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Spectrophotometric assay for the detection of antibodies to DNA
Immunology Letters, 7 (1983) 25-27
Elsevier
SPECTROPHOTOMETRIC
ASSAY
FOR THE DETECTION TO DNA
OF ANTIBODIES
Asif ALI and Rashid ALI Department of Biochemistry, Faculty of Medicine, Aligarh Muslim University, Aligarh 202001, India
(Received9 March 1983) (Modified versionreceived3 June 1983) (Accepted 6 June 1983)
1. Summary A spectrophotometric assay for the detection of anti-DNA antibodies in the sera of patients with various connective tissue diseases is described. The sensitivity of the method was compared with hemagglutination and enzyme-linked immunosorbent assay. The method is convenient, specific and suitable for use in clinical laboratories where facilities for radioimmunoassay do not exist.
2. Introduction Autoantibodies to intraceUular proteins and nucleic acids are a hallmark of certain autoimmune diseases which include systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), progressive systemic sclerosis (PSS) and Sjogren's syndrome (SS). Patients with SLE spontaneously produce antibodies directed against double-stranded DNA (dsDNA). A simple and sensitive laboratory procedure for the detection of circulating DNA antibodies in the sera of patients which SLE is of great clinical significance for the diagnosis and management of such patients. Various techniques have been developed for the measurement of these antibodies [ 1,2]. The most widely used technique is radioimmunoassay of Farr [3] involving the use of labelled DNA and sophisticated radioactive measurKey words: spectrophotometricassay- anti-DNA antibodies -
systemiclupus erythematosus 0165-2478/ 83/ $03.00 © ElsevierScience Publishers B.V.
ing equipment which limits its use in laboratories where all these facilities are not available. Recently we described a modified Farr technique for the measurement of anti-DNA antibodies in SLE and experimental animals using unlabelled antigen [4]. Here we describe a spectrophotometric procedure for the detection and quantitation of DNA antibodies in the sera of patients with various connective tissue diseases. The sensitivity of the method has been compared with hemagglutination and enzymelinked immunosorbent assay. The method described is simple, specific, reproducible and is highly economical.
3. Experimental 3.1. Sera
Normal human sera were obtained from healthy individuals. Sera of patients with autoimmune diseases were collected as described earlier [5]. The patients had features meeting preliminary criteria of the American Rheumatism Association for these diseases [6]. 3.2. Antigen Calf thymus DNA, purchased from Sigma Chemicals, U.S.A., was used as antigen. Single-stranded DNA was prepared by heat denaturation. 3.3. Equipment Measurements of absorbance at 260 nm were made by a single-beam spectrophotometer (Carl 25
Zeiss Jena VSU-2P, G.D.R.). ~00-
I
"
3.4. Passive hemagglutination and enzvme-finked
I
immunosorbent assay Passive hemagglutination (PHA) and enzymelinked immunosorbent assay (ELISA) were carried out as described earlier [7,8].
z
3.5. SpectrophotometHc assay The anti-DNA antibody assay was performed as follows: 50 tag of double- or single-stranded DNA in 0.9 ml of 0.15 M borate-sodium chloride buffer, pH 8.0, was mixed with 0.1 ml of decomplemented serum (56 °C for 30 rain). The mixture was incubated at 37 °C for 1 h followed by 16-24 h incubation at 4 ° C, One ml of saturated ammonium sulfate solution was added and the contents mixed thoroughly. The mixture was kept at 4 °C for the complete precipitation of immune complexes. The contents were centrifuged and supernatant separated. Absorbance of supernatant was recorded at 260 nm. Each test serum was run along with a control which contained all reagents except the antigen. The results were expressed in terms of percent DNA bound to anti-DNA antibodies, calculated according to the formula: Percent DNA bound :100
[ o...o°. ...
N 100
~40 •
20
oeo
eoo eee •
I
oe e • . .
• .
• •
o-o
•
.,.
eoo .
--I
734~220
PSS 138t35
SLE+PSS 156~I00
RA 113~57
o ° eeo~el I
"
.., leo
ee°• SLE
L ~,S 148"4.7
Nh 16C~69
Fig. 1. Levels of DNA binding activity in patients with various connective tissue diseases. The mean DNA binding values of positive SLE sera. normal human sera and sera of patients with other connective tissue diseases with standard deviations are indicated at the bottom of the figure.
]
O, D,50 #g DNA
3.6. Inhibition experiment The specificity of anti-DNA antibody measurement by the spectrophotometric assay was studied by inhibition studies. Varying concentrations of DNA were mixed with 0.1 ml of anti-DNA antibody positive SLE serum to a final concentration of 2-50 tag D N A / m l and incubated at 37 °C for I h and at 4 °C for 16 h. Incubated contents were used in the assay instead of serum.
