VOXSang. 28: 1-8 (1975)

HBs Antigen in Human Plasma Fractions P. LEVEQUE, J. DROUET,R. SCHMITTHAEUSLER, M. L. NORTH,P. AMOUCH and J. MALGRAS Centre de Transfusion Sanguine (Duecteur: Prof. R. WAITZ), Strasbourg, and Centre National de Transfusion Sanguine (Duecteur: Prof. J. P. SOULIER), Paris

Abstract, Therapeutic fractions were obtained by fractionating human plasma containing HBs antigen, by the Cohn ethanol technique modified by Nitschmann. Plasmas with various HBs antigen titers were selected. The antigen was sought in each fraction by the techniques of counterelectrophoresis and radioimmunological detection in liquid phase. With the exception of very pure albumins, all fractions were found to contain the HBs antigen, though in variable proportions. A technique for the purification of albumin, applicable on an industrial scale, is proposed. It makes it possible to obtain an albumin fraction in which the HBs antigen cannot be detected by the most sensitive techniques, even starting with a plasma which is very rich in HBs antigen (titer 1/64 by counterelectro phoresis).

A great deal of work has been done on the detection and location of HBs antigen in human blood fractionation products, but the findings have frequently diverged [20]. We thought it would be interesting to look for the HBs antigen in various therapeutic fractions and investigate the influence of the method of preparation, the degree of purity of the resultant products and the HBs antigen titer of the initial plasma on its distribution. The HBs antigen was sought by the classical technique of counterelectrophoresis (CEP), and also by the technique of radioimmunological detection (RID) in liquid phase. Equipment and Methods Plasma The plasmas came from blood determined as HBs-positive by CEP and were stored in ACD. Plasmas with titers ranging between 1/1 and 1/64, as determined by CEP, were studied. ~~~

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Received: April 29, 1974; accepted: June 12, 1974.

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LEVEQUE et al.

Fractionation Technique The fractionation technique employed is the Cohn method as modified by KISTLERand NITSCHMANN [12]. The y-globulins are fractionated by the technique of NITSCHMANN et al. [15]. The fibrinogen is prepared according to the method described by NITSCHMANN et al. [16], and the coagulant fractions by the technique of DEGGELER [8]. The method of preparing the coagulant fractions and the albumin was modified as follows. Coagulant fractions. Prior to elution of the active factors, the washing recommended [8] was carried out 3 times. Furthermore, centrifugation was replaced by by DEGGELER filtration to separate the two phases; a marked decrease is observed in the HBs antigen level when the washing liquids are separated from the resin by careful Buchner filtration instead of being simply centrifuged and decanted. Albumin. In order to obtain a very pure albumin (100 % byelectrophoreticand immunoelectrophoretic analysis), we reprecipitated the C precipitate [12]. The C precipitate is redissolved in the quantity of saline solution needed to give a protein concentration of 50 g/liter. The pH k i n g fixed at 5.85, the temperature is lowered to -8 "C and cold ethanol gradually added until a concentration of 40% is reached. The precipitate obtained by centrifuging at -8 "Cis eliminated. The supernatant is brought to a pH of4.8 by theaddition of acetic acid and cooled to -8°C. The precipitate obtained by centrifuging at -8°C consists of pure albumin. Detection of the HBs Antigen Tow techniques were employed: CEP [17] and RID in the liquid phase [l-31. The HBs antigen is sought at each fractionation stage by these two methods, using samples in which the protein concentration is brought down to an identical value, close to that of a normal plasma. A plasma shown to be HBs-negative by RID is fractionated under the same conditions and used as a negative control. Study of the Proteins in Each Fraction For each fraction we measured the total proteins and carried out an electrophoresis on cellulose acetate gel and immunoelectrophoresis by means of an antihuman globulin polyvalent equine immunoserun.

Results The overall results are summarized in table I. Cryoprecipitate The HBs antigen is found in all cryoprecipitates, although the CEP technique is not sensitive enough to detect the amounts of antigen present in the cryoprecipitates prepared from plasma with a low initial HBs antigen titer (lower than l/8 by CEP; table I).

HBs Antigen in Human Plasma Fractions

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Table I. Presence of HBs antigen in therapeuticfractions

CEP titer in plasma