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Immunological characterization of factor VIII inhibitors by a sensitive micro-ELISA method
THROMBOSIS RESEARCH 57; 897-908,199O 0049-3848/90 $3.00 + .OO Printed in the USA. Copyright (c) 1990 Pergamon Press pk. All rights reserved.
IMMUNOLOGICAL INHIBITORS
BY
Sanchez-Cuenca, Jesiis Villanueva
J.M. Maria
t
CHARACTERIZATION A SENSITIVE
Research Congenital
OF FACTOR VIII MICRO-ELISA METHOD
Elena and
Carmona, J.A. Aznar”
Center. La Fe Hospital. Coagulopathies Unit. Valencla. Spaln
(Received 16.8.1989, accepted in revised form 12.11 .1989 by Editor S. Coccheri) (Received
by Executive
Editorial Office 18.1.1990)
ABSTRACT
A micro-ELISA method for the immunological characterizaf Ion of factor VIII (F.VIll) Inhibitors is described. Microplates sensitized with purified monospecific anti-von Willebrand factor (vWF) IgG were firstly incubated with F.VIII concentrate a commercial and then with the samples containtng the F.VIII inhibitors to be character12 ed. Peroxidase conjugated monospecific antisera were used to determine the lg class and the light chain type. AlKal ine phosphatase conjugated monoclonal antibodies were employed to investigate the IgG subclasses. The F.VIII inhibitors from 8 hemophiliacs and 1 patient with systemi c I upus erythematosus (SLE) were characterized. All of them belonged to the IgG class but one case simultaneously exhibited another inhibitor of the IgA cl ass. Eigth inhibitors analyzed for light chains resul_t ed to be polyclonal. In 7 cases the inhibitors belonged solely to the IgG4 subclass while in the other 2 cases contained all the 4 IgG subclasses. The method appears to be simple, accurate and highly sensitive (detecting as low as 0.156 Bethesda Units/ml) for the immunological characterization of F.VIII inhibitors.
INTRODUCTION
The development of factor the most severe compllcatkon It is well Known that these against the ected either
KEY
WORDS:
F.VIII
Inhibitors,
VIII
(F.VIll) that can inhlbltors functional
F.VIII,
897
affect are part
inhibftors constitutes hemophil~c patients. really antibodies dirof the F.VIII or other
Hemophilia,
Micro-ELISA
898
FVlll INHIBITORS IMMUNO-TYPING
Vol. 57, No. 6
to this active part (1). However, most placed very close epitope do not share those properties which char the F.VIII inhibitors Of precipitate nor antibodies: they do not conventional acterize combining with their antigen (2). Because of when complement fix characteristics of these anthe immunological the study of this, increasing attention during the last two drj received tibodies has several biological For this purpose, methods have cades (314). developed (5). These entail the incubation of the patient’s been antisera specific for Ig class, type or sub different plasma with the inhibitor. This neutralization, neutralize class in order to its residual anti-F.VIII activity, will characterize measured by immunological point of view. By means of inhibitor from the the of F.VIII inhibitors have been cha methods a reduced number these racterized (5). Although there is no definite information with re of the F.VIII inhibigard to this, it has been claimed that most hemophiliacs display a reduced heterogeneicharacterized in tors subclass (reviewed in 5). The predominantly’ of the lgG4 being ty, not Known, but at least reduced heterogeneity is reason for this antibodies, functional features of these since for the account fixing complement. Furthermore, it for is devoid of capacity lgG4 (6) and van der Zee et been reported by Aalberse et al. has subclass are functionally antibodies of the al. (7) that 1gG4 precipitating immuneunable to form and therefore univalent explain hemophiliacs who Both reasons could complexes. these why suffer from complement-mediated inhibitors do not have developed diseases. such as Kidney or vascular immunological damage characterization of in cases the immunological However, many carried out by biological inhibitors cannot be directly F.VIII This to the following: a) only tests. can be due relatively high titre inhibitors can be directly tested by these methods, since low titre ones require a previous isolation of the inhibitor-containing fraction of plasma; and b) in some plasmas tlie total level of subclass or the Ig class, type to which the inhibitor becan be so high that the neutralization of the F.VIII inhik longs, itory activity cannot be achieved, maKing impossible the immunological characterization of the inhibitor. In order to avoid both these disadvantages, other non biological methods, such as immung blotting, have been applied Nonetheless, these techniques, (6). although more sensitive, are sophisticated and time-consuming pro cedures requiring highly qualified personnel. Consequently, they are only available for very specialized laboratories. In an attempt to approach the immunological characterization of these inhibitors from a practical point of view, a simple, sensitive and accurate method, based upon the micro-ELISA technology, has been developed in our laboratory. The aim of this paper is to describe this new method as well as to report the results obtained in it to the immunological characterization of a seapplying ries of plasmas from patients who have developed F.VIII inhibitors. HATERIAL Materials F.VIII concentrates
(HAEMATE,
AND
METHODS
Behring,
West
Germany);
Ortho-Phe-
Vol. 57, No. 6
FVlll INHIBITORS IMMUNO-TYPING
899
Para-Nitrophenyl Phosphate Sigma, USA) ; nylene-Diamine (CPD, H2SO4 (MercK, West Spain); H202 (Foret, USA) ; 30% (PNP, Sigma, Plasma containing West Germany); Standard Germany) ; NaOH (MercK, inhibitor of 50 Bethesda Units (B.U) per ml class F.VIII an IgG 9.8 (Sigma, USA). pH Austria); 10 mM Diethanol-amine, (lmmuno, (Pharmacia, Sweden). Freund’s Sephacryl-1000 and Sepharose 48 of OPD (IO complete adjuvants (Difco, USA). OPD solution: 1.5 ml mixed with 0.15 ml of H202 (l/l0 of the 30% H20) were mg/ml in solution) and 150 ml of 0.05 M citric acid-NaOH pH 5.1. PNP solu_t 10 mM Diethanolamine, pH 9.8. Rabbit antiion: PNP (lmg/ml) in Kappa and lambda monospecific anti human IgG, IgA, IgM, IgD, IgE, Monoclonal (mouse) anti-h9 sera were purchased from Melloy (USA), IgG3 and lgG4) monospecific antiman IgG subclasses (IgGl, IgG2, (calibrated against the reference WHO standard serum bodies 67/97) were supplied by Bio-Yeda (Israel). A polyclonal rabbit anti-mouse IgG antiserum was purchased from Serotec (United Kingdom) . Peroxidase conjugated anti-human IgG, IgA, IgM, IgD, kappa were suppl ied by and lambda monospecific antisera Dako, (Denmark) . Alkal ine phosphatase conjugated monoclonai (mouse) antihuman IgGl, IgG2, lgG3 and lgG4 monospecific antibodies (calibrat ed against the reference WHO standard serum 67/97) were