4. Results and Discussion Normal human sera and sera of patients with various connective tissue diseases were tested for the presence of anti-DNA antibodies by spectrophotometric method using double-stranded DNA as anti26
i
•
e
gen. Fig. 1 shows DNA binding activity in these sera. Sera showing DNA binding of more than 30% were considered as positive. Using this criteria, 37% of SLE patients were positive for anti-DNA antibodies. The range of percent DNA binding activity of these sera was 35-93, depending upon the severity of the disease. Threepatients with inactive SLE had a DNA binding activity of 3543%. Normal human sera and sera of patients with RA, PSS and SS were all negative for such antibodies. One out of seven SLE patients ~vith overlapping PSS showed a binding of 38%. Sera positive for anti-dsDNA antibodies were also found to be positive for antibodies against ssDNA with a varying degree of cross-reactivity (data not given). The specificity of antigen-antibody reaction in the assay was studied by an inhibition experiment. Percent inhibition obtained at different DNA concentrations using an anti-DNA antibody positive SLE serum is shown in Fig. 2. A high degree of specificity is evident since 70% inhibition is achieved with 20 tag of DNA. Besides being specific, the assay described is highly reproducible as is evident from the precision data with three anti-DNA positive SLE sera (Table l). The incidence of anti-DNA antibodies by the
Table 2 Comparison of incidence of anti-DNA antibodies in connective tissue diseases by various techniques
80-
~- 6 0 -
Sera
Number tested
~_ 4 0 -
Percent positive ELISA
PHA
Spectrophotometric assay
48 10 0 29 0 0
37 0 0 14 0 0
37 0 0 14 0 0
Z W
m 20-
0
I 10
/ 20
I 30
L 40
L 50
Fig. 2. Demonstration of specificity of spectrophotometric assay by antigen inhibition. Inhibition of antigen-antibody reaction was performed in the presence of varying concentrations of DNA. Other procedural details are given in the text.
present method was compared with two most commonly used techniques, P H A and ELISA. The results obtained are tabulated in Table 2.48% of SLE patients were positive for anti-DNA antibodies by ELISA. With P H A and spectrophotometric assay, the incidence was 37% individually. These results indicate that the present assay, although not as sensitive as ELISA, is comparable with the P H A technique. The method has, however, certain advantages over P H A as the latter is cumbersome, time consuming and the stability of antigen-coated cells is a major drawback of the assay. Moreover, the possibility of the presence of an agglutinating-inhibiting factor in some SLE sera, as has been reported in thyrotoxic serum [9], could not be ruled out. EL1SA, although more sensitive, is a multistep proceTable 1 Reproducibility data for anti-DNA antibody assay of 3 positive SLE sera Assay
Variation ± S.D. a
lntra-assay
84.4 ± 6.6 52.1 :t: 3.2 35.0 ± 1.50
Inter-assay
81.4 ± 10,1 55.0 ± 5.2 35,9 ± 2.8
Coefficient of variation (%) 7.80 6.07 4.23 12.41 9.45 7.80
Number of samples analyzed in each case is 10. a Standard deviation.
SLE PSS SS SLE + PSS RA Normal
30 10 6 7 12 20
dure involving costly biochemicals which limits its use in a number of laboratories~ In conclusion, the spectrophotometric assay described here is simple, specific, reproducible and can be carried out with rapidity and reliability in less well equipped laboratories for routine clinical investigation of SLE sera for the presence of anti-DNA antibodies.
Acknowledgements This work is supported by a grant from the Indian Council of Medical Research and Council of Scientific and Industrial Research, New Delhi.
R eferences [1] Greenwald, C. A., Peebles, C. L. and Nakamura, R. M. (1978) Lab. Med. 9, 19-27. [2] Vladutiu, A. O., Palumbo, D. A. and Asirwatham, J. E. (1979) Clin. Chem. 25,366-370. [3] Farr, R. S. (1958) J. Infect. Dis. 103,239-262. [4] Ali, A., Ishaq, M., Malaviya, A. N. and Ali, R. (1982) IRCS Med. Sci. 10, 890. [5] lshaq, M., Ali, A., Malaviya, A. N. and Ali, R. (1982) J. Biosci. 4, 499 506. [6] American Rheumatism Association on criteria for diagnosis and classification of rheumatic disease: Primer on the Rheumatic Diseases (1973) J. Am. Med. Ass. 224, 137 140. [7] Ali, A., Ishaq, M., Malaviya, A. N. and Ali, R. (1983) Ind. J. Med. Res. (in press). [8] Ali, A. and Ali, R. (1983) J. Immunol. Meth. 56, 341 346. [9] Wilkin, T. J., Swanson, B. J., Hayes, P. C., Ports, R. C. and Young, R. (1979) J. Clin. Endocrinol. 10, 507-514.
