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Detection of low avidity anti-dna antibodies in systemic lupus erythematosus.

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DETECTION OF LOW AVIDITY ANTI-DNA
ANTIBODIES IN SYSTEMIC LUPUS
ERYTHEMATOSUS
RICHARD L. RILEY, HUGH McGRATH, JR., and RONALD P. TAYLOR
The binding of sonicated, radiolabeled DNA by
systemic lupus erythematosus (SLE) sera was measured by a high sensitivity polyethylene glycol (PEG)
precipitation assay. This method is considerably more
sensitive than currently used techniques. The results
suggest that a significant concentration of low avidity
antibodies is present in SLE sera; however, these antibodies are not detected by conventional techniques.
Immune complexes of DNA and anti-DNA antibody are of considerable importance in the pathogenesis of systemic lupus erythematosus (SLE) (1-7). In patients with renal involvement, deposits of native and
single stranded DNA and anti-DNA antibodies have
been detected along the glomerular basement membranes (2-4). Recent observations suggest that DNAanti-DNA complexes are present in the circulation of
SLE patients (8,9) and it is believed that deposition of
From the Departments of Biochemistry and Rheumatology,
University of Virginia School of Medicine, Charlottesville, Virginia.
Supported by a grant from the Jones Foundation and National Institutes of Health grant 5-ROIAMl1766. Ronald P. Taylor is
a Research Career Development Awardee (1K04AI-00062-04) of the
National lnstitutes of Health.
Richard L. Riley, B.S.: predoctoral student, Department of
Biochemistry; Hugh McGrath, Jr., MD: Department of Rheumatology; Ronald P. Taylor, PhD: Associate Professor of Biochemistry, Department of Biochemistry.
Address reprint requests to Ronald P. Taylor, PhD, Department of Biochemistry, University of Virginia School of Medicine,
Charlottesville, Virginia 22908.
Submitted for publication July 21, 1978; accepted in revised
form October 30. 1978.
Arthritis and Rheumatism, Vol. 22, No. 3 (March 1979)
these circulating complexes along the glomerular basement membrane may be critical in the development of
lupus nephritis.
Although little is known concerning the molecular properties of the DNA-anti-DNA complexes in SLE,
it is apparent from numerous investigations that qualitative differences (e.g., avidity, specificity, and mode of
binding to DNA) among anti-DNA antibodies may influence the development of lupus nephritis (10-17). The
majority of these studies employ the ammonium sulfate
globulin precipitation (Farr) assay to detect the binding
of DNA by specific antibody; however, it has been suggested that this technique does not provide information
regarding the entire anti-DNA antibody population
typically found in SLE sera (18). There is some evidence that suggests that the high salt concentration used
in the assay may cause dissociation of all but those complexes that contain the highest avidity antibodies (1 8).
In addition, it is now recognized that this method is
most applicable in the study of the binding of antibodies to high molecular weight DNA (19,20). Recent
evidence suggests that low molecular weight DNA may
in fact play a significant role in the pathogenesis of SLE
(21,22). Unfortunately, Farr assays with DNA of low
molecular weight suffer from a significant loss of sensitivity which can lead to additional ambiguities if DNA
of a heterogeneous molecular weight distribution is employed in the assay (23).
In view of the problems associated with the Farr
assay and the potential importance of low molecular
h
weight DNA, the polyethylene glycol (PEG) immunecomplex precipitation technique (24) has been applied
to develop an assay that measures the binding of antibody to low molecular weight DNA with high sensitivity. With this assay one is able to detect a significant
amount of DNA binding activity in SLE sera which is
not demonstrable by the Farr assay. This observation is
most pronounced when low molecular weight double
stranded DNA (dsDNA) is used. The results suggest
that a large population of relatively low avidity antiDNA antibodies is present in SLE sera, but these antibodies are not detected in the Farr assay.
