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Detection of nuclear antigens DNA in normal and pathologic human fluids by quantitative complement fixation.

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Detection of Nuclear Antigens (DNA) in Normal and
Pathologic Human Fluids by
Quantitative Complement Fixation
By EUGENE
V. BARNGIT
antibodies have been detected in the sera of many patients
with collagen-vascular disease, particularly
those with systemic lupus erythematosus.1
One such class of antinuclear antibodies,
antibodies to deoxyribonucleic acid (antiDNA), are receiving increasing attention
because they are commonly found in severely ill patients with systemic lupus erythematosus and rarely detected in other
collagen-vascular disease^.^.^ Despite this
interesting correlation, the exact pathogenic
significance of such antinuclear antibodies
is at present unknown. It has been suggested that such antinuclear antibodies may
combine with nuclear antigens in the circulation of patients, and that the resultant
antigen-antibody complexes contribute to a
serum sickness-type illness that we describe
as lupus glomerulo-nephritis? This hypothesis received great support from a report
by Tan, Schur, Carr, and K ~ n k e lwho
, ~ detected DNA antigens in the sera of some
patients ill with systemic lupus erythematosus. This antigen was identified as DNA by
a precipitation reaction with other human
sera shown previously to contain precipitating antibody to DNA. By chemical tests,
others6 have detected DNA in human sera,
but these tests lack specificity and do not
identify DNA as an antigen capable of fixing complement with lupus antibody.
Studies in collaboration with Dr. John H.
A
Vaughanr have suggested that normal human sera may contain small amounts of
antigenic nuclear materials, since rabbits
immunized with whole human serum in
complete Freunds’ adjuvant commonly develop antinuclear antibodies, and occasionally develop complement-fixing antibodies
to DNA. The present report describes a
more direct serologic attempt to measure
nuclear antigens in normal and pathologic
human sera and synovial fluids.
Sera from 2 patients with systemic lupus
erythematosus were shown to contain complement-fixing antibodies to native and
single-strand DNA. These sera were used
in quantitative complement fixation tests to
detect and quantitate nuclear antigens
(DNA) in normal and pathologic human
sera and synovial fluids. Since sera from patients with systemic lupus erythematosus
contain many antibodies directed against a
variety of antigens in addition to DNA,
their use in tests designed to measure DNA
in “unknown” fluids may be criticized.
Therefore, confirmation of measurements
made utilizing lupus sera was attempted
by: 1) haptene inhibition by purines and
pyrimidines of reactions between human
specimens and the lupus sera; 2) use of
rabbit antibodies directed against DNA for
the measurement of antigens in human
fluids; and 3) extraction of human specimens with hot perchloric acid and meas-
From the Department of Medicine, UCLA Medical School, University of California, Los Angeles,
California 90024.
Presented in part before the American Rheumatism Association, 18 June 1966.
Supported in part by Research Grant N I H AM
10,000 and USPHS Training Grant AM 05068.
EUGENE
V . BARNETT, M.D.: Associate Professor
of Medicine and Senior Znuestigator of the Arthritis Foundation.
NTINUCLJUR
~
ARTHRITISAND
RHEUMATISXI, VOL.
11, N O . 3 (JUNE 1968)
407
EUGENE V. BARNEm
408
Fig. 1.-Complement fixation with Serum E. 1:300 a n d S.S.DNA (calf thymus)
diluted in normal human serum, and in phosphate buffered saline, pH 7.4 with and
without DNAse treatment.
protein (less than 0.5 per cent) was obtained from
Worthington Biochemical Corporation and from
Dr. Alexander Dounce of the Department of Biochemistry, University of Rochester School of MediMATERIAISAND METHODS
cine and Dentistry. Preparations of calf thymus
250 sera from patients with systemic lupus ery- nucleoprotein and histone, as yell as DNA, nucleothematosus were kindly supplied by Dr. Carl M. protein, and histone extracted from human and
Pearson. All these sera were shown to contain rabbit livers were also kindly supplied by Mr. Vinantinuclear antibodies against nuclear antigens in cent Agnello and Dr. Alexander Dounce.lzJ3 The
immunofluorescence tests using human leukocyte DNA content of these preparations was estimated
nuclei. These 250 sera were tested for precipitating by absorption at 260 mp. and in the Dephenylaantibody against single-strand DNA by double dif- mine test.14 T-4 phage DNA was kindly supplied
fusion in 1 per cent Agarose (0.15 M Phosphate, by Dr. Verne Schumacker of the Department of
pH 7.3) against single-strand calf thymus DNA at Chemistry, University of California at Los Angeles.