27
Elsevier
SPECTROPHOTOMETRIC
ASSAY
FOR THE DETECTION TO DNA
OF ANTIBODIES
Asif ALI and Rashid ALI Department of Biochemistry, Faculty of Medicine, Aligarh Muslim University, Aligarh 202001, India
(Received9 March 1983) (Modified versionreceived3 June 1983) (Accepted 6 June 1983)
1. Summary A spectrophotometric assay for the detection of anti-DNA antibodies in the sera of patients with various connective tissue diseases is described. The sensitivity of the method was compared with hemagglutination and enzyme-linked immunosorbent assay. The method is convenient, specific and suitable for use in clinical laboratories where facilities for radioimmunoassay do not exist.
2. Introduction Autoantibodies to intraceUular proteins and nucleic acids are a hallmark of certain autoimmune diseases which include systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), progressive systemic sclerosis (PSS) and Sjogren's syndrome (SS). Patients with SLE spontaneously produce antibodies directed against double-stranded DNA (dsDNA). A simple and sensitive laboratory procedure for the detection of circulating DNA antibodies in the sera of patients which SLE is of great clinical significance for the diagnosis and management of such patients. Various techniques have been developed for the measurement of these antibodies [ 1,2]. The most widely used technique is radioimmunoassay of Farr [3] involving the use of labelled DNA and sophisticated radioactive measurKey words: spectrophotometricassay- anti-DNA antibodies -
systemiclupus erythematosus 0165-2478/ 83/ $03.00 © ElsevierScience Publishers B.V.
ing equipment which limits its use in laboratories where all these facilities are not available. Recently we described a modified Farr technique for the measurement of anti-DNA antibodies in SLE and experimental animals using unlabelled antigen [4]. Here we describe a spectrophotometric procedure for the detection and quantitation of DNA antibodies in the sera of patients with various connective tissue diseases. The sensitivity of the method has been compared with hemagglutination and enzymelinked immunosorbent assay. The method described is simple, specific, reproducible and is highly economical.
3. Experimental 3.1. Sera
Normal human sera were obtained from healthy individuals. Sera of patients with autoimmune diseases were collected as described earlier [5]. The patients had features meeting preliminary criteria of the American Rheumatism Association for these diseases [6]. 3.2. Antigen Calf thymus DNA, purchased from Sigma Chemicals, U.S.A., was used as antigen. Single-stranded DNA was prepared by heat denaturation. 3.3. Equipment Measurements of absorbance at 260 nm were made by a single-beam spectrophotometer (Carl 25
Zeiss Jena VSU-2P, G.D.R.). ~00-
I
"
3.4. Passive hemagglutination and enzvme-finked
I
immunosorbent assay Passive hemagglutination (PHA) and enzymelinked immunosorbent assay (ELISA) were carried out as described earlier [7,8].
z
3.5. SpectrophotometHc assay The anti-DNA antibody assay was performed as follows: 50 tag of double- or single-stranded DNA in 0.9 ml of 0.15 M borate-sodium chloride buffer, pH 8.0, was mixed with 0.1 ml of decomplemented serum (56 °C for 30 rain). The mixture was incubated at 37 °C for 1 h followed by 16-24 h incubation at 4 ° C, One ml of saturated ammonium sulfate solution was added and the contents mixed thoroughly. The mixture was kept at 4 °C for the complete precipitation of immune complexes. The contents were centrifuged and supernatant separated. Absorbance of supernatant was recorded at 260 nm. Each test serum was run along with a control which contained all reagents except the antigen. The results were expressed in terms of percent DNA bound to anti-DNA antibodies, calculated according to the formula: Percent DNA bound :100
[ o...o°. ...
N 100
~40 •
20
oeo
eoo eee •
I
oe e • . .
• .
• •
o-o
•
.,.
eoo .
--I
734~220
PSS 138t35
SLE+PSS 156~I00
RA 113~57
o ° eeo~el I
"
.., leo
ee°• SLE
L ~,S 148"4.7
Nh 16C~69
Fig. 1. Levels of DNA binding activity in patients with various connective tissue diseases. The mean DNA binding values of positive SLE sera. normal human sera and sera of patients with other connective tissue diseases with standard deviations are indicated at the bottom of the figure.
]
O, D,50 #g DNA
3.6. Inhibition experiment The specificity of anti-DNA antibody measurement by the spectrophotometric assay was studied by inhibition studies. Varying concentrations of DNA were mixed with 0.1 ml of anti-DNA antibody positive SLE serum to a final concentration of 2-50 tag D N A / m l and incubated at 37 °C for I h and at 4 °C for 16 h. Incubated contents were used in the assay instead of serum.