MATERIALS AND METHODS
Preparation of 'H-DNA. A thymine deficient auxotroph of E coli was grown in minimal media containing 1 p g /
ml thymidine, 1% of which was [methyl-'HI thymidine (New
England Nuclear). The cells were harvested and the DNA extracted in accord with published procedures (25). The extracted DNA was chromatographed on a column of methylated albumin kieselguhr (26) and the dsDNA was eluted with
0.67 M NaCI-0.03 M borate buffer. pH 6.7, and dialyzed
against 0.03 M borate, 0.15 M NaCI, pH 7.8 (BS buffer). The
260:280 absorbance ratio of the dsDNA was 1.92 and the
specific activity was 193 cpm/ng by liquid scintillation counting (see below). This 'H-dsDNA was designated high molecular weight DNA. This 'H-dsDNA was somewhat heterogeneous by isokinetic sucrose gradient centrifugation.
However, 75% of the DNA was of molecular weight 5 X 10'
daltons or greater.
Low molecular weight dsDNA was prepared by sonicating aliquots of the E coli 'H-dsDNA for 2 minutes with a
Biosonik sonifier. The sonication was done at the maximum
setting compatible with the microtip used. Sonication was performed in an ice bath at 15 second intervals with cooling periods of 15 seconds after each sonication period. The sonicated
'H-dsDNA sedimented in isokinetic sucrose gradients as a
single peak with S , , , = 8.3 S, corresponding to a molecular
weight of about 5 X 10' daltons. Single stranded (ss) sonicated
3H-DNA was prepared by immersing aliquots of sonicated
'H-dsDNA in a boiling water bath for 10 minutes, followed
by rapid cooling in an ice bath.
The sonicated 'H-dsDNA was unreactive with specifically purified rabbit anti-adenosine monosphosphate and
anti-guanosine antibodies (4) in the PEG assay under conditions where these antibodies bound significant amounts of
sonicated 'H-ssDNA. By these criteria, the sonicated 'HdsDNA preparations consist essentially of double-stranded
DNA.
and then stored in small aliquots at -20°C until use. Sera
were tested for DNA binding ability (see below); any sera capable of spontaneously precipitating 'H-DNA in the absence
of added precipitant (i.e., PEG or ammonium sulfate) were
not included in this study.
Preparation of IgG subfractions of sera and aggregation of IgC. The gamma globulin fractions of SLE and normal
human sera were prepared by repeated precipitation of the
sera at final concentrations of 40% saturated ammonium sulfate. The gamma globulin fractions were then dialyzed extensively against BS buffer and chromatographed on G-200
Sephadex columns. The second major peak was shown to be
p r e d o m i n a n t l y IgG (greater t h a n 90%) by immunoelectrophoresis and by SDS-PAGE electrophoresis. All G200-IgG subfractions were concentrated to between 5 and 10
mg/ml. This G-200-IgG was further purified by DEAE chromatography and these DEAE-IgG preparations were shown
to consist entirely of IgG by immunoelectrophoresis.
Human 1gG isolated from NHS by DEAE cellulose
chromatography was aggregated by heating a 10 mg/ml solution of the IgG at 63°C for 30 minutes.
Measurement of DNA binding by sera in the PEG,
Farr, and Millipore filter assays. In the standard PEG assay,
25 pl of the serum to be tested and 15 ng of 'H-DNA in 75 p1
of BS buffer were mixed in a 0.4 ml disposable centrifuge
tube. The mixture was incubated for one hour at 37°C and
then 100 pl of chilled 7% PEG (MW = 6,000: Sigma Chemical
Co.) in BS buffer were added, followed by immediate mixing.
After a 2 hour incubation at 4°C. the mixture was centrifuged
for 15 minutes in a Beckman Model B microfuge (approximately 8,OOOg). One hundred microliters of the supernatant
were then counted in 1.5 ml of deionized water and 10 ml of a
scintillation cocktail consisting of 33% Triton X-100and 67%
toluene, plus 4 gm/liter 2,5-diphenyloxazole (PPO; Sigma
Chemical Co.). In later assays Beckman Ready Solve EP was
used with identical results. The Farr assays were performed in
exactly the same manner as the PEG assay, except that 100 pI
of chilled 70% saturated ammonium sulfate solution were
added to the serum-DNA mixture instead of the 7% PEG solution.
