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Chapter XI
DNA Antibodies
DR. SELIGMANN:Four or 5 years ago when we presented our work about
DNA antibodies in 1upus,1 many people thought we should have quotation
marks on both “DNA” and “antibodies.”As far as quotation marks on DNA are
concerned, I think everybody now agrees that the reaction is with DNA itself
and there is no need to discuss it; it is precisely because we are dealing with
a relatively pure substance that so much interest in the subject was raised in
different laboratories.
As for the antibody nature of these factors in lupus, I think (just as in the
case of the rheumatoid factor) that this meeting presents a good opportunity
for anyone who has strong evidence against the antibody hypothesis to give
it during the discussion. This is also a reason why I think it useful to report
briefly the different findings which we think confirm that anti-DNA antibodies
in lupus are true antibodies.
First, they are detectable by quite different immunologic technics such as
precipitation in liquid or gel medium,2v3 complement fixation4-0 and passive
cutaneous anaphylaxis reactions.1° Second, the precipitin curves that were
obtained if the pH was above 7 were quite analogous to those of an immunologic reaction and we could recover in the precipitate all the DNA in
the antibody excess zone.
Third, and we thought this the most important, it was possible to extract
by action of deoxyribonuclease the proteins (or at least a great part of the
proteins) linked to DNA in the washed precipitate, and it was easy to demonstrate that this protein was immunochemically a 7s y-globulin.ll This finding
DNA antiwas exactly confirmed by Deicher, Holman, and K ~ n k e l These
bodies, in all cases of lupus sera we studied, appeared to be immunologically
7 s 7-globulins and behaved as such on DEAE columns.
Now there are two disconcerting facts which well could be against the
antibody hypothesis: ( 1 ) The fact that apparently these antibodies lacked
specificity, in that we had similar positive reactions with human DNA, phage
DNA, bacterial DNA, and so on. In Ouchterlony plates we saw a continuous
line of precipitation with these different kinds of DNA.”
( 2 ) The fact that it was extremely difficult, if not impossible, to obtain DNA
antibodies in immunized animals.
We have some new results on these two subjects which I am sure will be
important topics for us to discuss.
The apparent lack of specificity for different kinds of DNA led us 2 or 3
years ago to try to look for the reactive sites on the DNA molecules.12I would
Iike to present some of these results even though they are rather old because
some of them show discrepancies with some of Dr. Levine’s results and it may
be stimulating for discussion.
g globulines cpg)
Serum No 1635
DNA dWnt6grb
par ultra Sons
DNAparmldeserum()LQ 1
Fig. 1.-Precipitin curve of DNA degraded by ultrasonic waves with fragments.
Although we did not use as sensitive technics, we saw, just as he did, that
considerably denatured and degraded DNA still reacted with the lupus antibodies. But when we studied DNA degraded by ultrasonic waves with fragments which should be approximately 300,000 molecular weight, we had a
positive complement fixation reaction and the precipitin curve (fig. 1) was
very similar to that obtained with native DNA except that, as you could expect, in the antigen excess zone it went down more quickly. This kind of
curve was found with four lupus sera. However, Dr. Levine only found inhibition with sonically treated DNA.
Now, you can notice on this curve the very high ratio of protein to DNA
in weight and this means a considerably high molecular ratio, although what
we used here is what we thought to be native calf thymus DNA. It may have
contacted some denatured DNA but in any case it is not easy to understand
this tremendous ratio.
Another discrepancy is that when we destroyed DNA by DNase, the end
products obtained with high amounts of DNase not only did not give us any
positive precipitin or complement fixation reactions but were unable to inhibit the reaction even when 500 times more than the amount of native DNA
required for absorbing all precipitating antibodies was added.
