THROMBOSIS RESEARCH Printed in Great Britain
Vol . 11, pp . 653-662, 1977 Pergamon Press, Ltd .
STUDIES OF HUMAN FACTOR IX BY A HIGH-TITRED SHEEP ANTISERUM AGAINST FACTOR IX Karen Helene orstavik 1 , Anne Marie Venner¢d 2 and Knut Laake 3
2 Institute of Medical Genetics, University of Oslo, Oslo, Norway,
Department of Pharmacology, Institute of Pharmacy, University of Oslo, Norway and Department 9, UllevA1 Hospital, Oslo, Norway
(Received 9 .8 .1977 ; in revised form 2 .9 .1977 . Accepted by Editor H .C . Godal)
ABSTRACT In order to produce a high-titred antiserum against factor IX a sheep was immunized with highly purified human factor IX . One ml of the antisera obtained from three different bleedings neutralized 90% of the activity of factor IX in 130-200 ml standard plasma . The antisera gave one main precipitin band and occasionally an additional weak precipitin band against normal plasma in double immunodiffusion . The purified factor IX preparation had a specific activity of 290 U per absorbancy unit at 280 nrl . However, it was not homogenous in various polyacrylamide gel electrophoresis techniques . In analytical polyacrylamide gel electrophoresis one main band and several additional weaker bands with lower electrophoretic mobility were seen . When this gel was submitted to electrophoresis into an agarose gel containing the antiserum against factor IX, one precipitin arc was seen corresponding to each of the bands . Reactions of identity was seen between the precipitin arcs, thus demonstrating the presence of factor IX antigen in all the bands . The minor bands with lower electrophoretic mobility disappeared on polyacrylamide disc gel electrophoresis in the presence of 10M urea and on SDS polyacrylamide electrophoresis . They may represent aggregates of factor IX, or complexes between factor IX and other proteins .
INTRODUCTION In an accompanying paper (1) we report on the purification of human factor IX for the purpose of producing a high-titred 653
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antiserum against factor IX . The purified factor IX had the same specific activity as that found by di Scipio (2) and a higher specific activity than that found by other workers (3,4,5) . The product was still not homogenous by various analytical polyacrylamide gel electrophoresis techniques . In the present paper we report on the production of a high-titred antiserum against factor IX by immunization with the purified factor .The antiserum was used to study the polyacrylamide electrophoresis gels by immunological techniques . MATERIALS AND METHODS A standard plasma was prepared by mixing equal volumes of plasma from 20 healthy male students . Aluminium hydroxide eluate was prepared from citrated plasma as described by Swart et al . '(6) . Purified factor IX was prepared as described in the accompanying paper (1) . Immunization schedule . A sheep and two rabbits were immunized with purified- factor IX . The factor IX solution was mixed with an equal volume of Freunds incomplete adjuvant and injected intradermally in multiples sites on the back of the animal . The sheep was given 400 ug factor IX the first time and 200 ug another three times during a period of four months and bled from the jugular vein repeatedly (Fig .l) . The rabbits were given 80 ug every 2-4 weeks five times and bled one week after the last immunization .
'j'
= Immunization
toBleeding
1 0
3
2
4
,1 1 1 t , S j
25
50
75 FIG .
1
100
125 Days
Immunization and bleeding schedule of sheep Immunological methods . Neutralization of the activity of coagulation factors by the antisera was carried out as described in the accompanying paper (1) Double immunodiffusion, crossed immunoelectrophoresis and electroimmunoassay (rocket immunoelectrophoresis) were performed as previously described (7,8) . Immunoelectrophoresis was performed
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6 55
witn 1% agarose in 0 .025M barbital buffer pH 8 .6 . The electrophoresis was run for one hour at 10 V/cm . Agarose was obtained from 1'Industrie Biologique Francaise, S .A . Genevilliers, France (Indubiose A 37) and from Litex, Glostrup, Denmark (Agarose HSB with low electroendosmosis for crossed immunoelectrophoresis) . Polyacrylamide gel electrophoresis was performed as previously described (9) . After electrophoresis the gels were sliced longitudinally in two halves and washed for one hour in large volumes of 0 .025M barbital buffer or for two hours if the gels contained sodium dodecyl sulfate (SDS) . A 3 .5 cm broad and 1 .5 mm thick layer of 1% agarose in 0 .025M barbital buffer was casted on glass plates 10x10 cm . Another 6 .5 cm broad layer of agarose containing antiserum was casted next to the first gel layer . The washed polyacrylamide gel half was embedded in the agarose gel without antibody and the electrophoresis was run for 16 hours at 1 V/cm . After drying the gels were stained with Coomassie Brilliant Blue R Immunofixation of the polyacrylamide gels (10) was also performed on longitudinally cut gel halves . The disc gels were washed for 10 minutes whereas the SDS gels were washed for two hours . After washing the gels were immersed in undiluted antiserum or preimmune serum ~n a small tube . The tubes were incubated for two hours at 37 C and for two to three days at room temperature . The gels were then washed in large volumes of saline and stained with Coomassie Brilliant Blue R according to the method of Fairbanks (11) Coagulation factor activities were determined as described in the accompanying paper (1) . RESULTS 1 .Characterizationof sheep and rabbit antisera . Sheep serum from bleedings 3, 3 and 4 were examined and behaved similarly in precipitation techniques . In double immunodiffusion tae sheep antiserum diluted 1/10 gave one precipitin band against normal plasma and purified factor IX (Fig . 