Immunology Today !1ol 8. NO 3, 1987 .
possibly by mast cells, attack target are not generally regarded as infeccells that bear complementary rec- tious, or allergic, including our main ognition factors (major and minor killer, acute myocardial infarction 7. A mutant growth control stem cell histocompatibility, tissue-specific and mosaic-specific antigens) to is, in effect, 'non self' by virtue of its cause 'autoaggressive ° disease. mutant status. Its contact with conThat acute and infectious diseases tiguous non-mutant, and therefore fall into this general category has a complementary, cells often causes particular significance in the present both types to be damaged, with the context. Reproducibility of the form release of self and, in effect, non-self of the age-pattern in high and low antigens. These antigens elicit a epidemic years and from country to humoral (IgM and IgG)immune recountry implies that host factors de- sponse and thus the emergence of termine whether or not an infected autoantibodies both in health (when person will deve!op the associated the mutant stem cells stay suppresdisease4.5. These factors are the sed) and in dis~3se (when mutant presence in the host of one or more stem cell~ propagate forbidden mutant growth-control cells that clone~). An autoaggressive attack on remain suppressed by the target cells often results in further immunoglobulin-based defence sys- antigenic debris and consequent imtem in the absence of a competing munoglobulin autoantibodies. In this microorganism (or allergen where unified theory of growth and disease allergic diseases are concerned) and immunoglobulin autoantibodies are hence fail to propagate pathogenic never the primary pathogens in forbidden clones. A similar mechan- autoaggressive (including autoism can be inferred for diseases that immune) disease; to the contrary,
which appears to play an important role in the binding to the ELISA plates. However, this ssDNA was not detected after coating under our Sir, technica! conditions and, in agreeThe claim by S. Ghcsh and A. M. ment with previous studies 1, $1 nucCampbell :immunoL Today, 1986, 7, lease treatment of the plates did not 355) that ELISA is unreliable for the modify the results of the anti-dsDNA measurement of antibodies to assay (except for plates that were double-stranded (ds) DNA because kept at 4°C without caution, where of the detection of antibodies to denaturation occurred within a few single-stranded (ss) DNA cannot be days). Inhibition experiments have left unanswered because of its im- led to the same conclusion 2. The portant practical implications in value of ELISA for the detection of clinical immunology. We have been anti-dsDNA antibodies is illustrated using such an ELISA routinely for by our results in more than 400 several years: plates coated with antinuclear antibody containing 5 i~g/ml calf thymus DNA are used sera. There was a very good correlaeither immediately or after short (less tion with the Farr assay except that than 2 months) storage at -80°C. ELISA was more sensitive in systemic The problem of anti-ssDNA anti- lupus erythematosus (SLE). Very high bodies was, of course, a major con- titer antibodies were found by ELISA cern and we examined it from four in active SLE exclusively. Antibodies approaches: (1) $1 nuclease treat- belonging to the three main imment of the DNA in solution before munoglobulin classes, to IgG and coating and of the coated plates; (2) IgM or IgG and IgA were usually comparison with ELISAplates coated present at high titer and virtually with ssDNA, (3) use of sera from restricted to SLE, except for antipatients with drug-induced lupus bodies from a few patients with acthat contained high titer anti-ssDNA tive chronic hepatitis, scleroderma, antibodies and none or virtually no rheumatoid arthritis or Sj0gren's anti-ds DNA antibodies as controls; syndrome 3.4. In contrast, we found and (4) comparison with the Farr low titer IgM antibodies in certain assay. patients without SLE, which is not The results showed that calf thy- surprising since normal lymphoid tismus DNA indeed contains ssDNA sue in vitro produce anti-dsDNA anti-
Anti-DNAantibodiesby ELISA
74
.
.
.
.
.
.
.
they often suppress the growth of forbidden clones.
P. R. J. Burch N. R. Rowell Departmentsof MedicalPhysicsand Dermatology, Universi~of Leeds,TheGeneral Infirmary, LeedsLS13EX,UK References 1 Burnet, F. M. (1959) The Clonal Selection Theory of Acquired Immunity, Vanderbilt UniversityPress,Nashville 2 Burch, P. R. J. and Rowell, N. R. (1963) Lancet ii, 507-513 3 Burch, P. R. J. and Rowell, N. R. (1968) Acta Dermatol. Venereol. Stockholm 48, 33--46 4 Burch, P. R. J. (1968)An Inquiry Concerning Growth, Diseaseand Ageing, Oliver & Boyd, Edinburgh 5 Burch, P. R. J. (1976) The Bio/ogy of Cancer. A New Approach, MPT Press, Lancaster 6 Burch, P. R. J. and Burwell, R. G. (1965) O. Rev. Biol. 40, 252-279 7 Burch, P. R. J. (1980) Cardiovasc. Res. 14, 307-338
bodies of the IgM but not of the IgG classs and since, as discussed by Isenberg et al. 6, hybridomas secreting IgM anti-dsDNA may be derived from normal subjects. Specificity of such IgM monoclonal antibodies derived from normal lymphocytes has been demonstrated by methods other than ELISA, such as inhibition radioimm,-noassay 7.