Preliminary experiments indicated that the degree of
precipitation of 'H-DNA-antibody complexes in the PEG assay increased with the time of incubation at 4°C. By 2 hours
the extent of precipitation was essentially at maximum. The
maximum precipitation of bound 'H-DNA in the Farr assay
was reached within the first 15 minutes of incubation at 4°C.
Additional incubation at 4°C for periods of up to 3 hours did
not lead to enhanced binding of 'H-DNA or to solubilization
of the precipitated antibody-'H-DNA complexes.
Normal human serum controls were performed in
each experiment. The percentage of antigen bound by an SLE
serum was calculated from the following formula:
% 'H-DNA bound =
Patients and sera
Sera were obtained from patients with SLE receiving
medical care at the University of Virginia Hospital. Normal
human serum (NHS) was obtained from healthy volunteers.
All sera were heated at 5 6 T for 25 minutes to inactivate complement, briefly centrifuged to remove aggregated material,
cpm in N H S supernatant - cpm in SLE supernatant
x 100%
cpm in NHS supernatant - background cpm
In the Farr assay, NHS bound about 5 to 10% of the
high molecular weight 'H-dsDNA and approximately 5% of
the sonicated 'H-ds or 'H-ssDNA present in the samples
tested. The binding of 'H-DNA by NHS in the PEG assay is
DETECTION OF ANTI-DNA ANTIBODIES
discussed below. The total amount of 'H-DNA added to each
tube in the PEG and Farr assays was determined by substituting a I% solution of bovine serum albumin for serum in
the assays.
Greater than 50% of high molecular weight 'H-ds or
ssDNA is precipitated by NHS in the PEG assay. However,
this unacceptable level of background binding can be reduced
in a number of ways. If the DNA is sonicated, the binding by
NHS is reduced to 27% and 14%for ds- and ssDNA respectively. Alternatively, if the serum is diluted fivefold with BS
buffer, the background binding is comparably reduced for
high molecular weight DNA. Finally, if the IgG subfractions
(see above) of NHS are used, the background binding for
both high molecular weight and sonicated DNA is reduced to
less than 10% in the PEG assay.
In order to eliminate possible sources of artifactual
binding by sera in the PEG assay, several control experiments
were performed. Appropriate dilutions of NHS containing up
to 1 mg/ml aggregated human IgG did not precipitate greater
amounts of sonicated 'H-DNA than did NHS samples without added IgG aggregates. Also, raising the total concentration of NHS protein to three times the normal concentration
did not lead to a significant increase in sonicated dsDNA
binding in the PEG assay.
The binding of 'H-DNA by SLE sera using the Millipore filter technique was done according to the literature
(27,28) except that the same amount of 'H-dsDNA (approximately 15 ng) was used as in the Farr and PEG assays. In
those cases where different assay methods were compared, the
same amount of serum was used. In the Millipore filter assay,
appropriately diluted sera and prefiltered 'H-dsDNA were incubated for one hour at 37°C to maintain comparable conditions with the other assays.
Reproducibility in the binding experiments (performed in duplicate) was generally f 7% or better. Approximately one half of the sera were tested at least twice in independent experiments.
Measurement of dissociation kinetics with the PEG
and Farr assays. The tendency of antibody-'H-DNA complexes to dissociate was determined in the following manner.
Fifteen nanograms of 'H-DNA in 50 p1 of BS buffer were
added to 25 pl of test serum in a 0.4 ml centrifuge tube. The
mixture was then incubated for one hour at 37°C. At the end
of the incubation period, 25 pl of unlabeled, calf thymus DNA
(approximately 0.16 mg/ml) or 25 p1 of borate saline buffer
were added to the mixture and the contents of the tube were
mixed thoroughly. After a 2 hour incubation at 24"C, 100 pl
of either 7% PEG solution or 70% saturated ammonium sulfate were added to the mixture, followed by rapid mixing.
This mixture was then incubated for 2 hours at 4°C. The contents of the tube were then centrifuged and 100 pl of supernatant were counted in exactly the same manner as stated previously for PEG and Farr DNA binding assays.
