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Journal of Clinical Sleep Medicine : JCSM : Official Publication of the American Academy of Sleep Medicine logoLink to Journal of Clinical Sleep Medicine : JCSM : Official Publication of the American Academy of Sleep Medicine
. 2024 Jun 1;20(6):941–946. doi: 10.5664/jcsm.11056

Cell-based vs enzyme-linked immunosorbent assay for detection of anti-Tribbles homolog 2 autoantibodies in Chinese patients with narcolepsy

Xianhui Zhong 1,2,*, Yuqing Yuan 1,2,*, Qingqing Zhan 1,2, Tiantian Yin 1,2, Chengxin Ku 1,2, Yuxin Liu 1,2, Fen Wang 1,2,3,4, Yongmin Ding 1,2,3,4, Liying Deng 1,2,3,4, Wei Wu 1,2,3,4,✉, Liang Xie 1,2,3,4,✉
PMCID: PMC11145039  PMID: 38318919

Abstract

Study Objectives:

Narcolepsy type 1 is attributed to a deficiency in cerebrospinal fluid orexin and is considered linked to autoimmunity. The levels of anti-Tribbles homolog 2 (TRIB2) autoantibodies are elevated in the sera of some patients with narcolepsy with cataplexy. Additionally, injecting mice with serum immunoglobulin from patients with narcolepsy with positive anti-TRIB2 antibodies can induce hypothalamic neuron loss and alterations in sleep patterns. Consequently, we hypothesized the existence of a potential association between anti-TRIB2 antibodies and narcolepsy. To test this possibility, we used cell-based assays (CBAs) and enzyme-linked immunosorbent assays (ELISAs) to detect the presence of anti-TRIB2 antibodies in Chinese patients with narcolepsy.

Methods:

We included 68 patients with narcolepsy type 1, 39 patients with other central disorders of hypersomnolence, and 43 healthy controls. A CBA and a conventional ELISA were used to detect anti-TRIB2 antibody levels in patients’ sera.

Results:

CBA was used to detect serum anti-TRIB2 antibodies in Chinese patients with narcolepsy, and the results were negative. However, when the ELISA was used, only 2 patients with narcolepsy type 1 had TRIB2 antibody titers higher than the mean titer plus 2 standard deviations of the healthy controls.

Conclusions:

In our study, ELISA identified TRIB2 autoantibodies in sera of patients with narcolepsy where CBA failed to demonstrate them. Contrary to our hypothesis, this intriguing finding deserves further research to elucidate the potential association between TRIB2 and narcolepsy type 1. Exploring the implications of TRIB2 autoantibodies in narcolepsy and disparate outcomes between ELISA and CBA could provide crucial insights.

Citation:

Zhong X, Yuan Y, Zhan Q, et al. Cell-based vs enzyme-linked immunosorbent assay for detection of anti-Tribbles homolog 2 autoantibodies in Chinese patients with narcolepsy. J Clin Sleep Med. 2024;20(6):941–946.

Keywords: narcolepsy, Tribbles homolog 2, TRIB2, cell-based assay, CBA, enzyme-linked immunosorbent assay, ELISA, autoantibodies

BRIEF SUMMARY

Current Knowledge/Study Rationale: Elevated anti-Tribbles homolog 2 (TRIB2) antibodies may be related to narcolepsy; however, this possibility has not yet been tested in Chinese patients with this condition. We employed a novel cell-based assay to detect anti-TRIB2 antibodies in Chinese individuals with narcolepsy and compared the results with those obtained using an enzyme-linked immunosorbent assay.

Study Impact: In Chinese patients with narcolepsy, the anti-TRIB2 antibody was not detected by cell-based assay. Notably, 2 patients with narcolepsy type 1 tested positive for the TRIB2 antibody in enzyme-linked immunosorbent assay experiments, highlighting the possibility that narcolepsy type 1 cases with enzyme-linked immunosorbent assay–based positivity for anti-TRIB2 represent a distinct subtype or phenotype. Exploring the implications of TRIB2 autoantibodies in narcolepsy and disparate outcomes between enzyme-linked immunosorbent assay and cell-based assay could provide crucial insights.

