Antibodies to natural killer stimulatory factor
a natural killer and stimulatory factor technology, applied in the field of antibodies to natural killer stimulatory factor, can solve the problems of hampered biochemical and biological identification and characterization of certain cytokines, and achieve the effect of enhancing natural killer cell functions and showing synergistic effects
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example 1
PREPARATION OF SERUM-FREE RPMI 8866 CELL-CONDITIONED MEDIUM
[0076] The human B-lymphoblastoid cell line RPMI 8866 was maintained in RPMI 1640 medium containing 5% heat-inactivated fetal calf serum (FCS). For preparation of serum free conditioned medium, cells were washed and suspended (106 cells / ml) in serum free RPMI 1640 medium containing 10−7 M phorbol-12-13-dibutvrate (PdBU) and cultured for 48 hours at 37° C., 5% CO2. The cell free supematants were harvested by filtration through a 0.2 μm filter [Durapore® hydrophilic cartridge filter, Millipore, Bedford, Mass. and Tween-20 and phenylmethylsulfonyl-fluoride (PMSF) were added to 0.02% and 0.1 mM, respectively. The cell conditioned medium was then concentrated 50 fold under pressure using an ultra-filtration cartridge [Spiral-Wound, S1, Amicon, Danvers, Mass.].
example 2
PURIFICATION OF NKSF FROM CONDITIONED MEDIUM
[0077] The following procedures are presently employed to obtain homogeneous NKSF protein from RPMI 8866 conditioned medium, as described in Example 1 above.
a. Anion Exchange Cartridge Chromatography
[0078] Two liters of the crude concentrated conditioned medium was diluted with distilled water to a conductivity of 6m Os / cm and adjusted to pH 8 with 1 M Tris-HCl buffer (pH 8). The concentrate was then applied to five QAE Zetaprep 250 cartridges [Pharmacia] connected in parallel and previously equilibrated with 0.1 M Tris-HCl buffer (pH 8) at a flow rate of 150 ml / min. Unless otherwise cited, all the buffers used for purification contained 0.02% Tween-20 and 0.1 mM PMSF. The cartridges were washed with 3 liters of 0.1 M Tris-HCI buffer (pH 6.8) followed by washing with 1.5 liters of 0.5 M NaCl in 0.1 M Tris-HCl buffer (pH 6.8) and 300 ml fractions were collected. The NKSF activity was eluted with the 0.5 M NaCl-containing wash.
b. Lentil-...
example 3
SODIUM DODECYL SULFATE-POLYACRYLAMIDE GEL ELECTROPHORESIS
[0085] SDS-PAGE was performed according to the method of Laemmli [Laemmli, U. K., Nature, 227:680-685 (1970)] on 10% acrylamide slab gels (0.75 mm thickness). After electrophoresis the gels were either stained by the silver-nitrate method using a silver staining reagents [BioRad] or cut into 2 mm slices and eluted in 0.5 m! RPMI medium for 4 hours at 24° C. and assayed for NKSF activity. Apparent molecular weight was determined with protein standards, phospholipase b (94 kD), bovine serum albumin (67 kD), ovalbumin (43 kD), carbonic anhydrase (30 kD), soybean trypsin inhibitor (20 kD) and lactalbumin (14.4 kD).
[0086] SDS-PAGE analysis (non-reducing conditions) of the Mono Q column fractions (Example 2, step (e)) beginning with several fractions which eluted before the NKSF activity, continuing right through the active fractions and ending with fractions which eluted after the peak of NKSF activity, revealed that the presence...
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