Jean-Louis Preud'Homme Laboratoryof Immunologyand Immunopathology, (O,'RSUA 1172),Poitiers JniversityHospital, .%021PoitiersCedex, France
References 1 Klotz, J. L., Minami, R. M. and Teplitz, R. L. (1979)J. ImmunoL Meth. 29, 155-165 2 Gripenberg, M. and Kurki, P. (1986) J. ImmunoL Meth. 92, 145-159 3 Stollar, B. D. (1981)Clin. ImmunoL Allergy 1,243-260 4 Tan, E. M. (1982)Adv. ImmunoL 33, 167-240 5 Cairns, E., Germain,J. and Bell, D. A. (1985)J. ImmunoL 135, 3839-3844 6 Isenberg,D., Dudeney,C. and Williams, W. (1986) Immunol. Today 12, 354--355 7 Hoch, S. and Schwaber,J. (1986) J. Immunol. 136, 892-897
~ 1987, ElsevierPubhcatlon5,Cambndge 0167 4919/87/$02 00
ImmunologyToday,vol. 8, No. 3, 1987
............................ ,ii; /i! iiii!!I¸/ This letter was shown to Drs Ghosh and Campbell who reply as follows: Sir, We have few points of disagreement with J.-L Preud'homme's detailed analysis (above) which deals with serum studies. As we pointed out in the first paragraph of our original article (/mmunoL Today, 1986, 7, 217-222), citing Talmage, serum may be regarded as a complex mixture of many monoclonal antibodies. If a substantial number of these react
with any form of DNA then this is clearly a clinically significant observation. It is where data from a single monoclonal antibody generated from one individual is used as a basis to reconstruct hypotheses relating to the aetiology of SLE that we feel the conclusions may be invalid. We see no reason why a carefully constructed ELISA assay such as he describes should not be a perfectly valid method for screening serum. However, we would like to adhere to our view that none of the mono-
and our results also suggest that much of the cross-reactivity may be explained in terms of rather low affinity (Group III) 'irrelevant' interactions. Harris and co-workers Sir, We were pleased to read both the apparently were the first group to recent incisive article by S. Ghosh come to a similar conclusion 2, and and A.M. Campbell on multispecific Eilat's work 3 also supports this point monoclonal antibodies (ImmunoL of view. The comments of Ghosh Today, 1986, 7, 217-222), and their and Campbell regarding methodoresponse to the letter" of Isenberg et logical problems in studies of crossaL (ImmunoL Today, 1986, 7, 354- reactivity were particularly well 355), regarding anti-DNA antibody founded in our opinion. Thus, ~'./e suggest that until careful solution specificity. We have examined the quantita- phase studies are done with approtive aspects of the cross-reactivity of priate quantitation 4, the significance bonafide high avidity IgG anti- and relevance to SLE of reports of dsDNA antibodies found in SLE sera 1 cross-reactivity of anti-dsDNA anti-
Cross-reactivityof anti-dsDNA antibodies
clonal IgM antibodies generated by this method of screening have been specific to the Watson Crick structure. Many of them react with double stranded DNA but also (and usually at higher affinity) with single stranded DNA and synthetic polynucleotides. Ref. 7 cited by Preud'homme describes three such rnonoclonal antibodies.
S. Ghosh A.M. Campbell Department of Biochemistry, University of Glasgow, GlasgowG12 8QQ, UK
bodies remains uncertain.