INTRODUCTION

Narcolepsy is a rare chronic neurological disorder characterized by severe and irresistible episodes of excessive daytime sleepiness, cataplexy, vivid sleep hallucinations, sleep paralysis, and fragmented nocturnal sleep.1,2 In the International Classification of Sleep Disorders, third edition, narcolepsy has been categorized into type 1 (NT1) and type 2 according to whether it is accompanied by a decrease in hypothalamic orexin-1 levels in the cerebrospinal fluid.3,4 Many studies have suggested that narcolepsy may be an autoimmune disease, and it is found to be closely related to the HLA-DQB1 gene.5,6 Furthermore, a genome-wide association study has revealed noteworthy associations between NT1 and gene polymorphisms such as TCR-α, P2RY11, CCR1/CCR3, TNFSF4, CTSH, ZNF365, and CPT1B/CHKB.1,7 The increased occurrence of narcolepsy may be relevant to infection with Streptococcus pyogenes,8,9 influenza A (H1N1) virus, and vaccination with influenza vaccine H1N1-AS03,10,11 indicating the presence of an immune trigger for narcolepsy. The autopsy findings of patients with narcolepsy with cataplexy demonstrated a specific and selective loss of hypothalamic orexin neurons, which may be caused by autoimmune mediation. However, this hypothesis remains unverified to date.12

To obtain evidence of autoimmune attack, several studies attempted to identify specific antibodies such as NMDAR, CASPR2, LGI1,13 Contactin2, Dopamine D2R,14 AMPA-R1/2, DPPX, GABAB-R1/2, LGI-2, CASPR-1, Hu, Ri, Yo, Ma1, GAD65,15 and Ma216 in people with narcolepsy, but no positive antibodies were detected.

There exists a potential correlation between the anti-Tribbles homolog 2 (TRIB2) antibody and the immunological mechanism underlying narcolepsy. TRIB2 protein belongs to a class of serine/threonine pseudokinases that play a crucial role in regulating various signaling pathways in vivo and participate in cellular immunity, growth, proliferation, apoptosis, and other physiological activities.17 Studies have confirmed that TRIB2 is an autoantigen of autoimmune uveitis.18 Cvetkovic-Lopes et al19 reported that TRIB2 transcripts were more than 3-fold enriched in hypothalamic hypocretin neurons of a transgenic mouse model. In 2010, 3 clinical studies on patients with narcolepsy from Europe,19 the United States,20 and Japan21 were performed to detect the serum levels of anti-TRIB2 antibodies by enzyme-linked immunosorbent assay (ELISA) or radioligand binding assay techniques. These study findings showed that 14–26% patients with narcolepsy with cataplexy had elevated levels of anti-TRIB2 antibodies. In 2013, Katzav et al22 extracted serum immunoglobulin from patients with narcolepsy with a positive HLA-DQB1*06:02 gene and positive anti-TRIB2 antibody in the serum and slowly injected these immunoglobulins into the ventricles of mice; they found that the mice showed narcolepsy behavior changes and selective loss of hypocretin neurons in the lateral hypothalamus. The hypocretin/ataxin-3 mice serve as an established animal model for narcolepsy, in which orexin neurons are lost with age. In a study conducted by Tanaka et al23 in 2017, this model was utilized to investigate the dynamics of anti-TRIB2 antibody titers in the plasma of mice as the ablation of hypocretin neurons followed, suggesting that an increase in anti-TRIB2 antibody titer is a consequence of the loss of hypothalamic orexin neurons.

To the best of our knowledge, no study has yet been conducted to detect the anti-TRIB2 antibody in Chinese patients with narcolepsy. To further explore whether anti-TRIB2 antibody is related to the pathogenesis of narcolepsy in the Chinese population, we used a cell-based assay (CBA) method, known for its high specificity and sensitivity, and conventional ELISA to determine the serum levels of anti-TRIB2 antibodies in Chinese patients with narcolepsy.

METHODS

Study groups

A total of 107 patients presenting with central disorders of hypersomnolence were recruited from the Second Affiliated Hospital of Nanchang University between November 2018 and August 2023. These patients were meticulously classified and diagnosed per strict adherence to the diagnostic criteria outlined in the International Classification of Sleep Disorders, third edition, published in 2014.3 Among the recruited patients, 68 were diagnosed with NT1, and the remaining 39 patients had other central disorders of hypersomnolence, including 16 patients with narcolepsy type 2, 14 with idiopathic hypersomnia, and 9 with other causes of excessive daytime sleepiness. None of the patients received any treatment before blood collection. We collected data on patients’ sex, age, body mass index, Epworth Sleepiness Scale, multiple sleep latency test, polysomnography disease duration, and clinical features (ie, disease duration, daytime sleepiness, cataplexy, hypnagogic hallucinations, and sleep paralysis).

