Detection of IgG antibody to deoxyribonucleic acid by the radioallergosorbent test M. Shimizu,
M.D.,
T. Yagura,
M.D.,
and Y. Yamamura,
M.D.
Osaka, Japan
IgG antibodies to heat-denatured calf thymu.s deoxyribonucleic stranded DNA :ss-DNA) in the sera of patients with’ systemic
add (DNA)
(tinglelzlpus erythematosus (SLE) could be deteoted by mans of a solid-ph,hape radi&nmunoa&%zy. Stiglestranded DNA cowpled oovalontly to m&oorystalline cellulose and leaI-labeled antibrnan y oh& were used for this test. The principle of th& test is known as the radioallergooorbent test (RAST). Thk paper desoribes the usefulness of the RAST for the detection. of human IgG antibody. Speoijicity of this test was examined by an inhibitim test using s&DNA and double-stranded DNA (&-DNA). A trial for the detection. of IgiK and IgA antibodies to ss-DNA was also attempted.
A solid-phase radioimmunoassay of antibodies to deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) using polynucleotides adsorbed to plastic cups and 3H-labeled polynucleotides has been reported by Tan and Epstein.l In 196’7, the radioallergosorbent test (RAST) for the detection of IgE antibodies against particular allergens was reported by Wide, Bennich, and Johansson.2 Theoretically, the RAST should be suitable for the detection of antibodies of all immunoglobulin classes. In the experimental animal system, this method has been used to determine IgG and IgM antibodies to dinitrophenol (DNP) hapten In the sera of atopic patients, however, IgG antibodies against allergens have not been successfully detected by the RAST, though the double-sandwich method using plastic tubes was reported by Thompson. 4 The aims of this investigation are as follows : 1. To detect autoantibodies of different innnunoglobulin classes against chemically defined autoantigens such as DNA by a solid-phase radioimmunoassay. 2. To evaluate the usefulness of the RAST for detection of IgG antibody using this DNA system. MATERIALS AND METHODS Sera Sera were obtained from 6 patients with systemic lupus erythematosus (SLE). These sera were all positive on anti-ss-DNA hemagglutination test.6 Four (A, B, C, and D) of From the Third Department of Internal Medicine, Osaka University Medical School. Presented in part at the Eighth International Congress of Allergology on Oct. 26, 18’3. Received for publication June 24, 1974. Reprint requests to: Dr. M. Shim&u, The Third Department of Internal Medicine, Osaka University Hospital, Fukushimaku, Osaka 553, Japan. Vol. 56, No. 5, pp. 356-363
VOLUME NUMBER
Detection
56 5
TABLE
I. Specificities
of ‘251-labeled
lmmunoglobulins coupled to cellulose particles’
antibodies
to human 1261-labeled
Anti-y
of
IgG
antibody
357
immunoglobulins antibodies
Anti-r
Anti-a
Anti-Fab
(IgO)
(cpm)
21,420
Co;;n;4;dded 12:087
21,732 Counts ;(f$
kG
kA
bound
to cellulose
(36.6%)t
;$j
( 1.6%)
;;;
( 3.3%)
E( ;$; f;;
Fab of IgG Rabbit 9.3
,;;
( 0.5%)
mg oi immogiobuiin-ceiluiose bound
to Ig-cellulose Counts
compiex
;7;
( 2.0%)
(70.6%)
;;
( 1.0%)
g’s’:
(41.5%)
(48.2%)
;*“4;;
(21.7%)
( 1.2%)
;;$;
(50.6%)
( 1.4%)
;;;;; :;;
and
;;
was added
( 0.5%)
f;“S ( 2.4%)
to each tube.
x 100.
added
them were found to be positive but the other 2 (E and F) were
Isolation
particles
;; ( 0.6%)
conjugates
6,642 6,516
0.6%)
;‘6 ( 0.6%)
IgG
tCounts
9,591 9,691
fragmentation
for antinuclear negative.
antibodies
by
the
immunofluorescent
method,
of immunoglobulins
Human IgG was obtained from pooled normal sera by diethylaminoethyl (DEAE) cellulose column chromatography. The IgG fraction eluted from the column with 0.0175 M phosphate buffer pH 6.3 was collected and concentrated. The protein was digested with crystalline papain (Sigma Chemical Co., St. Louis, MO.), and both Fc and Fab fragments were isolated by the method of Franklin and Prel1i.e Human IgM was obtained from the serum of a patient with Waldenstr6m macroglobulinemia by Sephadex G-200 gel filtration and digested by the hot trypsin method of Plaut and T0masi.r The Fe fragments were isolated by Sephadex G-200 gel filtration. Human IgA was obtained from the serum of an IgA myeloma patient (Hi) s by DEAE cellulose column chromatography. The IgA fraction eluted from the column with 0.01 M phosphate buffer pH 8.4 was collected and concentrated. Rabbit IgG was obtained from normal rabbit serum by DEAE cellulose column chromatography with 0.0175 M phosphate buffer pH 6.3.
Double-stranded
DNA and
single-stranded
DNA
Double-stranded calf thymus DNA (ds-DNA) from Miles-Seravac Ltd. (Maidenhead Birkshire, England) was used without further purification. Single-stranded calf thymus DNA (ss-DNA) was made from ds-DNA by heating. A solution of ds-DNA in citrate saline (3 mg/ml in 0.0015 M sodium citrate and 0.16 M NaCl) was heated at 100” C for 10 min. The solution was cooled quickly in ice water and used in the inhibition test.
