Vox Sang. 33: 321-323 (1977)
Anti-Hepatitis B Immunoglobulin For Clinical Use B . S . Combridge Blood Products Laboratory, Lister Institute of Preventive Medicine, Elstree
Abstract. Anti-hepatitis B immunoglobulin, prepared by a method which involves the initial removal of the fibrinogen fraction, has been reported to lack certain antibodies and fail to detect certain hepatitis B surface antigens when used as a detector reagent in counterimmunoelectrophoresis. The possibility exists that any passive immunity conferred by the immunoglobulin might not protect against these antigens. Anti-hepatitis B immunoglobulin prepared from plasma by a modified technique which omitted the precipitation of the fibrinogen fraction reacted against these antigens in gel precipitin tests. It is suggested that such a method is preferable for the production of anti-hepatitis B surface immunoglobulin.
Introduction Anti-hepatitis B immunoglobulin prepared from the plasma of haemophilic donors and used as the detector reagent for hepatitis B surface antigen (HBsAg) in routine screening of blood donors by the New York Blood Centre failed to react with three antigens (No. 220, 226 and 244) in the panel No. 2 of the Bureau of Biologics [l]. Before fractionation, the haemophilic plasma reacted with these antigens. The fractionation was carried out by the method of Nitschrnann et a/. [2] and it was shown that the particular antibodies separated with the fibrinogen The interest of Dr. W. &A. Maycock, Director of the Blood Products Laboratory, is greatly appreciated. 1
fraction and were therefore absent from the immunoglobulin. A similar immunoglobulin prepared for clinical use has also been used for several years in this laboratory as the detector reagent in routine screening for HBsAg by discontinuous counter immunoelectrophoresis (DCIE) because it was found to be a sensitive reagent which produced clear precipitin lines with both ad and ay subtype antigens. This immunoglobulin was made by the method of Kistler and Nistschmann [ 3 ] modified by omitting the initial precipitation of fraction I in order to increase the yield of immunoglobulin. It seemed possible that this immunoglobulin would contain those antibodies which might otherwise have been removed in fraction I.
322
Cornbridge
Table I. Titration of BOB antigens against hepatitis B immunoglobulin by DCIE lmmunoglobulin dilution
Antigen dilutions 220 undiluted
undiluted 1:2 1:5
l:l0 1:15 1 :20 1 :25 1:30 1 :35
+ + ++ ++ + + + +
-
226 1:2
1:4
f
-
-
&
-
-
1:8
undiluted 1:2 1:4
+ - +
+
&
-
-
-
+ - + - -
244
_ - -
Three batches of such immunoglobulin were tested by DCIE to assess their ability to react with the three antigens No. 220, 226 and 244.
Materials and Methods Anti-hepatitis B immunoglobulins (batches SHGS, G A l and GA2), prepared by the method of Kistler and Nitschmann [3] for clinical use, from a plasma pool of about 250 plasma donations containing hepatitis B surface antibody (anti-HBs), were used at a final concentration of 100mg protein per 1 ml. Panel No. 2 of reference antigens including No.220, 226 and 244 was obtained from the Bureau of Biologics (US Food and Drug Administration). counter-immunoelectrophoresis Di:.:nntinuous (CIE) u,; performed by the method of Combridge and Shaw [4]. The three batches of immunoglobulin were tested against the antigens of the panel. Slides were washed and stained with Coomassie Brilliant Blue. The tanned-cell haemagglutination test was performed by the method of Hopkins and Das 151 using human cells sensitized with HBsAg of subtypes d and y.
+ + + + ++ ++ + +
-
1.8
undiluted
++ ++ ++
-
-
-
-
-
-
-
-
_
-
-
-
f
-
-
+
-
+ - -
-
-
_
-
-
++ ++ ++ ++ + -
1:2
1.4
f
-
+ + + +
-
+
1.8 -
-
-
&
-
-
-
-
-
-
-
-
Results The tanned-cell haemagglutination test showed that the three immunoglobulin preparations contained both anti- ad and ay specificities. By DCIE all three immunoglobulin preparations gave precipitin lines with the antigens concerned. Table I shows the results of a titration using one batch of immunoglobulin, The intensity of the precipitin lines was graded k (weak) to ++ (strong). At a dilutionof 1:2, the antigens were readily detected but there was evidence of antibody excess with higher concentrations of immunoglobulin.
