Inhibitor-neutralisation assay and electro-immuno assay of human factor IX (Christmas factor)

275

Clinica Chimica Acta, 77 (1977) 275-286 @ Elsevier/North-Holland Biomedical Press

CGA 8454

INHIBITOR-NEUTRALISATION ASSAY OF HUMAN FACTOR

R.M. BERTINA

ASSAY AND ELECTRO-IMMUNO IX (CHRISTMAS FACTOR)

* and I.K. VAN DER LINDEN

Hemostasis and Thrombosis Research Unit, Department Leiden, Rijnsburgerweg 10, Leiden (The Netherlands) (Received

November

of Medicine,

University Hospital

22nd, 1976)

Summary A rabbit antibody specifically precipitating human factor IX has been used in the assay of factor IX antigen. The results obtained with two different methods (inhibitor-neutralisation assay and electro-immunoassay) have been compared in a group of healthy individuals and in a group of hemophilia B patients and carriers. In general, identical results are obtained with both methods, except in some hemophilia B’ carriers and patients, where the electroimmuno assay gives 1.5-2.0 times higher levels. Results obtained by electroimmuno assay are more accurate and reproducible than those obtained by inhibitor-neutralisation assay, which is of importance for its potential use in carrier detection.

Introduction In 1967 Hougie and Twomey [l] reported a new type of factor IX (Plasma Thromboplastin Component, Christmas factor) deficiency, characterized by the presumed presence of a biologically inactive factor IX molecule: hemophilia B,. Since then, several groups have been interested in the detection of these inactive molecules and their occurrence among hemophilia B patients (genetic variants) [2-9]. So far, different techniques have been reported for assay of the (inactive) factor IX molecules qualitatively or quantitatively: inhibitorneutralisation assay [2-9], an immunosorbens technique [lo] and an electroimmuno assay [ll]. All techniques have in common their use of antibodies against human factor IX (raised in rabbits or IX-deficient patients). Most reports are dealing with an inhibitor-neutralisation assay, which is partially due to a specific precipitating antibody against human factor IX being not readily available. * To whom correspondence should be addressed.

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In the present study we evaluate the quantitative results obtained in an electro-immuno assay (EIA) of factor IX antigen and those obtained in an inhibitor-neutralisation assay (INA). At the same time the levels of factor IX activity and antigen have been measured and compared in a group hemophilia B patients and carriers (both obligatory and possible), Dependent on the presence or absence of inactive factor IX molecules (cross reacting material) in their plasma, hemophilia B’ and B- variants will be discriminated [ 31. Materials and methods Human blood was collected either in 1150 volume 20% (w/v) or in l/l0 volume 3.2% sodium citrate. The platelet free plasma obtained after centrifugation at 20 000 X g (30 min, 4°C) was stored at -70°C. Normal plasma was gathered from the platelet free plasmas of 28 healthy volunteers (average age 30.2 years; 17 men and 11 women) and stored at -70°C. am-a~orbed plasma was prepared from platelet free plasma by stirring for 30 min at room temperature with l/10 volume of a 20% (w/v) Al(OH)3 suspension (BDH, moist gel) in 0.32% sodium citrate. Factor II, VII and X activities were measured by one stage assays in artificially depleted reagents, prepared according to Hemker et al. (123 (factor VII and X) and Koller et al. [13J (factor II). Factor IX activity was measured as described by Veltkamp et al. [14] using the plasma of a hemophilia B- patient as deficient plasma. 1 unit of activity is defined as the activity present in 1 ml normal plasma. Human factor IX was purified essentially as described by Andersson et al. [15]. The material recovered after the first hep~n-Seph~ose affinity chromatography step has been used for ~munization purposes (specific activity 27 U/mg protein; no detectable factor II, VII or X activity). The purified factor IX fraction used in the experiment of Fig. 2A was obtained after a further purification on Sephadex G-200 (specific activity > 30 U/mg protein). Anti-human factor IX serum was obtained from rabbits, previously immunized with a prothrombin-complex concentrate [16], adsorbed to heparinSepharose (Brigt, E., personal communication). After an 11-month rest period they were boosted by subcutaneous injection of a mixture of 1 ml factor IX preparation and 1 ml incomplete Freund adjuvant (Difco). 7 to 8 days later blood was collected from the ear vein and allowed to clot (16 h, 4°C). Supernatant serum was treated with BaS04 (100 mg/ml) and incubated 30 min at 56°C. For the inhibitor-neutralisation assay a crude immunoglobulin fraction was prepared by ammonium sulfate precipitation (50% saturation at 4°C); the precipitate was dissolved in 0.01 M kalium phosphate buffer (pH 6.8) and dialysed extensively against the same buffer. The material used in the electro-immuno assay was obtained after absorption of the rabbit antiserum with a mixture of A1(OH)3-adsorbed plasma and plasma of a factor IX deficient patient (Baccording to ~hibitor-neutr~isation assay with both rabbit and human anti factor IX). Immune precipitates were removed by cent~fugation (30 mm, 20 000 X g). After treatment of the supernatant mater&l with l/l0 vol. 20%

