The preparation of tumour specific antiserum leading to the purification of tumour associated antigen and studies of its role in the recognition of syngenetic cytotoxic lymphocytes
Creators: name: Al-Rammahy, Abdul Khaliq Abdullah
Publisher: name: University of British Columbia
Year: 2010

description: Considerable evidence indicates that events at the cell surface play a central role in the regulation of growth of normal and neoplastic cells. It is possible that modification of the surface molecules might be, in part, responsible for the processes that accompany tumorigenesis. These surface molecules are termed TSTA (tumor specific transplantation antigens) and TAA (tumor associated antigens). The purpose of this study is to raise xenogeneic antiserum which can recognize TAA. This antiserum, then, can be used as a tool to enrich or purify the antigen in question. Using the antibody feedback inhibition method which has been developed in our laboratory, an antiserum directed toward membrane components of DBA/2 mastocytoma P815 cells was raised. This antiserum was found to be specific for tumor cell extracts and had no reactivity with comparable extracts of normal cells when tested by complement fixation. This antiserum was also capable of killing P815 cells in the presence of guinea pig complement but had no reactivity with another DBA/2 tumor or a variety of normal DBA/2 cell preparations. When mixed with varying numbers of tumor cells and injected intraperitoneally into either DBA/2 or B6D2 Fl mice, the antiserum demonstrated a protective effect by either prolonging survival time or, apparently facilitating complete removal from the body of tumor cells when low numbers of cells were injected. The antiserum was used to monitor the purification of tumor associated antigen (TAA) of P815 cells. Membrane extracts of both P815 and normal DBA/2J spleen and peritoneal exudate cells were subjected to DEAE fractionation and gel filtration, following which fractions were tested for reactivity with the anti-P815 antiserum. Fractions from P815 extracts shown to be enriched for the TAA were used to raise a second antiserum specific for the tumor. This antiserum was also shown to have specificity for P815 and none for normal DBA/2J cells by ⁵¹Cr release assays in the presence of guinea pig complement and by surface labeling using peroxidase labeled sheep anti-rabbit immunoglobulin after treatment of either P815 or normal cells with the antiserum. This second antiserum (anti-P815-2), when allowed to react with ¹²⁵I-labeled TAA-enriched fractions of the P815 membrane extracts and passed over Sepharose-protein A columns, permitted the isolation of a single major component, detectable on autoradiographs of gradient acrylamide gels. This component was not present in equivalent normal DBA/2 tissue extracts, nor was it detectable when these tests were repeated using an antiserum raised against normal DBA/2 membrane preparations. It was thus concluded that this material constituted a TAA of the P815 mastocytoma. Then this major band was cut, eluted and used in CFA to immunize a rabbit. Three immunizations were needed to get antiserum which was specific to P815 cells and membrane extracts. P815 cells treated with this last antiserum were resistant to lysis by syngeneic cytotoxic cells but they were not when allogeneic cytotoxic cells were used. On the other hand, rabbit anti DBA/2 as well as mouse anti H-2d serum also blocked the syngeneic killing as well as having a blocking effect in the allogeneic killing. This suggests that this antiserum recognizes membrane molecules which are important for recognition by syngeneic cytotoxic cells.