Discussion Horowitz et al. [l] showed that during the preparation of anti-hepatitis B immunoglobulin from haemophilic plasma certain antibodies of the IgG class were removed in the cold ethanol fraction I and were absent
Anti-Hepatitis B Immunoglobulin for Clinical Use
from the y-globulin fraction 11. This immunoglobulin fraction failed to react with certain HBsAg although the plasma from which the immunoglobulin was derived produced precipitin lines with the antigens by CIE. The investigators were concerned that such immunoglobulin would not protect against these antigens and that immunoglobulin used for passive protection against HBsAg should include the fraction I antibodies. In this laboratory, hepatitis B immunoglobulin is normally prepared by the method of Kistler and Nitschmann [3] omitting the fraction I precipitation, in order to obtain a higher yield of y-globulin from whole plasma since some y-globulin is thought to be held back by protein-protein interaction with fibrinogen in fraction I. It is evident that the immunoglobulin thus prepared contains those antibodies which react with BOB antigens No. 220, 226 and 244 and it is suggested that a method of preparation which omits the initial separation of fraction I is the method of choice in the production of hepatitis B immunoglobulin.
323
References 1 Horowitz, M. S.; Woods, K. R.; Gersten, M., and Ehrich, C.: Separation of two hepatitis B human antibody fractions by cold-ethanol precipitation. Vox Sang. 30: 50 (1976). 2 Nitschmann, H.; Kistler, P., and Lergier, W.: Preparation of human albumin and y-globulin from blood plasma by alcoholic precipitation. Helv. chim. Acta 37: 866 (1954). 3 Kistler, P. and Nitschmann, H.: Large scale production of human plasma fractions. Eight years experience with the alcohol fractionation procedure of Nitschmann, Kistler and Lergier. Vox Sang. 7: 414 (1962). 4 Combridge, B . S . and Shaw, C.S.: Immunoelectroosmophoresis using discontinuous buffer system to detect Australia antigen in pooled human plasma. Lancet ii: 1184 (1971). 5 Hopkins, R. and Das, P. C.: Australia antigen (HB-Ag) subtyping by a sensitive tanned cell haemagglutination inhibition technique. Br. J . Haemat. 27: 501 (1974).
Received: November 1, 1976 Accepted: November 30, 1976 B. S . Combridge, Blood Products Laboratory, Lister Institute of Preventive Medicine, Elstree, Herts. (England)
Anti-Hepatitis B Immunoglobulin For Clinical Use B . S . Combridge Blood Products Laboratory, Lister Institute of Preventive Medicine, Elstree
Abstract. Anti-hepatitis B immunoglobulin, prepared by a method which involves the initial removal of the fibrinogen fraction, has been reported to lack certain antibodies and fail to detect certain hepatitis B surface antigens when used as a detector reagent in counterimmunoelectrophoresis. The possibility exists that any passive immunity conferred by the immunoglobulin might not protect against these antigens. Anti-hepatitis B immunoglobulin prepared from plasma by a modified technique which omitted the precipitation of the fibrinogen fraction reacted against these antigens in gel precipitin tests. It is suggested that such a method is preferable for the production of anti-hepatitis B surface immunoglobulin.
Introduction Anti-hepatitis B immunoglobulin prepared from the plasma of haemophilic donors and used as the detector reagent for hepatitis B surface antigen (HBsAg) in routine screening of blood donors by the New York Blood Centre failed to react with three antigens (No. 220, 226 and 244) in the panel No. 2 of the Bureau of Biologics [l]. Before fractionation, the haemophilic plasma reacted with these antigens. The fractionation was carried out by the method of Nitschrnann et a/. [2] and it was shown that the particular antibodies separated with the fibrinogen The interest of Dr. W. &A. Maycock, Director of the Blood Products Laboratory, is greatly appreciated. 1
fraction and were therefore absent from the immunoglobulin. A similar immunoglobulin prepared for clinical use has also been used for several years in this laboratory as the detector reagent in routine screening for HBsAg by discontinuous counter immunoelectrophoresis (DCIE) because it was found to be a sensitive reagent which produced clear precipitin lines with both ad and ay subtype antigens. This immunoglobulin was made by the method of Kistler and Nistschmann [ 3 ] modified by omitting the initial precipitation of fraction I in order to increase the yield of immunoglobulin. It seemed possible that this immunoglobulin would contain those antibodies which might otherwise have been removed in fraction I.