What we saw also in this study is that when we used acidified DNA at pH
1.7 without heating, we had no decrease of the immunologic reactivity. When
we used DNA which was first acidified-and then heated for increasing times,
we had no more reactivity and at the end we were unable to inhibit the reactions with an amount 80 times more than the amount of native DNA required for neutralizing the precipitating antibodies ( table 1). Therefore, we
thought that the purine bases were involved in the reactive sites. I may add
that RNA did not inhibit the reactions with DNA.
Perhaps some of these discrepancies mentioned are due to the fact that
from one serum to another and from one patient to another we are dealing
with different kinds of DNA antibodies.
Table 1.-Reactions with Acidified and Heated DNA
Reactions with Lupus Serum
Precipita- 70Purine
tion by GI04
H0,K N
Complement of precipitating
2xn =
3xn =
20xn =
80xn = 24h
80xn = 24h
*n = minimum amount of native DNA necessary for neutralization of the precipitating
Another important point in Dr. Levine’s studies is that in some way we
are able to see that the reaction with the different kinds of DNA is also different quantitatively from one DNA to another.
As far as the production of anti-DNA antibodies in immunized animals is
concerned, all our attempts in rabbits were unsuccessful regardless of the
method employed.
I think that until the work of Dr. Levine with T4 phage DNA, most of the
positive results in the literature were at least open to discussion. This work,
however, gives the first conclusive evidence that you could obtain DNA antibodies despite the fact that the antibodies were a particular kind of DNA
antibody specific to glucosylated DNA.13
Now, the fact that we are able to induce formation of anti-DNA antibodies
in animals gives stronger evidence that you may have true antibodies to DNA
in lupus but does not mean at all that these anti-DNA antibodies are pathogenic and noxious in the patients. As Dr. Dameshek noted, I do not agree
with the term “connective tissue disease,” but neither do I agree with the
term “autoimmune disorder.” I think that all we can say is that lupus is a
disease with autoantibodies and, among others in some cases, with anti-DNA
antibodies and nothing more.
But the studies on experimentally produced anti-DNA antibodies may be
very important and interesting for future research about the eventual pathogenic role of the anti-DNA antibodies in lupus. First of all, I think that with
these kinds of antibodies (even if they do not come from lupus patients) we
may perhaps have a definitive answer to this very important question: Is an
antinuclear antibody able to penetrate a living cell? I think we have, as far
as lupus antinuclear factors are concerned, much evidence against it, but this
question is still open to discussion and has not yet been definitely solved.
I think also that the soluble complexes eventually formed in vivo in lupus
patients (complexes between different antinuclear factors, and perhaps especially anti-DNA and the homologous antigens) may be pathogenic and
lead to formation of vascular lesions similar to those of Dr. Dixon’s model. I
think that it would be very important to detect the antigens in these lesions
where we find y-globulins and complement; among these antigens, it would
Table S.-Positive Reactions with DNA (per cent)
Systemic Lupus Erythematosus
(217 samples from 93 patients)
Before treatment
Under treatment but no real
Under treatment and great
Apparent clinical remission
Per cent patients giving, at laast once,
a positive reaction
Other Dkemes
Rheumatoid arthritis (73 patients)
Scleroderma, polyarteritis nodosa,
dermatomyositis, discoid lupus,
Miscellaneous (300 patients)
be perhaps relatively easy to detect DNA and the experimentally produced
DNA antibodies could be useful for this purpose. I would like to know, during the discussion, if anyone has some evidence for the presence of DNAanti-DNA soluble complexes in the blood of lupus patients. We have had
some evidence for it, but we could not draw from our experiments definitive
Aside from these very interesting theoretic problems, there are a few
practical questions we could well ask: Are DNA antibodies useful for the
diagnosis of lupus?
I would first like to show our results (table 2 ) . I think it is extremely important in lupus to give results in connection with the stage of the disease.
This is especially true for DNA antibodies. The spectrum of antibodies present
in lupus sera differs considerably if you have untreated patients or patients
already in treatment or patients in remission. That is why we give our results
by number of samples and according to the stage of the disease.