Single-strand DNA was prepared by heating
a concentration of 0.5 mg./ml. Fourteen sera were
shown to have precipitating antibodies to DNA. Of preparations at 100 C for 10 minutes and then
these 14, only 2 were shown to have complement- rapidly cooling in an ice bath.
fixing antibodies to DNA in higher titer than the
Specimens of normal and pathologic human sera
anti-complementary activity of the sera. These 2 and synovial fluids were tested for their content
sera were used as antibodies to detect nuclear an- of nuclear antigens in an initial dilution of 1:50
tigens in human fluids.
or 1:lOO. Less dilute specimens were found to be
Rabbits were immunized with single-strand calf anti-complementary in the quantitative complethymus DNA, complexed to methylated rabbit ment fixation test.7 The sera and synovial fluids
serum albumin.8~9 The antisera produced reacted tested were generally separated from the clot withexclusively with single-strand and native calf thy- in 8 hours of aspiration. The Supernatants were
mus DNA. Antibody was restricted to the gamma either tested directly following dilution or were
M class of rabbit immunoglobulins as determined first centrifuged at 4,000 rpm. for 20 minutes.
by DEAE chromatography,lo as previously re- Neither centrifugation nor hemolysis appeared to
ported with antibody elicited by immunization affect the results obtained in the quantitative comwith DNA-bovine serum albumin complexes.11 plement fixation test. Inhibition tests utilizing nuThe rabbit antibodies had much greater activity cleic acid bases were performed.15 Known nuclear
in the complement fixation test performed at 37 C. antigens as well as selected specimens of normal
than 4 C. Calf thymus DNA without detectable pnthologic human sera and fluids were digested
urement of the DNA content of the proteinfree material in the diphenylamine test.
DETECTION OF ANTIGENS BY COMPLEMENT FIXATION
Fig. 2.-Complement
fixation with Serum V. 1:200and S.S. and native
409
T-4phage
DNA.
with DNAse prior to testing in some of the complement fixation tests.7 Not all specimens were digested with DNAse or tested in inhibition tests
utilizing nucleic acid bases because of the limited
quantities of sera with complement-fixing antibodies to DNA. Plasma was treated with perchloric
acid16 and the extracts tested with the diphenylamine reagent for deoxyribose content.14
DNA was indeed DNA and not some contaminant, complement fixation tests were
repeated, utilizing single-strand DNA, previously digested with DNAse as well as
DNA preparation which had been similarly
incubated without the addition of DNAse.
Complement fixation was entirely abolished
RESULTS
when single-strand DNA was digested with
Serum from Patient V when diluted 1: DNAse prior to testing, whether the DNA
100 gave positive complement fixation with was added to undiluted human serum or
single-strand calf thymus DNA and calf diluted in buffer containing calcium and
thymus nucleoprotein; less complement fix- magnesium (Fig. 1). Identical quantities
ation with native calf thymus DNA; and of single-strand calf thymus DNA diluted
insignscant complement fixation with RNA in buffer or in normal human serum for 6
and histone.'T The serum from Patient V hours at 37 C. gave identical complement
when diluted 1:300 gave highly reproduc- fixation curves. These data suggest that
ible complement-fixationcurves with single- there were no significant inhibitors or DNA
strand calf thymus DNA.17 On multiple de- enzymes in the normal human serum that
terminations, 50 per cent complement fixa- would be active in vitro during that period.
tion was obtained with 0.03 to 0.45 pg. DNAse was shown to destroy the antigenic
single-strand calf thymus DNA/ml. In all activity.
experiments where human specimens were
Serum from Patient V was shown to give
tested for nuclear antigen content, a simul- complement fixation with single-strand hutaneous complement fixation curve was ob- man, and T-4 phage DNA as well as calf
tained with single-strand calf thymus DNA. thymus DNA. In every case the percent
To confirm that the antigen in the prepa- complement fixation was greater with
ration labeled single-strand calf thymus single-strand than native DNA (Fig. 2).
EUGENE V. BARNE'IT
410
Fig. 3.-Complement
fixation with Serum V. 1:300 and S.S. T-4 phage and S.S.
DNA (calf thymus).