purchase? from Bio-Yeda (Israel). Subjects The 8 hemophiliacs and 1 study included: patient suffering from systemic 1 upus erythematosus (SLE) , all of whom had developed F.VIIl inhibitors; 6 hemophiliacs without F.VIII inhibitors; 6 h_e mophiliacs without F.VllI inhibitors but with high levels of circulating immunecomplexes; 6 non-hemophilic patients exhibiting high levels of circulating immunecomplexes; 6 non-hemophilic patients showing high levels of rheumatoid factor and 12 normal human subjects. Blood samples Blood samples were collected by venipuncture into 0.38% trisodium citrate. Within 30 min after collection, plasmas were separated by centrifugation at 1,600 x g for 20 min, and stored at -70° C, F.VIII inhibitors were evaluated by the agarose gel method descr_i bed by Jorquera et al . (9) . The 9 positive cases ShOWed titres corresponding to 5 to 108 B.U./ml. Rheumatoid factor was detected by the RA-latex test (Hyland, California, USA) and immunecompl ex es were demonstrated by the SIA-Immune Complexes Test (Sigma Diag nostics, St. Louis, USA) Purification of rabbit anti-vWF monospecific IgG Purified von Willebrand factor (vWF) was obtained according to the method described by Zimmerman et al. (IO). Several 0.5 ml al i quots (100 ug of protein) of purified vWF were then emulsified (1:3) with Freund’s complete adjuvants and injected into two male rabbits which were bled one month after the first injection. The serum obtained was absorbed with normal human plasma and then an_a lyzed by double gel diffusion (DGD) (11) and immunoelectrophoresis (IEP) (12). The resultant antiserum showed a potent anti-human vWF activity and was completely devoid of contaminants, The specific anti-vWF IgG fraction was then isolated by means of an immunoadsorbent made of Sephacryl-1000 to which a commercial
FVlll INHIBITORS IMMUNO-TYPING
900
Vol. 57, No. 6
F.VIII concentrate had been coupled according to the method deselUate obtained in this Step was decribed by Hornsey (13). The monstrated to be (DGD, IEP and ELISA, 14) a pure rabbit IgG monoantiserum yielded 0.5 mg of specj specific for human vWF (1 ml of fit IgG). test (ICMET) micro-ELISA characterization The inhibitor 4 layers (Fig. 1): the first layer consisted The ICMET included in 100 ul of anti-vWF monospecif ic of purified lgG lgG (1 ug of well) coupled to bicarbonate pH 9.6 carbonate-sodium 0.1 M per microplates (Nunc, Denmark) which were filled with 100 ul/well of incubated at and F.VIII concentrate (second commercial layer), the incubation, plates were washed 5 tiz7o C for 60 min. After a 0.15 M phosphate-NaCl buffer containing 0.05% Tween 20 mes with then filled with 100 ul/well of the (TPB) and drained. They were analyzed (third layer), and fursamples to be different plasma Plates were then washed, for 1 hour at 37O C. ther incubated ul/well of either peroxidase with 100 duplicate in filled again Kappa or lambda rabbit anti-human conjugated lgA, lgD, lgG, lgM, incubated for and further (fourth antisera monospecific layer), and drained, plates washed after being Finally, 60 min at 37O C. prepared OPD soluwere filled with 100 ul per well of a freshly tion and left at room temperature for 20-30 min. The reaction was and the plates were recordstopped with 100 ul/well of 4 N H2SO4
NIti-L ati-H
Fig.
1.
chain + PO
chain + PO 1 mti-196
iub-clars
+ AP
Schematic the representation of test principle. Plates coated with anti-vWF specific (first layer) were lgG treated as follows: a second layer consisting of the addition of a commercial F.VIII concentrate; a third lainvolving the application of the sample plasma conyer taining the inhibitor; a fourth layer which included the incubation with peroxidase (PO) conjugated anti-heavy (H) or light (L) chains antisera or, alternatively, with alK_a line phosphatase (AP) conjugated anti-IgG subclasses mono clonal antibodies.
Vol. 57, No. 6
FVlll INHIBITORS IMMUNO-TYPING
901
Finland) at (Cul tek, ed in an multi-channel micro-ELISA recorder Controls were carried out as follows: a) a plate control 492 nm. sample control (blank) which included several empty Wells; b) a yl/well of TPB instead of the F.VIII concentrate; containing 100 c) a F.VIII concentrate control which consisted of 100 ul/well of and d) peroxidase samples: different plasma of the TPB instead each antiserum used) conjugated antisera controls (two wells per ul/well of TPB instead of both F.VIII concentrate containing 200 and plasma samples. In those cases in which the F.VIII inhibitor was found to belong alkaline phosphatase conjugated monoclonal anti to the IgG class, (monospecific for bodies anti-human IgG subclasses IgGt, IgGg, employed in the fourth layer of the test, A lgG4) were IgG3 or the reaction stopPNP solution was used as enzyme substrate and the plates being recorded at 405 nm in the muJ ped with 3 N NaOH, controls to the above ti-channel micro-ELISA recorder. Similar described were always employed. RESULTS
Standardization of the ICMET In order to standardize the test, a standard plasma containing an IgG class F.VIII inhibitor (50 B.U./ml) was used. The ICMET was carried out but 3 different concentrations (12.5, as described, 6.7 and 3.3 I.U./ml of PBS) of a commercial F.VIII concentrate were applied in duplicate in the second layer of the test. No sig nificant variations in the standard plasma titres were detected. a F.VIII concentrate dilution containing 3.3 I.U. of F.VIII Thus, per ml of PBS was selected as the routine reagent to be used in the second layer of the subsequent experiments. Sensitivity of the method Different dilutions (ranging from l/l0 to l/640) in PBS of the standard plasma containing an IgG F.VI I I inhibitor (50 B.U./ml) were assayed in triplicate by the ICMET and the highest dilution showing a clear positive reading was determined, which corresponded to the l/320 dilution. Since, in general terms, a direct correlation between the F.VIII inhibitory activity of the standard the amount pl asma and of its specific anti-F.VIII IgG could be presumed, as low as 0.156 B.U./ml of F.VIII inhibitory activity would be clearly detected by means of this test. However, it could be observed that 2 plasma samples containing a low F.VIII inhibitory activity showed, when analyzed by the ICMET, higher levels of specific anti-F.VIII antibodies than those expected (data not shown). Reproducibility The standard plasma was reassayed 20 times by applying it in double serial dilutions, ranging from i/20 to l/640, in the third the ICMET. The mean of the titres obtained was l/240. layer of Additionally, a regression analysis (fig. 2) was carried out by dilution (expressed as its log2) against the mean plotting every of its optical density at 492 nm. A value of r = - 0.922 was obtained (p < 0.0001).