Ronald P. Taylor Jeffrey C. Edberg Departmentof Biochemistry,Universityof VirginiaSchoolof Medicine,Charlottesville, Virginia22908, USA
References
1 Edberg,J.C. and Taylor, R.P.(1986) J. Immunol. 136, 4581-4587 2 Harris, E.N., Boey,M.L., MackworthYoung, C.G. et al. (1983) Lancet ii, 1211-1214 3 Eilat, D., Zlotnick, A.Y. and Fischel,R. (1986) Clin. Exp. Immunol. 65, 269-278 4 Emlen,W., Pisetsky,D. and Taylor, R.P. (1986) Arthritis Rheum. 29, 1417-1426
possibly by mast cells, attack target are not generally regarded as infeccells that bear complementary rec- tious, or allergic, including our main ognition factors (major and minor killer, acute myocardial infarction 7. A mutant growth control stem cell histocompatibility, tissue-specific and mosaic-specific antigens) to is, in effect, 'non self' by virtue of its cause 'autoaggressive ° disease. mutant status. Its contact with conThat acute and infectious diseases tiguous non-mutant, and therefore fall into this general category has a complementary, cells often causes particular significance in the present both types to be damaged, with the context. Reproducibility of the form release of self and, in effect, non-self of the age-pattern in high and low antigens. These antigens elicit a epidemic years and from country to humoral (IgM and IgG)immune recountry implies that host factors de- sponse and thus the emergence of termine whether or not an infected autoantibodies both in health (when person will deve!op the associated the mutant stem cells stay suppresdisease4.5. These factors are the sed) and in dis~3se (when mutant presence in the host of one or more stem cell~ propagate forbidden mutant growth-control cells that clone~). An autoaggressive attack on remain suppressed by the target cells often results in further immunoglobulin-based defence sys- antigenic debris and consequent imtem in the absence of a competing munoglobulin autoantibodies. In this microorganism (or allergen where unified theory of growth and disease allergic diseases are concerned) and immunoglobulin autoantibodies are hence fail to propagate pathogenic never the primary pathogens in forbidden clones. A similar mechan- autoaggressive (including autoism can be inferred for diseases that immune) disease; to the contrary,
which appears to play an important role in the binding to the ELISA plates. However, this ssDNA was not detected after coating under our Sir, technica! conditions and, in agreeThe claim by S. Ghcsh and A. M. ment with previous studies 1, $1 nucCampbell :immunoL Today, 1986, 7, lease treatment of the plates did not 355) that ELISA is unreliable for the modify the results of the anti-dsDNA measurement of antibodies to assay (except for plates that were double-stranded (ds) DNA because kept at 4°C without caution, where of the detection of antibodies to denaturation occurred within a few single-stranded (ss) DNA cannot be days). Inhibition experiments have left unanswered because of its im- led to the same conclusion 2. The portant practical implications in value of ELISA for the detection of clinical immunology. We have been anti-dsDNA antibodies is illustrated using such an ELISA routinely for by our results in more than 400 several years: plates coated with antinuclear antibody containing 5 i~g/ml calf thymus DNA are used sera. There was a very good correlaeither immediately or after short (less tion with the Farr assay except that than 2 months) storage at -80°C. ELISA was more sensitive in systemic The problem of anti-ssDNA anti- lupus erythematosus (SLE). Very high bodies was, of course, a major con- titer antibodies were found by ELISA cern and we examined it from four in active SLE exclusively. Antibodies approaches: (1) $1 nuclease treat- belonging to the three main imment of the DNA in solution before munoglobulin classes, to IgG and coating and of the coated plates; (2) IgM or IgG and IgA were usually comparison with ELISAplates coated present at high titer and virtually with ssDNA, (3) use of sera from restricted to SLE, except for antipatients with drug-induced lupus bodies from a few patients with acthat contained high titer anti-ssDNA tive chronic hepatitis, scleroderma, antibodies and none or virtually no rheumatoid arthritis or Sj0gren's anti-ds DNA antibodies as controls; syndrome 3.4. In contrast, we found and (4) comparison with the Farr low titer IgM antibodies in certain assay. patients without SLE, which is not The results showed that calf thy- surprising since normal lymphoid tismus DNA indeed contains ssDNA sue in vitro produce anti-dsDNA anti-
Anti-DNAantibodiesby ELISA
74
.
.
.
.
.
.
.
they often suppress the growth of forbidden clones.
P. R. J. Burch N. R. Rowell Departmentsof MedicalPhysicsand Dermatology, Universi~of Leeds,TheGeneral Infirmary, LeedsLS13EX,UK References 1 Burnet, F. M. (1959) The Clonal Selection Theory of Acquired Immunity, Vanderbilt UniversityPress,Nashville 2 Burch, P. R. J. and Rowell, N. R. (1963) Lancet ii, 507-513 3 Burch, P. R. J. and Rowell, N. R. (1968) Acta Dermatol. Venereol. Stockholm 48, 33--46 4 Burch, P. R. J. (1968)An Inquiry Concerning Growth, Diseaseand Ageing, Oliver & Boyd, Edinburgh 5 Burch, P. R. J. (1976) The Bio/ogy of Cancer. A New Approach, MPT Press, Lancaster 6 Burch, P. R. J. and Burwell, R. G. (1965) O. Rev. Biol. 40, 252-279 7 Burch, P. R. J. (1980) Cardiovasc. Res. 14, 307-338
bodies of the IgM but not of the IgG classs and since, as discussed by Isenberg et al. 6, hybridomas secreting IgM anti-dsDNA may be derived from normal subjects. Specificity of such IgM monoclonal antibodies derived from normal lymphocytes has been demonstrated by methods other than ELISA, such as inhibition radioimm,-noassay 7.