You can note that 75 per cent of the patients with definite lupus, prior to any
treatment, gave precipitin and complement fixation positive reactions; when
they are treated and improve (but not in remission), this incidence falls
to 10 or 12 per cent; in remission the reaction was never positive. Now, in
the other diseases we had altogether one single serum giving a weak positive
reaction with these technics: this was a rheumatoid arthritis patient who
had L.E. phenomenon but not its clinical nor pathologic features.
Since last year, and after the. paper of B o z i c e v i ~ h ,we
~ ~ have worked
with the bentonite flocculation test. We had much difficulty the first
months, but now we have reproducible results. This test does indeed increase
the sensitivity of the reaction and we have still some positive sera during
remission. The problem was to see if with this increase in sensitivity we had
more specificity for lupus. To the present we found only 5 per cent positive
results in cases of rkeumatoid arthritis (patients were selected on the basis
of also having positive L.E. phenomenon).
I am quite aware that we have a much higher percentage of positive reactions with DNA than most people in this country have reported. At the beginning I selected nontreated cases in order to study anti-DNA antibodies, but
now this contains a wider sampling and I can only present my data even if
they are not completely in accordance with yours. Our tests were made at
a pH of 7.5 or 7.8.
SPEAKER:Were these rheumatoid arthritis patients examined for L.E. cells
and for antinuclear factors?
Yes, they were examined for L.E. cells. The 5 per cent are
all patients with positive L.E. phenomenon and they all have antinuclear factors in fluorescence; the incidence of antinuclear factors in fluorescence is of
course much higher than these 5 per cent. The one patient giving a positive
reaction in complement fixation is a true rheumatoid arthritis patient with
L.E. phenomenon.
DR. LEVINE:While investigating" the immunochemical nature of T2 bacteriophage, it was found that rabbits immunized with alkali ruptured bacteriophage produced antibodies to DNA. The anti-DNA in these sera reacted
more effectively with denatured (single strand) than native (double strand)
DNA.13J6These bacteriophage DNA antibodies were specific for the DNA of
T2, T4, and T6 bacteriophage,16 and did not react with DNA preparations
from about 40 other sources. It was found subsequently that the antibodies
to phage DNA were directed in part to the glucosyl moieties17J8(either a, f3
or diglucosyl residues of the hydroxymethylcytosine unique to these DNAs ) .
It appeared that in the native, double strand, helical form, antigenic determinants of DNA were less available for reaction with their homologous antibodies. As a result of collapse of the double strand helix, these antigenic
determinants were unmasked and reacted more effectively with anti-DNA.
If the antigenic determinants are masked in the helicaI form of DNA, tlte
single strand state should be more reactive in other DNA immune systems.
Dr. David Stollar looked at the other DNA immune systems known at that
time; the DNA reaction with anti-DNA present in SLE sera4-6,9J2
just discussed
by Dr. Seligmann.
The data shown in figure 2 describes the complement ( C ) fixation of six
SLE sera with native and thermally denatured T4 phage DNA. It can be seen
that denatured DNA reacted more effectively than native DNA.l9 In general
most of the SLE sera gave these results, however, some of the SLE sera fixed
C' with native DNA (although more fixation was observed with denatured
D N A ) , and one SLE serum reacted as well with native as denatured DNA,
It must be remembered that this C' fixation technic20 used diluted antiserum
(1:500with serum C.C. in figure 2 ) . More concentrated SLE serum does
give C' fixation with native DNA.
"This is publication No. 207 from the Graduate Department of Biochemistry, Brandeis
University, Waltham, Mass. Aided in part by grants from the National Institutes of
Health (E-1940)and the American Cancer Society (E-222).
Serum Hu.
Serum N.A.