277

(w/v) Al(OH)J suspension and subjection to heat inactivation, an immunoglobulin fraction was prepared as described above. It should be noted that for the absorption of different batches of antiserum not always the plasma of the same B- patient was used. Antibody titers were determined as described by Bri& et al. [17]. 1 unit of anti-factor IX (II, VII, X) activity is defined as the highest dilution of antiserum that can still neutralize more than 98% of the activity present in 1 ml of normal plasma. Immunodiffusion was carried out according to Ouchterlony [ 181 using 1.2% agar (Agar Noble, Difco) in Tris-veronal buffer, pH 8.8 (Gelman, HRB, I 0.0028). Electra-immunoassay was carried out essentially as described by Laurel1 [ 191, using 1.2% agarose (Indubiose A37, Industrie Biologique Francaise S.A.) in Tris-veronal, pH 8.8. Gels contained 3.5-4.0% (v/v) of the immunoglobulin fraction of the absorbed rabbit anti human factor IX serum. Dimensions of the gels were 10 X 10 X 0.012 cm; lo-15+1 samples were applied in holes 4 mm in diameter. Electrophoresis time was 5 h at 8-10 volts/cm. Routinely, each plate contained normal plasma in 3 dilutions (undiluted, 1 in 2, 1 in 4) and 7 test samples (undiluted or 1 in 2). Samples were diluted either in Trisveronal buffer or Al(OH), -adsorbed plasma. Precipitated proteins were stained with 0.25% (w/v) Coomassie Brillant Blue R250 (Serva) in methanol/acetic acid/water (5 : 1 : 5, by vol.). The inhibitor-neutralisation assay of factor IX antigen was carried out as described by Briet et al. [17] for the determination of factor VII antigen. 0.015 ml of anti-factor IX was added to 0.085 ml normal plasma (in l/l, l/2, ‘l/4, l/8, l/16 and l/m dilution) or test sample (in two dilutions). After a 30min incubation at room temperature, a second incubation was started by the addition of 0.1 ml normal plasma to remove an eventual excess of antibody. Again 30 min later 1.8 ml dilution buffer was added, after which residual factor IX activity was measured in triplo on each sample. Results Properties of the rabbit anti human factor IX Due to their previous immunization with a prothrombin-complex concentrate, the rabbits were able to develop appreciable titers of anti-factor VII and anti-factor IX activity and to a lesser extent of anti-factor II and anti-factor X activity (cf. Denson [20]). After boosting with the purified factor IX preparation, the antiserum contained per ml 6-7 units anti-factor IX, 1 unit antifactor VII and no detectable anti-factor II or X. In the immunoglobulin fraction prepared after absorption of the antiserum, only the anti-factor IX activity was recovered (>3 units/ml). Fig. 1 shows that in a two-dimensional immunoelectrophoresis of normal plasma only one symmetrical peak appears when 4% (v/v) anti-factor IX is used in the second dimension. The nature of the antigen precipitated was further analysed in immunodiffusion experiments (Figs. 2A and 2B). In Fig. 2A it is shown that the precipitin line formed against a prothrombin-complex concentrate (a) fuses with those formed against a purified factor IX fraction (b),

