Subtractive antibody to a human immunosuppressive lymphokine affinity isolates a suppressive factor and blocks its function

Abstract

Hybridoma suppressor factor(s) (HSF), secreted by a human thymus hybridoma (8E-24) established in this laboratory, suppresses Ig as well as IL-2 synthesis by peripheral blood mononuclear cells (PBMC). To aid in the characterization of this lymphokine, we prepared a subtractive antibody to HSF using the products of the hybridoma parent cell line to generate antibodies to irrelevant proteins. The concentrated supernatant fluid of the hybridoma parent cell line (CEM) was used to generate rabbit antibodies and titers of anti-CEM were monitored by enzyme immunoassay (EIA). Next, to remove factors shared by both the parent cell line and hybridoma, the concentrated supernatant fluid of 8E-24 (crude HSF) was passed over an immunoaffinity column, composed of protein A beads coupled to anti-CEM. The ‘subtracted’ HSF, termed partially purified HSF, was shown to be suppressive in vitro and was then used to prepare a second rabbit antisera. Using partially purified HSF as antigen, the presence of specific antibody was monitored by EIA. This antibody (anti-HSF) was used to prepare another immunoaffinity column by covalently coupling this antibody to protein A beads. Factors bound and then eluted from this affinity column were shown to inhibit IL-2 production by PBMC in a manner similar to HSF. Specific activity of the affinity purified HSF was 50 times that of partially purified HSF. Furthermore, the suppressive activity of affinity purified HSF was abrogated in the presence of anti-HSF. Western blot analysis performed on the concentrated crude HSF, using both the anti-HSF and anti-CEM antibodies, revealed the presence of several bands that selectively reacted with anti-HSF and not anti-CEM. Each of these bands were present in the affinity purified HSF. Of particular interest due to their similar size to the suppressive agent are a band at 12 kDa that reacts selectively with anti-HSF and is detected in crude and affinity purified HSF and a band at 10 kDa. In summary, this protocol resulted in the detection and separation of hybridoma specific proteins within the predicted size range of the suppressive lymphokine.