Fig. 2.-C' fixation by LE sera and varying quantities of native and thermally
denatured DNA. 0- - 0,
native DNA; 0 - - 0, thermally denatured DNA.2f
The anti-DNA in SLE sera reacted with about 40 DNA preparations from
various sources,z1 confirming the earlier ~tudies.~J'
Quantitative differences
were observed, however, both in the extent of C fixation by a given serum
with denatured DNA of different origin and the amount of C' fixation by
diflerent SLE sera with native and denatured DNA. Again, with a single SLE
serum, denatured was a more effective antigen than native DNA.21 The
variation found in SLE sera was ascribed to differences in specificity of antibodies and the variation from DNA to DNA with respect to reactivity of
native and denatured DNA was probably due to variable extents of single
strand areas in our DNA preparations.
Dr. Stollar next attempted to identify the antigenic determinants of the
DNA, DNA digested by pancreatic deoxyribonuclease did not fix C with
the anti-DNA in SLE sera. The digests, however, did inhibit the homologous
DNA-anti-DNA reaction although the amount of enzymatic digest required
for inhibition varied from serum to serum. With two SLE sera, pyrimidine
oligonucleotides (prepared by treatment of DNA with diphenylamine and
formic acid) and the enzymatic digest were equally as effective for inhibition
of the DNA-anti-DNA reaction. It appeared that the antigenic determinants
Fig. 3.-C' fixation inhibition of serum M.M. and denatured BaciUus natto DNA
by increments of pyrimidine oligonucleotides.22
in DNA which reacted with the antibodies in these two SLE sera were
Pyrimidine oligonucleotides of varying chain lengths were isolated by
DEAE chromatography and tested for inhibition of an L.E. antibody. The
effectiveness of inhibition increases with increasing size of oligonucleotide, up
to the pentanucleotide.22The oligonucleotides from each chromatographic
peak were separated by paper chromatography according to cytosine and
thymine content. Inhibition by the separated pyrimidine tetranucleotides is
shown in figure 3. The effectiveness of inhibition reflects the thymine content
of the oligonucleotide. When polythymidylic acids of varying chain lengths
were tested for inhibition it was found that the pentathymidylic acid was
only slightly more effective than the tetrathymidylate.22
Most of the SLE sera we have studied are inhibited by enzymatic digests
much better than pyrimidine oligonucleotides,23suggesting that purine oligonucleotides or purine-pyrimidine oligonucleotides are antigenic determinants.
The earlier studies of Dr. Seligmann12 had also suggested the antigenic role
of the DNA bases. We are presently attempting to isolate and identify purine
oligonucleotides from apyrimidinic acid and oligonucleotides from enzymatic
The anti-DNA in one SLE serum (G.B.), which we thought might be
directed to a small antigenic determinant because its C' fixation response
resembled quantitatively that obtained by Butler et al.24with antibodies to
puronyl-conjugated bovine serum albumin, was investigated. These antibodies
in the serum (G.B.) were characterized as anti-DNA by their lability to
pancreatic deoxyribonuclease and their greater reactivity with denatured
Fig. 4 . 4 4 C' fixation by serum G. B. (1/200) and Bacdlus nutto DNA kept for
10 min. at O", 51", 65", 73",82' ( a ) ,91" ( A ) , and 100" ( O ) ,and then quickly
chilled. (b)Immunologic thermal denaturation profile of Bacil2us natto DNA as measured by serum G. B.
DNA.23It can be seen in figure 4 that the C' fixation can be used to follow
the transition of BaciEZus mtto DNA from the native double strand to single
strand state.
This serum was analyzed by inhibition with deoxyadenylic, deoxyguanylic, thymidylic and deoxycytidylic acids. The quantity of nucleotides
giving 50 per cent inhibition was found to be 3Q, 230, 400, and 1000 mpmoles
respectively, suggesting that adenylic acid was part of the antigenic determinant reacting with the anti-DNA in G.B. Further studies showed that the
adenine moiety of deoxyadenylic acid was the active inhibitor.