Forty-three individuals who were matched in terms of age, sex, and body mass index were recruited as the healthy controls (HCs). These individuals did not manifest any sleep disorders and underwent a standard health assessment at the hospital.

The study was approved by the ethics committee of the Second Affiliated Hospital of Nanchang University, China. All participants provided written informed consent to participate in the study.

Sample collection

Blood samples were collected via routine venipuncture from 68 patients with NT1, 39 participants with nonnarcolepsy type 1 (non-NT1), and 43 HCs. The collected blood samples were refrigerated at 4°C and then centrifuged at 3,000 × g at 4°C for 10 minutes. The centrifuged serum was carefully aliquoted and stored at −80°C until further analysis.

The Biobank Center of the Second Affiliated Hospital of Nanchang University conducted the collection, handling, and preservation of clinical specimens in accordance with Chinese Regulations on the Management of Human Genetic Resources, ensuring scientific and rigorous procedures and strict quality control of all samples.

CBAs

Antibody testing was conducted using a CBA for anti-TRIB2 antibody by Kingmed Diagnostics Co., Ltd., which is the largest College of American Pathologists–certified laboratory in China.24 HEK 293 cells were cultured in 96-well plates and cotransfected with a full-length human TRIB2 gene and pcDNA3.1-EGFP plasmid. The cells were fixed at 36 hours posttransfection with 4% paraformaldehyde for 20 minutes, followed by incubation in phosphate-buffered saline containing 0.25% Triton X-100 for 30 minutes to permeabilize the cells for antibody detection. Briefly, 1:10 dilution of serum in phosphate-buffered saline with 10% goat serum was used to incubate cells for 2 hours at room temperature (25°C). Subsequently, the cells were subjected to 3 30-minute washes in phosphate-buffered saline supplemented with 0.1% Tween-20. The plate was wrapped in foil to avoid light exposure and incubated for 1 hour with goat antihuman immunoglobulin G (1:500; Thermo Scientific, Waltham, MA) at room temperature. The cells were subsequently rinsed with phosphate-buffered saline supplemented with 0.1% Tween-20 and observed under immunofluorescence microscopy. Two independent blinded assessors, unaware of the experimental conditions, categorized each sample as either positive or negative based on the intensity of the immunofluorescence observed on the cell surface, in direct contrast to nontransfected cells and control samples. Following validation, the positively identified specimens were subsequently diluted in a serial manner, ranging from 1:10 to 1:1000, to ascertain the titers. The ultimate titer was determined as the dilution value of the sample at which specific fluorescence was minimally yet distinctly detectable and was subsequently denoted as the corresponding dilution value.

ELISA

TRIB2-specific antibody quantification in patients with narcolepsy and controls was performed using an ELISA kit (kt95566; MSKBIO, Wuhan, China) following the manufacturer’s instructions. Standard, blank, and sample holes were set, respectively. Standard wells contained 50 µl of diluted standards and sample wells contained 50 µl of test samples diluted 5-fold. The plates were then sealed with a plate-sealing membrane and incubated at 37°C for 30 minutes. Subsequently, the liquid was removed, the wells were dried by swinging, washing buffer was added to every well, the plate was left to stand for 30 seconds, and the buffer was removed; this procedure was repeated 5 times. Then, 50 µl of enzyme-labeled reagent was added to all but the blank wells and the cells were incubated at 37°C for 30 minutes. Subsequently, the liquid was removed, the wells were dried by swinging, washing buffer was added to every well, the plate was left to stand for 30 seconds, and the buffer was removed; this procedure was repeated 5 times. Chromogen A (50 µl) and chromogen B (also 50 µl) were sequentially added to each well, followed by mixing with gentle shaking, and incubation at 37°C in the dark for 15 minutes. Stop solution (50 µl) was then added to each well to stop the reaction (an immediate blue-to-yellow color change). The absorbance value (optical density) was detected at 450 nm using a microplate reader. For normalization and interplate calibration, 3 serum samples were repetitively quantified on each plate (intraassay coefficient of variation, < 6%; interassay coefficient of variation, < 11%).