2) . A reaction of identity was seen with a rabbit antiserum against factor IX prepared by immunization with immunoprecipitates (8) . One precipitin band was also seen against plasma from a patient with hemophilia B with a normal amount of factor IX antigen (hemophilia B+) whereas no precipitin band could be seen against plasma from a patient with hemophilia B and no detectable factor IX antigen (`emophilia B-) (12) . When undiluted antiserum was tested against normal plasma, an additional weak precipitin band was occasionally seen .In crossed immunoelectrophoresis one precipitin arc with the same electrophoretic mobility was seen against the purified factor IX and aluminium hydroxide eluate from normal plasma (Fig . 3) When plasma was used as antigen no precipitin arc could be seen . In one dimensional immunoelectrophoresis one precipitin arc was seen when aluminium hydroxide eluate from normal plasma was tested against antiserum diluted 1/2-1/20 . When undiluted antiserum was used an additional weak precipitin arc in the gamma region was occasionally seen against plasma as well as aluminium hydroxide eluate .
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STUDIES OF HUMAN FACTOR IX
FIG . 2
FIG .
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3
Fig . 2 : Double immunodiffusion slide showing a reaction of identity between precipitin bands produced by sheep and rabbit antisera against normal plasma, plasma from a patient with hemophilia B+ and purified factor IX . Unstained slide . Central well : Sheep antiserum against factor IX diluted 1/10 Well 1 : Purified factor IX Well 2 : Rabbit antiserum against factor IX Well 3 : Plasma from patient with hemophilia B+ Well 4 : Plasma from patient with hemophilia BWell 5 : Rabbit antiserum against factor IX Well 6 : Normal plasma Fig . 3 : Crossed immunoelectrophoresis showing single precipitin arc between aluminium hydroxide eluate from normal plasma (15 pl) and sheep antiserum (0 .67% in 1% agarose) . The arrow points to the position of bovine albumin after electrophoresis in the first dimension . The slide was stained with Coomassie Brilliant Blue .
The factor IX neutralizing activity of the antisera is seen in Fig . 4 . The dilution which neutralized 90% of the factor IX activity was read from the curve and the reciprocal value was defined as the titre of the antiserum . The titre was 200 for the sheep antiserum from bleeding 2 and 130 for the antisera from bleedings 3 and 4 . Essentially no reduction in the activity of factor IX was seen when the standard plasma was incubated with preimmune sheep serum . Undiluted sheep antiserum did not neutralize the activity of factors II, VII or X . After five immunizations the antisera from both rabbits gave one main precipitin band and an additional weaker precipitin band against normal plasma in double immunodiffusion .The titre of the rabbit antisera was 25 and 40 as seen in Fig . 4 . 2 . Immunological study of polyacrylamide elec trophoresis gels of purified factor IX . As reported in the accompanying paper (1) polyacrylamide disc gel electrophoresis of 20 pg of the purified factor IX resulted in one main band and four weaker bands with lower electrophoretic mobility . The specific activity of the purifed factor was about 290 U
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657
F II U/mi
Neutralization of factor IX activity by antisera
o---0 0--0
-y
Sheep antiserum bleeding Sheep antiserum bleeding Sheep antiserum bleeding Sheep antiserum bleeding Rabbit antiserum 1 Rabbit antiserum 2
1 2 3 4
Different dilutions of the antisera were inc8bated with an equal volume of standard plasma for one hour at 37 C and the remaining activity of factor IX was determined . per absorbency unit at 280 nm which is the same as that reported by di Scipio (2) and higher than that reported by other workers (3,4,5) . We considered it possible that the minor bands did not represent contaminants only . The gels were therefore submitted to electrophoresis into agarose containing the sheep antiserum . Five precipitin arcs with a position corresponding to the five bands were seen when the concentration of sheep antiserum in the agarose gel was low enough (Fig . 5A) . A reaction of identity was seen between the arcs . When the concentration of antiserum in the agarose was increased the peak of the main precipitin arc was seen as a homogenous arc, but then one or two of the minor bands disappeared . The height of the precipitin arcs seemed to correspond to the intensity of the colour in the bands . The same precipitation pattern was seen when the rabbit antiserum prepared by immunization with immunoprecipitates (8) was used . In order to confirm the presence of factor IX antigen in the minor bands the polyacrylamide disc gels were studied by the immuno-
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FIG . 5 Electrophoresis into agarose containing sheep antiserum of polyacrylamide electrophoresis gels of 20 ug purified factor IX . A photograph of a stained polyacrylamide gel was mounted on a photograph of the agarose gel . A) . Polyacrylamide disc gel . 0 .7% antiserum in the agarose . B) . Polyacrylamide disc gel run in the presence of 10M urea . 1% antiserum in the agarose . C) . SDS polyacrylamide gel . 0 .9% antiserum in the agarose .
