Antibodies to guanosine triphosphate misidentified as anti-double-stranded DNA antibodies in a patient with antinuclear antibody-negative lupus, due to buckling of insolubilized assay DNA.
OBJECTIVE
To investigate why the serum of a pediatric patient with systemic lupus erythematosus was persistently (>30 months) and strongly positive for antibodies to double-stranded DNA (dsDNA) as revealed by enzyme-linked immunosorbent assay (ELISA), but yielded negative results on the antinuclear antibody test (HEp-2 immunofluorescence [IF]).


METHODS
The patient's antibodies were isolated on dsDNA and single-stranded DNA (ssDNA) supports, which were then examined by dsDNA ELISA and HEp-2 IF. Tests included the use of various inhibitors to determine the fine specificity of the antibodies. Other tests performed included immunoblotting, immunoprecipitation, Crithidia luciliae IF, and neutrophil IF.


RESULTS
The antibodies isolated from the dsDNA and ssDNA supports were similar, in that they were of the IgG type, bound well in the dsDNA ELISA, and recognized a normally hidden nucleolar RNA antigen in HEp-2 cells. With both the dsDNA ELISA and nucleolar antigens, inhibition studies revealed that the epitope recognized was guanosine 5'-triphosphate (GTP). Binding of the antibodies was better to GTP than to guanosine 5'-monophosphate or cytidylyl (3'-5') guanosine, and, in turn, was better than to guanosine, while N7-methylated GTP was unreactive. The antibodies did not bind to dsDNA present in solution or in HEp-2 or Crithidia cells, but bound transfer RNA well and recognized a cytoplasmic RNA antigen in neutrophils.


CONCLUSIONS
A new problem in dsDNA ELISA is revealed in the occurrence of a hitherto-unknown and unusual buckling of the insolubilized DNA molecule, which, absent in dsDNA found in solution or in whole cells, presumably creates gaps of single-strandedness in the molecule. A new antibody specific for GTP is described in this patient, which may be clinically important.
Ma
Chun Hung
CH
Clinical Immunology Unit, Prince of Wales Hospital, The Chinese University of Hong Kong, Shatin, Hong Kong.
Hui
Joannie
J
Tang
Janet Tsui Ying
JT
Leung
Danny Tze Ming
DT
Chui
Yiu Loon
YL
Fok
Tai Fai
TF
Lim
Pak-Leong
PL
D000974:Antibodies, Antinuclear / Q000097:blood*; D000918:Antibody Specificity; D002648:Child; D004247:DNA / Q000276:immunology* / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay / Q000592:standards*; D005189:False Positive Reactions; D005260:Female; D006160:Guanosine Triphosphate / Q000276:immunology*; D006801:Humans; D008180:Lupus Erythematosus, Systemic / Q000175:diagnosis / Q000276:immunology*; D012995:Solubility
D000974:Antibodies, Antinuclear; D006160:Guanosine Triphosphate; D004247:DNA