Jean-Louis Preud'Homme Laboratoryof Immunologyand Immunopathology, (O,'RSUA 1172),Poitiers JniversityHospital, .%021PoitiersCedex, France
References 1 Klotz, J. L., Minami, R. M. and Teplitz, R. L. (1979)J. ImmunoL Meth. 29, 155-165 2 Gripenberg, M. and Kurki, P. (1986) J. ImmunoL Meth. 92, 145-159 3 Stollar, B. D. (1981)Clin. ImmunoL Allergy 1,243-260 4 Tan, E. M. (1982)Adv. ImmunoL 33, 167-240 5 Cairns, E., Germain,J. and Bell, D. A. (1985)J. ImmunoL 135, 3839-3844 6 Isenberg,D., Dudeney,C. and Williams, W. (1986) Immunol. Today 12, 354--355 7 Hoch, S. and Schwaber,J. (1986) J. Immunol. 136, 892-897
~ 1987, ElsevierPubhcatlon5,Cambndge 0167 4919/87/$02 00
ImmunologyToday,vol. 8, No. 3, 1987
............................ ,ii; /i! iiii!!I¸/ This letter was shown to Drs Ghosh and Campbell who reply as follows: Sir, We have few points of disagreement with J.-L Preud'homme's detailed analysis (above) which deals with serum studies. As we pointed out in the first paragraph of our original article (/mmunoL Today, 1986, 7, 217-222), citing Talmage, serum may be regarded as a complex mixture of many monoclonal antibodies. If a substantial number of these react
with any form of DNA then this is clearly a clinically significant observation. It is where data from a single monoclonal antibody generated from one individual is used as a basis to reconstruct hypotheses relating to the aetiology of SLE that we feel the conclusions may be invalid. We see no reason why a carefully constructed ELISA assay such as he describes should not be a perfectly valid method for screening serum. However, we would like to adhere to our view that none of the mono-
and our results also suggest that much of the cross-reactivity may be explained in terms of rather low affinity (Group III) 'irrelevant' interactions. Harris and co-workers Sir, We were pleased to read both the apparently were the first group to recent incisive article by S. Ghosh come to a similar conclusion 2, and and A.M. Campbell on multispecific Eilat's work 3 also supports this point monoclonal antibodies (ImmunoL of view. The comments of Ghosh Today, 1986, 7, 217-222), and their and Campbell regarding methodoresponse to the letter" of Isenberg et logical problems in studies of crossaL (ImmunoL Today, 1986, 7, 354- reactivity were particularly well 355), regarding anti-DNA antibody founded in our opinion. Thus, ~'./e suggest that until careful solution specificity. We have examined the quantita- phase studies are done with approtive aspects of the cross-reactivity of priate quantitation 4, the significance bonafide high avidity IgG anti- and relevance to SLE of reports of dsDNA antibodies found in SLE sera 1 cross-reactivity of anti-dsDNA anti-
Cross-reactivityof anti-dsDNA antibodies
clonal IgM antibodies generated by this method of screening have been specific to the Watson Crick structure. Many of them react with double stranded DNA but also (and usually at higher affinity) with single stranded DNA and synthetic polynucleotides. Ref. 7 cited by Preud'homme describes three such rnonoclonal antibodies.
S. Ghosh A.M. Campbell Department of Biochemistry, University of Glasgow, GlasgowG12 8QQ, UK
bodies remains uncertain.
Ronald P. Taylor Jeffrey C. Edberg Departmentof Biochemistry,Universityof VirginiaSchoolof Medicine,Charlottesville, Virginia22908, USA
References
1 Edberg,J.C. and Taylor, R.P.(1986) J. Immunol. 136, 4581-4587 2 Harris, E.N., Boey,M.L., MackworthYoung, C.G. et al. (1983) Lancet ii, 1211-1214 3 Eilat, D., Zlotnick, A.Y. and Fischel,R. (1986) Clin. Exp. Immunol. 65, 269-278 4 Emlen,W., Pisetsky,D. and Taylor, R.P. (1986) Arthritis Rheum. 29, 1417-1426