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STUDIES OF HUMAN FACTOR IX
FIG .
659
6
Immunofixation of polyacrylamide electrophoresis gels of 20 ug purified factor IX . After the electrophoresis the gel halves were immersed in sheep antiserum, washed in large volumes of saline and stained . A) Polyacrylamide disc gel B) Polyacrylamide disc gel run in the presence of 10M urea C) Polyacrylamide SDS gel
fixation technique . After incubation of the gels in antiserum for 30-60 minutes one band appeared in the position of the main band . After incubation for another two days no additional bands were seen before staining . After washing and staining five bands were seen in the same position as seen in the stained gel which had not been incubated in antiserum (Fig . 6A) . No bands were seen in corresponding gel halves incubated in preimmune sheep serum . In order to examine for the presence of factor IX activity in the minor bands the polyacrylamide disc gels were cut transversally in 20 equally large slices . Each slice was homogenized and tested for factor IX activity and for factor IX antigen by the electroimmunoassay technique with a rabbit antiserum against factor IX (8) . In three experiments factor IX activity and factor IX antigen were detected in slices corresponding to the major band . Factor IX antigen was detected in a slice corresponding to one of the minor bands in only one of the three experiments . Factor IX activity was not detected corresponding to the minor bands in any of the three experiments . In the presence of 10M urea one main band which appeared and two minor bands were seen in polyacrylamide disc gel phoresis (1) . Electrophoresis of these gels into agarose ing the sheep antiserum revealed one main orecinitin arc
double electrocontainand a
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smaller precipitin arc which was not completely separated from the main arc (Fig . 5B) . A reaction of identity was seen between the two precipitin arcs . Apparently the minor arc corresponded to one or both of the minor bands . With increased concentration of antiserum in the agarose gel the major arc was seen as one homogenous peak . Immunofixation of such gels gave one major band and one minor band close to the major band (Fig . 6B) . Polyacrylamide electrophoresis of purified factor IX in the presence of SDS had revealed one main band and a weaker secondary band (1) . Electrophoresis of these gels into agarose with antiserum gave precipitin arcs corresponding to these bands as seen in Fig . 5C, with a reaction of identity between the two arcs . Two bands with a position corresponding to these arcs were also seen with the immunofixation technique (Fig . 6C) . DISCUSSION The sheep antiserum neutralized factor IX activity, gave one precipitin band against plasma and purified factor IX, and gave a reaction of identity with a previously characterized rabbit antiserum against factor IX . The antiserum reacted with plasma from patients with hemophilia B+ and not with plasma from patients with hemophilia B- . The sheep antiserum was therefore directed against factor IX . The titre of the sheep antiserum as defined here was 130-200 which is significantly higher than the titre of previously reported rabbit antisera against factor IX . Denson (12) reported on the production of antisera with titre 5 . Elodi (13) produced antisera with titre 3 and the titres of the antisera produced by osterud and Flengsrud was 3 .5-5 (3) . Hypothetically, the reason for the high titre obtained here could be that sheep factor IX is more different from human factor IX than is rabbit factor IX, and that human factor IX therefore is more antigenic in sheep . than in rabbits . However, immunization of two rabbits with the same antigen also resulted in antisera with a higher titre (25 and 40 respectively) than previously reported . The method of immunization, intradermally in multiple sites, may therefore be important . This method was not used by the above mentioned authors . Our results seem to suggest that sheep may be well suited for the production of high-titred antisera against human factor IX . The purified factor IX gave more than one band both in polyacrylamide disc gel electrophoresis, in the presence and abscence of 10M urea, and in SDS polyacrylamide gel electrophoresis with and without reduction . The minor bands in the disc electrophoresis without urea could represent aggregates of factor IX, contaminants or complexes between factor IX and contaminants . Immunoelectrophoresis of the disc gels revealed that factor IX was present in all the minor bands in an amount apparently corresponding to the intensity of stain in the bands (Fig . 5A) . It cannot be excluded, however, that other proteins in complex with factor IX were present in the bands . Immunofixation of the disc gels without urea confirmed the presence of factor IX antigen in all the bands . This technique was