The dissociation of antibody-DNA complexes was
calculated with the following formula:
% 'H-DNA bound by serum after
- incubation with unlabeled DNA
'H-DNA binding- - % 'H-DNA bound by serum tested
in absence of unlabeled DNA
% residual
,oo%
22 1
The percentage of 'H-DNA bound by a serum was calculated
as described previously. Complete dissociation of antibodyDNA complexes would be indicated as zero percent residual
DNA binding; 100% residual DNA binding would indicate
that no dissociation had occurred.
In all kinetic experiments, controls were performed to
determine the "end points" of complex dissociation as follows:
test sera were incubated with excess unlabeled calf thymus
DNA for one hour at 37°C and then radiolabeled DNA was
added. Under these conditions, only negligible binding of 'HDNA was observed. It should be noted that the weight ratio of
unlabeled DNA to 'H-DNA in all of the dissociation experiments was approximately 200 : 1. Further control experiments
indicated that when cold E coli DNA was used in place of calf
thymus DNA, the results of the kinetic experiments were the
same.
RESULTS
Binding of dsDNA by SLE sera in the PEG,
Farr, and Millipore filter assays. When sonicated 'HdsDNA is used, the PEG assay is of considerably higher
sensitivity than the Farr assay (Table 1). Each SLE
serum tested bound considerably greater amounts of
sonicated dsDNA in the PEG assay; indeed, little, if
any, binding of the sonicated dsDNA could be detected
by the Farr assay. It should also be noted that enhanced
binding of sonicated dsDNA in the PEG assay was observed when the IgG subfractions of SLE sera were
used.
Although the high levels of nonspecific binding
exhibited by NHS precluded the use of high molecular
weight DNA in the above experiments (see the experimental section for a discussion of DNA binding by
NHS in the PEG assay) fivefold dilutions of NHS and
SLE sera reduced background binding by NHS to an
acceptable level. A preliminary comparison of the binding of nonsonicated 'H-dsDNA in the PEG and Farr assays indicated that with some sera enhanced binding of
dsDNA was observed with the PEG assay (Table 2).
However, the trends with high molecular weight
dsDNA may be more complicated. Our studies using
homogeneous 'H-PM-2 dsDNA (molecular weight approximately 6 10" daltons) indicate that certain sera
show less binding in the PEG assay (relative to the
Farr) whereas others show enhanced binding (RP Taylor and DJ Addis, in preparation). The binding of high
molecular weight 'H-dsDNA by diluted SLE sera in the
Millipore filter assay was enhanced relative to the Farr
assay and comparable to the binding observed in the
PEG assay. However, little binding of sonicated 'HdsDNA by undiluted SLE sera was observed with the
Millipore filter assay (Table 1). In certain instances
rather high (about 50% or more) binding of sonicated
-
RILEY ET AL
222
Table 1. Binding of sonicated 'H-dsDNA by SLE sera; comparison of
the PEG, Farr, and Millipore filter assay
Patient
co
Di
Cr
Sh
Wr
Jo
Mo
Wi
Sm
Be
Ta
Ri
Em
Br
G-200-IgG-Ri§
G-200-IgG-Em§
G-200-IgG-Tag
DEAE-IgG-Cag
DEAE-IgG-Fig
M e a n f SDI(
% 'H-dsDNA bound
PEG assay*
F a n assay?
52
55
27
40
32
46
52
53
78
50
38
54
32
70
49
34
17
86
60
48f 147
2
0
0
8
7
12
10
25
I1
10
2
11
8
2
5
6
2
26
8
t3* 71
Millipore
filter assay*
I
2
4
6
0
7
2
9
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
These values are all calculated relative to normal controls (NHS)
(see Materials and Methods). The actual binding by the NHS controls
(relative to a bovine serum albumin standard which showed no binding) was 27% f 9% for a total of 10 serum samples collected from 5
normal individuals. Sera from 7 patients with rheumatoid arthritis
were examined and their binding values were the same as the NHS
controls.
t Values are calculated relative to NHS controls (see above note).
The background binding by NHS in this assay was 5% f 5%.
*The background binding by NHS was less than 3%;ND = not determined.
