engleski

The improvement of human leukocyte interferon (HuIFN-alpha) production by eliminating of inhibitor from residual plasma of leukocytes

Human leukocyte interferons (HuIFN-alpha) are pleiotropic molecules showing the antiviral, antiproliferative and immunomodulatory activity. They are synthesized and released from leukocytes after Sendai virus induction. IFN-alpha derived from leukocytes consists from multiple subtypes, each with subtle differences in structure and biologic activity. HuIFN-alpha is being produced in a quantities mostly by standard Cantell' s protocol: two-step lysis of a erithrocytes, 2-hour priming of remained leukocytes (0.8 x 10^7/ml) with 200 l.U. of HuIFN-alpha and induction by 150 HAU/ml of Sendai virus followed by 18 hour incubation at 37^oC. Despite the constant use of this, an evident variability in IFN's titers could be seen. Antiviral activity was tested on the WISH cells with a VSV as a challenge. The range from: 21.6 - 1 90.3 x 10^3 I.U./ml was found. The only variables were the "buffy coat" bags. Due to this, the existence of IFN's inhibitor in some of them can be speculated. To eliminate the inhibitor 10 bags were taken. Half of each was carried out by standard procedure, a second half, was a first washed out in excesse of PBS (Phosphate buffer saline) and than being pooled. The concentration of leukocytes before priming was 0.1 x 10^7/ml. To enhance the IFN yield the primer was added directly into the leukocyte pool (60.000 l.U/bag) before the further procedure. In the control experiments the normal 200 l.U./ml priming was excluded. The obtained data shows: (1) Washing of the leukocytes probably remove the inhlbtor, and consequently 3-5 times higher titers were found; (2) Direct priming could additionally increase the titer; (3) Addition of the primer directly into the leukocyte pool could not replace the standard priming, Using all these improvements the production become more uniform, with higher titers in a range of 115.7 - 165.9 x 10^3/ml.

Human leukocyte interferon alpha (HuIFN-alpha); leukocyte; production

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