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as sensitive in detecting factor IX as was the direct, unspecific detection by staining of the gels, or the specific detection by immunoelectrophoresis . Furthermore, the immunoelectrophoresis was rather sensitive to the concentration of antiserum in the agarose, as a too high concentration of antiserum lead to loss of the minor bands . For the immunofixation, undiluted antiserum could be used in all the experiments . In the homogenized disc gel slices corresponding to the minor bands, factor IX antigen was usually not and factor IX activity never detected . The reason for this was probably that the concentration of factor IX in the final solution was too small to be detected . This method, was therefore not suited for the study of factor IX activity in the minor bands and no conclusions can be drawn from the lack of detectable factor IX activity in the slices corresponding to the minor bands . Immunoelectrophoresis of polyacrylamide disc gels which had been run in the presence of urea revealed that factor IX antigen was present in at least one of the minor bands (Fig . 5B) . One of these bands might therefore represent activated factor IX . The other minor band could represent a contaminant . If factor IX was present in this band it might merge with factor IX in the main band during the immunoelectrophoresis and appear as a homogenous arc . The minor band seen in SDS polyacrylamide electrophoresis also contained factor IX antigen (Fig . 5C) and may also represent activated factor IX . It can therefore be concluded that the multiple bands found in different kinds of polyacrylamide electrophoresis represented various physico-chemical states of factor IX, possibly with the exception of one minor band found in the urea disc . Further studies are necessary to clarify whether the factor IX aggregation indicated in the polyacrylamide disc electrophoresis occurs in plasma, or is a result of the purification procedure or the conditions during the analytical disc electrophoresis . ACKNOWLEDGEMENTS This work was supported by grants from the Norwegain Research Council for Science and the Humanities to Karen Helene orstavik and professor K$re Berg, M .D . REFERENCES 1 . VENNER(D, A . M ., ORSTAVIK, K . H ., LAAKE, K ., FAGERHOL, M . K . and LY, 3 . Purification of human factor IX . Thromb . Res . (in press) . 2 . DI SCIPIO, R . G ., HERMODSON, M . A ., YATES, S . G . and DAVIE, E . W . A . A comparison of human prothrombin, factor IX (Chrismas factor), factor X (Stuart factor) and protein S . Biochemistry, 16 698, 1977 . 3 . OSTERUD, B . and FLENGSRUD, R . Purification and some characteristics of the coagulation factor IX from human plasma . Biochem . J . 145, 469, 1975 .
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4 . ANDERSSON, L-O ., BORG, H . and MILLER-ANDERSSON, M . Purification and characterization of human factor IX . Thromb . Res . 7, 451, 1975 . 5 . SUOMELA, H . Human coagulation factor IX . Isolation and characterization . Eur . J . Biochem . 71, 145, 1976 . 6 . SWART, A . C . W ., KLAASSEN, B . H . M, BLOYS-VAN TRESLONG-DE GROOT, C . H . F . and HEMKER, H . C . The adsorption of blood coagulation factors II, VII, IX and X from human plasma to aluminium hydroxide . Thromb . Diath . Haemorrh . 27, 490, 1972 . 7 . ORSTAVIK, K . H ., OSTERUD, B ., PRYDZ, H . and BERG, K . Electroimmunoassay of factor IX in hemophilia B . Thromb . Res . 7, 373, 1975 . 8 . ORSTAVIK, K . H . Comparison between precipitating antisera against normal and abnormal factor IX . Thromb . Res . 10, 731, 1977 . 9 . LAAKE, K . and ELLINGSEN, R . Purification and some characteristics of factor VII in human citrated plasma, glass-activates serum,and cold-activated plasma . Thromb . Res . 5, 539, 1974 . 10 . STUMPH, W . E ., ELGIN, S . C . R . and HOOD, L . Antibodies to proteins dissolved in sodium dodecyl sulfate . J . immunol . 113, 1752, 1974 . 11 . FAIRBANKS, G ., STECK, T . L . and WALLACH, D . H . F . Electrophoretic analysis of the major polypeptides of the human erythrocyte membrane . Biochemistry, 10, 2606, 1971 . 12 . DENSON, K . W . E . Editorial . Molecular variants of haemophilia B . Thromb . Diath . Haemorrh . 29, 217, 1973 . 13 . ELODI, S . and PUSKAS, E . Variants of haemophilia B . Thromb . Diath . Haemorrh . 28, 489, 1972 .