Activation
of microcrystalline
cellulose
by cyanogen
bromide
Four grams of microcrystalline cellulose (Sigmacell 38 from Sigma Chemical Co., Louis, MO.) was activated by cyanogen bromide (CNBr) by the modified methods Axen, Porath and Ernback.10 The activated particles were suspended in borate-buffered saline pH 8.0 (0.2 M H,BO, _ 0.04 M NaOH - 0.16 M NaCl) for the next procedure.
St. of
358
Shim&,
Yagura,
CNBr-activated Sweden.
Coupling
and
Sepharose
of S-DNA
J. ALLERGY
Yamamura
4B
was
to activated
purchased
from
Pharmacia
microcrystalline
Fine
CLIN. IMMUNOL. NOVEMBER 1975
Chemicals,
Gppsala,
cellulose
A covalent coupling of deoxyand ribonucleic acids to agarose-derived matrix activated with CNBr has been reported by Poonian, Schlabach, and Weisshach.ll The coupling of ssDNA to activated microcrystalline cellulose was performed with some modifications. Twenty milliliters of the heated solution containing 60 mg of ss-DSA were added dropwise to the cooled suspension (0” C) containing about 500 mg of activated particles. The pH of the) mixture was adjusted to 8.4 with 4 N NaOH. The mixture was rotated end over end gtntlg for 24 hr at 4’ C. Three washing cycles were used to remove unbound substances. Each cycle consisted of a wash at pH 4.0 (0.1 M acetate buffer containing 1. M NaCl) and a wash at pH 8.0 (0.1 M borate buffer containing 1 M NaCl).
Coupling of IgG, IgM, microcrystalline cellulose
IgA,
Fab
of IgG,
rabbit
IgG
to activated
Immunoglobulins and the Fab fragment of IgG were coupled to activated microcrystalline cellulose to examine specificities of izaI-labeled anti-immunoglobulin antibodies. Fifty milligrams of each protein was added to suspension containing about 500 mg of activated microcrystalline cellulose and the mixture was rotated gently for 24 hr at 4” C, pH 8.4. Particles were washed three times, as described previously herein.
Coupling
of purified
immunoglobulins
to CNBr-activated
Sepharose
48
Immunoglobulins and the Fab fragment of IgG were coupled to CNBr-activated Sepharose 4B for purification of antisera. Five grams of CNBr-activated Sepharose 4B was suspended in 50 ml of borate saline. With continuous magnetic stirring, about 150 mg of human IgG, IgM, IgA, or Fab fragment of human IgG in 5 ml of borate saline was added dropwise to each suspension. The pH was raised up to 8.4 with 4 N NaOH. Each mixture> was rotated gently for 24 hr at 4” C and washed three times as we described previously.
Antibodies Four groups of rabbits were immunized by serial injections of the Fe fragment of IgG, the Fab fragment of IgG, and t,he Fe fragments of IgM and IgA (undigested) in complete Freund’s adjuvant, respectively. The animals were bled 2 weeks after the last immunization. The antibodies specific for y chain, p chain, LY chain, and Fab portion of IgG were prepared using the immunosorbent columns (CNBr-activated Sepharose 4B coupled with each immunoglobulin or the Fab fragment of IgG). Anti-‘y. Antiserum against Fe of IgG was passed through a SepharoseFab column to remove contaminating anti-Fab antibodies from the antiserum, and then passed through a column of Sepharose-TgG. The antibody specific for y chain was eluted with 0.17 M glycine buffer pH 3.0. Anti-p. Antiserum against Fe portion of IgM was absorbed with a Sepharose-IgG column and passed through a Sepharose-IgM column. The antibody specific for Jo chain was eluted at acid pH. Ant&. Anti-IgA serum was absorbed with a Sepharose-IgG column and passed through a Sepharose-IgA column and eluted at acid pH. Anti-Fob. Anti-Fab serum was passed through a Sepharose-Fab column and eluted at acid pII.
lodination Purified labeled with
anti-y chain, anti-p chain, anti-a 1251 by the method of McConahey
chain, and and Dixon.12
anti-Fab
of
IgG
antibodies
were
VOLUME NUMBER
56 5
Detection
of
IgG
antibody
359
4 I
1:32
DILUTION
1:4
1:256
@
1
1:32
1:256
DILUTION
FIG. 1. A, Detection of IgG antibody to ss-DNA by the RAST. Sera from Patients A (0) and 6 (xl, and from 6 healthy persons were tested for IgG antibody to ss-DNA at 3 different dilutions; 1:4, 1:32, 1:256 (final dilutions of them in the test tubes were 1:40, 1:320, 1:2,560, respectively.) The shaded area shows mean f 2 x SD of the counts of sera from the 6 healthy persons. Radioactivity added: 9,381 cpm. B, Detection of IgG antibody to ss-DNA by the RAST. Sera from Patients A (o), C, D, E. and F (0) and pooled serum from 6 healthy persons [P.S., 0-O) were tested for IgG antibody to ss-DNA at 3 different dilutions. The pooled serum denotes a mixture composed of equal amount of sera from each healthy person. Radioactivity added: 9154 cpm.
