Goldberg J S, Wagenknecht D R, McIntyre J A
Methodist Center for Reproduction and Transplantation Immunology, Methodist Hospital of Indiana, Indianapolis 46202, USA.
J Clin Lab Anal. 1996;10(5):243-9. doi: 10.1002/(SICI)1098-2825(1996)10:5<243::AID-JCLA3>3.0.CO;2-9.
Interlaboratory inconsistencies in antiphospholipid antibody (aPA) solid phase assays have prompted controversy in clinical laboratory testing for aPA. We found that the aPA ELISA can be influenced by the type of microtiter plate utilized and by the conditions in which the plates are stored. By exposing 96-well, flat-bottom polystyrene microtiter plates to short wave UV light (254 nm), the aPA ELISA signal decreased in a UV dose-dependent manner. No effect was seen with long wave UV light (366 nm). These results were independent of the antibody isotype under study or the phospholipid (PL) antigen used: anionic phosphatidylserine (PS) and cardiolipin (CL), or zwitterionic phosphatidylethanolamine (PE). Purified human beta 2-glycoprotein I (beta 2 GPI), a known cofactor for anionic PL, and rabbit anti-beta 2 GPI antisera were used to demonstrate that beta 2 GPI bound equally to UV treated and untreated microtiter plates. In contrast, recognition of beta 2 GPI on an anionic PL surface was decreased on UV treated plates, suggesting that UV exposure alters the lipid binding properties of the microliter plate. To determine whether UV exposure inhibited PL binding directly or caused a change in the way the PL was bound, the amount of PL bound to UV treated and untreated plates was measured by using fluorescent labeled PS and a fluorimeter. PS binding was decreased by 53% in UV treated wells as compared to untreated wells. These data show that short wave UV exposure reduces PL binding to polystyrene microtiter plates, thereby reducing the amount of beta 2 GPI bound to PL coated ELISA plates. Thus by using UV exposed microtiter plates, decreased or false-negative a PA ELISA results may be obtained for aPA positive plasmas.
抗磷脂抗体（aPA）固相检测中实验室间的不一致性引发了aPA临床实验室检测的争议。我们发现，aPA酶联免疫吸附测定（ELISA）会受到所用微量滴定板类型以及板储存条件的影响。通过将96孔平底聚苯乙烯微量滴定板暴露于短波紫外线（254 nm）下，aPA ELISA信号以紫外线剂量依赖性方式降低。长波紫外线（366 nm）则未见影响。这些结果与所研究的抗体亚型或所用的磷脂（PL）抗原无关：阴离子磷脂酰丝氨酸（PS）、心磷脂（CL）或两性离子磷脂酰乙醇胺（PE）。纯化的人β2糖蛋白I（β2GPI）是一种已知的阴离子PL辅因子，使用兔抗β2GPI抗血清来证明β2GPI与紫外线处理和未处理的微量滴定板的结合能力相同。相比之下，在紫外线处理过的板上，阴离子PL表面上β2GPI的识别能力下降，这表明紫外线照射改变了微量滴定板的脂质结合特性。为了确定紫外线照射是直接抑制PL结合还是导致PL结合方式发生变化，通过使用荧光标记的PS和荧光计测量了与紫外线处理和未处理板结合的PL量。与未处理的孔相比，紫外线处理的孔中PS结合减少了53%。这些数据表明，短波紫外线照射减少了PL与聚苯乙烯微量滴定板的结合，从而减少了与PL包被的ELISA板结合的β2GPI量。因此，通过使用紫外线照射过的微量滴定板，对于aPA阳性血浆可能会获得降低的或假阴性的aPA ELISA结果。