Vol . 11, pp . 653-662, 1977 Pergamon Press, Ltd .
STUDIES OF HUMAN FACTOR IX BY A HIGH-TITRED SHEEP ANTISERUM AGAINST FACTOR IX Karen Helene orstavik 1 , Anne Marie Venner¢d 2 and Knut Laake 3
2 Institute of Medical Genetics, University of Oslo, Oslo, Norway,
Department of Pharmacology, Institute of Pharmacy, University of Oslo, Norway and Department 9, UllevA1 Hospital, Oslo, Norway
(Received 9 .8 .1977 ; in revised form 2 .9 .1977 . Accepted by Editor H .C . Godal)
ABSTRACT In order to produce a high-titred antiserum against factor IX a sheep was immunized with highly purified human factor IX . One ml of the antisera obtained from three different bleedings neutralized 90% of the activity of factor IX in 130-200 ml standard plasma . The antisera gave one main precipitin band and occasionally an additional weak precipitin band against normal plasma in double immunodiffusion . The purified factor IX preparation had a specific activity of 290 U per absorbancy unit at 280 nrl . However, it was not homogenous in various polyacrylamide gel electrophoresis techniques . In analytical polyacrylamide gel electrophoresis one main band and several additional weaker bands with lower electrophoretic mobility were seen . When this gel was submitted to electrophoresis into an agarose gel containing the antiserum against factor IX, one precipitin arc was seen corresponding to each of the bands . Reactions of identity was seen between the precipitin arcs, thus demonstrating the presence of factor IX antigen in all the bands . The minor bands with lower electrophoretic mobility disappeared on polyacrylamide disc gel electrophoresis in the presence of 10M urea and on SDS polyacrylamide electrophoresis . They may represent aggregates of factor IX, or complexes between factor IX and other proteins .
INTRODUCTION In an accompanying paper (1) we report on the purification of human factor IX for the purpose of producing a high-titred 653
654
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antiserum against factor IX . The purified factor IX had the same specific activity as that found by di Scipio (2) and a higher specific activity than that found by other workers (3,4,5) . The product was still not homogenous by various analytical polyacrylamide gel electrophoresis techniques . In the present paper we report on the production of a high-titred antiserum against factor IX by immunization with the purified factor .The antiserum was used to study the polyacrylamide electrophoresis gels by immunological techniques . MATERIALS AND METHODS A standard plasma was prepared by mixing equal volumes of plasma from 20 healthy male students . Aluminium hydroxide eluate was prepared from citrated plasma as described by Swart et al . '(6) . Purified factor IX was prepared as described in the accompanying paper (1) . Immunization schedule . A sheep and two rabbits were immunized with purified- factor IX . The factor IX solution was mixed with an equal volume of Freunds incomplete adjuvant and injected intradermally in multiples sites on the back of the animal . The sheep was given 400 ug factor IX the first time and 200 ug another three times during a period of four months and bled from the jugular vein repeatedly (Fig .l) . The rabbits were given 80 ug every 2-4 weeks five times and bled one week after the last immunization .
'j'
= Immunization
toBleeding
1 0
3
2
4
,1 1 1 t , S j
25
50
75 FIG .
1
100
125 Days
Immunization and bleeding schedule of sheep Immunological methods . Neutralization of the activity of coagulation factors by the antisera was carried out as described in the accompanying paper (1) Double immunodiffusion, crossed immunoelectrophoresis and electroimmunoassay (rocket immunoelectrophoresis) were performed as previously described (7,8) . Immunoelectrophoresis was performed
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witn 1% agarose in 0 .025M barbital buffer pH 8 .6 . The electrophoresis was run for one hour at 10 V/cm . Agarose was obtained from 1'Industrie Biologique Francaise, S .A . Genevilliers, France (Indubiose A 37) and from Litex, Glostrup, Denmark (Agarose HSB with low electroendosmosis for crossed immunoelectrophoresis) . Polyacrylamide gel electrophoresis was performed as previously described (9) . After electrophoresis the gels were sliced longitudinally in two halves and washed for one hour in large volumes of 0 .025M barbital buffer or for two hours if the gels contained sodium dodecyl sulfate (SDS) . A 3 .5 cm broad and 1 .5 mm thick layer of 1% agarose in 0 .025M barbital buffer was casted on glass plates 10x10 cm . Another 6 .5 cm broad layer of agarose containing antiserum was casted next to the first gel layer . The washed polyacrylamide gel half was embedded in the agarose gel without antibody and the electrophoresis was run for 16 hours at 1 V/cm . After drying the gels were stained with Coomassie Brilliant Blue R Immunofixation of the polyacrylamide gels (10) was also performed on longitudinally cut gel halves . The disc gels were washed for 10 minutes whereas the SDS gels were washed for two hours . After washing the gels were immersed in undiluted antiserum or preimmune serum ~n a small tube . The tubes were incubated for two hours at 37 C and for two to three days at room temperature . The gels were then washed in large volumes of saline and stained with Coomassie Brilliant Blue R according to the method of Fairbanks (11) Coagulation factor activities were determined as described in the accompanying paper (1) . RESULTS 1 .Characterizationof sheep and rabbit antisera . Sheep serum from bleedings 3, 3 and 4 were examined and behaved similarly in precipitation techniques . In double immunodiffusion tae sheep antiserum diluted 1/10 gave one precipitin band against normal plasma and purified factor IX (Fig . 