A method for the inactivation and removal of dengue virus from biological samples
A dengue virus, virus inactivation technology
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Embodiment 1
[0026] Example 1: Dengue virus inactivation and removal by immunoglobulin process
[0027] Step 1: Virus Removal Filtration
[0028] The preliminarily purified immunoglobulin solution was passed through a 0.22 μm or 0.1 μm filter (Millipore Steritop TM ) pre-filtered to remove viral aggregates. The immunoglobulin solution was kept at room temperature and under a constant pressure of 80kPa, and then a 35nm filter (Asahi KaseiPlanova ) for virus removal filtration.
[0029] Step 2: Treatment with Organic Solvents and Detergents
[0030] The virus-removed and filtered immunoglobulin solution was heated to 28°C, and then octylphenol polyoxyethylene ether (Triton X-100 ) and tri-n-butyl phosphate to final concentrations of 1% and 0.3%. Viral inactivation of the immunoglobulin solution was maintained for sixteen hours at 28°C-30°C.
[0031] Step 3: Ion Exchange Chromatography
[0032] The cross-linked agarose resin with carboxymethyl groups (Pharmacia CM Sepharose) was re...
Embodiment 2
[0061] Example 2: Preliminary purification of immunoglobulins
[0062] Plasma fractions II+III were obtained from frozen human plasma by frozen ethanol precipitation (Cohn et al., J. Am. Chem. Soc. 1946; 68: 459-475). Human plasma was first subjected to 8.0% ethanol precipitation at pH 7.1, the resulting supernatant was subjected to 19.0% ethanol precipitation at pH 5.85, and fractions II+III were obtained by centrifugation at 2,300 xg. Add 0.8M sodium acetate / 4.0M acetate buffer (pH 3.9) to the dissolved fraction II+III to adjust to pH 5.1, then add 95% ethanol to a final concentration of 15.0%, and at -5°C to -5.5°C Gentle agitation in the range for 1.5 hours. The pellet was removed by centrifugation at 2,300 x g to obtain a supernatant containing immunoglobulins.
Embodiment 3
[0063] Example 3: Purification of human plasma albumin free from infectious dengue virus
[0064] Step 1: Precipitation of Component IV
[0065] As described in Example 2, supernatants II+III were obtained by two consecutive steps of ethanol precipitation (8.0% and 19.0%). 95% ethanol was then added to the supernatant to a final concentration of 40.0% and gentle stirring was performed in the range of -5°C to -5.5°C for 1 hour. Fraction IV was removed by centrifugation at 2,300 xg to obtain supernatant IV containing albumin.
[0066] Step 2: Pasteurization
[0067] The purified albumin solution was diafiltered with 8 volumes of purified water using Millipore's 30kD ultrafiltration membrane package, and then concentrated to a final albumin concentration of 22%. Pharmaceutical grade sodium caprylate was added to the concentrated albumin to a final concentration of 0.032M, then adjusted to pH 6.8 and final albumin concentration of 20%. Next, the 20% albumin solution and the st...
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