Fig. 4.-corn lement fixation with Serum E. 1:ZOO and Serum V. 1:300, vs.
S.S. DNA (calf thpymus) .
Single-strand calf thymus and T-4 phage
DNA (Fig. 3 ) gave the greatest percent
complement fixation per microgram of antigen.
Serum V at a dilution of 1:300 in a complement fixation test utilizing 0.05 pg./ml.
of single-strand calf thymus DNA, was
completely inhibited by 300 millimicromoles per ml. of cytidylic acid. Insufficient
quantities of serum V were available for
further inhibition studies.
Serum from the second patient with systemic lupus erythematosus, Patient E, when
diluted 1:200 gave similar complement fixation curves with single-strand DNA as obtained with the serum from Patient V
diluted 1:300 (Fig. 4). The sera from Patient E diluted 1:200 and Patient V, 1:300
411
DETECTION OF ANTIGENS BY COMPLEMENT FIXATION
Table 1.-Complement Fixation by Serum E. 1:300 and Synovial
Fluid Specimens Treated with DNAse
Specimen No.
JF-1
JF-3
JF-5
JF-8
JF-9
Dh&%O&
Result
R. A.
Anti-complementary
Destroyed by DNAse
Enhanced by DNAse
Destrayed by DNAse
Destroyed by DNAse
Gout
Infection
Gout
R. A.
Table 2.-Complement Fixation Inhibition of Serum E. 1:300 and Specimens
of Synovial Fluid, JF-3, JF-9 and Abnormal Sera, Ab-14, Ab-21 and SingleStrand Calf Thymus DNA by 300 m,um./ml. of Cytidylic Acid
Specimen No.
JF-3
JF-9
Ab-14
Ab-21
ss-calf thymus
-
DNA
% Inhibition
DissnOSis
Gout
R. A.
S. L. E.
S. L. E.
60
5
25
0
-
50
Table 3.-Nuclear Antigen Content of Synovial Fluid Specimens
Disease
NumberTtsted
RA
SLE
Gout
Reiter’s
Infection
gave no complement fixation with specimens of histone or RNA but did detect
calf thymus nucleoprotein. At these dilutions neither sera fixed complement in the
presence of DNA or nucleoprotein that had
been previously digested with DNAse. The
complement fixation of serum E in a dilution of 1:300 in the presence of 0.05 pg./ml.
single-strand calf thymus DNA was inhibited up to 76 per cent by 300 millimicromoles per ml. of cytidylic acid.
Synovial fluid specimens from two patients with gout were tested in dilutions
from 1:100 to 1:2700. Significant complement fixation was obtained with both specimens.17
Complement fixation was abolished with
Specimens No. JF-3, JF-8, and JF-9, but
not with Specimens JF-1 and JF-5,when
the specimens were digested with DNAse
prior to testing (Table 1).The addition of
( “SS calf thymus DNA” )
74.3, 31.8, 7.6
37.8
42.6, 0.4
2.9
<0.2
300 mpm./ml. of cytidylic acid markedly inhibited complement fixation by serum E, at
dilution of 1:300 and JF-3 and abnormal
serum, Ab-14 (Table 2). JF-3 was from a
patient with gout and serum Ab-14 was a
serum from a patient with systemic lupus
erythematosus. Complement fixation with
JF-9 (patient with rheumatoid arthritis)
and with abnormal serum Ab-21 (systemic
lupus erythematosus) was not inhibited
with 300 mpm./ml. of cytidylic acid.
Eight synovial fluids were tested for the
content of nuclear antigen. In 7 of these,
nuclear antigen was detected in high concentration (Table 3). There was no correlation between the synovial leukocyte count
and the quantity of antigen detected.
Twenty-seven sera from patients with
various diseases were tested for their content of nuclear antigen (Table 4). Seven of
these patients gave complement fixation
412
EUGENE V. BARNETT
Table I.-Nuclear
Antigen Content of Abnormal Human Sera
Disease
Number
Tested
RA
SLE
Nephritis
SLE without Nephritis
Systemic Mastocytosis
Uremia with Chronic
Glomerulonephritis
2
1
2
RA
SLE
4
5
5
1
1
2
1
+
+ Nephritis
SLE without Nephritis
Vasculitis, Local
Rheumatic Heart
Psoriasis with RA
Gout
Scleroderma
Units/ml.
