Development of in-house materials for the verification of specific gravity measurements: Homogeneity, stability, and proficiency studies.

We developed and evaluated the properties of in-house urine reference materials for the verification of laboratory refractometers, which are frequently used in clinical chemistry and doping testing laboratories. Urine was gathered from 26 healthy volunteers (16 male 30 ± 5 years old and 10 female 29 ± 4 years old), from which two urine batches were obtained: one with a low specific gravity (1.012± 0.003) and the other with a high specific gravity (1.027 ± 0.003). Homogeneity studies were conducted over 20 consecutive days. For short-term stability studies, aliquots of both urine batches were stored at -20 ± 2°C; 3 ± 2°C; 20 ± 2°C; 45 ± 2°C for 0, 2, 7, 14 and 35 days, under both light and dark conditions. Similarly, another study was conducted to measure the long-term stability of urine at -20 ± 2°C, over a 24-month evaluation period. Our data showed that the urine was homogeneous and stable at -20 ± 2°C, 3 ± 2°C, 20 ± 2°C, and 45 ± 2°C under both light and dark conditions. In all cases, the urine was evaluated by specific gravity and no statistically significant differences (p ≤ 0.05) were recorded. Additionally, a proficiency test was conducted in collaboration with 15 ISO/IEC 17025 accredited laboratories, and z-scores and performance factors were evaluated. These data indicate that this material could be used for the verification of refractometers.

Desarrollo de una Vacuna Inactivada Oleosa con Cepa Venezolana del Virus de la Encefalitis Equina del Este / Oil Inactivated Vaccine Development with Venezuelan Strain of Eastern Equine Encephalitis Virus

Se utilizó la cepa El Pao de encefalitis del este venezolana, aislada en el 2002 (EE VE02 EL PAO) como antígeno, para producir una vacuna inactivada oleosa contra el Virus de la Encefalitis Equina del Este (VEEE) en tres formulaciones, a saber: formulación I (F1): antígeno-fase oleosa; formulación II (F2): antígeno-fase oleosa-medio de mantenimiento y formulación III (F3): antígeno-fase oleosa-antígeno. La suspensión de virus se obtuvo en monocapa de cultivo de células de riñón del mono verde africano. Como inactivante se usó la etilenimina binaria (BEI) con adyuvante incompleto de Freund. Se realizaron pruebas de identidad, carga viral, esterilidad, fisicoquímica, inocuidad, potencia, inmunidad y viabilidad. Todas estas pruebas de control de calidad fueron satisfactorias. Cuando las formulaciones se administraron a cobayos, estimularon títulos de anticuerpos por inhibición de la hemoaglutinación ≥ 20 y de seroneutralización ≥ 40, a los 21 d postvacunación con 1 dosis de 1 mL por vía subcutánea. No se observaron diferencias estadísticamente significativas (P>0,05) en la detección de anticuerpos entre las formulaciones a los 21 días posvacunación, aunque la F3 presentó mayores títulos, seguida de la F2 y la F1, respectivamente (P<0,01). Los anticuerpos neutralizaron in vitro las cepas virulentas del VEEE, y los cobayos vacunados no desarrollaron viremia ni enfermedad neurológica con el desafío de virus vivo, tanto por vía intracerebral como por vía intraperitoneal, en las pruebas de potencia. Se concluye que la vacuna es segura y provee una excelente protección contra la infección del VEEE.

The Pao of eastern encephalitis venezuelan isolated in 2002 (EE VE02 EL PAO) strain was used as an antigen to produce an inactivated oil vaccine against the Eastern Equine Encephalitis Virus (EEEV) in three formulations, as follows: Formulation I (F1): antigen in oil phase; Formulation II (F2): antigen in oil phase-maintenance medium; and Formulation III (F3): antigen-oil phase-antigen. The virus suspension was obtained from African green monkey kidney monolayer cell cultures. The binary ethylenimine (BEI) was used to inactivate the virus, along with an incomplete Freund´s adjuvant. The following quality control tests were performed: identity, viral load, sterility, physicochemical, safety, potency, immunity, and viability. All these tests were satisfactory. The administration to guinea pigs of a single subcutaneous dose of 1 mL of the formulations, caused stimulated antibody titers by hemagglutination inhibition (≥ 20) and serum neutralization (≥ 40), respectively, at 21 days post-vaccination. No statistically significant differences (P> 0.05) in the detection of antibodies between formulations at 21 days post-vaccination were observed, although F3 had higher titles, followed by F2 and F1, respectively (P<0.01). The antibodies neutralized EEEV virulent strains in vitro and the vaccinated guinea pigs did not develop viremia or neurological disease, when challenged with live virus, both intracerebrally and intraperitoneally in potency tests. It is concluded that the vaccine is safe and provides excellent protection against EEEV infection.