Characterization of DNA used to assay sera for anti DNA antibodies; determination of the specificities of anti DNA antibodies in SLE and non SLE rheumatic disease states

J. D. Locker, M. E. Medof, Robert (Rob) Bennett, S. Sukhupunyaraksa

Research output: Contribution to journalArticle

68 Citations (Scopus)

Abstract

Commercial 14C labeled KB cell DNA, widely used to assay sera for anti DNA antibodies, was chromatographed on benzoylated naphthoylated DEAE cellulose (BNDC) and on hydroxyapatite (HAP). On BNDC, only 25% of the 14C label eluted with 1 M NaCl (KB fraction I) characteristic of ds DNA. Fifty five percent of the label eluted with 50% formamide 1 M NaCl (KB fraction II) characteristic of ss or denatured DNA. On HAP, however, none of the 14C label eluted with 0.2 M phosphate buffer as anticipated for ss DNA, but, rather, all of the 14C label eluted with 0.4 M phosphate, characteristic of ds DNA. After pretreatment with S1 endonuclease of Aspergillus oryzae, which selectively digests ss regions, however, 42% of the 14C label was lost from the 0.4 M phosphate peak. These results indicated that more than half of this 14C KB cell DNA preparation was ds DNA with ss regions which was undetectable by HAP chromatography. 3H ds DNA and circular 3H ss DNA prepared from T7 and phi X174 bacteriophage, respectively, were found to be chromatographically pure on both BNDC and HAP. None of 10 non SLE sera (rheumatoid arthritis 3, mixed connective tissue disease 4, scleroderma 1, ulcerative colitis 1, and pulmonary fibrosis with chronic active hepatitis 1), previously believed to contain anti ds DNA antibodies on the basis of KB cell DNA testing had detectable antibodies against KB fraction I or T7 DNA: all of 10 KB cell DNA positive SLE sera had antibodies against both. Additionally, none of the 10 non SLE sera had antibodies against KB cell DNA when retested with DNA that had been pretreated with S1 endonuclease. Seven of these 10, however, as well as all 10 SLE sera, had antibodies against phi X174 DNA, KB fraction II DNA and alkali denatured T7 DNA. The data support the conclusions that (1) false positive tests for anti ds DNA antibodies can result from contamination of ds DNA with ds DNA having ss regions, and (2) non SLE sera do not contain antibodies specific for ds DNA at levels comparable to those found in SLE sera but rather contain high levels of antibodies reacting with ss regions or mixed DNA.

Original languageEnglish (US)
Pages (from-to)694-701
Number of pages8
JournalJournal of Immunology
Volume118
Issue number2
StatePublished - 1977
Externally publishedYes

Fingerprint

Antinuclear Antibodies
Rheumatic Diseases
DNA
Serum
KB Cells
Durapatite
Antibodies
Endonucleases
Phosphates
Mixed Connective Tissue Disease
Aspergillus oryzae

ASJC Scopus subject areas

  • Immunology

Cite this

Characterization of DNA used to assay sera for anti DNA antibodies; determination of the specificities of anti DNA antibodies in SLE and non SLE rheumatic disease states. / Locker, J. D.; Medof, M. E.; Bennett, Robert (Rob); Sukhupunyaraksa, S.

In: Journal of Immunology, Vol. 118, No. 2, 1977, p. 694-701.

Research output: Contribution to journalArticle

@article{f1775b3f57804bd48b053e0978954179,
title = "Characterization of DNA used to assay sera for anti DNA antibodies; determination of the specificities of anti DNA antibodies in SLE and non SLE rheumatic disease states",
abstract = "Commercial 14C labeled KB cell DNA, widely used to assay sera for anti DNA antibodies, was chromatographed on benzoylated naphthoylated DEAE cellulose (BNDC) and on hydroxyapatite (HAP). On BNDC, only 25{\%} of the 14C label eluted with 1 M NaCl (KB fraction I) characteristic of ds DNA. Fifty five percent of the label eluted with 50{\%} formamide 1 M NaCl (KB fraction II) characteristic of ss or denatured DNA. On HAP, however, none of the 14C label eluted with 0.2 M phosphate buffer as anticipated for ss DNA, but, rather, all of the 14C label eluted with 0.4 M phosphate, characteristic of ds DNA. After pretreatment with S1 endonuclease of Aspergillus oryzae, which selectively digests ss regions, however, 42{\%} of the 14C label was lost from the 0.4 M phosphate peak. These results indicated that more than half of this 14C KB cell DNA preparation was ds DNA with ss regions which was undetectable by HAP chromatography. 3H ds DNA and circular 3H ss DNA prepared from T7 and phi X174 bacteriophage, respectively, were found to be chromatographically pure on both BNDC and HAP. None of 10 non SLE sera (rheumatoid arthritis 3, mixed connective tissue disease 4, scleroderma 1, ulcerative colitis 1, and pulmonary fibrosis with chronic active hepatitis 1), previously believed to contain anti ds DNA antibodies on the basis of KB cell DNA testing had detectable antibodies against KB fraction I or T7 DNA: all of 10 KB cell DNA positive SLE sera had antibodies against both. Additionally, none of the 10 non SLE sera had antibodies against KB cell DNA when retested with DNA that had been pretreated with S1 endonuclease. Seven of these 10, however, as well as all 10 SLE sera, had antibodies against phi X174 DNA, KB fraction II DNA and alkali denatured T7 DNA. The data support the conclusions that (1) false positive tests for anti ds DNA antibodies can result from contamination of ds DNA with ds DNA having ss regions, and (2) non SLE sera do not contain antibodies specific for ds DNA at levels comparable to those found in SLE sera but rather contain high levels of antibodies reacting with ss regions or mixed DNA.",
author = "Locker, {J. D.} and Medof, {M. E.} and Bennett, {Robert (Rob)} and S. Sukhupunyaraksa",
year = "1977",
language = "English (US)",
volume = "118",
pages = "694--701",
journal = "Journal of Immunology",
issn = "0022-1767",
publisher = "American Association of Immunologists",
number = "2",

}

TY - JOUR

T1 - Characterization of DNA used to assay sera for anti DNA antibodies; determination of the specificities of anti DNA antibodies in SLE and non SLE rheumatic disease states

AU - Locker, J. D.