902
FVlll INHIBITORS IMMUNO-TYPING
0-D.
<4?2
Vol. 57, No. 6
nm>
600.00
550.00
Fig.
Regression 1 ine obtained by plotting (ordinate) s_e rial dilutions (expressed as their IogB) of a stand ard plasma containing an IgG class F.VIII inhibitor (50 B.U. per ml) against (abscissa) their optical densities at 492 nm (mean t/-one standard deviation of 20 different tests)
500.00
450.00
400.00
350.00
2.
-
r
=
-0.922
n
-
120
p < 0.0001
I
300-B@ 1.00
2.10 1/<10
3.20 x
4.30 log,
5.40
6.30
dilution>
Specificity to evaluate the specificity of the test, the following In order experiments were carried out: Half a part of five microplates IgG was incubated with 100 ul sensitized with anti-vWF specific per well of a F.VIII concentrate dilution containing 3.3 I.U. of F.VI I I per PBS and the other half with TPB instead of the ml of F.VIII concentrate. The following undiluted reagents were then applied to both parts of each plate in the third layer of the a) 12 normal human plasmas negative for test: rheumatoid factor; b) 6 plasmas from hemophiliacs without F,VIII inhibitors; c) 6 plasmas from hemophiliacs, without F.VIII inhibitors, Known to contain high levels of circulating immunecomplexes (CIC); d) 6 human non-hemophilic plasmas Known to contain high levels of CIC and e) 6 human non-hemophilic plasmas Known to contain high levels of rheumatoid factor. The a), c) and d) controls mentioned (see Material and Methods) were carried plates. Additionally, 3 serial dilutions the standard plasma containing an IgG per ml) were also included.
in the ICMET description out in each half of the 5 (l/5, l/10 and i/25) of F.VIII inhibitor (50 B.U.
FVlll INHIBITORS IMMUNO-TYPING
Vol. 57, No. 6
903
plasmas from hemoNone of the 12 normal human plasmas nor the 6 F.VIII inhibitors resulted positive when analyphiliacs without or in the absence of F.VIII conce_n zed either in the presence of, non-hemophilic plasmas containing CIC resulted the 6 trate. Al I hemophiliacs containing plasmas from Two out of the 6 negative. CIC exhibited a clear positive reaction to the anti-IgG antiserum which was exclusively localized in the part of the plate in which All the 6 plasmas contai_n the F.VIII concentrate was not applied. the polycloing rheumatoid factor showed a positive reaction of and anti-lambda antisera), in (to both the anti -kappa nal type both the F.VIII concentrate treated and the TPB treated halves of with both the anti-IgG (2 cases) or anti-IgM (4 cases) the plate, peroxidase conjugated antisera. Immunological characterization of F.VIII inhibitors by the ICMET 9) were characAll the inhibitors completely analyzed (6 out of A detailed description subcl ass and type. terized for Ig class, of all of them is shown in table I.
TABLE lmnunological
I
characterization
of
9 F.VIII
inhibitors
Patient
Inh.
P.
R.
38
B.U.
A,
M.
23
B.U.
1SIG4
K+L
V.
J.
27
B.U.
1gG4
K+L
J,
F.
108
B.U.
1gG4
K+L
J.
V.
40
B.U.
1gGq
K+L
G,
c.
5 B.U.
1gG4
K+L
I.
F.
15
B.U.
1gG4
K+L
J.
D. (SLE)
14
B.U.
IgGl,
s.
I.
50
B.U.
1gGq
n.
d.
= not
H chain
Tit.
IN
+ IgG1,
L chain
IN+,
lgG2,
lgG3 8, I@4
lgG3
& IgGq
K+L
K+L n.
d.
done
As can be observed in this table, all the inhibitors fully charas terized were found to be polyclonal (containing both kappa and lambda light chains) and belonged to the IgG class. It is notewor one plasma from a hemophiliac simultaneously showed anthy that other anti-F.VIII antibody of the IgA class. Furthermore, the IgG class inhibitor of this pl asma was demonstrated to contain all
904
FVlll INHIBITORS IMMUNO-TYPING
the 4 known IgG subclasses. case (15) will be the subject
The of
details concerning a separate publication
Vol. 57, No. 6
this
unusual
.
inhibitors from hemophiliacs were found other 6 Conversely, the subclass, while that correspto exclusively belong to the lgG4 onding to the SLE patient was demonstrated to contain IgGl, lgG2, determinants when analyzed chain antigenic lgG5 and IgG4 heavy for IgG subclasses. DISCUSSION
for the immunological characterization of A micro-ELISA method The test has proven to be simple, F.VIII inhibitors is described. accurate and highly specific for the study of this type of antib_o no non-specific reactions were observed when nordies. In fact, plasmas from hemophiliacs without F.VIII inhi mal human plasmas, non-hemophilic plasmas containing CIC were assayed by bitors or 2 out of the 6 plasmas from hemophiliacs However, this test. high levels of CIC showed a false positive reacwhich contained tion when assayed in absence of F.VIII concentrate. Nevertheless, it did not occur in those wells in which a F.VIII COnCet'Itrate had been previously applied in the second layer of the test. Thus, a containing TPB instead of the F.VIII concentrate simple control certainly detect the appearance of false positive results would CIC. In any case, this fact would hemophiliacs containing in suggest the possibility of detecting CIC containing vWF/F.VIII this method. and anti-F.VIII anti bodies by In order to assess further experiments are this attractive hypothesis, currently in our I aboratory . in All the plasma samples containing progress of rheumatoid factor demonstrated a false positive high titres both the previously F.VIII concentrate incubated and reaction in TPB incubated wells. Nevertheless, this false positive reaction can be certainly expected as a background phenomenon occurring in all those ELISA methods which employ coupled IgG, from either human or animal origin, in the first layer of the test. Indeed, the appearance of such a positive reaction will strongly suggest the rheumatoid factor presence of in the analyzed sample and will therefore make impossible its immunological analysis by means of the ICMET. On the other hand, the ICMET could be easily applied for the simultaneous characterization, in a single assay, of both the heavy (class and subc I ass) and light (kappa or lambda type) chains of all the F.VIII inhibitors tested. However, since it is not a funs tional test, it cannot substitute the biological assays for the evaluation of F.VIII inhibitors. In fact, the demonstration that in 2 plasmas exhibiting low titres of F.VIII inhibitory activithe ICMET was able to reveal the presence of quantities of ty, anti-F.VIII specific antibodies notably higher than those expectstrongly suggested that these plasmas might contain antied, F.VIII antibodies other than those exhibiting F.VIII inhibitory activity. These non-inhibitor antibodies are thought to be proba bly directed against some epitomes distanced from the functional part of the F.VIII structure (5j and have been demonstrated in some hemophilic patients, as well as in certain auto-immune conditions such as SLE (5).