The data in table 3 show the inhibition of serum G.B.and denatured calf
thymus DNA by purine derivatives, histidine and chloroquin. The more effective inhibition by some of the purine analogues and especially theobromine
and theophylline raises the possibility that the DNA-anti-DNA in G.B.serum
is really a cross-reaction. For example, if G.B.had produced antibodies to
theophylline of theobromine, the C' fixation with denatured DNA could be
due to the structural similarity of adenine and these purine analogs.
Chloroquin is also an effective inhibitor of the DNA-anti-DNA in G.B.serum (50 per cent inhibition with 50 mpmoles of chloroquin). Unlike the
nucleotides and nucleosides, chloroquin also inhibits the DNA-anti-bacteriophage DNA reaction. Dr. Stollar has demonstrated that chloroquin inhibits
the DNA-anti-DNA reaction by binding not to the anti-DNA but to the
DNA.25The binding was measured by equilibrium dialysis, fluorescence, and
spectrophotometric technics and by inhibition of bacterial DNA transformation. Holman has shown that chloroquin inhibits the L.E. cell phenomenon.2*
SELIGMANN:Could you tell us how many different reactive groups you
were able to find for such a number of sera?
Second, I wonder if it wouldn't be useful to perform with strong sera
Table 3.-C% Inhibition of Serum G.B. (1/200) and Denatured Calf Thymus DNA
b y Purine Derivatives, Histidine, and Chloroquin
mpmoles Inhibitor Required
for 60% Inhibition
precipitin curves both with native DNA and with Sins-Heimer +x-174 single
strand DNA. We were only able to see that it gives strong reactions but we
did not have enough of the material to perform quantitative studies.
Third, have you studied reactions between the DNA antibodies of a particular patient and his own DNA extracted from his cells? This might be
quite interesting because I wonder if this is the homologous DNA.*
DR. LEVINE:With the L.E. system we have never performed quantitative
precipitin experiments. We have looked at the DNA from +x-174 bacteriophage. This DNA is in the single strand state as isolated from the purified
phage. This DNA gave identical fixation with native and thermally treated
DNAz1when tested with an SLE serum such as in figure 2.
Have you tried any synthetic derivatives of either DNA or
Yes, we have. The anti-DNA in serum G.B. is inhibited by
polyadenylic acid. It was no more effective than monomeric adenylic acid in
terms of the number of purine residues required for a given degree of inhibition. With the anti-DNA in LE sera directed to pentathymidylic acid, poly
AT was not inhibitory. Apparently the inhibitor must have iininterrupted
thymidine tracts.
We have not performed inhibition studies with Kharana’s synthetic poly-T‘s..
What do you mean by single strand? I know that your meltDR. TOMASI:
ing temperatures look like Doty’s melting temperatures. Is this coiled in a
OHijmans and Klein (unpublished results) have recently had the opportunity to answer
this question. Liver and spleen were obtained within 2 hours after death of a patient
suffering from SLE whose serum contained antibodies precipitating with DNA. DNA was
prepared from these organs by the detergent method of Zamenhof. Calf thymus DNA was
prepared by a similar method. Figure 5 shows the precipitin curves (at pH 8.5) obtained
when the serum of this patient reacted with autologous and heterologous DNA, before and
after ultrasonic breakdown. The shape of the different curves in the region of antigen excess,
quite different from those of figure 1, remains unexplained. In any case, there are no indications from this experiment that autologous DNA has a greater affinity for the antibody
than heterologous DNA.
.* = CALF
L -
Fig. 5.-Precipitation of LE serum B with DNA.
single strand helix? Or do you think this is a random specific distribution in
terms of tertiary structure?
That is a good question. The temperature profile of DNA obtained immunologically is identical to that obtained by Doty et aL2' using
physical measurements. There is a 2-3" displacement to lower temperature
when compared to the physical data obtained at ambient temperatures. The
nature of the DNA along this temperature profile is thought to be ( 1 ) a
double strand helix; ( 2 ) a state best described as double strand with single
strand areas or bubbles; and ( 3 ) completely separated strands. The completely separated strands on quick chilling can coil over on itself, but all of
the antigenic determinants are not masked in this state. Formaldehyde prevents the intramolecular bonding and increases the serologic response of the
denatured DNA slightly.28 The DNA containing bubbles may be metastable.