Statistical analysis

All measurements are presented as mean ± standard deviation (SD), and categorical variables are expressed as n (%). Comparison between the 2 groups was carried out by Mann–Whitney U test (for nonnormally distributed data) or t test (for normally distributed data). Comparisons among 3 or more groups were analyzed by 1-way analysis of variance or Kruskal–Wallis H test. Categorical variables are compared by chi-squared test. Statistical significance was defined as P < .05. Mean titer plus 2 SD of 43 controls (536.14 ng/l) was employed as the cut-off anti-TRIB2 autoantibody index value when using ELISA to assay the serum of participants; Cvetkovic-Lopes et al19 and Kawashima et al20 employed the same statistical approach to set cut-off values in their respective studies. All statistical analyses were performed using SPSS software version 26.0 (IBM Corp., Armonk, New York).

RESULTS

Baseline demographics and sleep status are summarized in Table 1.

Table 1.

Comparison of demographic characteristics, clinical features, polysomnography (PSG), and multiple sleep latency test (MSLT) date of patients and HC.

Variables NT1 (n = 68) non-NT1 (n = 39) HC (n = 43)
Sex (male/female) 44/24 23/16 22/21
Age (years) 18.31 ± 9.73 18.15 ± 8.75 19.77 ± 5.25
Onset age (years) 13.48 ± 9.38 14.89 ± 8.51 NA
Disease duration (years) 5.49 ± 5.12 3.98 ± 4.57 NA
BMI (kg/m2) 24.94 ± 4.91* 22.50 ± 4.42 21.17 ± 3.07###
ESS, scores 15.77 ± 4.34* 12.43 ± 4.18&&& 2.81 ± 4.42###
Clinical presentation
 EDS (%) 68 (100%) 39 (100%) NA
 Cataplexy (%) 68 (100%)*** 0 (0%) NA
 SP (%) 30 (44.1%) 10 (25.6%) NA
 HH (%) 29 (42.6%)** 7 (17.9%) NA
 Duration of cataplexy symptoms (years) 4.61 ± 4.66 NA NA
PSG testing
 TST (minutes) 461.59 ± 75.73 465.10 ± 79.29 431.93 ± 59.06
 SL (minutes) 3.85 ± 4.56*** 9.18 ± 8.70 12.07 ± 7.26#
 SE (%) 84.56 ± 8.92 86.23 ± 12.29 84.31 ± 12.16
 REML (minutes) 60.10 ± 82.90** 99.92 ± 69.60 160.36 ± 86.19#
 N1 (%) 24.20 ± 10.32*** 14.49 ± 12.07 16.60 ± 5.72
 N2 (%) 33.90 ± 10.44*** 43.91 ± 9.59 46.17 ± 8.60#
 N3 (%) 21.96 ± 9.42 21.37 ± 6.22 19.43 ± 6.83
 REM (%) 19.95 ± 7.14 20.21 ± 5.25 17.77 ± 5.03
 SOREMP on PSG (%) 35 (51.47%)*** 4 (10.26%) NA
MSLT
 SL (minutes) 1.91 ± 1.60*** 5.59 ± 3.02 10.43 ± 3.23###
 SOREMPs 4.29 ± 0.95 *** 1.41 ± 1.62 0.29 ± 0.76###
Laboratory testing
 HLA DQB1*06:02 (positive/tested) 68/68 10/39 2/15
 CSF orexin-A level (pg/ml) 27.95 ± 17.03*** (51/68) 320.29 ± 122.08 (26/39) NA
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All NT1 and non-NT1 patients and 7 HC participants completed the PSG and MSLT. NT1 vs non-NT1: *P < .05, **P < .01, ***P < .001; NT1 vs HC: #P < .05, ###P < .001; non-NT1 vs HC: &&&P < .001. BMI = body mass index, CSF = cerebrospinal fluid, EDS = excessive daytime sleepiness, ESS = Epworth Sleepiness Scale, HC = healthy control, HH = hypnagogic hallucinations, NA = not assessed, NT1 = narcolepsy type 1, REM = rapid eye movement sleep, REML = rapid eye movement sleep latency, SE = sleep efficiency, SL = sleep latency, SOREMPs = numbers of sleep onset rapid eye movement periods, SP = sleep paralysis, TST = total sleep time.

All sera were first tested by the CBA method. The results show that anti-TRIB2 antibody was not detected in any participants.

We then tested sera from the same patients by ELISA and found that the TRIB2 antibody titer in patients with NT1 was significantly higher than that in the HCs (P < .05); no significant differences in TRIB2 antibody titers were detected between the non-NT1 group and the other 2 groups. Two patients with NT1 and 2 HCs had TRIB2 antibody titers greater than the mean titer plus 2 SD of the HCs (Figure 1).