Vol. 57, No. 6
In a
any case, this sensitive highly
FVlll INHIBITORS IMMUNO-TYPING
would fact for method
indicate
that
the detection the against
905
ICMET
the
of
seems
extremely
to
low
be
am-
independently of F.VIII, directed antibodies of useful very appears to be a Thus, it activity. biological their antiall types of characterization of immunological the tool for anti-F.VIII specificity. In fact, 9 anti-F.VIII showing bodies antibodies (8 from hemophiliacs and 1 from an SLE patient) Of va8 out characterized. In able to be titre were inhibitory riable all the 8 investigated and light chain was the type of of them, found to be polyclonal (namely, containing both kappa were cases with other observations contrast This is in chains). lambda and (5,8,16), which claimed a marked predominance of the kappa light studied. This could be a the inhibitors majority of chain .in the sensitivity of the biological tests which weak consequence of the had been generally used for this purpose (5) or due to a lack of antibodies employed (8). Furthe anti-light chain reliability of cannot be ruled out the existence of some racial or thermore, it constitutional variations.
ounts
inhibitors of the class. However, All the 9 plasmas exhibited lgG them contained a double anti-F.VIII is noteworthy that one of it activity: one of the IgG class and the other of the IgA one. This could not be detected when unusual antibody the plasma was IgA conventional biological but it analyzed by a method, was unexpect This fact was probably due to the follow_ edly found by the ICMET. circumstances: firstly, because of the existence of a ing marked the low inhibitory activity of the discordance between class IgA anti-F.VIII antibody and the large amount of specific (15) lgA was which could be demonstrated when this plasma analyzed by the because the inhibitor was with an ICMET; secondly, associated IgA which other potent inhibitor of the class, probably masked IgG IgA-carried inhibitory activity when biologically the assayed. Both circumstances emphasize by themselves some of the advantages ICMET over the conventional biological of the tests. An additionadvantage al of this method would be its application to the eventual detection of anti-F.VIII non-inhibitor antibodies that other completely wise would have remained unobserved in the biological assays. the Two out of 9 cases studied (the hemophiliac containing the inhibitor and the SLE patient) exhibited a F.VIII inhibitor lgA which was composed of the IgG1, lgG2, lgG5 and lgG4 subclasses of the human IgG. Some descriptions of F .VIII inhibitors containing IgGg have been reported (5, 8) but, to our knowledge, these are the 2 first in which cases anti-F.VIII has been found. The lgG2 hemophilic patient (w) showing such an heterogeneous anti-F.VIII response (15) will be the subjet of a separate publication.
(*I
FOOTNOTE: When the plasma samples from this patient were taken, he was undergoing a massive administration of human F.VIII concentra tes because of a severe hemorrhage which finally his led to death.
FVIII INHIBITORS IMMUNO-TYPING
Vol. 57, No. 6
The other 7 F.VIII inhibitors from hemophilic patients were desolely belong to the IgGq subclass. This lgG4 resmonstrated to triction of the F.VIII inhibitors has been reported in other sim_i lar observations (reviewed in 5). Furthermore, immunological reshave also ponses with a marked lgG4 predominance been described as it is the case of hemophiliacs in other situations in which, acontinuous and prolonged immutreated with F.VIII concentrates, In fact, it has been also reported in bee nization often occurs. suffering from filariasis (17) or schisKeepers (7) 1 in patients as well as in allergic patients undergoing speci tosomiasis (la), this striking lgG4 fit hyposensitization (~9,20). The reason for predominance is not known. However, as suggested by Aalberse et is tempting a.1 . (6) and van der Zee et al. to believe (7), it that an immunological response switched to the production of a v,e ry homogeneous lgG4 subclass antibody could be considered as a d_e fensive attempt by the immunological system to avoid the problems which could certainly arise from a continuous and prolonged antigenie challenge. This would be specially emphasized by the fact of complement fixing activity that lgG4 is devoid and does not form precipitating immunecomplexes (6, 7). Therefore, complementimUrteCOfnpleinduced damage and other consequences of phlogistic obviously be avoided by this type of IgGq-mediated imxes would mune responses. In agreement with these arguments would be our findings of a polyclonal (namely, containing both Kappa and lambda light chains) type of inhibitors, since normal, defensive imunological responses are always of such a type. On the contrary, homogenqous antibody responses of both heavy and light chain restriction are the main characteristic of those immunological situations which usua_l ly lead to the monoclonal gammopathies. And these monoclonal responses are associated, in the vast majority of cases, with unusually abnormal conditions such as some malignant neoproliferative disorders.
ACKNOWLEDGMENTS We gratefully acKnowledge Reganon for their helpful typescript and to Dr. F. Research studies described by a grant (No. 86/1096) of Health”, Spain.
to Dr. A. Gonzalez tlolina and Dr E. discussion and thoughtful review of the Llopis for his excellent suggestions. in this paper were supported in part from “The FIS of the National Institute
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ISKANDER, R., DAS, P.K. and AALBERSE, with schistosomiasis. Egyptian patients Immunol., 66, 200-207, 1981. APP~.