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Fig. 1. Crossed immunoelectrophorwis of human plasma. Second dimension: 4% (v/v) immunoglobulin fraction of absorbed anti human factor IX serum. 16 nl normal plasma was applied in a 4-mm hole.

an Al(OH)3 eluate of normal plasma (c) and the 1 M NaCl eluate of heparinSepharose previously mixed with the plasma of a patient shown to be of the type BM (Thrombotest@ : 88 set) (d). No precipitation occurs against Al(OH)3 adsorbed plasma (f) and purified factor IX fraction treated with Al(OH)3 (e). Fig. 2B further shows that fusing precipitin lines are formed against prothromA

B

Fig. 2. Precipitating characterlstlcs of absorbed anti human factor IX in immunodiffusion. A. Protbrombincomplex concentrate (a), purified human factor IX fraction (b). 0.25 M Wum phosphate AhOH) eluate of normal plasma (c), 1 M NaCl eluate of heparln-Sepharose treated with the Plasma of a hemoPhiha B patient classified as a BM (Thrombotest @: 88 set) (d). Al(OH)3 adsorbed normal plasma (f) and b after treatment with Al(OH)3 (e). B. Prothrombln-complex concentrate (a, c, e), 0.25 M kallum phosphate AHOH) eluate of hemophilia B patients classified as B+ (b, d) or B (f) in an inhibitor-neutralisation assay: the central holes contained the absorbed anti human factor IX (immunoglobulin fraction).

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bin-complex concentrate (a, c, e) and the Al(OH)3 eluate of the plasma of IXdeficient patients, previously shown to be B’ in the inhibitor-neutralisation assay (b, d). No precipitin line is visible against the eluate of the plasma of a patient shown to be B- (f). To exclude the eventual presence of precipitating material in the patients’ plasma, not adsorbable to Al(OH)3, plasmas were tested directly in an electro-immunoassay. The results in Fig. 3 confirm and extend those of Figs. 2A and 2B in arguing that the precipitin pattern of Fig. 1 is directed against antigens on the factor IX molecule. Fluid phase inhibitor-neutralisation assay of factor IX antigen The principle of an inhibitor-neutralisation assay is that during a first incu-

bation, antigen is allowed to react with an inhibitor (e.g. antibodies), after which unreacted inhibitor is neutralized during a second incubation with a known excess of antigen. Finally the residual free antigen can be measured by its associated activity. Plotting of the antigen concentration of a series of normal plasma dilutions against the corresponding residual activity (both on

Fig. 3. Precipitating characteristics of absorbed anti human factor IX in electio-immune assay. Eleetroimmuno assay wes carried out as described under methods. Normal plasma dilutions: 1 U/ml (g), 0.75 U/ml (f). 0.5 U/ml (e). 0.26 U/ml (d); plssma of hemophilia B patients: B+ (b). T (c. h), BM (j). Al(OH)3 adsorbed plasma (i); (a) contains the Plasma of a carrier from a B* family.

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a logarithmic scale) then provides us with a calibration curve from which the antigen content of a test sample can be obtained by interpolation (see Fig. 4). It should be realized, however, that the result is only independent of the activity/antigen ratio (ranging from 0.26 unit/ml. So, on the basis of the experiment in Table I, the calibration curve of Fig. 4 may be used for the quantitative estimation of factor IX antigen, independent of its inherent factor IX activity. When the residual factor IX activity is measured in triplicate, the error in the mean value is 17%, when <0.35 unit/ml, and 12% when >0.35 unit/ml. The intra-assay coefficient of variation (S/x) amounts 5.6% (n = 6), while the interassay coefficient was calculated to be 18% (n = 6-13). The lower detection limit of the method depends on the difference in residual factor IX activity measured in the case of 0 (
I