AU - Medof, M. E.

AU - Bennett, Robert (Rob)

AU - Sukhupunyaraksa, S.

PY - 1977

Y1 - 1977

N2 - Commercial 14C labeled KB cell DNA, widely used to assay sera for anti DNA antibodies, was chromatographed on benzoylated naphthoylated DEAE cellulose (BNDC) and on hydroxyapatite (HAP). On BNDC, only 25% of the 14C label eluted with 1 M NaCl (KB fraction I) characteristic of ds DNA. Fifty five percent of the label eluted with 50% formamide 1 M NaCl (KB fraction II) characteristic of ss or denatured DNA. On HAP, however, none of the 14C label eluted with 0.2 M phosphate buffer as anticipated for ss DNA, but, rather, all of the 14C label eluted with 0.4 M phosphate, characteristic of ds DNA. After pretreatment with S1 endonuclease of Aspergillus oryzae, which selectively digests ss regions, however, 42% of the 14C label was lost from the 0.4 M phosphate peak. These results indicated that more than half of this 14C KB cell DNA preparation was ds DNA with ss regions which was undetectable by HAP chromatography. 3H ds DNA and circular 3H ss DNA prepared from T7 and phi X174 bacteriophage, respectively, were found to be chromatographically pure on both BNDC and HAP. None of 10 non SLE sera (rheumatoid arthritis 3, mixed connective tissue disease 4, scleroderma 1, ulcerative colitis 1, and pulmonary fibrosis with chronic active hepatitis 1), previously believed to contain anti ds DNA antibodies on the basis of KB cell DNA testing had detectable antibodies against KB fraction I or T7 DNA: all of 10 KB cell DNA positive SLE sera had antibodies against both. Additionally, none of the 10 non SLE sera had antibodies against KB cell DNA when retested with DNA that had been pretreated with S1 endonuclease. Seven of these 10, however, as well as all 10 SLE sera, had antibodies against phi X174 DNA, KB fraction II DNA and alkali denatured T7 DNA. The data support the conclusions that (1) false positive tests for anti ds DNA antibodies can result from contamination of ds DNA with ds DNA having ss regions, and (2) non SLE sera do not contain antibodies specific for ds DNA at levels comparable to those found in SLE sera but rather contain high levels of antibodies reacting with ss regions or mixed DNA.

AB - Commercial 14C labeled KB cell DNA, widely used to assay sera for anti DNA antibodies, was chromatographed on benzoylated naphthoylated DEAE cellulose (BNDC) and on hydroxyapatite (HAP). On BNDC, only 25% of the 14C label eluted with 1 M NaCl (KB fraction I) characteristic of ds DNA. Fifty five percent of the label eluted with 50% formamide 1 M NaCl (KB fraction II) characteristic of ss or denatured DNA. On HAP, however, none of the 14C label eluted with 0.2 M phosphate buffer as anticipated for ss DNA, but, rather, all of the 14C label eluted with 0.4 M phosphate, characteristic of ds DNA. After pretreatment with S1 endonuclease of Aspergillus oryzae, which selectively digests ss regions, however, 42% of the 14C label was lost from the 0.4 M phosphate peak. These results indicated that more than half of this 14C KB cell DNA preparation was ds DNA with ss regions which was undetectable by HAP chromatography. 3H ds DNA and circular 3H ss DNA prepared from T7 and phi X174 bacteriophage, respectively, were found to be chromatographically pure on both BNDC and HAP. None of 10 non SLE sera (rheumatoid arthritis 3, mixed connective tissue disease 4, scleroderma 1, ulcerative colitis 1, and pulmonary fibrosis with chronic active hepatitis 1), previously believed to contain anti ds DNA antibodies on the basis of KB cell DNA testing had detectable antibodies against KB fraction I or T7 DNA: all of 10 KB cell DNA positive SLE sera had antibodies against both. Additionally, none of the 10 non SLE sera had antibodies against KB cell DNA when retested with DNA that had been pretreated with S1 endonuclease. Seven of these 10, however, as well as all 10 SLE sera, had antibodies against phi X174 DNA, KB fraction II DNA and alkali denatured T7 DNA. The data support the conclusions that (1) false positive tests for anti ds DNA antibodies can result from contamination of ds DNA with ds DNA having ss regions, and (2) non SLE sera do not contain antibodies specific for ds DNA at levels comparable to those found in SLE sera but rather contain high levels of antibodies reacting with ss regions or mixed DNA.

UR - http://www.scopus.com/inward/record.url?scp=0017613695&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0017613695&partnerID=8YFLogxK

M3 - Article

C2 - 300090

AN - SCOPUS:0017613695

VL - 118

SP - 694

EP - 701

JO - Journal of Immunology

JF - Journal of Immunology

SN - 0022-1767

IS - 2

ER -