2) . A reaction of identity was seen with a rabbit antiserum against factor IX prepared by immunization with immunoprecipitates (8) . One precipitin band was also seen against plasma from a patient with hemophilia B with a normal amount of factor IX antigen (hemophilia B+) whereas no precipitin band could be seen against plasma from a patient with hemophilia B and no detectable factor IX antigen (`emophilia B-) (12) . When undiluted antiserum was tested against normal plasma, an additional weak precipitin band was occasionally seen .In crossed immunoelectrophoresis one precipitin arc with the same electrophoretic mobility was seen against the purified factor IX and aluminium hydroxide eluate from normal plasma (Fig . 3) When plasma was used as antigen no precipitin arc could be seen . In one dimensional immunoelectrophoresis one precipitin arc was seen when aluminium hydroxide eluate from normal plasma was tested against antiserum diluted 1/2-1/20 . When undiluted antiserum was used an additional weak precipitin arc in the gamma region was occasionally seen against plasma as well as aluminium hydroxide eluate .
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FIG . 2
FIG .
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Fig . 2 : Double immunodiffusion slide showing a reaction of identity between precipitin bands produced by sheep and rabbit antisera against normal plasma, plasma from a patient with hemophilia B+ and purified factor IX . Unstained slide . Central well : Sheep antiserum against factor IX diluted 1/10 Well 1 : Purified factor IX Well 2 : Rabbit antiserum against factor IX Well 3 : Plasma from patient with hemophilia B+ Well 4 : Plasma from patient with hemophilia BWell 5 : Rabbit antiserum against factor IX Well 6 : Normal plasma Fig . 3 : Crossed immunoelectrophoresis showing single precipitin arc between aluminium hydroxide eluate from normal plasma (15 pl) and sheep antiserum (0 .67% in 1% agarose) . The arrow points to the position of bovine albumin after electrophoresis in the first dimension . The slide was stained with Coomassie Brilliant Blue .
The factor IX neutralizing activity of the antisera is seen in Fig . 4 . The dilution which neutralized 90% of the factor IX activity was read from the curve and the reciprocal value was defined as the titre of the antiserum . The titre was 200 for the sheep antiserum from bleeding 2 and 130 for the antisera from bleedings 3 and 4 . Essentially no reduction in the activity of factor IX was seen when the standard plasma was incubated with preimmune sheep serum . Undiluted sheep antiserum did not neutralize the activity of factors II, VII or X . After five immunizations the antisera from both rabbits gave one main precipitin band and an additional weaker precipitin band against normal plasma in double immunodiffusion .The titre of the rabbit antisera was 25 and 40 as seen in Fig . 4 . 2 . Immunological study of polyacrylamide elec trophoresis gels of purified factor IX . As reported in the accompanying paper (1) polyacrylamide disc gel electrophoresis of 20 pg of the purified factor IX resulted in one main band and four weaker bands with lower electrophoretic mobility . The specific activity of the purifed factor was about 290 U
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657
F II U/mi
Neutralization of factor IX activity by antisera
o---0 0--0
-y
Sheep antiserum bleeding Sheep antiserum bleeding Sheep antiserum bleeding Sheep antiserum bleeding Rabbit antiserum 1 Rabbit antiserum 2
1 2 3 4
Different dilutions of the antisera were inc8bated with an equal volume of standard plasma for one hour at 37 C and the remaining activity of factor IX was determined . per absorbency unit at 280 nm which is the same as that reported by di Scipio (2) and higher than that reported by other workers (3,4,5) . We considered it possible that the minor bands did not represent contaminants only . The gels were therefore submitted to electrophoresis into agarose containing the sheep antiserum . Five precipitin arcs with a position corresponding to the five bands were seen when the concentration of sheep antiserum in the agarose gel was low enough (Fig . 5A) . A reaction of identity was seen between the arcs . When the concentration of antiserum in the agarose was increased the peak of the main precipitin arc was seen as a homogenous arc, but then one or two of the minor bands disappeared . The height of the precipitin arcs seemed to correspond to the intensity of the colour in the bands . The same precipitation pattern was seen when the rabbit antiserum prepared by immunization with immunoprecipitates (8) was used . In order to confirm the presence of factor IX antigen in the minor bands the polyacrylamide disc gels were studied by the immuno-
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FIG . 5 Electrophoresis into agarose containing sheep antiserum of polyacrylamide electrophoresis gels of 20 ug purified factor IX . A photograph of a stained polyacrylamide gel was mounted on a photograph of the agarose gel . A) . Polyacrylamide disc gel . 0 .7% antiserum in the agarose . B) . Polyacrylamide disc gel run in the presence of 10M urea . 1% antiserum in the agarose . C) . SDS polyacrylamide gel . 0 .9% antiserum in the agarose .