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Fig. 4. CaHbration curve for the inbibitor~eutmlisati~~

no factor

IXantigen

1

281 TABLE I EFFECT OF FACTOR COMPLEX

IX ANTIGEN

ON THE DISSOCIATION

OF THE FACTOR

IX-ANTIBODY

Residual factor IX activity (U/ml)

Addition

Al(OH13-addsorbed plasma Hemophilia B+ plasma

Rabbit anti-IX

Human anti-IX

0.010 0.012

0.018 0.012

The experiment was carried out exactly as an inhibitor-neutralisation assay except that the addition of 0.1 ml normal plasma as a start of the second incubation was replaced by the respective additions mentioned in the first column. The human anti-factor IX plasma was a gift of Dr. H.R. Roberts, Chapel Hill, N.C.. USA to Dr. J.J. Veltkamp.

Electra-immuno assay of factor-IX antigen Fig. 5 shows a calibration curve for the electro-immuno assay of factor IX antigen, which was obtained by plotting the height of the precipitation cone (mm) against the antigen concentration in the test sample (units/ml) in a double logarithmic plot. The nature of this relationship over a larger interval of antigen levels was studied by testing several dilutions of a prothrombincomplex concentrate (factor IX antigen t30 units/ml). shows that linearity is obtained at The second curve in Fig. 5 (X -X) least between antigen levels of 0.15 units/ml till 2.0 units/ml, which is satisfactory for practical purposes. The accuracy of the technique is mainly determined by sample application and endpoint determination (see e.g. Fig. 6). This will result in a potential error of 12% in the lower antigen regions (-CO.35 ,

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I

.2

1

.;

Factor IX antigen

,

1

1

;

( u /ml )

Fig. 5. Calibration curve for the electro-lmmuno sssay of factor IX antigen. Normal plasma (O-) and prothrombin-complex concentrate (X -X) were electrophoresed on the same plate. The latter preparation was assumed to contain 30 U/ml factor IX antigen (the factor IX activity was assayed to be 30 U/ml). All dilutions were made in Al(OH)s-adsorbed plasma.

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Fig. 6. Electra-immune assay of factor IX. Positions l-3 and F-10 contain 12-M plasma samples of 6 normal individuals. Positions 4-6 contain normal plasma dilutions (1 in 1.3 in 4. and 1 in 2). The top of each “rocket” is marked by a black dot.

units/ml) and of 6% at the higher antigen levels (>0.35 units/ml). The lower detection limit of the assay is fixed by the diameter of the sample application hole, which generally would correspond with 0.01-6.02 units antigen/ml. The intra-assay coefficient of variation is 3.2% (n = 3) while the inter-assay variation amounts to 10% (n = 3). Comparison of factor IX antigen levels measured by inhibitor-neutralisation (INA) and electro-immuno assay (EIA) Table II shows the mean levels and distribution of factor IX activity and antigen as determined in a group of 33 healthy volunteers. The same batch of normal plasma was used throughout the experiment. Fig. 7 compares the factor IX antigen levels measured by both methods in a large group of hemophilia B patients, carriers and suspected carriers. In general there is a good correlation between both assays, certainly at the lower antigen concentrations (
283

Factor

IX Antigen (

ELectrolmmum

Assay

) (u/ml)

Fig. 7. Comparison of inhibitor-neutralisation assay and electro-immuno assay of factor IX antigen. (*) Hemophilia B patients and carriers (both obligatory and possible): (0) Hemophilia B+ patients and carriers. It should be noted that the black dot in the origin represents 13 different patients. Regression line, calculated for 64 experimental points (0 excluded): y = 0.917x + 0.046. r = 0.945 (- - - - - -).

terized by occurrence of hemophilia B’ (open squares in Fig. 7). Neglecting the squares, regression analysis results in the straight line, dotted in Fig. 7 (slope 0.92; r = 0.945; n = 64). All patients observed to be B- (or B’) by one of the factor IX antigen assays gave the same result in the other assay. From the 21 hemophilia B patients studied 13 showed to be B- and 8 B’ (antigen ranging from 0.45 to 1.60 units/ml). The latter result is included in Fig. 8 that correlates factor IX activity with factor IX antigen (assayed by EIA) for about the same group of individuals used in Fig. 7. The triangles represent patients apparently of the B’ type, while the open squares stand for obligate B’ carriers. When the individuals belonging to B’ families are excluded a linear relationship TABLE II PLASMA (n = 33)

