Platelets and human serum have been evaluated as sources for extraction of soluble HL-A antigens. 3 M KC1 was found to efficiently solubilize HL-A antigens from platelets with a recovery ranging between 50 and 100 %. However, because of the low density of HL-A determinants on platelets, the yield of sbluble antigens is low, as only 1 mg of protein can be recovered from 1 x l o 9 platelets. Thus, while it is difficult to solubilize sufficient antigens from platelets for chemical characterization, it is possible t o use these materials for biological applications such as pretreatment of kidney recipients. Soluble HL-A antigen is present in human serum, since following extensive ultracentrifugation, it can still effectively inhibit the cytotoxic activity of HL-A alloantisera. The same HL-A specificities were found to be present in varying amounts among different donors. Partial purification of such antigens can be achieved by ion-exchange chromatography of serum on QAE-Sephadex.

No abstract

No abstract

Human transplantations performed during the last decade have shown that the success of these procedures depends largely on two genetic systems, the ABO blood group system and the HL-A (human leukocyte locus A) system. Essentially all strong transplan tat ion antigens affecting the survival of allografts belong to these two systems. Progress in successful selection of compatible donors and in a better understanding of the genetics and molecular biology of the HL-A system has been made largely through the extensive application of histocompatibility typing utilizing the lymphocytotoxic technique.The results of direct typing by specific cytotoxic HL-A alloantisera in the presence of complement, which causes the lysis of leukocytes, can be improved by typing by absorption which overcomes a major disadvantage of the cytotoxic tests, i.e., the CYNAP ( cy totoxici ty-negat ive-absorption-posi tive ) reaction responsible for false negative results ( 1). Moreover, quantitiative absorption of HL-A antisera with human leukocytes permits quantitition of HL-A determinants on these cells and evaluation of gene dose effects ( 2 ) to differentiate between homo-and heterozygotes. By quantitative absorption, yields of soluble antigens from cultured cells can be compared meaningfully among different extraction procedures. This is particularly appropriate as cultured cell lines are being used more frequently since they provide the only adequate, miform genetic source for isolation of large quantities of soluble HL- A antigens ( 3 , 4 ) .The application of quantitative absorption techniques has, however, been hampered by lack of sufficient amounts of cells and monospecific HL-A alloantisera. To overcome this difficulty, we have developed a simple, reproducible microabsorption technique for HL-A typing of both human cultured cells and peripheral human leukocytes. Materids and U e t h o d s . H L . 4 alloantisera.Sera were obtained from the serum bank at the National Tnstitute of Allergy and Infectious Diseases and from Professor R. Ceppellini. Each typing serum was titrated against lymphocytes containing the corresponding antigen and was used for the absorption test at a dilution corresponding to 1 cytotoxic unit, i.e., a twofold greater concentration than zero cytotoxic units which, in turn, corresponds to the 9570 lytic end point.Absorbing CSZZS. Peripheral leukocytes were obtained from healthy subjects by sedimentation of heparinized blood with plasmageI. The human cultured lymphoid cell lines were RPAlI 1788, RPRII 4098, RPMI 7249, RMPI 8866, and WI-L2. Both peripheral leukocytes and cultured human lymphoid cells were washed three times with PBS (0.005 Ad phosphate buffer, 0.15 M NaCl, pH 7.1) and then suspended at the desired concen tration.Cytotoxic test. The slightly modified eosin method of Mittal et aZ. ( 5 ) was employed with both peripheral lymphocytes and cultured human lymphoid cells (6, 7 ) . At least three typing sera were used for each HL-A 484 by guest on July 25, 2015 ebm.sagepub.com Downloaded from