4. Results and Discussion Normal human sera and sera of patients with various connective tissue diseases were tested for the presence of anti-DNA antibodies by spectrophotometric method using double-stranded DNA as anti26
i
•
e
gen. Fig. 1 shows DNA binding activity in these sera. Sera showing DNA binding of more than 30% were considered as positive. Using this criteria, 37% of SLE patients were positive for anti-DNA antibodies. The range of percent DNA binding activity of these sera was 35-93, depending upon the severity of the disease. Threepatients with inactive SLE had a DNA binding activity of 3543%. Normal human sera and sera of patients with RA, PSS and SS were all negative for such antibodies. One out of seven SLE patients ~vith overlapping PSS showed a binding of 38%. Sera positive for anti-dsDNA antibodies were also found to be positive for antibodies against ssDNA with a varying degree of cross-reactivity (data not given). The specificity of antigen-antibody reaction in the assay was studied by an inhibition experiment. Percent inhibition obtained at different DNA concentrations using an anti-DNA antibody positive SLE serum is shown in Fig. 2. A high degree of specificity is evident since 70% inhibition is achieved with 20 tag of DNA. Besides being specific, the assay described is highly reproducible as is evident from the precision data with three anti-DNA positive SLE sera (Table l). The incidence of anti-DNA antibodies by the
Table 2 Comparison of incidence of anti-DNA antibodies in connective tissue diseases by various techniques
80-
~- 6 0 -
Sera
Number tested
~_ 4 0 -
Percent positive ELISA
PHA
Spectrophotometric assay
48 10 0 29 0 0
37 0 0 14 0 0
37 0 0 14 0 0
Z W
m 20-
0
I 10
/ 20
I 30
L 40
L 50
Fig. 2. Demonstration of specificity of spectrophotometric assay by antigen inhibition. Inhibition of antigen-antibody reaction was performed in the presence of varying concentrations of DNA. Other procedural details are given in the text.
present method was compared with two most commonly used techniques, P H A and ELISA. The results obtained are tabulated in Table 2.48% of SLE patients were positive for anti-DNA antibodies by ELISA. With P H A and spectrophotometric assay, the incidence was 37% individually. These results indicate that the present assay, although not as sensitive as ELISA, is comparable with the P H A technique. The method has, however, certain advantages over P H A as the latter is cumbersome, time consuming and the stability of antigen-coated cells is a major drawback of the assay. Moreover, the possibility of the presence of an agglutinating-inhibiting factor in some SLE sera, as has been reported in thyrotoxic serum [9], could not be ruled out. EL1SA, although more sensitive, is a multistep proceTable 1 Reproducibility data for anti-DNA antibody assay of 3 positive SLE sera Assay
Variation ± S.D. a
lntra-assay
84.4 ± 6.6 52.1 :t: 3.2 35.0 ± 1.50
Inter-assay
81.4 ± 10,1 55.0 ± 5.2 35,9 ± 2.8
Coefficient of variation (%) 7.80 6.07 4.23 12.41 9.45 7.80
Number of samples analyzed in each case is 10. a Standard deviation.
SLE PSS SS SLE + PSS RA Normal
30 10 6 7 12 20
dure involving costly biochemicals which limits its use in a number of laboratories~ In conclusion, the spectrophotometric assay described here is simple, specific, reproducible and can be carried out with rapidity and reliability in less well equipped laboratories for routine clinical investigation of SLE sera for the presence of anti-DNA antibodies.
Acknowledgements This work is supported by a grant from the Indian Council of Medical Research and Council of Scientific and Industrial Research, New Delhi.
R eferences [1] Greenwald, C. A., Peebles, C. L. and Nakamura, R. M. (1978) Lab. Med. 9, 19-27. [2] Vladutiu, A. O., Palumbo, D. A. and Asirwatham, J. E. (1979) Clin. Chem. 25,366-370. [3] Farr, R. S. (1958) J. Infect. Dis. 103,239-262. [4] Ali, A., Ishaq, M., Malaviya, A. N. and Ali, R. (1982) IRCS Med. Sci. 10, 890. [5] lshaq, M., Ali, A., Malaviya, A. N. and Ali, R. (1982) J. Biosci. 4, 499 506. [6] American Rheumatism Association on criteria for diagnosis and classification of rheumatic disease: Primer on the Rheumatic Diseases (1973) J. Am. Med. Ass. 224, 137 140. [7] Ali, A., Ishaq, M., Malaviya, A. N. and Ali, R. (1983) Ind. J. Med. Res. (in press). [8] Ali, A. and Ali, R. (1983) J. Immunol. Meth. 56, 341 346. [9] Wilkin, T. J., Swanson, B. J., Hayes, P. C., Ports, R. C. and Young, R. (1979) J. Clin. Endocrinol. 10, 507-514.
27