A new ELISA for the detection of anti-heparan sulfate reactivity, using photobiotinylated antigen.
Autoantibodies reacting with a great variety of autoantigens are characteristic for the autoimmune disease systemic lupus erythematosus (SLE). Although reactivity with heparan sulfate (HS) in sera of patients with SLE is found in association with the occurrence of nephritis, the aetiological significance of this association is not clear. The assay which is generally used to measure anti-HS reactivity is subject to false-positive results, as a consequence of the binding of negatively charged moieties within immune complexes to the precoat employed (protamine sulfate). Therefore, we have developed a new ELISA in which photobiotinylated HS is efficiently and reproducibly bound to streptavidin-coated wells. We compared the new ELISA with the classical anti-HS ELISA by testing culture supernatants of 20 murine monoclonal antibodies (mAb) to DNA (containing free anti-DNA and anti-DNA/nucleosome immune complexes) and preparations of these mAb (containing only free anti-DNA), purified under dissociating conditions. In the classical anti-HS ELISA, 14 out of 20 of the culture supernatants reacted positively with HS; after purification no reactivity remained. The discrepancy must be due to anti-DNA/nucleosome immune complexes present in the culture supernatants. In the new ELISA only four out of 20 culture supernatants and one of the purified preparations reacted with HS. This latter reactivity is probably not specific, since this mAb also reacted with streptavidin alone. To find out whether there is a correlation between the occurrence of nephritis and anti-HS reactivity, measured in this new anti-HS ELISA, we tested sera of patients with a renal- or non-renal exacerbation of SLE in the newly developed anti-HS ELISA. We observed a correlation between anti-HS reactivity and nephritis.
Hylkema
M N
MN
Department of Autoimmune Diseases, Central Laboratory of the Netherlands Red Cross Blood Transfusion Service (C.L.B.), Amsterdam.
Kramers
C
C
Van der Wal
T J
TJ
Van Bruggen
M C
MC
Swaak
A J
AJ
Berden
J H
JH
Smeenk
R J
RJ
D000818:Animals; D000974:Antibodies, Antinuclear / Q000032:analysis; D000911:Antibodies, Monoclonal; D001323:Autoantibodies / Q000032:analysis*; D001710:Biotin; D003429:Cross Reactions / Q000276:immunology; D004247:DNA / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006497:Heparan Sulfate / Q000276:immunology*; D006801:Humans; D008180:Lupus Erythematosus, Systemic / Q000175:diagnosis / Q000276:immunology; D008181:Lupus Nephritis / Q000175:diagnosis / Q000276:immunology; D051379:Mice
D000974:Antibodies, Antinuclear; D000911:Antibodies, Monoclonal; D001323:Autoantibodies; D001710:Biotin; D004247:DNA; D006497:Heparan Sulfate