof. Dr. Márcio Fernando BergaminiCoorientador: Prof. Dr. Luiz Humberto Marcolino JúniorDissertação (mestrado) - Universidade Federal do Paraná, Setor de Ciências Exatas, Programa de Pós-Graduação em Química. Defesa : Curitiba, 23/02/2017Inclui referências: p.63-74Resumo: Sistemas microfluídicos são dispositivos que se baseiam em princípios similares aos de sistemas de análise por injeção em fluxo. Esses dispositivos permitem o baixo consumo de reagentes e amostras, o baixo tempo de análise e a possibilidade de portabilidade. Diversos avanços têm sido relatados ao utilizar dispositivos de baixo custo baseados em fios de algodão como canais microfluídicos, em que o transporte da solução ocorre devido à capilaridade dos fios de algodão, com auxílio da gravidade. No presente trabalho, foi demonstrado a versatilidade de um dispositivo eletroanalítico baseado em fios têxteis (?TED), construídos utilizando fios de algodão como microcanais para o transporte de solução e eletrodos impressos (SPE) modificados com nanotubos de carbono (CNT), como detectores na determinação amperométrica do hormônio estriol em amostras farmacêuticas e aquosas. Os parâmetros envolvidos na determinação amperométrica e no sistema microfluídico foram estudados e otimizados, em que se utilizou das condições experimentais que apresentaram melhores respostas (vazão do sistema de 1,25 ?L s-1, 2,0 ?L de volume de injeção e potencial de detecção de 0,75 V) para a obtenção de uma curva analítica, abrangendo concentrações de 1,0 a 1000 ?mol L-1. Os limites de detecção (LOD) e quantificação (LOQ) foram 0,53 e 1,77 ?mol L-1, respectivamente, e a frequência analítica foi de 44 injeções por hora. A metodologia proposta foi aplicada para a determinação do hormônio estriol em amostras farmacêuticas, sendo os resultados obtidos comparados com os obtidos a partir da técnica espectrofotométrica (metodologia oficial). Os resultados obtidos foram concordantes com 95 % de confiança. Palavras - chave: Sistemas microfluídicos, dispositivo baseado em fios de algodão, determinação de hormônios, eletrodos impressos, nanotubos de carbono.Abstract: Microflow systems are powerful analytical tools that explore similar principles of typical flow injection analysis driven to in a microfluidic device. Generally, microfluidic devices can promote a low consumption of reagents and samples, high speed of analysis and possibility of portability. Several advances have been reached applying a simple and low cost device based on cotton thread as microfluidic channel where the transportation of solutions is based on capillary force helped by gravity. In the present work, we have demonstrated the versatility of thread-based electroanalytical devices (?TED) constructed using a cotton thread as the solution channel and screen-printed electrodes (SPE) surface modified with carbon nanotubes (CNT) as electrochemical detectors for the amperometric determination of estriol hormone in pharmaceutical samples. The parameters involved in the amperometric detection and microflow system were studied and optimized, using the best experimental conditions (flow rate of 1.25 ?L s-1, 2.0 ?L of volume of injection and potential of detection of 0.75 V) a linear response was observed for concentration range (LDR) of 1.0 to 1000 ?mol L-1 with limits of detection (LOD) and quantification (LOQ) of 0.53 ?mol L-1 and 1.77 ?mol L-1, respectively, and sampling rate of 44 analyses/ per hour. The proposed methodology was applied for determination of estriol in commercial samples. The results were compared with those provided by spectrophotometric method (official methodology). The obtained results are in agreement at a 95% of confidence level. Keywords: Microfluidic systems, thread-based electroanalytical device, hormone determination, screen printed electrodes, carbon nanotubes

Immuno-pcr Detection Of Lyme Borreliosis

Lyme borreliosis, more commonly referred to as Lyme disease, is the fastest growing zoonotic disease in North America with approximately 30,000 confirmed cases and 300,000 estimated infections per year. In nature, the causative agent of Lyme disease, the bacterium Borrelia burgdorferi, cycles between Ixodes sp. ticks and small mammals. Humans become infected with Lyme disease after being bitten by an infected tick. The primary indicator of a Borrelia burgdorferi infection is a bull’s eye rash typically followed by flu-like symptoms with treatment consisting of a 2-4 week course of antibiotics. If not treated, later stages of the disease can result in arthritis, cardiovascular and neurological symptoms. Diagnosis of Lyme disease is challenging and currently requires a complex laboratory diagnostic using indirect detection of host-generated antibodies by a two-tiered approach consisting of an enzyme linked immunosorbent assay (ELISA) followed by IgM and IgG immunoblots. Although two-tier testing has provided an adequate approach for Lyme disease diagnosis, it has weaknesses including subjective analysis, complex protocols and lack of reagent standardization. Immuno-PCR (iPCR) is a method that combines ELISA-based detection specificity with the sensitivity of PCR signal amplification and has demonstrated increased sensitivity for many applications such as detection of disease biomarkers but has yet to be applied for diagnosis of Lyme disease. Herein, using iPCR and recombinant B. burgdorferi antigens, an assay for both the direct and the indirect detection of Lyme disease was developed iv and demonstrated improved sensitivity for detection of B. burgdorferi antibodies using a murine model. Moreover, we present evidence using human Lyme disease patient serum samples that iPCR using both multiple antigens and a unique single hybrid antigen is capable of achieving increased sensitivity and specificity compared to existing methodology. These data represent the first demonstration of iPCR for Lyme disease diagnosis and support the replacement of two-tier testing with a more simplified and objective approach