Background/Purpose : Better risk prediction is needed to identify patients who will benefit from immunosuppressive therapy with idiopathic membranous nephropathy (MN) due to the variable natural course of the disease–spontaneous remission occurs in 40–50% of patients. It has recently been reported that many patients with idiopathic MN, but not patients with secondary MN, have circulating antibodies against the M-type phospholipase A2 receptor (PLA2R), a transmembrane protein located on podocytes. Although measuring anti–PLA2R levels has also been suggested to be a method to follow and predict response to treatment, widely available published measuring methods are only Western blot and a semi-quantitative immunofluorescence test. We aimed to develop cell–based enzyme–linked immunosorbent assay (ELISA) for the quantification of anti–PLA2R antibodies and investigate its clinical usefulness in patients with MN. We also aimed to validate the absence of anti–PLA2R antibodies in patients with systemic lupus erythematosus (SLE).

Methods : The synthesized human PLA2R gene was transfected into the HEK293T cells using the pcDNA3.1/Hygro (+) vector. Stable cell line expressing PLA2R was generated through limiting dilution and evaluated by flow cytometry and Western blot. Using this cell line, we developed a quantitative cell–based ELISA for anti–PLA2R antibodies as follows. HEK293T cells stably expressing PLA2R were seeded and cultured in wells of a flat–bottomed 96–well tissue culture plate coated with poly–D–Lysine. After the cells were fixed, serum samples were added. Subsequently, the peroxidase–conjugated anti–human IgG and TMB were added in order, and color development was measured. The usefulness of this test was studied in 26 patients with biopsy–proven primary MN, and 16 SLE patients with pure MN. Western blots using the lysates of HEK293T cells stably expressing PLA2R were also performed with these samples. Clinical data of these patients were retrospectively evaluated. Treatment was determined by physician preference in each individual based on clinically available information without prior knowledge of anti–PLA2R antibody positivity.

Results : Stable expression of PLA2R was detected in the HEK293T cells by flow cytometry and Western blot. Anti–PLA2R antibodies were positive in 7/26 (27%) by cell–based ELISA and 7/26 (27%) by Western blot. Cohen’s κ coefficient of the 2 tests was 0.61 which means there is substantial agreement. Retrospective analyses revealed that all of the 7 patients who were anti–PLA2R positive by cell–based ELISA were treated with immunosuppressive therapy. In contrast, only 5 out of 26 patients with negative results received immunosuppressive therapy. Thus, the results of cell–based ELISA were associated with physicians’ decision on immunosuppressive therapy (p< 0.001). Renal function did not decline in any of the anti–PLA2R negative patients. All of the 16 SLE patients with pure MN were negative for both cell–based ELISA and Western blot.

Conclusion : This study showed that our cell–based ELISA is reliable and clinically useful examination for MN. Anti–PLA2R antibodies may serve as predictive biomarker in MN. The absence of anti–PLA2R antibodies in patients with SLE was reconfirmed.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/development-of-cell-based-enzyme-linked-immunosorbent-assay-for-the-quantification-of-anti-m-type-phospholipase-a-receptor-antibodies-and-its-clinical-u/