THROMBOSIS RESEARCH Printed in Great Britain
Vol .
10, PP . 731-'r41, 1917 Pergamon Press, Ltd .
COMPARISON OF PRECIPITATING ANTISERA AGAINST NORMAL AND ABNORMAL FACTOR IX
Karen Helene brstavik Institute of Medical Genetics, University of Oslo, Oslo 3, Norway
Blindern,
(Received 27 .1 .1977 ; in revised form 23 .3 .1977 . Accepted by Editor H .C . Godal)
ABSTRACT Precipitating rabbit antisera were prepared against normal factor IX and factor IX from a patient with hemophilia B by immunization with immunoprecipitates . These were pNpared by crossed immunoelectrophoresis between aluminium hydroxide eluate from plasma and rabbit antiserum against purified factor IX . The antisera did not reveal anv difference between normal factor IX antigen and factor IX antigen from the patient with hemophilia B . Both antisera neutralized factor IX activity in normal plasma . A reaction of identity was seen between precipitin bands produced by the two antisera when tested in double immunodiffusion against normal plasma and plasma from the patient with hemophilia B . Factor IX antigen was quantitated in plasma from 23 patients with hemophilia B by an electroimmunoassay with both antisera . In 9 of these patients factor IX antigen was detected and good agreement was found between the amount of antigen determined by the two antisera . Plasma samples from the 23 patients were also tested in double immunodiffusion . By this technique factor IX antigen could be detected only in the three patients with 50% or more antigen . INTRODUCTION Immunological methods using antibodies against coagulation factor IX have distinguished between hemophilia B patients with normal or reduced amounts of plasma factor IX antigen (hemophilia B+), and patients with no detectable factor IX antigen (hemophilia B-) . A third variant, hemophilia B M is characterized by a prolonged prothrombin time with bovine thromboplastin (1) . Studies with a precipitating rabbit antiserum against 731
732
ANTISERA AGAINST FACTOR IX
Vol .10,No .5
purified human factor IX demonstrated a reaction of identity between normal factor IX antigen and factor IX antigen from five patients with hemophilia B+ and one patient with hemophilia B in the fused rocket immunoelectrophoresis technique indicating a close immunochemical relationship between the normal and the abnormal factor IX antigens ( 2) . Abnormal factor Ix may further be studied by preparing antisera against the factor . However, purification of factor IX is difficult and needs large volumes of Plasma which may be difficult to obtain from patients with severe hemophilia . The abnormal factor IX may easily be isolated from small volumes of plasma by means of precipitating antibodies in gel precipitation techniques . Immunization with immunoprecipitates has been reported to be a simple way of obtaining monospecific antisera against proteins which are difficult to isolate (3, 4) . This method was used for the preparation of antisera against normal factor IX and factor IX from a patient with hemophilia B respectively, and the antisera thus obtained were compare .
MATERIALS AND METHODS Plasmasamples . Blood was collected into 1/10 volume of 0 .1M sodium citrate . Plasma was isolated by centrifugation at 2000g for 30 min at 4 °C and stored at -90 ° C until used . Astandardplasmapool was prepared by mixing equal volumes of plasma from 20 healthy fasting male students . This pool was chosen to contain 100% factor IX activity and 100% factor IX antigen . Aluminiumhydroxide eluate was prepared from citrated plasma as described by Swart et al . (5) . PurifiedfactorIX for use in gel precipitation tests was prepared by Anne Marie Vennerod and Knut Laake . The activity of the purified factor was about 75 units/ml . (Normal plasma contains one unit/ml) . Clottingassays, . Factor IX activity was determined in a one stage assay using plasma from a patient with severe hemophilia and "Cephotest" Nyco as activating substance . Factor VII activity was assayed by means of plasma from a patient with congenital deficiency of factor VII and rabbit thromboplastin . Factor II and X were determined with test kits from Behringwerke AG, Marburg . Rabbit antiserum against purified factor IX which was used for the production of immunoprecipitates for immunization, was a gift from Bjarne osterud . The properties of this antiserum was as previously reported (2, 6) . Immunological techniques . Double immunodiffusion was performed as described by Ouchterlony (7) with 1% agarose in 0 .075M barbital buffer pH 8 .6 containing 10 mM EDTA . The wells were 4
Vo 1 .1o,tio .5
ANTISERA AGAINST FACTOR IX
733
mm in diameter and were filled three times with 10 pl sample . The slides were left for three days at 20 C, washed in saline and stained with Coomassie Brilliant Blue R . Crossed immunoelectrophoresis was carried out as described by Laurell (8) at pH 8 .6 . The electrophoresis was run for one hour at 10 V/cm in the first dimension and for 1.6 hours at 2 V/cm in the second dimension . Immunoprecipitates for immunization by method II (see below) were prepared by electrophoresis for 40 hours in the second dimension . Electroimmunoassay of factor IX antigen was performed by the method of Laurell (9) . The lower limit of detectability by the electroimmunoassay was about 20% factor IX antigen relative to the standard plasma pool .Values below this were detected but the rockets were faint and were not always apparent . Preparationofnormal andabnormalfactorIXimmunoprecipitates forimmunization .Precipitin bands for immunization were prepared by crossed immunoelectrophoresis . Aluminium hydroxide eluate was used as antigen and was prepared from a plasma pool from several normal persons and from plasma from a patient with hemophilia B . This patient had factor IX activity
Vol .