§These are IgG subfractions prepared from the sera of patients
(see Materials and Methods).
11 The IgG subfractions were not included in the calculations.
1The differences in the two assays are significant at a level of P <
0.001.
DNA by certain SLE sera in the Millipore filter assay
was observed. However, these sera were later shown to
contain precipitating antibodies and therefore were excluded from these studies.
Binding of sonicated ssDNA by the PEG and
Farr assays. Although every SLE serum tested showed
enhanced binding of sonicated dsDNA in the PEG assay, increased binding of sonicated ssDNA was not observed with all SLE sera. Indeed, two groups of sera
could be identified: in one group, an enhancement of
binding was observed in the PEG assay (Table 3); in the
other group, roughly equal amounts of antigen were
bound in the PEG and Farr assays (Table 3).
Measurement of DNA-anti-DNA dissociation
rates by PEG and Farr assays. The dissociation of complexes of anti-DNA antibody and either high molecular
weight or sonicated dsDNA upon exposure to a 200fold
excess of calf thymus DNA was studied at 24°C with
the PEG assay (Table 4). In each experiment, essentially complete reversal of binding was achieved within
2 hours. Comparison of these results with those obtained with the Farr assay was not possible since 1) the
binding of sonicated 'H-dsDNA cannot be adequately
detected in the Farr assay (see Table l), and 2) levels of
nonsonicated 'H-dsDNA binding by diluted SLE sera
as determined with the Farr assay were too low to allow
the use of this system in the kinetic experiments (Table
2). However, by employing a high titer G-200-IgG subfraction (IgG-Ri), the tendency of high molecular
weight 'H-dsDNA-antibody complexes to dissociate
could be measured by both the PEG and Farr assays. In
the PEG assay, the IgG-Ri-'H-dsDNA complexes were
81% dissociated within 2 hours at 24°C. In contrast,
these complexes were observed to be relatively stable
(only 14% dissociation) when the extent of dissociation
was measured with the Farr assay (Table 4).
DISCUSSION
Numerous investigators have used PEG in radioimmunoassays to detect immune complexes (24,29,30),
including low molecular weight ssDNA-antibody complexes (31). In the present study, the PEG assay has
proved to be more sensitive than the Farr assay in the
detection and quantitation of complexes of antibody
and sonicated DNA. Of course, the enhanced binding
of this DNA observed in the PEG assays could be the
result of artifactual binding by immune aggregates present in the SLE sera tested. However, the presence of aggregated IgG in concentrations up to 1 mg/ml failed to
increase nonspecific binding of sonicated DNA by NHS
Table 2. Binding of high molecular weight 'H-dsDNA by SLE sera;
comparison of the PEG, Farr, and Millipore filter assays*
% 'H-dsDNA bound
Patient
PEG assay?
Farr assay?
Jo
Wi
Be
Sm
46
52
20
60
23
7
23
10
Millipore
assayt*
47
49
40
ND
* Sera were diluted fivefold in these assays (see experimental section).
t Values are calculated relative to NHS controls. The background
binding by NHS in this assay was 5% 5%
$ The background binding by NHS was less than 3%.ND = not determined.
*
DETECTION OF ANTI-DNA ANTIBODIES
in the PEG assay. One should also note that nonspecific
binding in the PEG assay is not enhanced by increased
concentrations of serum protein. Finally, the results of
experiments conducted with the IgG subfractions of
sera were similar to those in which whole serum was
employed.