FACTOR

IX ACTIVITY

IX activity (U/ml)

Mean + S.D. Range

0.98 + 0.15 0.72-1.30

AND ANTIGEN

IN A GROUP OF HEALTtiY

IX antigen (U/ml) EIA

INA

0.95 f 0.12 0.75-1.20

0.98 f 0.16 0.74 - 1.30

VOLUNTEERS

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0.8

0.40

0.60

h

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0.00

Factor IX Antigen

**

13

1.m

(

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a0

I.*

EbctmImmupK) Assay

1.60

1

1.m

2.m

2.20

2.‘0

lulti)

Fig. 8, Comxw!ieon of factor IX activity and factor IX antigen (as measured by HA). About the same population of IndIvidueIs was used as in Fig. 7. The proven B+ families have been divided inti carriers (0) and patients (A). Regression line caIcuI&d for the black dots (66): Y = l.OQx + 0.29. r= 0.958 ( ___.__ ).

between factor IX activity and antigen exists, defined by a slope 1.00 (r = 0.956, n = 56). Discussion As emphasized in the introduction, the need for an assay of factor IX antigen arose from an interest in the frequency of occurrence of variants of hemophilia B, possessing functionally inactive factor IX molecules. For the detection of these inactive molecules an anti-factor IX is preferred composed of antibodies directed against many different antigenic loci on the factor IX molecule. In general, heterologous antisera will respond better to this condition than homologous, although the latter certainly are important for further classification studies. Our anti-factor IX serum, raised against a factor IX fraction showing 3 bands in polyacrylamide gel electrophoresis, is free of antibodies against factors II, V, VIII, X, XI and XII. Because of these observations it was thought permissible to use it in an inhibitor-neutralisation assay of factor IX antigen. A crude immunoglobulin fraction of the antiserum was used to avoid con-specific effects. The experiment summarized in Table I justifies the use of calibration curves as shown in

285

Fig. 4 for the determination of factor IX antigen levels independent of their inherent biological activity. The lack of data on this matter in the available literature certainly hampers a proper comparison of results. The use of “precipitating” properties of anti human factor IX serum has been reported earlier by Denson et al. (immunodiffusion) [20], Nat&on and Coltman, Jr. (radial immunodiffusion) [21], and orstavik et al. (electro-immuno assay) [ll]. Denson et al. used an absorbed anti bovine factor IX [ 31. Natelson and Coltman, Jr. [21] and Orstavik et al. used anti purified human factor IX [ 111. The antiserum of the last group is the best characterized one, although it remains unclear whether the antiserum used in the electro-immuno assay of factor IX precipitates purified factor IX (cf. refs. 11, 12 and 23). It should be noted that the appearance of a precipitin-line against both normal plasma and its Al(OH)3 eluate is in itself insufficient evidence for the identification of factor IX as the precipitated antigen. With our antiserum, for instance, the absorption with hemophilia B- plasma additional to Al( OH), adsorbed plasma showed necessary to remove a second precipitin line present in plasma and Al(OH)3 eluates but not in purified factor IX fractions. The experiments shown in Figs. 1-3 strongly indicate that the material precipitated by our absorbed antiserum is factor IX; it is always present in normal plasma and relatively purified factor IX fractions; it is removed in both cases by Al(OH)J, and can be eluted from that by 0.25 M kahum phosphate (while it is absent in the washes). Finally it is always absent in plasma and Al(OH)3 eluate of hemophilia B- patients (characterized as such by INA), while it is always present in the corresponding fractions of B’ patients. Aforementioned observations make it highly unlikely that we fail to measure factor IX antigen in the electro-immuno assay. Direct comparison of the results obtained with both inhibitor-neutralisation and electro-immuno assay leads us to the following conclusions: (1) There is in general a good correlation between the data obtained in both assays (see Fig. 7), (2) the electro-immuno assay is significantly more accurate and shows lower inter- and intra-assay variation, and (3) the standard deviation in the factor IX level in a population of normal individuals is lowest for the electro-immuno assay (cf. Table II) and can be further decreased by assaying samples in duplicate (which already is done in activity assay and INA). This may be of interest when the use of the electroimmuno assay in carrier detection is considered. Of the 21 severe hemophilia B patients, 8 were observed to have crossreacting material (0.45-1.6 unit/ml) both in INA and EIA. The correlation between the actual values determined by both assays was excellent in 6 of the cases, while 2 (related) patients showed 1.5 and 2.0 times higher antigen levels when measured with the EIA. The latter observation is related to the remarkable phenomenon shown in Fig. 7 that 10 of the 19 hemophilia B’ patients and carriers (open squares in Fig. 7) have significant higher antigen values when measured in the EIA than in the INA. These 10 individuals belong to 3 different families where the incidence of this phenomenon is rather high: 3(4); 4(4) and 3(4). At this moment the reason for this discrepancy is not known; practically, it faciliates appreciably the carrier detection in those families.