Procedures
of the
RAST
The R,AST was performed according to the method described by Wide, Bennich, and Johanssonz in 1967, with slight modifications. Step 1. One-tenth milliliter of diluted human serum (1:4, 1:32, 1:256), 0.1 ml of a suspension containing about 25 pg of ss-DNA-cellulose conjugates, and 0.9 ml of boratebuffered saline pH 7.4 containing 0.3% bovine serum albumin and 1% Tween-20 solution were mixed in a test tube (50 x 10 mm) and incubated for 15 hr at 4” C with slow vertical rotation of the tubes. The suspension was centrifuged at 3,000 rpm for 5 min and washed 3 times with borate-buffered saline. Step 8. The sensitized particles were mixed with 0.1 ml of IzaI-anti-y antibodies. The mixture was incubated, centrifuged, and washed as described in step 1. The radioactivity bound to the particles was measured in a gamma counter. All the determinations were carried out in duplicate. Inhibition
test
The inhibition tests mere performed mixing ss-DNA or ds-DNA serum just prior to the addition of ss-DNA-cellulose conjugates. The the same as already described.
solution suhsequrnt
with diluted steps were
360
Shimizu,
Yagura,
and
J. ALLERGY
Yamamura
CLIN. IMMUNOL. NOVEMBER 1975
I
0
5% ss-DNA
50ng or
500ng
5Pri!
ds-DNA/tube
FIG. 2. Inhibition tests by ss-DNA and ds-DNA. One-tenth milliliter of diluted sera (1:32) from Patients C and D were mixed with solutions containing ss-DNA (&---A for Patient C, 0-O for Patient D) or ds-DNA (A- - - -A for Patient C, O- - - -0 for Patient D) just prior to the incubation with ss-DNA cellulose conjugates. The diluted pooled serum from healthy persons was also mixed with ss-DNA or ds-DNA, but no significant differences between them were observed (Cl--0). Radioactivity added: 7,340 cpm.
RESULTS Specificities
of radiolabeled
antibodies
As a preliminary experiment, the maximum binding capacities and the specificities of radiolabeled antibodies were examined. Radiolabeled antibodies (0.1 ml) and immunoglobulin-microcrystalline cellulose conjugates (0.3 mg) were incubated in each tube for 15 hr at 4O C with slow vertical rotation. This amount was preliminarily confirmed to be enough against added ?I-labeled antibodies. The precipitates were then washed three times with borate saline. The radioactivity attached to the particles was measured in a gamma counter. The radioactivity added and that recovered on the immunoglobulin-coated particles are presented in Table I. Each number in parentheses represents t,he percentage of the counts attached to the immunoglobulin cellulose conjugates to the counts added. lz51-labeled anti-y, anti-p, and anti-cl antibodies were found to react specifically with each corresponding immunoglobulin. IgG antibody
to H-DNA
Fig. 1, A shows results of the determinations for IgG antibodies to ss-DNA on sera from 2 SLE patients and 6 healthy persons. The determinations were carried out at 3 different dilutions of the sera (final dilutions were 1:40, 1:320, 1:2,560). The shaded area in Fig. 1, A shows the normal range from sera of the 6 healthy persons (mean t 2 x SD). The counts of diluted sera from Patients A and B were found to be beyond the normal range.
VOLUME NUMBER
56 5
Detection
e
of
IgG
antibody
361
2000
?i z a 0 1000
1:4
1:32
11256
DILUTION FIG.
3.
(x) were the dilution 6 healthy
Detection tested of persons.
of for 1:4.
IgM
IgM The
antibody
antibody shaded
Radioactivity
to to area added:
ss-DNA.
ss-DNA. shows 5,467
Diluted
The mean
sera
from
Patients
serum from Patient B was rt 2 x SD of the counts of
A not sera
(0)
and
tested from
B at the
cpm.
Fig. 1, B shows results from the determinations on sera of the other 4 patients and the pooled serum from the healthy persons. The diluted sera from these 4 patients (C, D, E, F) showed much higher counts than the diluted pooled serum. The serum from Patient A was examined in both experiments, and the results were in a fairly good accordance. Inhibition
test
Fig. 2 shows results from the inhibition test by ss-DNA and ds-DNA. Diluted sera (1:32) from Patient D was inhibited to bind to ss-DNA-cellulose conjugates by both ss-DNA and ds-DNA. The antibody in the serum from Patient C was inhibited by ss-DNA but not by ds-DNA. The diluted pooled serum from the healthy persons was mixed with ss-DNA or ds-DNA, but neither significant inhibitions nor different results between them were observed. IgM
antibody
to S-DNA
The RAST for the detection of IgM antibody to ss-DNA has been carried out the same as in the ease of IgG antibody to ss-DNA, using ‘251-anti-p instead of lz51- anti-y. Results are shown in Fig. 3. The counts of diluted sera from Patient A were found to be beyond the normal range, but those of Patient B were in the normal range.