It is probable that the PEG assay detects antibody-DNA complexes that are labile under the high salt
conditions in the Farr assay. The experiments employing both G-200-IgG and DEAE-IgG subfractions of
sera suggest that a significant proportion of the antibodies involved are IgG. Aarden has suggested that
only antibody-DNA interactions of relatively high avidity are detected in the Farr assay (32,33). Indeed, experiments using the Farr assay conducted both in our laboratory (34) and by Aarden (18) show that complexes of
antibody and 'H-dsDNA do not dissociate significantly
upon incubation with excess unlabeled DNA at 24°C
over time periods of about one day. In fact, the results
of numerous studies using the Farr assay indicate that
even at 37°C complexes of anti-ds DNA antibody and
dsDNA dissociate rather slowly with half-lives in excess
Table 3. Binding of sonicated 'H-ssDNA by SLE sera; comparison of
the PEG and Farr assays
% 'H-ssDNA bound
Patient
He
Sh
Wr
Br
Ch
Ri
s4*
s5*
Mean* SD
Em
lo
Mo
Mr
Sm
Be
Ka
M e a n f SD
PEG assay*
Enhanced Binding
25
Farr assayj
22
66
56
0
10
0
30
1
50
25
44
29
23
3 9 f 175
Comparable Binding
14
31
49
6
72
29
14
31 f 2311
0
0
8 + 125
46
29
36
20
71
28
16
35 f 1911
Values are calculated relative to NHS controls (see Table 1). The
actual binding by NHS controls was 14% f 6% for a total of 10 sera
from 7 normal individuals.
t Values are calculated relative to NHS (see above). Actual binding
was 5 & 5% for NHS controls.
S4 and S5 are pooled samples of several SLE sera. These sera were
not used individually in this study.
8 Significant at the level of P < 0.001.
11 Not statistically significant at the P < 0.10 level.
*
223
Table 4. Dissociation kinetics of complexes of 'H-dsDNA and
antibody at 24'C
Patient
Be
Wi
Jo (di1uted)t
G-200-IgG-Ri
G-200-IgG-Ri
G-200-IgG-RQ
Wi
Jo
Nature of dsDNA*
Sonicated
Sonicated
High molecular weight
Sonicated
High molecular weight
High molecular weight
High molecular weight
High molecular weight
* Approximately
% residual
binding after
a 2 hour
Assay
reversal
PEG
PEG
PEG
PEG
PEG
Farr
Farr
15
12
4
15
19
86
92
92
15 ng of 'H-DNA were used in the experiments.
1 : 5 with borate saline buffer.
t Serum Jo was diluted
+
Complexes of high molecular weight 'H-dsDNA and IgG-Ri
were also allowed to reverse for 2 hours at 24°C and 2 hours at 4OC
prior to addition of ammonium sulfate and subsequent measurement
of DNA bound. Similar results to those shown were obtained in these
experiments.
of 3 hours (18,34). The rapid dissociation at 24°C of 'HdsDNA-antibody complexes observed with the PEG assay under conditions where little dissociation is seen
with the Farr assay suggests that a significant proportion of the antibody-DNA interactions detected in the
PEG assay are of rather low avidity. At the present
time, we cannot determine whether these antibodies
have intrinsically lower affinities for epitopes on the
DNA molecule or whether the mode of binding (e.g.
monogamous binding versus cross-linking of DNA
molecules by antibody) leads to the lower avidity interactions.
The role of low avidity antibodies in SLE is unknown, but certainly the presence of low avidity antiDNA antibodies in SLE would be expected to influence
the size and degree of lattice formation manifested by
antibody-DNA complexes. The size of the complexes,
in turn, could have a significant influence on their
pathogenic properties. It should be noted that the predisposition of particular strains of mice to produce
lower avidity antibodies to protein and nucleic acid antigens has been observed to increase their susceptibility
to immune complex disease (15, 35-37).
Several reports suggest that DNA molecules
found in the blood or in the extravascular spaces may
have molecular weights of roughly 104-106 daltons
(7,8,21-22,3840). It is possible that in vitro complexes
of antibody and sonicated DNA (5 X 10' daltons) will
have properties similar to those of antibody-DNA complexes formed in the circulation of patients with SLE.
The PEG assay, unlike the Farr and Millipore assays, is
RILEY ET AL
224
capable of detecting complexes of antibody and sonicated DNA; the PEG assay therefore should be the
method of choice for studying the properties of these
immune complexes.
ACKNOWLEDGMENTS
We wish to thank Mr. Jin Chun and Mr. Jeff Tarrand
for performing the isokinetic gradient experiments. We also
wish to thank Ms Sylvia Riley for performing the PAGE electrophoresis of IgG samples and for helpful discussions.
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