Performance evaluation of a radioimmunoprecipitation assay for the detection of N-type voltage-gated calcium channel antibodies

https://doi.org/10.1016/j.jim.2021.113102Get rights and content

Highlights

  • N-VGCC antibody test can contribute to the diagnosis of LEMS

  • Relevance of antibody in routine evaluation is limited by availability of testing

  • This study demonstrates comparable performance for two N-VGCC antibody assays

  • Discrepant low positive N-VGCC results were associated with autoimmunity but were not specific for LEMS

  • Efforts to harmonize assays for optimal patient categorization are needed

Abstract

Background

In this study, we assessed the performance characteristics of a laboratory-developed radioimmunoassay (RIA) to detect N-type voltage-gated calcium channel (N-VGCC) antibodies found in several autoimmune neurologic diseases.

Methods

Four hundred and forty-five (n = 445) sera were evaluated, including 156 sera (50 positive and 106 negative for N-VGCC antibodies) previously tested at Mayo Clinic Laboratories (MCL) and 289 controls (n = 187 disease and n = 102 healthy). Specimens were analyzed with the RIA using N-VGCC labeled with 125I-ω-conotoxin GVIA. The RIA was compared to the predicate MCL assay using a tiered positive predictive value (PPV) approach. Other performance characteristics evaluated included specificity, precision, interference, and stability.

Results

Qualitative inter-laboratory agreement based on tiered PPVs was 100% for results >1.00 nmol/L (71% PPV), 48% for results of 0.10–0.99 nmol/L (24% PPV) and 22% for results of 0.04–0.10 nmol/L (19% PPV). Negative results showed 90% agreement (n = 106). Specificity in controls positive for other neural autoantibodies and healthy controls were 87% and 100%, respectively. Acceptable results were observed for other performance characteristics.

Conclusions

Inter-laboratory correlations demonstrate equivalence between assays with some discrepancies between low positive results. Collaborative efforts aimed at assessing the clinical spectrum associated with these antibodies and consensus for harmonizing test performance are required for optimal categorization of patients.

Introduction

Antibodies against the N-type voltage-gated calcium channel (N-VGCC) are the second most prevalent antibody in patients with Lambert-Eaton myasthenic syndrome (LEMS); detected in 33–73% of patients diagnosed with paraneoplastic LEMS (Lennon et al., 1995; Motomura et al., 1995, Motomura et al., 1997; Zalewski et al., 2016). N-VGCC autoantibodies are also detected in association with a variety of inflammatory neurologic autoimmune disorders including limbic encephalitis, neurodegenerative, psychiatric, seizure and movement disorders (Bekircan-Kurt et al., 2015; Zalewski et al., 2016; Totzeck et al., 2016; Gozzard et al., 2015). Detection of N-VGCC antibodies can inform treatment of neurological symptoms and prompt efforts to detect potential underlying neoplasms (Hülsbrink and Hashemolhosseini, 2014; Motomura et al., 2016).

The prevalence of N-VGCC antibodies is rare. Of 78,889 sera tested at Mayo Clinic Laboratories (MCL) for N-VGCC and 14 other paraneoplastic neural autoantibodies, N-VGCC antibodies were detected in 1.13% (n = 889) (Horta et al., 2014). Similarly, of 5241 sera referred by ARUP Laboratories (ARUP) for testing at MCL for N-VGCC antibodies and other paraneoplastic antibodies using the panel described above or related panels (Encephalopathy, Epilepsy, Dementia, Autoimmune Dysautonomia and Autoimmune Gastrointestinal Dysmotility), N-VGCC antibodies were detected in 2.23% (n = 117) of the specimens. Association with autoimmune neurologic symptoms (encephalopathy, neuromuscular junction disorder, ataxia, neuropathy, myelopathy and myopathy) differs based on the level of N-VGCC antibodies reported by MCL (Zalewski et al., 2016). Patients with low positive N-VGCC antibody results (0.03–0.09 nmol/L) had a 19% positive predictive value (PPV) for an autoimmune neurologic disorder, patients with medium positive results (0.10–0.99 nmol/L) had a 24% PPV and those with high positive results (>1.00 nmol/L) had a 71% PPV. Of the 117 N-VGCC antibody positive results detected in specimens referred to MCL by ARUP, 41% (48/117) had low positive, 58% (68/117) had medium positive and 1% (1/117) had high positive results. There are limited or no studies performed outside MCL that have examined the relationships between the relative concentration of these antibodies and their positive predictive values for the spectrum of clinical diseases.

While N-VGCC antibodies have been associated with both LEMS as well as other autoimmune neurologic diseases and may confer diagnostic and prognostic value, testing for this analyte is only offered by a few laboratories in the United States. Validation of this test for use at additional clinical laboratories could lead to better understanding and categorization of N-VGCC antibody associated disease. The purpose of this study was to evaluate the performance characteristics of a laboratory-developed radioimmunoprecipitation assay (RIA) for the serologic detection of autoantibodies directed against N-VGCC.

Section snippets

Patients and Controls

Four hundred and forty-five (n = 445) sera were evaluated. These included 156 sera (50 positive and 106 negative for N-VGCC antibodies) previously tested at Mayo Clinic Laboratories (MCL) for N-type VGCC and other paraneoplastic or autoimmune encephalitis related neural autoantibodies. These antibodies included the anti-neuronal nuclear antibodies (ANNA)-1, ANNA-2, ANNA-3, Purkinje cell cytoplasmic antibodies (PCA)-1, PCA-2, PCA-Tr, amphiphysin, collapsin response-mediator protein-5 (CRMP-5),

Performance characteristics of RIA for the detection of N-VGCC antibodies

The performance of the N-VGCC RIA was compared to results obtained for 139 sera previously tested at MCL based on a tiered positive predictive value (PPV) of the predicate assay. Comparison of N-VGCC antibody results obtained at ARUP with those of the predicate assay (MCL) using the reference intervals established as described above (<70.0 pmol/L = negative, 70.0–110.0 pmol/L = indeterminate, >110.0 pmol/L = positive) showed good overall agreement (Fig. 1). However, a few discrepancies were

Discussion

The presence of N-VGCC antibodies are associated with a variety of inflammatory neurologic autoimmune disorders including LEMS. While the prevalence of N-VGCC antibodies is rare, these antibodies were the fifth or sixth most frequent of 15 antibodies in a panel for paraneoplastic antibody evaluation (Horta et al., 2014; Tebo et al., 2016) and have been reported to have some prognostic value in addition to their diagnostic significance (Lennon et al., 1995; Motomura et al., 1995, Motomura et

Conclusion

N-VGCC antibodies are relatively frequent in a variety of inflammatory neurologic autoimmune disorders including LEMS and may confer diagnostic and prognostic value. Thus, increased accessibility of testing is needed. This evaluation addresses this immediate clinical need. Future collaborative efforts aimed at optimizing the performance of N-VGCC antibody tests for more optimal patient characterization and categorization of clinical associations are required. The use of smaller disease-specific

Declaration of Competing Interest

Reagents used for the detection of N-VGCC antibodies were provided free of charge by Kronus Inc. and RSR Limited. BKL, TRH, AET and LKP do not have any other declarations of interest.

Acknowledgements

The authors would like to thank Kronus for providing reagents for the development and validation of the N-VGCC assay at ARUP Laboratories. This study was funded by the ARUP Institute for Clinical and Experimental Pathology and the Department of Pathology, University of Utah School of Medicine, Salt Lake City, UT.

References (17)

There are more references available in the full text version of this article.

Cited by (0)

View full text