If you allow the separated DNA strands to incubate at conditions which
favor recombination to double strands (renaturation), you lose C' fixing
capacity. The similarity of denaturation and renaturation of DNA measured
immunologically and physically are two of several criteria we use to identify
the antibody as anti-DNA.
DR. SELIGMANN:When you say 'lose immunologic activity," it is not a
complete loss?
DR.LEVME:Under the conditions of our C fixation assay, loss or gain of
serologic activity is demonstrated by comparison of C' fixed by the completely separated strands ( a calibration curve) and the C' fixed by the DNA
preparations subjected to various experimental conditions. The amount of
denaturation is determined by the ratio or thermally denatured (loo" for
10 minutes ) to the experimentally treated sample required to give equivalent
fixation in the region of antibody excess.
DR. HOLMAN:When you cool rapidly you get separate strands-do they
remain separated?
DR. LEVINE:Yes. We routinely separate the strands by boiling DNA at
about 5 pg. per ml. at 0.15M salt concentratian and pH 7.5. The solution is
rapidly chilled in an ice bath. The strands remain separated by this procedure. With DNA preparations of high guanine and cytosine content one may
have to increase the temperature or decrease the salt concentration or both
to get separate strands rather than double strand DNA with “bubbles.”
If you use tetrathymidylic acid and it reacts with the antiDR. HEIMER:
body like a hapten, as in this case, do hapten antibody reactions fix complement?
I do not think a hapten antibody reaction in which a three-dimensional
complex is not formed will fix complement.
DR. LEVINE:In the precipitin analyses of Dr. Seligmann, if you use calf
thymus DNA, and assume it is 6 million molecular weight, you would have
a mole ratio in extreme antibody excess of about 50,000 antibodies per DNA
molecule. Is this not out of proportion?
We have made the same calculation and have found in
the extreme antibody excess zone a molar ratio of 3,000 to 5,000 with the
different sera which have been studied and at the equivalence zone a molar
ratio cf 1,200 to 2,500. I suppose you mean 5,000 to 1. Nevertheless, we were
astcnished by this high ratio in these precipitin curves performed at a pH
above 8.
I remember that in Dr. Deicher’s quantitative precipitin analyses showing
the recovery of DNA in the precipitin, the mole ratios were also very high.
May I intrude on a hematologic note in which DNA
hypersensitivity appears to be the central feature. In a patient with many
bouts of a peculiar hemorrhagic eruption, Levin and PinkuP demonstrated
a hypersensitivity to DNA. More recently we studied a similar patient30 with
the help of Dr. Levine. In our patient there was an extraordinary skin lesion
which eventually resulted in disability, She would break out with enormous
hemorrhagic welts; these were finally traced to an unusual DNA hypersensitivity. The lesions could be reproduced very quickly with tiny amounts
of DNA of various types; there was no reaction to RNA, and DNase acting
upon DNA stopped the sensitivity reaction. There were no humoral antibodies.
Apparently, these were cell bound antibodies and the reactions occurred
only on the arms and legs. We transplanted skin from the arms or legs to the
back and there was no reaction there; but, on the other hand, skin from the
back transplanted to the forearms did result in a reaction. The patient was
treated with chloroquin with a remarkable response and she is now well.
DR. BUTLER:The studiesZ4 I would like to discuss were carried out in the
laboratories of Dr. Sam M. Beiser and Dr. Stuart W. Tanenbaum in the Department of Microbiology at Columbia University. Like other investigators,
Dr. Beiser has been interested for the past decade in making antibodies to
DNA experimentally and to date he also has been unsuccessful.
Before I arrived at Columbia, Dr. Beiser along with Drs. Erlanger and
Tanenbaum had tried to elicit antibodies to certain purines and pyrimidines
but without much success in terms of specificity.
