Hylkema M N, Kramers C, Van der Wal T J, Van Bruggen M C, Swaak A J, Berden J H, Smeenk R J
Department of Autoimmune Diseases, Central Laboratory of the Netherlands Red Cross Blood Transfusion Service (C.L.B.), Amsterdam.
J Immunol Methods. 1994 Nov 10;176(1):33-43. doi: 10.1016/0022-1759(94)90348-4.
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.
与多种自身抗原发生反应的自身抗体是自身免疫性疾病系统性红斑狼疮（SLE）的特征。尽管在SLE患者血清中发现与硫酸乙酰肝素（HS）的反应性与肾炎的发生有关，但这种关联的病因学意义尚不清楚。通常用于测量抗HS反应性的检测方法容易出现假阳性结果，这是由于免疫复合物中的带负电荷部分与所用预包被物（硫酸鱼精蛋白）结合所致。因此，我们开发了一种新的酶联免疫吸附测定（ELISA），其中光生物素化的HS能高效且可重复地结合到链霉亲和素包被的孔中。我们通过检测20种针对DNA的鼠单克隆抗体（mAb）的培养上清液（含有游离抗DNA和抗DNA/核小体免疫复合物）以及这些mAb的制剂（仅含有游离抗DNA），在解离条件下进行纯化，将新的ELISA与经典的抗HS ELISA进行了比较。在经典的抗HS ELISA中，20种培养上清液中有14种与HS发生阳性反应；纯化后不再有反应性。这种差异一定是由于培养上清液中存在抗DNA/核小体免疫复合物。在新的ELISA中，20种培养上清液中只有4种以及纯化制剂中的1种与HS发生反应。后一种反应性可能不具有特异性，因为这种mAb也单独与链霉亲和素发生反应。为了弄清楚在这种新的抗HS ELISA中测量的肾炎发生与抗HS反应性之间是否存在相关性，我们在新开发的抗HS ELISA中检测了SLE肾脏或非肾脏病情加重患者的血清。我们观察到抗HS反应性与肾炎之间存在相关性。