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lgG4 antibodies Arch. Allergy
V. DIEGUES, P.H., KNUL-BRETLOVA, a blocKing van LEEUWEN, J. lgG4 as I, 289-295, 1983.
in
VOORE, P., antibody.
19.
AALBERSE, R.C., AALBERSE, M. and Rev. Allergy, Cl in.
20.
and clinical significance subc I ass nature R. The DJURUP, the IgG antibody response in patients undergoing allergen-specific immunotherapy. Allergy, 40, 469-486, 1985.
of
IMMUNOLOGICAL INHIBITORS
BY
Sanchez-Cuenca, Jesiis Villanueva
J.M. Maria
t
CHARACTERIZATION A SENSITIVE
Research Congenital
OF FACTOR VIII MICRO-ELISA METHOD
Elena and
Carmona, J.A. Aznar”
Center. La Fe Hospital. Coagulopathies Unit. Valencla. Spaln
(Received 16.8.1989, accepted in revised form 12.11 .1989 by Editor S. Coccheri) (Received
by Executive
Editorial Office 18.1.1990)
ABSTRACT
A micro-ELISA method for the immunological characterizaf Ion of factor VIII (F.VIll) Inhibitors is described. Microplates sensitized with purified monospecific anti-von Willebrand factor (vWF) IgG were firstly incubated with F.VIII concentrate a commercial and then with the samples containtng the F.VIII inhibitors to be character12 ed. Peroxidase conjugated monospecific antisera were used to determine the lg class and the light chain type. AlKal ine phosphatase conjugated monoclonal antibodies were employed to investigate the IgG subclasses. The F.VIII inhibitors from 8 hemophiliacs and 1 patient with systemi c I upus erythematosus (SLE) were characterized. All of them belonged to the IgG class but one case simultaneously exhibited another inhibitor of the IgA cl ass. Eigth inhibitors analyzed for light chains resul_t ed to be polyclonal. In 7 cases the inhibitors belonged solely to the IgG4 subclass while in the other 2 cases contained all the 4 IgG subclasses. The method appears to be simple, accurate and highly sensitive (detecting as low as 0.156 Bethesda Units/ml) for the immunological characterization of F.VIII inhibitors.
INTRODUCTION
The development of factor the most severe compllcatkon It is well Known that these against the ected either
KEY
WORDS:
F.VIII
Inhibitors,
VIII
(F.VIll) that can inhlbltors functional
F.VIII,
897
affect are part
inhibftors constitutes hemophil~c patients. really antibodies dirof the F.VIII or other
Hemophilia,
Micro-ELISA
898
FVlll INHIBITORS IMMUNO-TYPING
Vol. 57, No. 6
to this active part (1). However, most placed very close epitope do not share those properties which char the F.VIII inhibitors Of precipitate nor antibodies: they do not conventional acterize combining with their antigen (2). Because of when complement fix characteristics of these anthe immunological the study of this, increasing attention during the last two drj received tibodies has several biological For this purpose, methods have cades (314). developed (5). These entail the incubation of the patient’s been antisera specific for Ig class, type or sub different plasma with the inhibitor. This neutralization, neutralize class in order to its residual anti-F.VIII activity, will characterize measured by immunological point of view. By means of inhibitor from the the of F.VIII inhibitors have been cha methods a reduced number these racterized (5). Although there is no definite information with re of the F.VIII inhibigard to this, it has been claimed that most hemophiliacs display a reduced heterogeneicharacterized in tors subclass (reviewed in 5). The predominantly’ of the lgG4 being ty, not Known, but at least reduced heterogeneity is reason for this antibodies, functional features of these since for the account fixing complement. Furthermore, it for is devoid of capacity lgG4 (6) and van der Zee et been reported by Aalberse et al. has subclass are functionally antibodies of the al. (7) that 1gG4 precipitating immuneunable to form and therefore univalent explain hemophiliacs who Both reasons could complexes. these why suffer from complement-mediated inhibitors do not have developed diseases. such as Kidney or vascular immunological damage characterization of in cases the immunological However, many carried out by biological inhibitors cannot be directly F.VIII This to the following: a) only tests. can be due relatively high titre inhibitors can be directly tested by these methods, since low titre ones require a previous isolation of the inhibitor-containing fraction of plasma; and b) in some plasmas tlie total level of subclass or the Ig class, type to which the inhibitor becan be so high that the neutralization of the F.VIII inhik longs, itory activity cannot be achieved, maKing impossible the immunological characterization of the inhibitor. In order to avoid both these disadvantages, other non biological methods, such as immung blotting, have been applied Nonetheless, these techniques, (6). although more sensitive, are sophisticated and time-consuming pro cedures requiring highly qualified personnel. Consequently, they are only available for very specialized laboratories. In an attempt to approach the immunological characterization of these inhibitors from a practical point of view, a simple, sensitive and accurate method, based upon the micro-ELISA technology, has been developed in our laboratory. The aim of this paper is to describe this new method as well as to report the results obtained in it to the immunological characterization of a seapplying ries of plasmas from patients who have developed F.VIII inhibitors. HATERIAL Materials F.VIII concentrates
(HAEMATE,
AND
METHODS
Behring,
West
Germany);
Ortho-Phe-
Vol. 57, No. 6
FVlll INHIBITORS IMMUNO-TYPING
899
Para-Nitrophenyl Phosphate Sigma, USA) ; nylene-Diamine (CPD, H2SO4 (MercK, West Spain); H202 (Foret, USA) ; 30% (PNP, Sigma, Plasma containing West Germany); Standard Germany) ; NaOH (MercK, inhibitor of 50 Bethesda Units (B.U) per ml class F.VIII an IgG 9.8 (Sigma, USA). pH Austria); 10 mM Diethanol-amine, (lmmuno, (Pharmacia, Sweden). Freund’s Sephacryl-1000 and Sepharose 48 of OPD (IO complete adjuvants (Difco, USA). OPD solution: 1.5 ml mixed with 0.15 ml of H202 (l/l0 of the 30% H20) were mg/ml in solution) and 150 ml of 0.05 M citric acid-NaOH pH 5.1. PNP solu_t 10 mM Diethanolamine, pH 9.8. Rabbit antiion: PNP (lmg/ml) in Kappa and lambda monospecific anti human IgG, IgA, IgM, IgD, IgE, Monoclonal (mouse) anti-h9 sera were purchased from Melloy (USA), IgG3 and lgG4) monospecific antiman IgG subclasses (IgGl, IgG2, (calibrated against the reference WHO standard serum bodies 67/97) were supplied by Bio-Yeda (Israel). A polyclonal rabbit anti-mouse IgG antiserum was purchased from Serotec (United Kingdom) . Peroxidase conjugated anti-human IgG, IgA, IgM, IgD, kappa were suppl ied by and lambda monospecific antisera Dako, (Denmark) . Alkal ine phosphatase conjugated monoclonai (mouse) antihuman IgGl, IgG2, lgG3 and lgG4 monospecific antibodies (calibrat ed against the reference WHO standard serum 67/97) were purchase? from Bio-Yeda (Israel). Subjects The 8 hemophiliacs and 1 study included: patient suffering from systemic 1 upus erythematosus (SLE) , all of whom had developed F.VIIl inhibitors; 6 hemophiliacs without F.VIII inhibitors; 6 h_e mophiliacs without F.VllI inhibitors but with high levels of circulating immunecomplexes; 6 non-hemophilic patients exhibiting high levels of circulating immunecomplexes; 6 non-hemophilic patients showing high levels of rheumatoid factor and 12 normal human subjects. Blood samples Blood samples were collected by venipuncture into 0.38% trisodium citrate. Within 30 min after collection, plasmas were separated by centrifugation at 1,600 x g for 20 min, and stored at -70° C, F.VIII inhibitors were evaluated by the agarose gel method descr_i bed by Jorquera et al . (9) . The 9 positive cases ShOWed titres corresponding to 5 to 108 B.U./ml. Rheumatoid factor was detected by the RA-latex test (Hyland, California, USA) and immunecompl ex es were demonstrated by the SIA-Immune Complexes Test (Sigma Diag nostics, St. Louis, USA) Purification of rabbit anti-vWF monospecific IgG Purified von Willebrand factor (vWF) was obtained according to the method described by Zimmerman et al. (IO). Several 0.5 ml al i quots (100 ug of protein) of purified vWF were then emulsified (1:3) with Freund’s complete adjuvants and injected into two male rabbits which were bled one month after the first injection. The serum obtained was absorbed with normal human plasma and then an_a lyzed by double gel diffusion (DGD) (11) and immunoelectrophoresis (IEP) (12). The resultant antiserum showed a potent anti-human vWF activity and was completely devoid of contaminants, The specific anti-vWF IgG fraction was then isolated by means of an immunoadsorbent made of Sephacryl-1000 to which a commercial
FVlll INHIBITORS IMMUNO-TYPING
900
Vol. 57, No. 6
F.VIII concentrate had been coupled according to the method deselUate obtained in this Step was decribed by Hornsey (13). The monstrated to be (DGD, IEP and ELISA, 14) a pure rabbit IgG monoantiserum yielded 0.5 mg of specj specific for human vWF (1 ml of fit IgG). test (ICMET) micro-ELISA characterization The inhibitor 4 layers (Fig. 1): the first layer consisted The ICMET included in 100 ul of anti-vWF monospecif ic of purified lgG lgG (1 ug of well) coupled to bicarbonate pH 9.6 carbonate-sodium 0.1 M per microplates (Nunc, Denmark) which were filled with 100 ul/well of incubated at and F.VIII concentrate (second commercial layer), the incubation, plates were washed 5 tiz7o C for 60 min. After a 0.15 M phosphate-NaCl buffer containing 0.05% Tween 20 mes with then filled with 100 ul/well of the (TPB) and drained. They were analyzed (third layer), and fursamples to be different plasma Plates were then washed, for 1 hour at 37O C. ther incubated ul/well of either peroxidase with 100 duplicate in filled again Kappa or lambda rabbit anti-human conjugated lgA, lgD, lgG, lgM, incubated for and further (fourth antisera monospecific layer), and drained, plates washed after being Finally, 60 min at 37O C. prepared OPD soluwere filled with 100 ul per well of a freshly tion and left at room temperature for 20-30 min. The reaction was and the plates were recordstopped with 100 ul/well of 4 N H2SO4
NIti-L ati-H
Fig.
1.
chain + PO
chain + PO 1 mti-196
iub-clars
+ AP
Schematic the representation of test principle. Plates coated with anti-vWF specific (first layer) were lgG treated as follows: a second layer consisting of the addition of a commercial F.VIII concentrate; a third lainvolving the application of the sample plasma conyer taining the inhibitor; a fourth layer which included the incubation with peroxidase (PO) conjugated anti-heavy (H) or light (L) chains antisera or, alternatively, with alK_a line phosphatase (AP) conjugated anti-IgG subclasses mono clonal antibodies.
Vol. 57, No. 6
FVlll INHIBITORS IMMUNO-TYPING
901
Finland) at (Cul tek, ed in an multi-channel micro-ELISA recorder Controls were carried out as follows: a) a plate control 492 nm. sample control (blank) which included several empty Wells; b) a yl/well of TPB instead of the F.VIII concentrate; containing 100 c) a F.VIII concentrate control which consisted of 100 ul/well of and d) peroxidase samples: different plasma of the TPB instead each antiserum used) conjugated antisera controls (two wells per ul/well of TPB instead of both F.VIII concentrate containing 200 and plasma samples. In those cases in which the F.VIII inhibitor was found to belong alkaline phosphatase conjugated monoclonal anti to the IgG class, (monospecific for bodies anti-human IgG subclasses IgGt, IgGg, employed in the fourth layer of the test, A lgG4) were IgG3 or the reaction stopPNP solution was used as enzyme substrate and the plates being recorded at 405 nm in the muJ ped with 3 N NaOH, controls to the above ti-channel micro-ELISA recorder. Similar described were always employed. RESULTS
Standardization of the ICMET In order to standardize the test, a standard plasma containing an IgG class F.VIII inhibitor (50 B.U./ml) was used. The ICMET was carried out but 3 different concentrations (12.5, as described, 6.7 and 3.3 I.U./ml of PBS) of a commercial F.VIII concentrate were applied in duplicate in the second layer of the test. No sig nificant variations in the standard plasma titres were detected. a F.VIII concentrate dilution containing 3.3 I.U. of F.VIII Thus, per ml of PBS was selected as the routine reagent to be used in the second layer of the subsequent experiments. Sensitivity of the method Different dilutions (ranging from l/l0 to l/640) in PBS of the standard plasma containing an IgG F.VI I I inhibitor (50 B.U./ml) were assayed in triplicate by the ICMET and the highest dilution showing a clear positive reading was determined, which corresponded to the l/320 dilution. Since, in general terms, a direct correlation between the F.VIII inhibitory activity of the standard the amount pl asma and of its specific anti-F.VIII IgG could be presumed, as low as 0.156 B.U./ml of F.VIII inhibitory activity would be clearly detected by means of this test. However, it could be observed that 2 plasma samples containing a low F.VIII inhibitory activity showed, when analyzed by the ICMET, higher levels of specific anti-F.VIII antibodies than those expected (data not shown). Reproducibility The standard plasma was reassayed 20 times by applying it in double serial dilutions, ranging from i/20 to l/640, in the third the ICMET. The mean of the titres obtained was l/240. layer of Additionally, a regression analysis (fig. 2) was carried out by dilution (expressed as its log2) against the mean plotting every of its optical density at 492 nm. A value of r = - 0.922 was obtained (p < 0.0001).
