Formulation and Validation of Recombinant Antigens CFP10 and ESAT6 for Tuberculosis Diagnosis

Abstract The rapid and accurate diagnosis of tuberculosis (TB), especially considering limited resources, is still a challenge. Development of new methodologies and tests are needed to overcome several disadvantages of the available standard tests. We evaluated the diagnostic potential of two antigens specific for Mycobacterium tuberculosis, the CFP10 and ESAT6 recombinant proteins, and developed stable formulations thereof. Sensitivity and specificity of the delayed-type hypersensitivity (DTH) skin testing and the induction of gamma interferon production (IFN-γ) by lymphocytes, as a non-invasive test, were evaluated using the CFP10 and ESAT6 protein formulations. The recombinant proteins produced by our group presented a high DTH response and the ability to differentiate between tuberculosis infection, BCG vaccination, and the contact with non-tuberculous mycobacteria (NTM). The production of IFN-γ by stimulation with individual and combined proteins was detected in a panel of 40 individuals and showed a specificity of 100% and a sensitivity of 90% when the two proteins were used together. Lyophilized formulations were stable under all conditions, while soluble formulations were stable under freezing at -20 ºC and -80 ºC. The proposed formulations containing the ESAT6 and CFP10 recombinant antigens constitute satisfactory tools for TB testing, suitable to be developed and implemented in a large-scale trial.

Process parameters optimization to produce the recombinant protein CFP10 for the diagnosis of tuberculosis.

The aim of this study was to evaluate the parameters that affect the production of the recombinant 10 kDa culture filtrate protein (CFP10), a promising reagent of high specificity for intradermoreaction and other antigen-based methods used in the diagnosis of tuberculosis. Conditions of Escherichia coli growth temperature, induction temperature and IPTG-inducer concentration were evaluated in shake flasks and dissolved O2 concentrations of 15 and 30% were evaluated in a bioreactor. The process parameters defined on small scale were: growth temperature between 30 and 37 °C, induction temperature of 26 °C and IPTG concentration of 0.12 mM. The process conducted with 15% dissolved O2 presented a recombinant protein yield of 78.6 mg g-1 biomass and a proportion of recombinant protein (insoluble fraction) in relation to total insoluble protein of 72%, at the time of maximum productivity. The operation with 30% dissolved O2 resulted in lower recombinant protein yields of 62.9 mg g-1 biomass and 20% in relation to total insoluble protein, but in higher overall concentration in the culture broth (69.2 mg L-1versus 48.3 mg L-1). The protein identity was confirmed by mass spectrometry, showing high similarity to CFP10, 10 kDa of Mycobacterium tuberculosis H37Rv (score 95), and the purified antigen presented reactivity by the Western blotting assay.

Recovery of phytase produced by solid-state fermentation on citrus peel

The extraction of phytase produced by solid-state fermentation of citrus peel was studied employing a multistage leaching process. It was observed that the extracts containing EDTA retained over 90 percent of phytase activity at room temperature after 24 h after the leaching. A fractional design 2² (with 4 replicates at the central point) was carried out for testing the pH and agitation as process independent factors. Only the interaction between the pH and agitation showed a significant influence. These factors were optimized with a central composite design. Agitation at 300 rpm and pH at 5.0 were the best conditions to extract the enzyme from solid matrix. The modeling of the process indicated that diffusivity of the enzyme in the solvent was the controlling mechanism. The corresponding kinetic constant and saturation concentration in this process were 0.89 min-1 and 4.0 IU/mL, respectively. The multistage process indicated that after two steps, it was possible to recover 85 percent of total enzyme produced.

A extração de fitases produzidas por fermentação em estado sólido de polpa cítrica foi estudada utilizando um processo de extração sólido-líquido em varias etapas. A adição de EDTA permite manter durante 24 horas a temperatura ambiente 90 por cento da atividade inicial do caldo com a enzima extraída. Um planejamento fatorial 2², com 4 replicas no ponto central, foi desenvolvido para testar os valores de ph e agitação convenientes para a extração das enzimas. A interação entre ambos os fatores foi estadisticamente significativa. A atividade da enzima foi otimizada nos valores onde o pH (5.0) e a agitação (350 rpm) resultaram ser as melhores condições para extrair a enzima da matriz sólida. O ajuste do modelo matemático obtido mostra que é possível considerar a difusividade como o mecanismo que controla o processo de transferência de massa. A constante cinética que descreve este processo e a concentração de saturação foram 0.039 min-1 e 4.01 IU/mL respectivamente. A extração em varias etapas mostrou que nas duas primeiras etapas é possível recuperar 85 por cento da fitase produzida.