362
Shimizu,
Yagura,
and
J. ALLERGY
Yamamura
1:4
1:32
CLIN. IMMUNOL. NOVEMBER 1975
1:256
DI LUTI ON FIG. 4. Detection of IgA antibody to Z.-DNA. Diluted were tested for IgA antibody to ss-DNA. The shaded counts of sera from the 6 healthy persons. Radioactivity
IgA antibody
sera from Patients A (0) and B (x) area shows mean + 2 x SD of the added: 6,448 cpm.
to s-DNA
The RAST for the detection of IgA antibody has also been carried out the sameas in the casesof IgG and IgM antibodies to ss-DNA. In this case, lZ”I-antiO(was used. Both sera from Patients A and B were regarded as positive for IgA antibody to ss-DNA because the counts of them were found to be beyond the normal range, as shown in Fig. 4. DISCUSSION
From the results shown in Fig. 1, A and B, all sera from Patients A, B, C, D, E, and F were considered to contain IgG antibodies to ss-DNA. Results from the inhibition test suggest that these antibodies have reacted specifically with SSDNA on the microcrystalline cellulose particles since the uptakes of radioactivity by diluted sera from Patients C and D were inhibited by ss-DNA. From these experiments, it was revealed that the R.AST is applicable to detect IgG antibody to ss-DNA or other polynucleotides that are able to be attached to polysaccharide particles. On the detection of IgM and IgA antibodies to ss-DNA, however, some problems still remain. In the experiment to detect IgM antibody to ss-DNA, the normal range was wider than in the case of IgG antibody (Figs. 1, A and 3). These high counts of diluted normal sera suggest that ?I-anti-p antibodies used in this experiment might have contained antibodies directed to some serum components other than immunoglobulins. In the experiment to detect IgA antibody to ss-DNA, as well as in the experiment to detect IgG antibody to SSDNA, the diluted normal sera showed very low uptakes of radioactivity (Figs. 1, A and 4). The counts of the 1:4 diluted sera from Patients A and B were about 4.9% and 9.5% of added counts, respectively. These ratios are much lower
VOLUME NUMBER
56 5
Detection
of
IgG
antibody
363
than those seen in Fig. 1, A. However, they are significantly higher than the maximum binding capacity of lz51-anti-a to IgG or IgM (Table I). This low reactivity of lZ51-anti-a in this system may imply that it contained mainly antibodies to idiotypic antigenicities of the myeloma protein because undigested IgA was used for immunization and the absorption was made with normal IgG. The RAST has been used widely to detect IgE antibodies against particular allergens.13-I5 It is expected that IgG antibodies to allergens might be measured in sera of atopic patients by the RAST. Our results suggest this possibility. Comparative studies on sensitivities and specificities between this method and others for the detection of antibody to DNA and other polynucleotides arc now in progress. From the preliminary experiments on the detection of IgM and IgA antibodies to ss-DNA, we have obtained promising results for further developement of this test. REFERENCES Tan, M., and Epstein, W. V.: A solid-phase immunoassay for antibody to DNA and RNA, J. L’ab. Clin. Med. 81: 122, 1973. Diagnosis of allergy by an in-vitro test Wide, L., Bennich, H., and Johansson, S. G. 0.: for allergen antibodies, Lancet 2: 1105, 1967. Kishimoto, T., and Ishizaka, K.: Regulation of antibody response in-vitro. I. Suppression of secondary response by anti-immunoglobulin heavy chains, J. Immunol. 107: 1567, 1971. Thompson, R. A.: Specific antibodies in allergic subjects, Int. Arch. Allergy 45: 170, 1973. Clark, C., Bell, D. A., and Vaughan, J. H.: Anti-DNA plaque-forming cells (PFC) and rosette-forming cells (RFC) in spleens of NZB/W F1 female (B/W) mice, J. Immunol. 109: 1143, 1972. Franklin, E. C., and Prelli, F.: Structural units of human 7X gamma globulin, J. Clin. Invest 39: 1933, 1960. Plaut, A. G., and Tomasi, T. B.: Immunoglobulin M: Pentameric Fcp fragments’ released by trypsin at higher temperatures, Proc. Natl. Acad. Sci. 65: 318, 1970. Matsuoka, Y., Takata, M., and Kitagawa, M.: Idiotypic determinants of immunoglobulins. I. Antigenic homology between the heavy and light polypeptide chains from a yA myeloma protein, Immunochemistry 8: 759, 1971. 9 Yunginger, J. W., and Gleich, G. J.: Comparison of the protein-binding capacities of cyanogen bromide-activated polysaccharides, J. ALLERGY CLIN. IMMUNOL. 50: 109, 1972. 10 Axen, R., Porath, J., and Emback, S.: Chemical coupling of peptides and proteins to polysaccharides by means of cyanogen halides, Nature 214: 1302, 1967. 11 Poonian, M. S., Schlabach, A. J., and Weissbach, A.: Covalent attachment of nucleic acid to agarose for affinity chromatography, Biochemistry 10: 424, 1971. 12 McConahey, P. J., and Dixon, F. J.: A method of trace iodination of proteins for immunologic studies, Int. Arch. Allergy 29: 185, 1966. 13 Aas, K., and Johansson, S. G. 0.: The radioallergosorbent test in the in vitro diagnosis of multiple reaginic allergy, J. ALLERGY CLIN. IMMUNOL. 48: 134. 1971. 14 Berg, T., Bennich, H., and Johansson, S. G. 0.: In-vitro di&nosis of atopic allergy I. A comparison between provocation tests and the radioallergosorbent test, Int. Arch., Allergy 40: 770, 1971. 15 Lichtenstein, L. M., Ishizaka, K., Norman, P. S., Sobotka, A. K., and Hill, B. M.: IgE antibody measurements in ragweed hay fever, J. Clin. Invest. 52: 472, 1973.