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STUDIES OF HUMAN FACTOR IX
FIG .
659
6
Immunofixation of polyacrylamide electrophoresis gels of 20 ug purified factor IX . After the electrophoresis the gel halves were immersed in sheep antiserum, washed in large volumes of saline and stained . A) Polyacrylamide disc gel B) Polyacrylamide disc gel run in the presence of 10M urea C) Polyacrylamide SDS gel
fixation technique . After incubation of the gels in antiserum for 30-60 minutes one band appeared in the position of the main band . After incubation for another two days no additional bands were seen before staining . After washing and staining five bands were seen in the same position as seen in the stained gel which had not been incubated in antiserum (Fig . 6A) . No bands were seen in corresponding gel halves incubated in preimmune sheep serum . In order to examine for the presence of factor IX activity in the minor bands the polyacrylamide disc gels were cut transversally in 20 equally large slices . Each slice was homogenized and tested for factor IX activity and for factor IX antigen by the electroimmunoassay technique with a rabbit antiserum against factor IX (8) . In three experiments factor IX activity and factor IX antigen were detected in slices corresponding to the major band . Factor IX antigen was detected in a slice corresponding to one of the minor bands in only one of the three experiments . Factor IX activity was not detected corresponding to the minor bands in any of the three experiments . In the presence of 10M urea one main band which appeared and two minor bands were seen in polyacrylamide disc gel phoresis (1) . Electrophoresis of these gels into agarose ing the sheep antiserum revealed one main orecinitin arc
double electrocontainand a
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smaller precipitin arc which was not completely separated from the main arc (Fig . 5B) . A reaction of identity was seen between the two precipitin arcs . Apparently the minor arc corresponded to one or both of the minor bands . With increased concentration of antiserum in the agarose gel the major arc was seen as one homogenous peak . Immunofixation of such gels gave one major band and one minor band close to the major band (Fig . 6B) . Polyacrylamide electrophoresis of purified factor IX in the presence of SDS had revealed one main band and a weaker secondary band (1) . Electrophoresis of these gels into agarose with antiserum gave precipitin arcs corresponding to these bands as seen in Fig . 5C, with a reaction of identity between the two arcs . Two bands with a position corresponding to these arcs were also seen with the immunofixation technique (Fig . 6C) . DISCUSSION The sheep antiserum neutralized factor IX activity, gave one precipitin band against plasma and purified factor IX, and gave a reaction of identity with a previously characterized rabbit antiserum against factor IX . The antiserum reacted with plasma from patients with hemophilia B+ and not with plasma from patients with hemophilia B- . The sheep antiserum was therefore directed against factor IX . The titre of the sheep antiserum as defined here was 130-200 which is significantly higher than the titre of previously reported rabbit antisera against factor IX . Denson (12) reported on the production of antisera with titre 5 . Elodi (13) produced antisera with titre 3 and the titres of the antisera produced by osterud and Flengsrud was 3 .5-5 (3) . Hypothetically, the reason for the high titre obtained here could be that sheep factor IX is more different from human factor IX than is rabbit factor IX, and that human factor IX therefore is more antigenic in sheep . than in rabbits . However, immunization of two rabbits with the same antigen also resulted in antisera with a higher titre (25 and 40 respectively) than previously reported . The method of immunization, intradermally in multiple sites, may therefore be important . This method was not used by the above mentioned authors . Our results seem to suggest that sheep may be well suited for the production of high-titred antisera against human factor IX . The purified factor IX gave more than one band both in polyacrylamide disc gel electrophoresis, in the presence and abscence of 10M urea, and in SDS polyacrylamide gel electrophoresis with and without reduction . The minor bands in the disc electrophoresis without urea could represent aggregates of factor IX, contaminants or complexes between factor IX and contaminants . Immunoelectrophoresis of the disc gels revealed that factor IX was present in all the minor bands in an amount apparently corresponding to the intensity of stain in the bands (Fig . 5A) . It cannot be excluded, however, that other proteins in complex with factor IX were present in the bands . Immunofixation of the disc gels without urea confirmed the presence of factor IX antigen in all the bands . This technique was