286

References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

Hougie, C. and Twomey. J.J. (1967) Lancet i. 698-700 Roberts, HR., Grizzle. J.E., McLester. W.D. and Penick. G.D. (1968) J. Clin. Invest. 47, 360-365 Denson. K.W.E.. Biggs. R. and Mannucci. P.M. (1968) J. CIIn. Pathol. 21,160-165 Brown, P.E., Hougle, C. and Roberta, H.R. (1970) New EngI. J. Med. 283.61-64 Meyer. D. and LarrIeu, M.J. (1971) Eur. J. ClIn. Invest. 1.425432 Meyer, D.. BidweII. E. and Larrieu, M.J. (1972) J. Clin. Pathol. 25.433436 EIiidi. S. and Pusk&. E. (1972) Thromb. Diath. Haemorrh. 28.489495 RI&ii, S. (1976) Min. Med. 66.2037-2043 PfueIIer. S.. Somer. J.B. and Cast&Ii, P.A. (1969) Coagulation 2. 213-219 Neal, W.R.. Tayloe, Jr., D.T.. Cederbaum. AI. and Roberts, H.R. (1973) Br. J. Haematol. 25.6348 f$rstavik. K.H.. @sterud. B.. Prydr, H. and Berg, K. (1975) ‘Ihromb. Res. 7.373-382 Hemker. H.C.. Swart. A.C.W. and AIink. A.J.M. (1972) Thromb. Diath. Haemorrh. 27, 206212 KoIIer. F.. Loeiiger, E.A. and Duckert, F. (1951) Acta Haematol. 6.1-18 Veltkamp. J.J., Drion. E.F. and LoeIiger. E.A. (1968) Thromb. Diath. Haemorrh. 19, 279-303 Andersson, L.-O., Borg, H. and Miller-Andersson. M. (1975) Thromb. Res. 7.451459 BrunIng. P.F. and LoeIIger. E.A. (1971) Br. J. Haematol. 21.377-398 B&t. E., LoeIiger. E.A., van TiIburg. N.H. and Veltkamp. J.J. (1976) Thrombos. Haemostas. 35. 289-294 18 Guchterloney. 0. (1962) Progr. AIIergy 6. 30-154 19 Laurell, C.-B. (1972) Scand. J. Clin. Lab. Invest. 29. SUPP~. 124. 21-37 20 Denson, K.W.E. (1967) The use of antibodies in the study of blood coagulation. Blackwell Scientific Publications, Oxford and Edinburgh 21 Nat&on. E.A. and Coltman. Jr., C.A. (1972) CIin. Res. 20. 495 22 (bsterud, B., Laake. K. and Prydz, H. (1976) Thromb. Diath. Haemorrh. 33.553-563 23 @sterud. B. and FIengsrud. R. (1976) Biochem. J. 145.469474