M.D.,
T. Yagura,
M.D.,
and Y. Yamamura,
M.D.
Osaka, Japan
IgG antibodies to heat-denatured calf thymu.s deoxyribonucleic stranded DNA :ss-DNA) in the sera of patients with’ systemic
add (DNA)
(tinglelzlpus erythematosus (SLE) could be deteoted by mans of a solid-ph,hape radi&nmunoa&%zy. Stiglestranded DNA cowpled oovalontly to m&oorystalline cellulose and leaI-labeled antibrnan y oh& were used for this test. The principle of th& test is known as the radioallergooorbent test (RAST). Thk paper desoribes the usefulness of the RAST for the detection. of human IgG antibody. Speoijicity of this test was examined by an inhibitim test using s&DNA and double-stranded DNA (&-DNA). A trial for the detection. of IgiK and IgA antibodies to ss-DNA was also attempted.
A solid-phase radioimmunoassay of antibodies to deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) using polynucleotides adsorbed to plastic cups and 3H-labeled polynucleotides has been reported by Tan and Epstein.l In 196’7, the radioallergosorbent test (RAST) for the detection of IgE antibodies against particular allergens was reported by Wide, Bennich, and Johansson.2 Theoretically, the RAST should be suitable for the detection of antibodies of all immunoglobulin classes. In the experimental animal system, this method has been used to determine IgG and IgM antibodies to dinitrophenol (DNP) hapten In the sera of atopic patients, however, IgG antibodies against allergens have not been successfully detected by the RAST, though the double-sandwich method using plastic tubes was reported by Thompson. 4 The aims of this investigation are as follows : 1. To detect autoantibodies of different innnunoglobulin classes against chemically defined autoantigens such as DNA by a solid-phase radioimmunoassay. 2. To evaluate the usefulness of the RAST for detection of IgG antibody using this DNA system. MATERIALS AND METHODS Sera Sera were obtained from 6 patients with systemic lupus erythematosus (SLE). These sera were all positive on anti-ss-DNA hemagglutination test.6 Four (A, B, C, and D) of From the Third Department of Internal Medicine, Osaka University Medical School. Presented in part at the Eighth International Congress of Allergology on Oct. 26, 18’3. Received for publication June 24, 1974. Reprint requests to: Dr. M. Shim&u, The Third Department of Internal Medicine, Osaka University Hospital, Fukushimaku, Osaka 553, Japan. Vol. 56, No. 5, pp. 356-363
VOLUME NUMBER
Detection
56 5
TABLE
I. Specificities
of ‘251-labeled
lmmunoglobulins coupled to cellulose particles’
antibodies
to human 1261-labeled
Anti-y
of
IgG
antibody
357
immunoglobulins antibodies
Anti-r
Anti-a
Anti-Fab
(IgO)
(cpm)
21,420
Co;;n;4;dded 12:087
21,732 Counts ;(f$
kG
kA
bound
to cellulose
(36.6%)t
;$j
( 1.6%)
;;;
( 3.3%)
E( ;$; f;;
Fab of IgG Rabbit 9.3
,;;
( 0.5%)
mg oi immogiobuiin-ceiluiose bound
to Ig-cellulose Counts
compiex
;7;
( 2.0%)
(70.6%)
;;
( 1.0%)
g’s’:
(41.5%)
(48.2%)
;*“4;;
(21.7%)
( 1.2%)
;;$;
(50.6%)
( 1.4%)
;;;;; :;;
and
;;
was added
( 0.5%)
f;“S ( 2.4%)
to each tube.
x 100.
added
them were found to be positive but the other 2 (E and F) were
Isolation
particles
;; ( 0.6%)
conjugates
6,642 6,516
0.6%)
;‘6 ( 0.6%)
IgG
tCounts
9,591 9,691
fragmentation
for antinuclear negative.
antibodies
by
the
immunofluorescent
method,
of immunoglobulins
Human IgG was obtained from pooled normal sera by diethylaminoethyl (DEAE) cellulose column chromatography. The IgG fraction eluted from the column with 0.0175 M phosphate buffer pH 6.3 was collected and concentrated. The protein was digested with crystalline papain (Sigma Chemical Co., St. Louis, MO.), and both Fc and Fab fragments were isolated by the method of Franklin and Prel1i.e Human IgM was obtained from the serum of a patient with Waldenstr6m macroglobulinemia by Sephadex G-200 gel filtration and digested by the hot trypsin method of Plaut and T0masi.r The Fe fragments were isolated by Sephadex G-200 gel filtration. Human IgA was obtained from the serum of an IgA myeloma patient (Hi) s by DEAE cellulose column chromatography. The IgA fraction eluted from the column with 0.01 M phosphate buffer pH 8.4 was collected and concentrated. Rabbit IgG was obtained from normal rabbit serum by DEAE cellulose column chromatography with 0.0175 M phosphate buffer pH 6.3.