During the past year Dr. Sasson Cohen and Dr. Aaron Bendich at the
Sloan-Kettering Institute synthesized a purine which has proved quite useful
in our work. The compound which they used was 6-trichloromethyl purine.
This compound is very readily synthesized from &methyl purine by a straightforward chemical reaction.
In studying this compound in glycine HCl buffers, Dr. Cohen noted that
it reacted very readily with amino groups under slightly alkaline conditions
and at room temperature as shown below:
c1 -c 4 1
When R is an amino acid, the product in the above reaction is a purinoyl
amino acid. In other words there is now a carboxyl group attached to the
6-carbon of the purine ring and linked by a peptide-like bond to the amino
group of the amino acid.
It became apparent that this compound might serve our purposes quite
nicely in terms of conjugating it to a protein carrier. And it turns out that
in bovine serum albumin (BSA), there are about 59 available epsilon-amino
residues of lysine to which it might be possible to conjugate the 6-trichloromethyl purine.
Accordingly, we added this compound to BSA under slightly alkaline conditions at room temperature and, without going into the chemistry of it, we
were able to get on about 25 residues of our purinoyl compound per molecule
of BSA. The conjugate with human serum albumin (HSA) was also prepared
in a similar manner.
The reason we were not satisfied with using a compound which will be
linked to protein in the 6-position is because in DNA the purine is linked to
the sugar by the &position. However, we felt, since we had a conjugate with
which we could work, that if we could elicit antibodies to this hapten, this
would be encouraging for further studies whereby we might investigate
compounds that are linked at the 9-position.
The purinoyl-BSA conjugate in Freund's adjuvant was injected into rabbits
in three weekly injections. All animals injected developed large amounts of
antibody which precipitated both with purinoyl-BSA and purinoyl-HSA. Surprisingly little of this antibody precipitated either with BSA or HSA alone
which made us believe that the antibody being elicited had great specificity
for the purinoyl residues. This was confirmed by appropriate hapten inhibition studies in which amino acids were quite effective as inhibitors of this
precipitin reaction.
We then studied these antisera with DNA from various sources, both native and denatured. In precipitin reactions we have been totally unsuccessful
in detecting any precipitation whatever with any of the DNA preparations
studied to date.
However, with Dr. Levine’s complement fixation technic, on addition of
thermally denatured pneumococcic DNA or denatured DNA from a number
of bacterial sources to anti-purinoyl sera, we observed complement fixation.
Furthermore upon the addition of unheated single strand +x-174 DNA we
got complement fixation.
For example, in a typical antiserum we observed the fixation of complement by a 1:400 serum dilution with denatured pneumococcic DNA. There
was essentially no reaction with native pneumococcic DNA at a 1:400 dilution of this serum. With this particular antiserum we did get slight amounts
of fixation with native preparations of pneumococcic DNA at a dilution of
1:lOO. It required 4 pg. of native DNA to get about 70 per cent fixation of
complement, whereas, with the 1:100 dilution of this particular antiserum,
we got similar fixation of complement with 0.03 pg. of the denatured DNA.
To date we have detected no reaction with DNA either by precipitation
or by complement fixation reactions. It may be that the molecular weight of
the RNA is too small to evince fixation in this system. We have tried various
methods of “denaturation” of the RNA according to some protocols suggested by Dr. Bendich, but as yet we have detected no complement fixation
I might also mention that this complement fixation reaction is inhibited
quite specifically by our purinoyl amino acids. We found essentially 100 per
cent inhibition of a typical complement fixation reaction by less than 1/100
of a pmole of purinoyl epsilon-amino caproic acid which is structurally the
closest derivative to purinoyl epsilon-amino lysine that we have available at
the current time. We get less striking inhibition by adenine and other purines
and very poor inhibition by pyrimidines.
L.E. preparations have not yet been performed on these antisera.