( “SS calf thymus DNA”)
15.0, 2.3
8.6
16.0, 13.5
1
4.9
1
9.0
less than 0.2
1
Table 5.-Nuclear Antigen Content of Normal Human Sera
source
R.K.
G. D.
G. w.
D. A.
D. C.
D. G .
8 others
Pool of “Normal” Serum
greater than that achieved with 0.2 pg./ml.
of single-strand calf thymus DNA. Although
it is particularly unlikely that these specimens contain single-strand calf thymus
DNA, the nuclear antigen content of these
specimens is expressed in units equal to
u./ml. of single-strand calf thymus DNA
that would be capable of achieving the percent complement fixation obtained by the
specimen with either serum E diluted 1:300
or serum V diluted 1:200. Twenty of these
specimens gave no complement fixation
when diluted 150 or 1:100, and thus have
less than the equivalent of 0.2 u./ml. of
single-strand calf thymus DNA in the undiluted specimen. The diagnoses of the 27
patients studied are listed. There was no
obvious correlation between the diagnoses
and nuclear antigen content, nor was there
UllitS/d.
(equivalent to SS calf thymus DNA)
3.50
2.60
1.94
0.80
0.28
0.26
<0.26
- not measurable
1.50
between the clinical severity or peripheral
white count of these patients.
The sera of 14 normal people were
promptly tested following phlebotomy and
separation of the serum from the clot
(Table 5). Nuclear antigen was detected
in 6 of these. In only one specimen did the
quantity of antigen detected approach that
found in 7 of the 27 pathologic sera.
The serum from rabbit no. 44 fixed complement with native DNA of calf thymus
origin at 4 C., but to a greater degree with
greater reproducibility at 37 C. (Fig. 5 ) . It
gave comparable complement fixation with
single-strand DNA of human liver origin,
but less complement fixation with rat liver
nucleoprotein and native DNA of human
liver origin. Greatest complement fixation
was achieved with single-strand calf thy-
DETECTION OF ANTIGENS BY COMPLEMENT FIXATION
413
%
C'
fix.
Fig. 5.-Complement fixation with Rabbit Antibody #44 1:400 and Native DNA
(calf thymus) tested at 37" C.
ALF THYMUS
1.0
0.1
0.01
u p S.S.
0.001
DNA
Fig. 6.-Complement fixation with Rabbit Antibody #44 1:400 and single-strand
DNA (calf thymus) tested at 37O C. and T-4phage.
mus DNA and least complement fixation
with single-strand DNA from T-4 phage
(Fig. 6), and no complement fixation with
RNA. In haptene inhibition tests (Table 6)
serum from rabbit no. 44 was best inhibited
by cytidylic acid of the ribonucleotides used
and by cytosine of the nucleic acid bases.
Seventeen serum specimens were available for repeat testing with rabbit antibody
no. 44. Table 7 compares results of complement fixation with human antibody E in a
dilution of 1:300 at 4 C. and rabbit antibody no. 44 at 37 c . Of 17 specimens measured utilizing human antibody E, 5 had detectable amounts of antigen. Seven of 17
tested with rabbit antibody had detectable
amounts of antigen. In not a single case did
both human and rabbit antibodies give a
positive reaction with the same specimen.
The plasma was separated from two units
of outdated blood obtained from the UCLA
Blood Bank. The plasma was treated with
EUGENE V. BARNETT
414
Table 6.-Complement Fixation Inhibition b y Haptenes of Rabbit Antibody # 44
(1:400) at 37" C. with 0.006 pg./ml. Single-Strand Calf Thymus D N A
~
Millimicromoles/ml.
of Haptene
% Inhibition
Haptene
Cytidylic acid
Adenylic acid
Guanylic acid
Cytosine
Adenine
Guanine
Thymine
100
100
100
1000
1000
1000
1000
84
43
0
45
0
0
0
Antigen Content of Abnormal Sera as Detected
by Human and Rabbit Antibodies
Table 7.-Nuclear
Human h t i b o d y E 1:300 (4OC.)
Serum
No.
Ab-1
Ab-2
AB-3
Ab-5
Ab-6
Ab-7
Ab-8
Ab-10
Ab-14
Ab-15
Ab-17
Ab-19
Ab-20
Ab-21
Ab-22
Ab-35Ab-36
Rabbit A n t i i y #44 (37OC.)
SerUm
Dilution*
C' Fix.
1:loo
1:loo
N.D.