902
FVlll INHIBITORS IMMUNO-TYPING
0-D.
<4?2
Vol. 57, No. 6
nm>
600.00
550.00
Fig.
Regression 1 ine obtained by plotting (ordinate) s_e rial dilutions (expressed as their IogB) of a stand ard plasma containing an IgG class F.VIII inhibitor (50 B.U. per ml) against (abscissa) their optical densities at 492 nm (mean t/-one standard deviation of 20 different tests)
500.00
450.00
400.00
350.00
2.
-
r
=
-0.922
n
-
120
p < 0.0001
I
300-B@ 1.00
2.10 1/<10
3.20 x
4.30 log,
5.40
6.30
dilution>
Specificity to evaluate the specificity of the test, the following In order experiments were carried out: Half a part of five microplates IgG was incubated with 100 ul sensitized with anti-vWF specific per well of a F.VIII concentrate dilution containing 3.3 I.U. of F.VI I I per PBS and the other half with TPB instead of the ml of F.VIII concentrate. The following undiluted reagents were then applied to both parts of each plate in the third layer of the a) 12 normal human plasmas negative for test: rheumatoid factor; b) 6 plasmas from hemophiliacs without F,VIII inhibitors; c) 6 plasmas from hemophiliacs, without F.VIII inhibitors, Known to contain high levels of circulating immunecomplexes (CIC); d) 6 human non-hemophilic plasmas Known to contain high levels of CIC and e) 6 human non-hemophilic plasmas Known to contain high levels of rheumatoid factor. The a), c) and d) controls mentioned (see Material and Methods) were carried plates. Additionally, 3 serial dilutions the standard plasma containing an IgG per ml) were also included.
in the ICMET description out in each half of the 5 (l/5, l/10 and i/25) of F.VIII inhibitor (50 B.U.
FVlll INHIBITORS IMMUNO-TYPING
Vol. 57, No. 6
903
plasmas from hemoNone of the 12 normal human plasmas nor the 6 F.VIII inhibitors resulted positive when analyphiliacs without or in the absence of F.VIII conce_n zed either in the presence of, non-hemophilic plasmas containing CIC resulted the 6 trate. Al I hemophiliacs containing plasmas from Two out of the 6 negative. CIC exhibited a clear positive reaction to the anti-IgG antiserum which was exclusively localized in the part of the plate in which All the 6 plasmas contai_n the F.VIII concentrate was not applied. the polycloing rheumatoid factor showed a positive reaction of and anti-lambda antisera), in (to both the anti -kappa nal type both the F.VIII concentrate treated and the TPB treated halves of with both the anti-IgG (2 cases) or anti-IgM (4 cases) the plate, peroxidase conjugated antisera. Immunological characterization of F.VIII inhibitors by the ICMET 9) were characAll the inhibitors completely analyzed (6 out of A detailed description subcl ass and type. terized for Ig class, of all of them is shown in table I.
TABLE lmnunological
I
characterization
of
9 F.VIII
inhibitors
Patient
Inh.
P.
R.
38
B.U.
A,
M.
23
B.U.
1SIG4
K+L
V.
J.
27
B.U.
1gG4
K+L
J,
F.
108
B.U.
1gG4
K+L
J.
V.
40
B.U.
1gGq
K+L
G,
c.
5 B.U.
1gG4
K+L
I.
F.
15
B.U.
1gG4
K+L
J.
D. (SLE)
14
B.U.
IgGl,
s.
I.
50
B.U.
1gGq
n.
d.
= not
H chain
Tit.
IN
+ IgG1,
L chain
IN+,
lgG2,
lgG3 8, I@4
lgG3
& IgGq
K+L
K+L n.
d.
done
As can be observed in this table, all the inhibitors fully charas terized were found to be polyclonal (containing both kappa and lambda light chains) and belonged to the IgG class. It is notewor one plasma from a hemophiliac simultaneously showed anthy that other anti-F.VIII antibody of the IgA class. Furthermore, the IgG class inhibitor of this pl asma was demonstrated to contain all
904
FVlll INHIBITORS IMMUNO-TYPING
the 4 known IgG subclasses. case (15) will be the subject
The of
details concerning a separate publication
Vol. 57, No. 6
this
unusual
.
