Haghighi A Z, Cathcart M K
Section of Immunology, Cleveland Clinic Foundation, OH 44195.
Immunopharmacology. 1992 Jul-Aug;24(1):65-76. doi: 10.1016/0162-3109(92)90071-j.
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.
本实验室建立的一株人胸腺杂交瘤（8E-24）分泌的杂交瘤抑制因子（HSF）可抑制外周血单个核细胞（PBMC）的免疫球蛋白（Ig）以及白细胞介素-2（IL-2）的合成。为了有助于对这种淋巴因子进行特性鉴定，我们利用杂交瘤亲本细胞系的产物制备了一种针对HSF的消减抗体，以产生针对无关蛋白的抗体。杂交瘤亲本细胞系（CEM）的浓缩上清液用于制备兔抗体，并通过酶免疫测定（EIA）监测抗CEM的效价。接下来，为了去除亲本细胞系和杂交瘤共有的因子，将8E-24的浓缩上清液（粗制HSF）通过一个免疫亲和柱，该柱由与抗CEM偶联的蛋白A珠组成。这种“消减”后的HSF，称为部分纯化的HSF，在体外显示出抑制作用，然后用于制备第二份兔抗血清。以部分纯化的HSF作为抗原，通过EIA监测特异性抗体的存在。该抗体（抗HSF）通过将其与蛋白A珠共价偶联用于制备另一个免疫亲和柱。从该亲和柱上结合然后洗脱的因子显示出以类似于HSF的方式抑制PBMC产生IL-2。亲和纯化的HSF的比活性是部分纯化的HSF的50倍。此外，在抗HSF存在的情况下，亲和纯化的HSF的抑制活性被消除。使用抗HSF和抗CEM抗体对浓缩的粗制HSF进行蛋白质印迹分析，结果显示存在几条与抗HSF选择性反应而不与抗CEM反应的条带。这些条带中的每一条都存在于亲和纯化的HSF中。由于它们的大小与抑制因子相似而特别令人感兴趣的是一条12 kDa的条带，它与抗HSF选择性反应，在粗制和亲和纯化的HSF中均能检测到，以及一条10 kDa的条带。总之，该方案导致在抑制性淋巴因子的预测大小范围内检测和分离出杂交瘤特异性蛋白。