<30%
<30%
N.D.f
1:loo
1:loo
1:loo
1:100
1:100
1:loo
1:loo
1:loo
1:loo
1:loo
1:lOO
1:loo
1:1350
1:1350
<a%
3.0pLS
<30%
<30%
<30%
9.op
<30%
<30%
<m
<30%
16.0~
<30%
8.6~
13.5~
Serum
Dilution
1:25
1:200
1:50
1:50
1:25
1:50
1:50
1:50
1:100
1:25
1:225
1:s
1:25
1:loo
1:50
1:50
1:50
+ 1:400
C' Fix.
<30%
0.46~
0.28~
0.09rr
<30%
<30%
0.08~
0.13:~
<30%
0.07~
<m
<30%
0.05~
<30%
<30%
<30%
<30%
'Dilution of abnormal serum yielded complement fixation with either human or rabbit antibodies.
Less dilute specimens were anti-complementary.
fN. D.
not tested.
$Units equal pg. of S. S. calf thymus DNA that would yield percent complement fixation obtained
with the human or rabbit antibodies.
-
perchloric acid and the protein-free mate- treated with hot perchloric acid and the
rial measured in the dinhenylamine test.
supernatants tested in both diphenylamine
The values obtained ranged from 0.15 to tests and comolement fixation tests, the
0.35 micrograms per ml. on specimens that amount of DNA detected in both tests
had been concentrated by pressure dialysis. equaled the amount of DNA added to sera
In aliquots of outdated aged whole blood or plasma.
and the hemolyzed plasma treated with hot
DISCUSSION
perchloric acid, the DNA in the proteinfree material measured 0.53 micrograms
A sensitive serologic technique has been
per ml. in the diphenylamine tests. When applied for the detection and quantitation
known amounts of calf thymus DNA were of nuclear antigens in serum and synovial
added to normal and abnormal plasma or fluid specimens. The antigens detected in
sera and these mixtures subsequently these specimens are most likely DNA or
DETECTION OF ANTIGENS BY COMPLEMENT FIXATION
DNA-containing material, in that the antisera in the dilutions used in these tests detected only native, single-strand DNA or
nucleo-protein. The antigens in both the
control specimens of DNA and the human
specimens were destroyed by DNAse in
nearly every case tested. The only exception
was JF-5 from the case of infectious arthritis. Inhibition of complement fixation was
achieved with both the control DNA antigen and with 2 of 4 of the human specimens tested by the addition of 300 mpm./
ml. nucleotides. This sensitive technique
has confirmed observations obtained by the
use of chemical tests which suggested that
DNA was present in human sera.
Considering the different techniques,
Tan, Schur, Carr, and Kunkel suggested
that normal sera may contain up to 3 micrograms per ml. of DNA.16 The present
report, using different antisera and different techniques, would support this estimate. Furthermore, the experiments utilizing perchloric acid treatment of plasma in
testing for DNA in the diphenylamine tests
suggest that no more than a microgram of
DNA per ml. of serum could be accounted
for by in uitro destruction of blood cells.
This latter figure is probably a gross overestimation, since the material tested was
the concentrated extract of plasma from 250
cc. of whole, aged, bank blood. As suggested by earlier work,16 DNA measurements
of human serum obtained by the usual techniques without gross hemolysis measure
quantities of DNA present in uiuo. The detection of small amounts of nuclear antigens in normal sera by the serologic test is
in accord with suggestions made as a result
of previous work, where it was shown that
immunization of rabbits with normal human
serum or freshly separated plasma may result in antibodies to DNA.? Much greater
quantities in a range of 8-12 mg. per cent
of DNA have been detected by chemical
tests6 The much lower amounts of nuclear
415
material detected in the present study suggest that: 1) a significant proportion of nuclear material present in human specimens
is not detectable as antigen by the antisera
used in the present study, possibly because
the nuclear material is in part bound to
serum proteins or antibodies; and/or 2) the
chemical tests detected materials other than
DNA in human specimens.
Using precipitin tests for DNA, Tan,
Schur, Carr and Kunkel detected nuclear
antigens in human sera. Since they utilized
other human antisera as reagents, the difference in quantities detected may well be
explained by differences in specificity of the
human antibodies used, as well as by differences in sensitivities of the tests employed.