322
Cornbridge
Table I. Titration of BOB antigens against hepatitis B immunoglobulin by DCIE lmmunoglobulin dilution
Antigen dilutions 220 undiluted
undiluted 1:2 1:5
l:l0 1:15 1 :20 1 :25 1:30 1 :35
+ + ++ ++ + + + +
-
226 1:2
1:4
f
-
-
&
-
-
1:8
undiluted 1:2 1:4
+ - +
+
&
-
-
-
+ - + - -
244
_ - -
Three batches of such immunoglobulin were tested by DCIE to assess their ability to react with the three antigens No. 220, 226 and 244.
Materials and Methods Anti-hepatitis B immunoglobulins (batches SHGS, G A l and GA2), prepared by the method of Kistler and Nitschmann [3] for clinical use, from a plasma pool of about 250 plasma donations containing hepatitis B surface antibody (anti-HBs), were used at a final concentration of 100mg protein per 1 ml. Panel No. 2 of reference antigens including No.220, 226 and 244 was obtained from the Bureau of Biologics (US Food and Drug Administration). counter-immunoelectrophoresis Di:.:nntinuous (CIE) u,; performed by the method of Combridge and Shaw [4]. The three batches of immunoglobulin were tested against the antigens of the panel. Slides were washed and stained with Coomassie Brilliant Blue. The tanned-cell haemagglutination test was performed by the method of Hopkins and Das 151 using human cells sensitized with HBsAg of subtypes d and y.
+ + + + ++ ++ + +
-
1.8
undiluted
++ ++ ++
-
-
-
-
-
-
-
-
_
-
-
-
f
-
-
+
-
+ - -
-
-
_
-
-
++ ++ ++ ++ + -
1:2
1.4
f
-
+ + + +
-
+
1.8 -
-
-
&
-
-
-
-
-
-
-
-
Results The tanned-cell haemagglutination test showed that the three immunoglobulin preparations contained both anti- ad and ay specificities. By DCIE all three immunoglobulin preparations gave precipitin lines with the antigens concerned. Table I shows the results of a titration using one batch of immunoglobulin, The intensity of the precipitin lines was graded k (weak) to ++ (strong). At a dilutionof 1:2, the antigens were readily detected but there was evidence of antibody excess with higher concentrations of immunoglobulin.
Discussion Horowitz et al. [l] showed that during the preparation of anti-hepatitis B immunoglobulin from haemophilic plasma certain antibodies of the IgG class were removed in the cold ethanol fraction I and were absent
Anti-Hepatitis B Immunoglobulin for Clinical Use
from the y-globulin fraction 11. This immunoglobulin fraction failed to react with certain HBsAg although the plasma from which the immunoglobulin was derived produced precipitin lines with the antigens by CIE. The investigators were concerned that such immunoglobulin would not protect against these antigens and that immunoglobulin used for passive protection against HBsAg should include the fraction I antibodies. In this laboratory, hepatitis B immunoglobulin is normally prepared by the method of Kistler and Nitschmann [3] omitting the fraction I precipitation, in order to obtain a higher yield of y-globulin from whole plasma since some y-globulin is thought to be held back by protein-protein interaction with fibrinogen in fraction I. It is evident that the immunoglobulin thus prepared contains those antibodies which react with BOB antigens No. 220, 226 and 244 and it is suggested that a method of preparation which omits the initial separation of fraction I is the method of choice in the production of hepatitis B immunoglobulin.
323
References 1 Horowitz, M. S.; Woods, K. R.; Gersten, M., and Ehrich, C.: Separation of two hepatitis B human antibody fractions by cold-ethanol precipitation. Vox Sang. 30: 50 (1976). 2 Nitschmann, H.; Kistler, P., and Lergier, W.: Preparation of human albumin and y-globulin from blood plasma by alcoholic precipitation. Helv. chim. Acta 37: 866 (1954). 3 Kistler, P. and Nitschmann, H.: Large scale production of human plasma fractions. Eight years experience with the alcohol fractionation procedure of Nitschmann, Kistler and Lergier. Vox Sang. 7: 414 (1962). 4 Combridge, B . S . and Shaw, C.S.: Immunoelectroosmophoresis using discontinuous buffer system to detect Australia antigen in pooled human plasma. Lancet ii: 1184 (1971). 5 Hopkins, R. and Das, P. C.: Australia antigen (HB-Ag) subtyping by a sensitive tanned cell haemagglutination inhibition technique. Br. J . Haemat. 27: 501 (1974).
Received: November 1, 1976 Accepted: November 30, 1976 B. S . Combridge, Blood Products Laboratory, Lister Institute of Preventive Medicine, Elstree, Herts. (England)