We have actually been interested in these antisera for purposes other than
the study of lupus erythematosus. Globulin fractions from these antisera have
already been shown to inhibit DNA-dependent bacterial transformation in a
pneumococci. Drs. Galis and Beiser have carried out this study. Drs. Bendich,
Rosenkrantz, Borenfreund and others are studying the possible effects of
these antibodies on various viral and tumor DNA preparations. We have also
instituted some immunohistochemical studies.
Obviously, the extension of this type of work is to conjugate better haptens
to proteins, compounds that have more resemblance to the structures in DNA.
This is not going to be easy; however, it is a highly promising area for further
You said that you had no complement fixation with RNA.
Did you look for inhibition?
DR. BUTLER:We have not completed these studies but we have not detected any inhibition so far.
DR. SELIGMANN:Did you refer to studies with fluorescent antibodies or
nuclei? And would you comment on the pyrimidine conjugates with which
you are now studying?
Dr. Pappas has been conducting some preliminary studies and
has observed some fluorescent antinuclear staining but he is not yet prepared
to comment on its specificity and significance.
Dr. Tanenbaum has prepared another conjugate, a pyrimidine-protein conjugate. Antisera to this conjugate also react with denatured DNA. (These
antisera are currently being studied by Drs. Tanenbaum and Beiser.)
How do you know that your site of attachment is a protein?
Have you actually evidence it is attached to an amino group? Could it not
attach to a serine hydroxyl group?
Dr. Cohen does not feel that this particular reaction occurs.
He does feel that there is some possibility that it may react with other groups
but he thinks that the reaction with amino groups is by far the most important. Also, the fact that the purinoyl epsilon-amino caproic acid is the
most effective inhibitor in both the precipitation and complement fixation
systems would indicate that this is the predominant means of attachment.
Spectrophotometric data also point to purinoyl-amino residues in the conjugate.
Do you know how many groups you had on your antigens
that were so antigenic?
Both by estimation from spectrophotometric measurements
and by actual alkaline digestion of the conjugate, we have determined that
both the BSA and HSA conjugates contain about 25 purinoyl residues per
protein molecule.
We do Folin reactions which I have to convert each time and it strikes
me that we have got probably 800 pg, of antibody nitrogen per ml. and most
of anti-purinoyl; less than 5 per cent in most antisera is anti-protein. It is
quite unusual in that respect. With some of the other haptens studied in our
laboratory we had not observed this high a percentage of antibody directed
against our hapten.
I think the answer to Dr. Tomasi's question is that the conjugation occurs in alkali and in esters. An ester would be formed in alkaline
wnditions and esters are not stable in alkali. That is probably the reason
why amides could be stable under those conditions but esters could not.
I am curious why you did not get a lysine derivative.
Six-trichloromethyl purine reacts with both amino groups of
lysine. We have tried to block the alpha-amino group in an attempt t o prepare the epsilon-amino derivative. We have not yet been successful, but expect to synthesize this compound in due time. The available hapten which
most closely resembles purinoyl epsilon-amino lysine is purinoyl epsilonamino caproic acid. This derivative can be readily synthesized and is an extremely effective inhibitor of anti-purinoyl sera.
W e have tried copper chelate but this did not work. It should be remem-
bered that copper has some peculiar interactions with purines, so that failure
to obtain the epsilon-purinoyl derivative from the copper chelate is not too
DR. SELIGMANN:Does anyone have new data or ideas about the eventual
pathogenicity of antinuclear antibodies in lupus patients?
DR. KAPLAN: I can report a negative experiment. Just as you might- localize rheumatoid factor in the tissues by using a labeled reactant, we have
labeled isolated beef thymus-nucleoprotein-in various ratios with fluorescein-hoping that this product might be useful for the localization of at least
antinucleoproteinfactors in the tissues. It turns out that at all ratios of labeling fluorescein to nucleoprotein, the nucleoprotein loses its reactivity completely with L.E. sera. So, whatever the reason, the resulting labeled material
is no longer reactive with L.E. sera.
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23. -,
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