Circulating NELL1 antibodies

Serum antibodies to NELL1 are present in patients with NELL1-associated MN, as demonstrated by seroreactivity against recombinant NELL1 in immunoblot under nonreducing conditions [11–13]. No reactivity was observed under reducing conditions, suggesting that anti-NELL1 autoantibodies recognize conformation-dependent epitopes [5,11].

Serial serum samples were available from before and during follow-up for one patient [11]. In this individual anti-NELL1 antibodies disappeared with immunosuppressive treatment, while proteinuria persisted [11]. This is in keeping with immunological remission preceding clinical remission, as
seen with anti-PLA2R-associated MN [11].

Second most common type of MN

NELL1-associated MN represents a new defined serological subtype and accounts for approximately 10% of MN cases overall [3]. In PLA2R-negative cohorts, the proportion of NELL1-positive cases exceeds that of THSD7A-positive cases [11,13]. These data suggest that NELL1 is the second most common target antigen in MN after PLA2R [3,11,13].

Although most cases of anti-NELL1 MN seem to be primary, NELL1 antibodies have also been identified in secondary MN associated with malignancy, alpha-lipoic use or mercury exposure [3,5]. NELL1-associated MN may be more frequently linked to malignancy than other MN subtypes, with tumour types including prostate, lung and breast carcinomas [12].

Serological diagnostic assays for anti-PLA2R and anti-THS7DA

Serological methods for detection of autoantibodies in MN diagnostics include indirect immunofluorescence assay (IFA), enzyme-linked immunosorbent assay (ELISA) and chemiluminescence immunoassay (ChLIA).

The IFA is a highly sensitive method that can detect very low antibody titres [14,15]. In the procedure, patient sera are incubated on slides with transfected cells that over-express the PLA2R or THS7DA target antigen [4,10,16]. The autoantibodies are detected using fluorescein-labelled antihuman antibodies and visualized with a fluorescence microscope [4,10,14]. Results are evaluated as positive/negative or semiquantitatively based on the dilution factor [7,10,14]. Using BIOCHIP Mosaics, PLA2R and THS7DA substrates can be analysed in parallel in one test [10].

ELISA and ChLIA enable quantitative antibody measurements of anti-PLA2R antibodies, as required for monitoring disease activity and therapy responses [15,16]. The clinical sensitivities for MN amount to 73.5% for ELISA and 83.9% for ChLIA, compared to 83.2% for IFA, with all assays demonstrating specificities of at least 99.4% [15]. In the ELISA, antibody levels of 14 RU/ml or more are classed as positive [4,7]. Values greater than 50 RU/ml are a criterion for a high risk of progressive loss of kidney function [7], whereas values greater than 150 RU/ml are associated with a lower likelihood of spontaneous remission or response to low-dose rituximab therapy [7,17].

New IFA for NELL1 (RUO)

To aid detection of anti-NELL1 autoantibodies, a novel anti-NELL1 IFA (for research use only) has recently been developed. Analogous to the established assays for anti-PLA2R and anti-THSD7A, the anti-NELL1 IFA uses transfected cells expressing the target antigen on their surface. Furthermore, a BIOCHIP Mosaic IFA combining PLA2R-, THSD7A- and NELL1-expressing substrates (research use only) enables multiparametric detection of all three autoantibodies in a single assay (Fig. 3).

Outlook – expanding understanding of MN

The identification of NELL1 as a target antigen in MN and its recognition as the second most common antigen after PLA2R have brought this parameter into the spotlight of MN research. Future studies will aim to clarify whether anti-NELL1 autoantibodies are directly pathogenic and to explore their role in disease mechanisms [12]. In addition, the potential correlation of anti-NELL1 antibody titres with proteinuria or underlying malignancy warrants further investigation [12].

Alongside NELL1, a plethora of additional MN target antigens has been discovered in the last few years, for example semaphorin  3B (SEMA3B), protocadherein 7 (PCDH7), serine protease HTRA1, exostosin 1 and exostosin 2, (EXT1/EXT2) and neural cell-adhesion molecule 1 (NCAM1) [3,4,5,9,17]. Many of these target antigens are associated with distinct clinical and pathological findings [3,4,5,9,17]. Elucidating the role of each antigen in MN disease pathogenesis may eventually allow more precision medicine to be used in MN disease management [3].

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