Detection of the anti-PLA2R antibody significantly improved early diagnosis and appropriate clinical treatment of IMN. Here, we developed a novel POCT test (QD-ICA) for the quantitative detection of PLA2R autoantibodies and compared its performance with a commercially available ELISA. Compared with other assays targeting anti-PLA2R antibody detection, QD-ICA was highly sensitive and faster, with an observed clinical sensitivity and specificity at 80.9 and 100%, respectively (both exceeding ELISA results). Furthermore, QD-ICA LoD was as low as 2.86 RU/mL, further demonstrating QD-ICA superiority to ELISA.
The titers of anti-PLA2R antibodies change throughout the IMN-treatment period [25]. Increasing levels of PLA2R autoantibodies predict the exacerbation of symptoms, whereas decreased levels correlate with disease remission [26, 27]. Therefore, it is necessary to detect autoantibodies quantitatively. Previous studies used western blotting, which requires electrophoresis and immunoblotting, as well as the use of naturally purified glomerular glycoprotein or extracts from cells overexpressing PLA2R, which may not be available in conventional clinical laboratories. Similarly, immunofluorescence testing methods are semiquantitative and observer dependent, making them unsuitable for clinical testing.
Several methods have been developed for quantifying PLA2R autoantibodies based on the use of recombinant human PLA2R, including CBA-IFA, ELISA, ALBIA, TRFIA, LIPS, and ChLIA [18, 20, 21, 28]. Positive rates of anti-PLA2R detection using QD-ICA (80.9%) were similar to those reported previously for the other assays [ELISA (50.0–71.8%) [26, 29], ALBIA (51.5–66.9%) [18, 30], TRFIA (71.0%) [28], LIPS (53.3%) [20], and ChLIA (83.9%) [21]]. Although the positive rates of western blotting (53.0–81.7%) [31, 32] and CBA-IFA (48.0–82.3%) [33, 34] are also comparable to those of quantitative assays, the semiquantitative nature of these methods hinders their clinical application.
The EUROIMMUN ELISA is currently a widely used method in clinical laboratories. The cut-off value as per the manufacturer instructions is recommended at 20 RU/mL, and values exceeding this are considered positive and those < 14 RU/mL are considered negative. However, a value between 14 and 20 RU/mL represents an uncertain outcome. Moreover, a value of 20 RU/mL results in a specificity ranging from 89.7 to 100% [18, 29, 35–39]. Some studies used different customized thresholds (e.g., 2, 2.6, or 14 RU/mL) to increase sensitivity [18, 36, 37, 39], although these sometimes can adversely affect specificity. In the present study, ROC analysis showed an optimal cut-off value for ELISA at 17.84 RU/mL, which resulted in maximum sensitivity and specificity of 72.1 and 98.5%, respectively, which was still lower than the results obtained using the QC-ICA. Bobart et al. found that cases tested using ELISA with results ranging from 2 to 20 RU/mL and positive via IFA were confirmed as MN via biopsy. However, 57.5% of cases tested using ELISA with results ranging from 2 to 20 RU/mL and negative via IFA were confirmed as MN via biopsy [40]. Therefore, Bobart et al. suggested that samples with ELISA results ≥ 2 RU/mL and ≤ 20 RU/mL should be confirmed via IFA before performing MN-related biopsies to improve diagnostic sensitivity. Among our 68 patients with IMN, confirmed using renal biopsy, the samples that tested positive for anti-PLA2R antibodies using ELISA were also positive in the QD-ICA test. However, anti-PLA2R antibodies from six patients with IMN were not detected in the ELISA, suggesting that QD-ICA was more sensitive than ELISA and did not require a confirmation test.
QD-ICA belongs to the lateral flow assay-based POCT devices category and is performed over a strip that consists of a sample pad, marker-binding pad, absorption pad, and NC membrane. Compared to ELISA, this method is easy to operate and has low operating costs [41]. The advantages offered by QD-ICA, including simplicity, user-friendly format, cost-effectiveness, the ability to quantify results, and high reagent stability, would be a breakthrough for IMN diagnosis. QD-ICA exhibited improved sensitivity due to its reliance upon labeling using aqueous QDs rather than macromolecular enzymes, thereby allowing a more efficient labeling. Additionally, the use of aqueous QDs increases the Stoker shift between the excitation and emission wavelengths, thus, effectively avoiding environmental interference due to a relatively long fluorescence-emission period. There were some limitations to this study. All patients enrolled in the study were from a single center, and a limited number of cases included in this study. In addition, only the performance of QD-ICA was compared with ELISA for the detection of PLA2R autoantibodies. To determine the potential advantages and disadvantages of QD-ICA further, additional detection studies are needed for comparative analysis.
In summary, this study constructed a rapid, quantitative, and highly sensitive detection assay of anti-PLA2R antibodies using a CdSe/ZnS-based immunosorbent assay. The novel QD-ICA has the advantages of faster detection time, fewer steps, wider testing scenarios, and greater overall feasibility for detecting PLA2R autoantibodies compared with ELISA. These findings suggest that QD-ICA can improve IMN diagnosis and monitoring of patient response to treatment. We will further optimize our assay and use a larger number of clinical samples to confirm the performance of the assay in hopes of achieving clinical implementation.