Double-stranded
DNA and
single-stranded
DNA
Double-stranded calf thymus DNA (ds-DNA) from Miles-Seravac Ltd. (Maidenhead Birkshire, England) was used without further purification. Single-stranded calf thymus DNA (ss-DNA) was made from ds-DNA by heating. A solution of ds-DNA in citrate saline (3 mg/ml in 0.0015 M sodium citrate and 0.16 M NaCl) was heated at 100” C for 10 min. The solution was cooled quickly in ice water and used in the inhibition test.
Activation
of microcrystalline
cellulose
by cyanogen
bromide
Four grams of microcrystalline cellulose (Sigmacell 38 from Sigma Chemical Co., Louis, MO.) was activated by cyanogen bromide (CNBr) by the modified methods Axen, Porath and Ernback.10 The activated particles were suspended in borate-buffered saline pH 8.0 (0.2 M H,BO, _ 0.04 M NaOH - 0.16 M NaCl) for the next procedure.
St. of
358
Shim&,
Yagura,
CNBr-activated Sweden.
Coupling
and
Sepharose
of S-DNA
J. ALLERGY
Yamamura
4B
was
to activated
purchased
from
Pharmacia
microcrystalline
Fine
CLIN. IMMUNOL. NOVEMBER 1975
Chemicals,
Gppsala,
cellulose
A covalent coupling of deoxyand ribonucleic acids to agarose-derived matrix activated with CNBr has been reported by Poonian, Schlabach, and Weisshach.ll The coupling of ssDNA to activated microcrystalline cellulose was performed with some modifications. Twenty milliliters of the heated solution containing 60 mg of ss-DSA were added dropwise to the cooled suspension (0” C) containing about 500 mg of activated particles. The pH of the) mixture was adjusted to 8.4 with 4 N NaOH. The mixture was rotated end over end gtntlg for 24 hr at 4’ C. Three washing cycles were used to remove unbound substances. Each cycle consisted of a wash at pH 4.0 (0.1 M acetate buffer containing 1. M NaCl) and a wash at pH 8.0 (0.1 M borate buffer containing 1 M NaCl).
Coupling of IgG, IgM, microcrystalline cellulose
IgA,
Fab
of IgG,
rabbit
IgG
to activated
Immunoglobulins and the Fab fragment of IgG were coupled to activated microcrystalline cellulose to examine specificities of izaI-labeled anti-immunoglobulin antibodies. Fifty milligrams of each protein was added to suspension containing about 500 mg of activated microcrystalline cellulose and the mixture was rotated gently for 24 hr at 4” C, pH 8.4. Particles were washed three times, as described previously herein.
Coupling
of purified
immunoglobulins
to CNBr-activated
Sepharose
48
Immunoglobulins and the Fab fragment of IgG were coupled to CNBr-activated Sepharose 4B for purification of antisera. Five grams of CNBr-activated Sepharose 4B was suspended in 50 ml of borate saline. With continuous magnetic stirring, about 150 mg of human IgG, IgM, IgA, or Fab fragment of human IgG in 5 ml of borate saline was added dropwise to each suspension. The pH was raised up to 8.4 with 4 N NaOH. Each mixture> was rotated gently for 24 hr at 4” C and washed three times as we described previously.
Antibodies Four groups of rabbits were immunized by serial injections of the Fe fragment of IgG, the Fab fragment of IgG, and t,he Fe fragments of IgM and IgA (undigested) in complete Freund’s adjuvant, respectively. The animals were bled 2 weeks after the last immunization. The antibodies specific for y chain, p chain, LY chain, and Fab portion of IgG were prepared using the immunosorbent columns (CNBr-activated Sepharose 4B coupled with each immunoglobulin or the Fab fragment of IgG). Anti-‘y. Antiserum against Fe of IgG was passed through a SepharoseFab column to remove contaminating anti-Fab antibodies from the antiserum, and then passed through a column of Sepharose-TgG. The antibody specific for y chain was eluted with 0.17 M glycine buffer pH 3.0. Anti-p. Antiserum against Fe portion of IgM was absorbed with a Sepharose-IgG column and passed through a Sepharose-IgM column. The antibody specific for Jo chain was eluted at acid pH. Ant&. Anti-IgA serum was absorbed with a Sepharose-IgG column and passed through a Sepharose-IgA column and eluted at acid pH. Anti-Fob. Anti-Fab serum was passed through a Sepharose-Fab column and eluted at acid pII.
lodination Purified labeled with
anti-y chain, anti-p chain, anti-a 1251 by the method of McConahey
chain, and and Dixon.12
anti-Fab
of
IgG
antibodies
were
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4 I
1:32
DILUTION
1:4
1:256
@
1
1:32
1:256
DILUTION
FIG. 1. A, Detection of IgG antibody to ss-DNA by the RAST. Sera from Patients A (0) and 6 (xl, and from 6 healthy persons were tested for IgG antibody to ss-DNA at 3 different dilutions; 1:4, 1:32, 1:256 (final dilutions of them in the test tubes were 1:40, 1:320, 1:2,560, respectively.) The shaded area shows mean f 2 x SD of the counts of sera from the 6 healthy persons. Radioactivity added: 9,381 cpm. B, Detection of IgG antibody to ss-DNA by the RAST. Sera from Patients A (o), C, D, E. and F (0) and pooled serum from 6 healthy persons [P.S., 0-O) were tested for IgG antibody to ss-DNA at 3 different dilutions. The pooled serum denotes a mixture composed of equal amount of sera from each healthy person. Radioactivity added: 9154 cpm.
