500 Reference Units/ml

RPMI-1640; human serum albumin (2 mg/ml)

13.1 x 106 Reference Units (RU)/mg (before addition of albumin)

-70oC. Freezing and thawing apparently do not affect this IL-2 preparation. However, it is recommended that this material be aliquoted upon first thawing so that subsequent samples are thawed only once more before further use. Further dilution of this material should be done in serum-containing medium.

This material is intended as a reference reagent only and should not be considered as a source of IL-2 for experimentation. It is suggested that you use this material to occasionally quantitate the activity of your laboratory standard IL-2. It is requested that you furnish the BRB with the data and dose curves from the comparison between your laboratory standard and this reference reagent as well as details of the assay. These data will help us continue to evaluate the performance of this reference reagent.

This IL-2 was prepared from a subclone of the human leukemia T-cell line JURKAT (1). The cells were stimulated in serum-free medium with phytohemagglutinin and phorbol 12-myristate 12-acetate, and the supernatant medium was harvested following 15-20 hr of incubation at 37oC. The IL-2 was obtained by immunoaffinity column purification (2).

The murine monoclonal antibody used in the purification selects for a particular glycosylated form of human IL-2 (3). This form, which contains a single residue of N-acetyl-D-galactosamine attached by )-glycosidic linkage to threonine in position #3 of the polypeptide chain, is the major component of JURKAT-derived IL-2 but is a minor component of IL-2 made from tonsil or peripheral blood cells. IL-2 derived from the latter sources is typically more extensively glycosylated and often contains sialic acid. Studies to date, however, indicate that the various glycosylated and unglycosylated forms of IL-2 all have the same specific activity whether assayed in murine or human systems. Moreover, each appears equally capable of maintaining long-term growth of IL-2-dependent murine cell lines.

This IL-2 preparation was judged to be 99% pure by the criteria of 2-dimensional gel electrophoresis, reverse-phase HPLC, and N-terminal amino acid sequence analysis. The amino acid composition also closely conforms to that predicted by the amino acid sequence.

This preparation has <0.14 ng/ml of endotoxin as measured by the chromogenic LAL assay. It has <50 IU/ml of interferon-gamma or interferon-alpha as measured by the viral inhibition assay. It has no apparent MAF, MIG, or BCGF activity and no IL-1 activity was detected on CRL-1445 fibroblasts.

One Reference Unit of IL-2 was defined for this preparation as the amount of IL-2 in 1 ml that will induce IL-2-dependent murine T cells to incorporate 3H-TdR at 50% of their maximum level after 24 hours of incubation. Data were analyzed by probit transformation for determination of the 50% endpoint (4). Maximum stimulation occurred at approximately 10 Reference Units/ml.

The 50% endpoint is affected by the type of IL-2-dependent cell line used and the assay conditions. However, the 50% endpoint for most IL-2-dependent cells will be within a factor of 4.0-0.25 Reference Units) of the value we have assigned as one Reference Unit.

Medium - RPMI-1111640
Serum - 5% Fetal Calf Serum
Cell Concentration - 4 x 104 cells/ml
Pulse Concentration - 5 Ci/ml
Specific A
Time - Last 4 Hr.
Culture Duration - 24 Hr.

(1) Kaplan, J., Tilton, J. and Peterson, W.D., Jr.
(1976) AM. J. Hematol. 1:219-225

(2) Robb, R.J., Kutny, R.M. and Chowdhry, V. (1983)
Proc. Natl. Acad. Sci. (USA) 80:5990-5594

(3) Robb, R.J., Kutny, R.M. Panico, M., Morris, H.,
DeGrado, W.F. and Chowdhry, V. (1983) Biochem.
Biophys. Res. Comm. 116:1049-1055

(4) Gillis, S., Ferm, M.M., Ou, W. and Smith, K.A. (1978)
J. Immunol. 120:2027-2032

This reagent is available in small amounts (approximately 0.05 microgram/sample) for use in the calibration of in vitro bioassays and in-house standards only and are not to be used for experimental purposes. Reference reagents are available to investigators with peer-reviewed support, at institutions or at commercial establishments and are limited to one sample of each reagent per year.