10, PP . 731-'r41, 1917 Pergamon Press, Ltd .
COMPARISON OF PRECIPITATING ANTISERA AGAINST NORMAL AND ABNORMAL FACTOR IX
Karen Helene brstavik Institute of Medical Genetics, University of Oslo, Oslo 3, Norway
Blindern,
(Received 27 .1 .1977 ; in revised form 23 .3 .1977 . Accepted by Editor H .C . Godal)
ABSTRACT Precipitating rabbit antisera were prepared against normal factor IX and factor IX from a patient with hemophilia B by immunization with immunoprecipitates . These were pNpared by crossed immunoelectrophoresis between aluminium hydroxide eluate from plasma and rabbit antiserum against purified factor IX . The antisera did not reveal anv difference between normal factor IX antigen and factor IX antigen from the patient with hemophilia B . Both antisera neutralized factor IX activity in normal plasma . A reaction of identity was seen between precipitin bands produced by the two antisera when tested in double immunodiffusion against normal plasma and plasma from the patient with hemophilia B . Factor IX antigen was quantitated in plasma from 23 patients with hemophilia B by an electroimmunoassay with both antisera . In 9 of these patients factor IX antigen was detected and good agreement was found between the amount of antigen determined by the two antisera . Plasma samples from the 23 patients were also tested in double immunodiffusion . By this technique factor IX antigen could be detected only in the three patients with 50% or more antigen . INTRODUCTION Immunological methods using antibodies against coagulation factor IX have distinguished between hemophilia B patients with normal or reduced amounts of plasma factor IX antigen (hemophilia B+), and patients with no detectable factor IX antigen (hemophilia B-) . A third variant, hemophilia B M is characterized by a prolonged prothrombin time with bovine thromboplastin (1) . Studies with a precipitating rabbit antiserum against 731
732
ANTISERA AGAINST FACTOR IX
Vol .10,No .5
purified human factor IX demonstrated a reaction of identity between normal factor IX antigen and factor IX antigen from five patients with hemophilia B+ and one patient with hemophilia B in the fused rocket immunoelectrophoresis technique indicating a close immunochemical relationship between the normal and the abnormal factor IX antigens ( 2) . Abnormal factor Ix may further be studied by preparing antisera against the factor . However, purification of factor IX is difficult and needs large volumes of Plasma which may be difficult to obtain from patients with severe hemophilia . The abnormal factor IX may easily be isolated from small volumes of plasma by means of precipitating antibodies in gel precipitation techniques . Immunization with immunoprecipitates has been reported to be a simple way of obtaining monospecific antisera against proteins which are difficult to isolate (3, 4) . This method was used for the preparation of antisera against normal factor IX and factor IX from a patient with hemophilia B respectively, and the antisera thus obtained were compare .
MATERIALS AND METHODS Plasmasamples . Blood was collected into 1/10 volume of 0 .1M sodium citrate . Plasma was isolated by centrifugation at 2000g for 30 min at 4 °C and stored at -90 ° C until used . Astandardplasmapool was prepared by mixing equal volumes of plasma from 20 healthy fasting male students . This pool was chosen to contain 100% factor IX activity and 100% factor IX antigen . Aluminiumhydroxide eluate was prepared from citrated plasma as described by Swart et al . (5) . PurifiedfactorIX for use in gel precipitation tests was prepared by Anne Marie Vennerod and Knut Laake . The activity of the purified factor was about 75 units/ml . (Normal plasma contains one unit/ml) . Clottingassays, . Factor IX activity was determined in a one stage assay using plasma from a patient with severe hemophilia and "Cephotest" Nyco as activating substance . Factor VII activity was assayed by means of plasma from a patient with congenital deficiency of factor VII and rabbit thromboplastin . Factor II and X were determined with test kits from Behringwerke AG, Marburg . Rabbit antiserum against purified factor IX which was used for the production of immunoprecipitates for immunization, was a gift from Bjarne osterud . The properties of this antiserum was as previously reported (2, 6) . Immunological techniques . Double immunodiffusion was performed as described by Ouchterlony (7) with 1% agarose in 0 .075M barbital buffer pH 8 .6 containing 10 mM EDTA . The wells were 4
Vo 1 .1o,tio .5
ANTISERA AGAINST FACTOR IX
733
mm in diameter and were filled three times with 10 pl sample . The slides were left for three days at 20 C, washed in saline and stained with Coomassie Brilliant Blue R . Crossed immunoelectrophoresis was carried out as described by Laurell (8) at pH 8 .6 . The electrophoresis was run for one hour at 10 V/cm in the first dimension and for 1.6 hours at 2 V/cm in the second dimension . Immunoprecipitates for immunization by method II (see below) were prepared by electrophoresis for 40 hours in the second dimension . Electroimmunoassay of factor IX antigen was performed by the method of Laurell (9) . The lower limit of detectability by the electroimmunoassay was about 20% factor IX antigen relative to the standard plasma pool .Values below this were detected but the rockets were faint and were not always apparent . Preparationofnormal andabnormalfactorIXimmunoprecipitates forimmunization .Precipitin bands for immunization were prepared by crossed immunoelectrophoresis . Aluminium hydroxide eluate was used as antigen and was prepared from a plasma pool from several normal persons and from plasma from a patient with hemophilia B . This patient had factor IX activity