Procedures
of the
RAST
The R,AST was performed according to the method described by Wide, Bennich, and Johanssonz in 1967, with slight modifications. Step 1. One-tenth milliliter of diluted human serum (1:4, 1:32, 1:256), 0.1 ml of a suspension containing about 25 pg of ss-DNA-cellulose conjugates, and 0.9 ml of boratebuffered saline pH 7.4 containing 0.3% bovine serum albumin and 1% Tween-20 solution were mixed in a test tube (50 x 10 mm) and incubated for 15 hr at 4” C with slow vertical rotation of the tubes. The suspension was centrifuged at 3,000 rpm for 5 min and washed 3 times with borate-buffered saline. Step 8. The sensitized particles were mixed with 0.1 ml of IzaI-anti-y antibodies. The mixture was incubated, centrifuged, and washed as described in step 1. The radioactivity bound to the particles was measured in a gamma counter. All the determinations were carried out in duplicate. Inhibition
test
The inhibition tests mere performed mixing ss-DNA or ds-DNA serum just prior to the addition of ss-DNA-cellulose conjugates. The the same as already described.
solution suhsequrnt
with diluted steps were
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CLIN. IMMUNOL. NOVEMBER 1975
I
0
5% ss-DNA
50ng or
500ng
5Pri!
ds-DNA/tube
FIG. 2. Inhibition tests by ss-DNA and ds-DNA. One-tenth milliliter of diluted sera (1:32) from Patients C and D were mixed with solutions containing ss-DNA (&---A for Patient C, 0-O for Patient D) or ds-DNA (A- - - -A for Patient C, O- - - -0 for Patient D) just prior to the incubation with ss-DNA cellulose conjugates. The diluted pooled serum from healthy persons was also mixed with ss-DNA or ds-DNA, but no significant differences between them were observed (Cl--0). Radioactivity added: 7,340 cpm.
RESULTS Specificities
of radiolabeled
antibodies
As a preliminary experiment, the maximum binding capacities and the specificities of radiolabeled antibodies were examined. Radiolabeled antibodies (0.1 ml) and immunoglobulin-microcrystalline cellulose conjugates (0.3 mg) were incubated in each tube for 15 hr at 4O C with slow vertical rotation. This amount was preliminarily confirmed to be enough against added ?I-labeled antibodies. The precipitates were then washed three times with borate saline. The radioactivity attached to the particles was measured in a gamma counter. The radioactivity added and that recovered on the immunoglobulin-coated particles are presented in Table I. Each number in parentheses represents t,he percentage of the counts attached to the immunoglobulin cellulose conjugates to the counts added. lz51-labeled anti-y, anti-p, and anti-cl antibodies were found to react specifically with each corresponding immunoglobulin. IgG antibody
to H-DNA
Fig. 1, A shows results of the determinations for IgG antibodies to ss-DNA on sera from 2 SLE patients and 6 healthy persons. The determinations were carried out at 3 different dilutions of the sera (final dilutions were 1:40, 1:320, 1:2,560). The shaded area in Fig. 1, A shows the normal range from sera of the 6 healthy persons (mean t 2 x SD). The counts of diluted sera from Patients A and B were found to be beyond the normal range.
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2000
?i z a 0 1000
1:4
1:32
11256
DILUTION FIG.
3.
(x) were the dilution 6 healthy
Detection tested of persons.
of for 1:4.
IgM
IgM The
antibody
antibody shaded
Radioactivity
to to area added:
ss-DNA.
ss-DNA. shows 5,467
Diluted
The mean
sera
from
Patients
serum from Patient B was rt 2 x SD of the counts of
A not sera
(0)
and
tested from
B at the
cpm.
Fig. 1, B shows results from the determinations on sera of the other 4 patients and the pooled serum from the healthy persons. The diluted sera from these 4 patients (C, D, E, F) showed much higher counts than the diluted pooled serum. The serum from Patient A was examined in both experiments, and the results were in a fairly good accordance. Inhibition
test
Fig. 2 shows results from the inhibition test by ss-DNA and ds-DNA. Diluted sera (1:32) from Patient D was inhibited to bind to ss-DNA-cellulose conjugates by both ss-DNA and ds-DNA. The antibody in the serum from Patient C was inhibited by ss-DNA but not by ds-DNA. The diluted pooled serum from the healthy persons was mixed with ss-DNA or ds-DNA, but neither significant inhibitions nor different results between them were observed. IgM
antibody
to S-DNA
The RAST for the detection of IgM antibody to ss-DNA has been carried out the same as in the ease of IgG antibody to ss-DNA, using ‘251-anti-p instead of lz51- anti-y. Results are shown in Fig. 3. The counts of diluted sera from Patient A were found to be beyond the normal range, but those of Patient B were in the normal range.