inhibitors from hemophiliacs were found other 6 Conversely, the subclass, while that correspto exclusively belong to the lgG4 onding to the SLE patient was demonstrated to contain IgGl, lgG2, determinants when analyzed chain antigenic lgG5 and IgG4 heavy for IgG subclasses. DISCUSSION
for the immunological characterization of A micro-ELISA method The test has proven to be simple, F.VIII inhibitors is described. accurate and highly specific for the study of this type of antib_o no non-specific reactions were observed when nordies. In fact, plasmas from hemophiliacs without F.VIII inhi mal human plasmas, non-hemophilic plasmas containing CIC were assayed by bitors or 2 out of the 6 plasmas from hemophiliacs However, this test. high levels of CIC showed a false positive reacwhich contained tion when assayed in absence of F.VIII concentrate. Nevertheless, it did not occur in those wells in which a F.VIII COnCet'Itrate had been previously applied in the second layer of the test. Thus, a containing TPB instead of the F.VIII concentrate simple control certainly detect the appearance of false positive results would CIC. In any case, this fact would hemophiliacs containing in suggest the possibility of detecting CIC containing vWF/F.VIII this method. and anti-F.VIII anti bodies by In order to assess further experiments are this attractive hypothesis, currently in our I aboratory . in All the plasma samples containing progress of rheumatoid factor demonstrated a false positive high titres both the previously F.VIII concentrate incubated and reaction in TPB incubated wells. Nevertheless, this false positive reaction can be certainly expected as a background phenomenon occurring in all those ELISA methods which employ coupled IgG, from either human or animal origin, in the first layer of the test. Indeed, the appearance of such a positive reaction will strongly suggest the rheumatoid factor presence of in the analyzed sample and will therefore make impossible its immunological analysis by means of the ICMET. On the other hand, the ICMET could be easily applied for the simultaneous characterization, in a single assay, of both the heavy (class and subc I ass) and light (kappa or lambda type) chains of all the F.VIII inhibitors tested. However, since it is not a funs tional test, it cannot substitute the biological assays for the evaluation of F.VIII inhibitors. In fact, the demonstration that in 2 plasmas exhibiting low titres of F.VIII inhibitory activithe ICMET was able to reveal the presence of quantities of ty, anti-F.VIII specific antibodies notably higher than those expectstrongly suggested that these plasmas might contain antied, F.VIII antibodies other than those exhibiting F.VIII inhibitory activity. These non-inhibitor antibodies are thought to be proba bly directed against some epitomes distanced from the functional part of the F.VIII structure (5j and have been demonstrated in some hemophilic patients, as well as in certain auto-immune conditions such as SLE (5).
Vol. 57, No. 6
In a
any case, this sensitive highly
FVlll INHIBITORS IMMUNO-TYPING
would fact for method
indicate
that
the detection the against
905
ICMET
the
of
seems
extremely
to
low
be
am-
independently of F.VIII, directed antibodies of useful very appears to be a Thus, it activity. biological their antiall types of characterization of immunological the tool for anti-F.VIII specificity. In fact, 9 anti-F.VIII showing bodies antibodies (8 from hemophiliacs and 1 from an SLE patient) Of va8 out characterized. In able to be titre were inhibitory riable all the 8 investigated and light chain was the type of of them, found to be polyclonal (namely, containing both kappa were cases with other observations contrast This is in chains). lambda and (5,8,16), which claimed a marked predominance of the kappa light studied. This could be a the inhibitors majority of chain .in the sensitivity of the biological tests which weak consequence of the had been generally used for this purpose (5) or due to a lack of antibodies employed (8). Furthe anti-light chain reliability of cannot be ruled out the existence of some racial or thermore, it constitutional variations.
ounts
inhibitors of the class. However, All the 9 plasmas exhibited lgG them contained a double anti-F.VIII is noteworthy that one of it activity: one of the IgG class and the other of the IgA one. This could not be detected when unusual antibody the plasma was IgA conventional biological but it analyzed by a method, was unexpect This fact was probably due to the follow_ edly found by the ICMET. circumstances: firstly, because of the existence of a ing marked the low inhibitory activity of the discordance between class IgA anti-F.VIII antibody and the large amount of specific (15) lgA was which could be demonstrated when this plasma analyzed by the because the inhibitor was with an ICMET; secondly, associated IgA which other potent inhibitor of the class, probably masked IgG IgA-carried inhibitory activity when biologically the assayed. Both circumstances emphasize by themselves some of the advantages ICMET over the conventional biological of the tests. An additionadvantage al of this method would be its application to the eventual detection of anti-F.VIII non-inhibitor antibodies that other completely wise would have remained unobserved in the biological assays. the Two out of 9 cases studied (the hemophiliac containing the inhibitor and the SLE patient) exhibited a F.VIII inhibitor lgA which was composed of the IgG1, lgG2, lgG5 and lgG4 subclasses of the human IgG. Some descriptions of F .VIII inhibitors containing IgGg have been reported (5, 8) but, to our knowledge, these are the 2 first in which cases anti-F.VIII has been found. The lgG2 hemophilic patient (w) showing such an heterogeneous anti-F.VIII response (15) will be the subjet of a separate publication.
(*I
FOOTNOTE: When the plasma samples from this patient were taken, he was undergoing a massive administration of human F.VIII concentra tes because of a severe hemorrhage which finally his led to death.
FVIII INHIBITORS IMMUNO-TYPING
Vol. 57, No. 6
The other 7 F.VIII inhibitors from hemophilic patients were desolely belong to the IgGq subclass. This lgG4 resmonstrated to triction of the F.VIII inhibitors has been reported in other sim_i lar observations (reviewed in 5). Furthermore, immunological reshave also ponses with a marked lgG4 predominance been described as it is the case of hemophiliacs in other situations in which, acontinuous and prolonged immutreated with F.VIII concentrates, In fact, it has been also reported in bee nization often occurs. suffering from filariasis (17) or schisKeepers (7) 1 in patients as well as in allergic patients undergoing speci tosomiasis (la), this striking lgG4 fit hyposensitization (~9,20). The reason for predominance is not known. However, as suggested by Aalberse et is tempting a.1 . (6) and van der Zee et al. to believe (7), it that an immunological response switched to the production of a v,e ry homogeneous lgG4 subclass antibody could be considered as a d_e fensive attempt by the immunological system to avoid the problems which could certainly arise from a continuous and prolonged antigenie challenge. This would be specially emphasized by the fact of complement fixing activity that lgG4 is devoid and does not form precipitating immunecomplexes (6, 7). Therefore, complementimUrteCOfnpleinduced damage and other consequences of phlogistic obviously be avoided by this type of IgGq-mediated imxes would mune responses. In agreement with these arguments would be our findings of a polyclonal (namely, containing both Kappa and lambda light chains) type of inhibitors, since normal, defensive imunological responses are always of such a type. On the contrary, homogenqous antibody responses of both heavy and light chain restriction are the main characteristic of those immunological situations which usua_l ly lead to the monoclonal gammopathies. And these monoclonal responses are associated, in the vast majority of cases, with unusually abnormal conditions such as some malignant neoproliferative disorders.
ACKNOWLEDGMENTS We gratefully acKnowledge Reganon for their helpful typescript and to Dr. F. Research studies described by a grant (No. 86/1096) of Health”, Spain.
to Dr. A. Gonzalez tlolina and Dr E. discussion and thoughtful review of the Llopis for his excellent suggestions. in this paper were supported in part from “The FIS of the National Institute
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