Indeed, the inhibition of the antisera used
by cytidylic acid points out the marked differences in specscity of different human
anti-DNA antibodies, since Stollar studied
human DNA antibodies that were not inhibited by cytidylic acid.15 It is of particular
interest that the human antibody which was
capable of reacting with single-strand DNA
of T-4 phage origin detected antigens in entirely digerent human sera as were detected by the rabbit antibody which showed
little or no reaction with single-strand T-4
phage DNA. It thus appears that the antigens in human fluids may be as heterogenous as the antinuclear antibodies previously described.
Since no elevation of serum DNA was
detected in 5 of 6 S.L.E. patients with neph
ritis, it is unlikely that DNA-anti-DNA
complexes with DNA in antigen excess
played a role in these 5 patients’ disease at
the particular time in their disease when
the specimens were tested. This in no way
excludes the possible pathogenetic role for
antigen-antibody complexes in lupus nephritis. Indeed, Tan, Schur, Carr and KunkeP6 have described marked fluctuations in
the DNA content of the serum of individual
patients. Their study suggests that the appearance of DNA in the serum of some,
EUGENE V. BAR"IT
416
but not a majority, of patients with systemic
lupus may signal the onset of glomerulonephritis. These observations should perhaps encourage us to search for antigenantibody complexes other than DNA-antiDNA as instrumental in the etiology of
lupus glomerulo-nephriti~.~
In the present study nuclear antigen or
DNA content of synovial fluids was markedly elevated regardless of the diagnosis.
No studies were made to test the possibility that synovial fluid leukocytes in vitro
when lyzed might release large amounts of
DNA. However this appears unlikely since
so little DNA was released from the leukocytes of 500 ml. of whole blood kept at refrigeration or at room temperature for long
periods.
The approach described in the present
study shows promise for the detection of
nuclear antigens, both DNA as well as
other antigens in fractions of normal and
abnormal sera. Such approaches may shed
light on the possible serum binding by various plasma proteins of the nuclear material
in various disease states. It is possible that
abnormalities in transport for nuclear antigens due to differences in serum binding of
nuclear antigens may be instrumental in
the induction of auto-antibodies to nuclear
material as seen in systemic lupus erythematosus and other collagen-vascular diseases. Furthermore, this approach shows
promise for the detection of nuclear antigens in diseases of lower animals. One such
situation is Aleutian Disease of Mink.
We have detected nuclear antigens in
serum from mink with Aleutian Disease but
not in normal mink serum nor in sera from
15 other species.l'J* This nuclear antigenemia may have some relationship to the
viremia postulated in Aleutian disease.
ACKNOWLEDGMENTS
The author gratefully acknowledges the technical assistance of Miss Margaret Alspaugh and Miss
Dorothy Corey.
SUMMARY
A sensitive serologic technique is described which shows promise for the quantitation
of nuclear antigens, particularly DNA in serum and synovial fluid specimens. The
nuclear antigen or DNA content of synovial fluids tested was markedly elevated regardless of the diagnoses. Six of 14 normal human sera had small but detectable
amounts of nuclear antigen. Seven of 27 athologic human sera had markedly increased
amounts of nuclear antigen (DNA). Adough antigen was detected in human sera by
rabbit antibody to DNA, in no case in this study did the human and rabbit antibodies
give a positive result with the same human serum. There was no correlation between
clinical severity, diagnosis, or peripheral white count and the level of nuclear antigens
detected.
SUMMARIO
IN INTERLINGUA
Es describite un sensibile technica serologic de promissa pro le quantification de
antigenos nucleari, particularmente de ADN in sero e liquid0 synovial. Le antigeno
nucleari o le contento de ADN del liquidos synovial testate esseva marcatemente
elevate sin reguardo a1 diagnose del casos individual. Sex de 14 normal seros human
habeva micre sed detegibile quantitates de antigeno nucleari. Septe de 27 pathologic
seros human habeva marcatemente augmentate de antigeno nucleari (ADN). Ben que
antigeno esseva detegite in seros human per anticorpore de conilio anti ADN, in nulle
del casos studiate esseva notate que le anticorpores de conilio o human rendeva resultatos positive con le mesme sero human. Esseva trovate nulle correlation inter le
severitate clinic, le diagnose, o le peripheric numeration leucocytic e le nivello del antigenos nucleari detegite.
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DETECTION OF ANTIGENS BY COMPLEMENT FIXATION
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complement, nuclear, fixation, pathologic, detection, antigen, dna, norman, human, fluid, quantitative
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