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as sensitive in detecting factor IX as was the direct, unspecific detection by staining of the gels, or the specific detection by immunoelectrophoresis . Furthermore, the immunoelectrophoresis was rather sensitive to the concentration of antiserum in the agarose, as a too high concentration of antiserum lead to loss of the minor bands . For the immunofixation, undiluted antiserum could be used in all the experiments . In the homogenized disc gel slices corresponding to the minor bands, factor IX antigen was usually not and factor IX activity never detected . The reason for this was probably that the concentration of factor IX in the final solution was too small to be detected . This method, was therefore not suited for the study of factor IX activity in the minor bands and no conclusions can be drawn from the lack of detectable factor IX activity in the slices corresponding to the minor bands . Immunoelectrophoresis of polyacrylamide disc gels which had been run in the presence of urea revealed that factor IX antigen was present in at least one of the minor bands (Fig . 5B) . One of these bands might therefore represent activated factor IX . The other minor band could represent a contaminant . If factor IX was present in this band it might merge with factor IX in the main band during the immunoelectrophoresis and appear as a homogenous arc . The minor band seen in SDS polyacrylamide electrophoresis also contained factor IX antigen (Fig . 5C) and may also represent activated factor IX . It can therefore be concluded that the multiple bands found in different kinds of polyacrylamide electrophoresis represented various physico-chemical states of factor IX, possibly with the exception of one minor band found in the urea disc . Further studies are necessary to clarify whether the factor IX aggregation indicated in the polyacrylamide disc electrophoresis occurs in plasma, or is a result of the purification procedure or the conditions during the analytical disc electrophoresis . ACKNOWLEDGEMENTS This work was supported by grants from the Norwegain Research Council for Science and the Humanities to Karen Helene orstavik and professor K$re Berg, M .D . REFERENCES 1 . VENNER(D, A . M ., ORSTAVIK, K . H ., LAAKE, K ., FAGERHOL, M . K . and LY, 3 . Purification of human factor IX . Thromb . Res . (in press) . 2 . DI SCIPIO, R . G ., HERMODSON, M . A ., YATES, S . G . and DAVIE, E . W . A . A comparison of human prothrombin, factor IX (Chrismas factor), factor X (Stuart factor) and protein S . Biochemistry, 16 698, 1977 . 3 . OSTERUD, B . and FLENGSRUD, R . Purification and some characteristics of the coagulation factor IX from human plasma . Biochem . J . 145, 469, 1975 .
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4 . ANDERSSON, L-O ., BORG, H . and MILLER-ANDERSSON, M . Purification and characterization of human factor IX . Thromb . Res . 7, 451, 1975 . 5 . SUOMELA, H . Human coagulation factor IX . Isolation and characterization . Eur . J . Biochem . 71, 145, 1976 . 6 . SWART, A . C . W ., KLAASSEN, B . H . M, BLOYS-VAN TRESLONG-DE GROOT, C . H . F . and HEMKER, H . C . The adsorption of blood coagulation factors II, VII, IX and X from human plasma to aluminium hydroxide . Thromb . Diath . Haemorrh . 27, 490, 1972 . 7 . ORSTAVIK, K . H ., OSTERUD, B ., PRYDZ, H . and BERG, K . Electroimmunoassay of factor IX in hemophilia B . Thromb . Res . 7, 373, 1975 . 8 . ORSTAVIK, K . H . Comparison between precipitating antisera against normal and abnormal factor IX . Thromb . Res . 10, 731, 1977 . 9 . LAAKE, K . and ELLINGSEN, R . Purification and some characteristics of factor VII in human citrated plasma, glass-activates serum,and cold-activated plasma . Thromb . Res . 5, 539, 1974 . 10 . STUMPH, W . E ., ELGIN, S . C . R . and HOOD, L . Antibodies to proteins dissolved in sodium dodecyl sulfate . J . immunol . 113, 1752, 1974 . 11 . FAIRBANKS, G ., STECK, T . L . and WALLACH, D . H . F . Electrophoretic analysis of the major polypeptides of the human erythrocyte membrane . Biochemistry, 10, 2606, 1971 . 12 . DENSON, K . W . E . Editorial . Molecular variants of haemophilia B . Thromb . Diath . Haemorrh . 29, 217, 1973 . 13 . ELODI, S . and PUSKAS, E . Variants of haemophilia B . Thromb . Diath . Haemorrh . 28, 489, 1972 .