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1:4
1:32
CLIN. IMMUNOL. NOVEMBER 1975
1:256
DI LUTI ON FIG. 4. Detection of IgA antibody to Z.-DNA. Diluted were tested for IgA antibody to ss-DNA. The shaded counts of sera from the 6 healthy persons. Radioactivity
IgA antibody
sera from Patients A (0) and B (x) area shows mean + 2 x SD of the added: 6,448 cpm.
to s-DNA
The RAST for the detection of IgA antibody has also been carried out the sameas in the casesof IgG and IgM antibodies to ss-DNA. In this case, lZ”I-antiO(was used. Both sera from Patients A and B were regarded as positive for IgA antibody to ss-DNA because the counts of them were found to be beyond the normal range, as shown in Fig. 4. DISCUSSION
From the results shown in Fig. 1, A and B, all sera from Patients A, B, C, D, E, and F were considered to contain IgG antibodies to ss-DNA. Results from the inhibition test suggest that these antibodies have reacted specifically with SSDNA on the microcrystalline cellulose particles since the uptakes of radioactivity by diluted sera from Patients C and D were inhibited by ss-DNA. From these experiments, it was revealed that the R.AST is applicable to detect IgG antibody to ss-DNA or other polynucleotides that are able to be attached to polysaccharide particles. On the detection of IgM and IgA antibodies to ss-DNA, however, some problems still remain. In the experiment to detect IgM antibody to ss-DNA, the normal range was wider than in the case of IgG antibody (Figs. 1, A and 3). These high counts of diluted normal sera suggest that ?I-anti-p antibodies used in this experiment might have contained antibodies directed to some serum components other than immunoglobulins. In the experiment to detect IgA antibody to ss-DNA, as well as in the experiment to detect IgG antibody to SSDNA, the diluted normal sera showed very low uptakes of radioactivity (Figs. 1, A and 4). The counts of the 1:4 diluted sera from Patients A and B were about 4.9% and 9.5% of added counts, respectively. These ratios are much lower
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than those seen in Fig. 1, A. However, they are significantly higher than the maximum binding capacity of lz51-anti-a to IgG or IgM (Table I). This low reactivity of lZ51-anti-a in this system may imply that it contained mainly antibodies to idiotypic antigenicities of the myeloma protein because undigested IgA was used for immunization and the absorption was made with normal IgG. The RAST has been used widely to detect IgE antibodies against particular allergens.13-I5 It is expected that IgG antibodies to allergens might be measured in sera of atopic patients by the RAST. Our results suggest this possibility. Comparative studies on sensitivities and specificities between this method and others for the detection of antibody to DNA and other polynucleotides arc now in progress. From the preliminary experiments on the detection of IgM and IgA antibodies to ss-DNA, we have obtained promising results for further developement of this test. REFERENCES Tan, M., and Epstein, W. V.: A solid-phase immunoassay for antibody to DNA and RNA, J. L’ab. Clin. Med. 81: 122, 1973. Diagnosis of allergy by an in-vitro test Wide, L., Bennich, H., and Johansson, S. G. 0.: for allergen antibodies, Lancet 2: 1105, 1967. Kishimoto, T., and Ishizaka, K.: Regulation of antibody response in-vitro. I. Suppression of secondary response by anti-immunoglobulin heavy chains, J. Immunol. 107: 1567, 1971. Thompson, R. A.: Specific antibodies in allergic subjects, Int. Arch. Allergy 45: 170, 1973. Clark, C., Bell, D. A., and Vaughan, J. H.: Anti-DNA plaque-forming cells (PFC) and rosette-forming cells (RFC) in spleens of NZB/W F1 female (B/W) mice, J. Immunol. 109: 1143, 1972. Franklin, E. C., and Prelli, F.: Structural units of human 7X gamma globulin, J. Clin. Invest 39: 1933, 1960. Plaut, A. G., and Tomasi, T. B.: Immunoglobulin M: Pentameric Fcp fragments’ released by trypsin at higher temperatures, Proc. Natl. Acad. Sci. 65: 318, 1970. Matsuoka, Y., Takata, M., and Kitagawa, M.: Idiotypic determinants of immunoglobulins. I. Antigenic homology between the heavy and light polypeptide chains from a yA myeloma protein, Immunochemistry 8: 759, 1971. 9 Yunginger, J. W., and Gleich, G. J.: Comparison of the protein-binding capacities of cyanogen bromide-activated polysaccharides, J. ALLERGY CLIN. IMMUNOL. 50: 109, 1972. 10 Axen, R., Porath, J., and Emback, S.: Chemical coupling of peptides and proteins to polysaccharides by means of cyanogen halides, Nature 214: 1302, 1967. 11 Poonian, M. S., Schlabach, A. J., and Weissbach, A.: Covalent attachment of nucleic acid to agarose for affinity chromatography, Biochemistry 10: 424, 1971. 12 McConahey, P. J., and Dixon, F. J.: A method of trace iodination of proteins for immunologic studies, Int. Arch. Allergy 29: 185, 1966. 13 Aas, K., and Johansson, S. G. 0.: The radioallergosorbent test in the in vitro diagnosis of multiple reaginic allergy, J. ALLERGY CLIN. IMMUNOL. 48: 134. 1971. 14 Berg, T., Bennich, H., and Johansson, S. G. 0.: In-vitro di&nosis of atopic allergy I. A comparison between provocation tests and the radioallergosorbent test, Int. Arch., Allergy 40: 770, 1971. 15 Lichtenstein, L. M., Ishizaka, K., Norman, P. S., Sobotka, A. K., and Hill, B. M.: IgE antibody measurements in ragweed hay fever, J. Clin. Invest. 52: 472, 1973.