Figure 1. Determination of TRIB2-specific antibodies in sera by enzyme-linked immunosorbent assay.

Figure 1

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Each symbol corresponds to serum of a single participant. The dotted horizontal line (536.14 ng/l) indicates the mean TRIB2-specific antibody titer plus 2 standard deviations (SD) of the 43 healthy controls (HCs) as the cut-off point. Values above this line are considered positive. The anti-TRIB2 antibody titer in patients with NT1 was significantly higher than that in HCs (*P < .05); no significant differences in anti-TRIB2 antibody titers were detected between the non-NT1 group and the other 2 groups. Two patients with NT1 and 2 HCs had TRIB2 antibody titers greater than the mean titer plus 2 SD for the HCs. ns = not statistically significant, NT-1 = narcolepsy type 1, TRIB2 = Tribbles homolog 2.

DISCUSSION

By using CBA in our study, we found that anti-TRIB2 antibodies were absent in the sera of Chinese patients with narcolepsy. In addition, we tested the same sera of patients by ELISA and found that TRIB2 antibody titers were significantly higher in patients with NT1 than in HCs. The ELISA assay identified 2 patients with NT1 with TRIB2 antibody titers exceeding the mean titer plus 2 SD of the HCs.

In 2010, a study conducted by Cvetkovic-Lopes et al19 in Switzerland used ELISA to examine the serum of 119 patients with narcolepsy with cataplexy. The results showed that 14% of patients exhibited increased levels of TRIB2 antibodies. Furthermore, it was observed that the antibody titer showed a more significant increase during the early stages of the disease, gradually declining within a span of 2–3 years. Additionally, a positive correlation was established between the severity of cataplexy and the TRIB2 antibody titer. Kawashima et al20 used radioligand binding assay to investigate 90 patients with narcolepsy with cataplexy in the United States. Their findings revealed that 21% of participants exhibited increased levels of serum TRIB2 antibodies. Notably, the presence of anti-TRIB2 antibodies was significantly correlated with the occurrence of recent cataplexy, specifically within 2.3 years following the onset of cataplexy symptoms. Similarly, Toyoda et al21 used the same methodology to investigate Japanese patients and found that 26.1% of these individuals had elevated TRIB2 antibody levels. Lind et al25 in 2014 and Wallenius et al26 in 2019 used radiobinding assays to examine the existence of TRIB2 antibodies in patients with narcolepsy who received the Pandemrix vaccine. Notably, no TRIB2 antibodies were observed in these vaccinated individuals, which is consistent with our CBA experimental results.

The findings from our ELISA align with the research of Cvetkovic-Lopes et al,19 namely, that TRIB2 antibody titers were notably elevated in patients with NT1 relative to those in HCs. Notably, however, in the study conducted by Cvetkovic-Lopes et al, 14% of the patients exhibited TRIB2 antibody levels surpassing the mean titer plus 2 SD of the HC group. In contrast, in our ELISA analysis, only 2 patients (2.9%) displayed TRIB2 antibody levels exceeding the same threshold. However, 2 HCs also exhibited antibody concentrations surpassing the mean plus 2 SD of the control group, as also observed in the study of Cvetkovic-Lopes et al.19 Interestingly, no anti-TRIB2 antibodies were detected in these individuals when the CBA was employed. Several factors may have contributed to this phenomenon. First,27–29 insufficient washing of some wells during the ELISA may have resulted in excessive binding of the solid phase carrier to nonspecific proteins, resulting in elevated antibody titer. The presence of numerous nontarget antibodies in serum increases the likelihood of cross-reactivity with nonspecific antigens. When the ELISA detection method fails to differentiate this nonspecific binding, a false positive result will ensue. Second,30 this discrepancy may be attributed to high-fidelity antigen preservation in a membrane-dependent, 3-dimensional structure in the CBA assay, in contrast to that seen with the ELISA and radioligand binding assay methods, where the antibodies react with a TRIB2 antigen fragment rather than the entire TRIB2 antigen in its complete spatial configuration.

In recent years, there has been a gradual increase in the use of CBA for antibody detection. The antibodies identified through CBA are capable of attaching themselves to the target antigen expressed in its original conformation on the external surface of transfected cells. This approach offers the benefits of preserving the antigen’s conformation, thereby enabling a more precise representation of the antigen–antibody binding characteristics.31,32 However, ELISA has low sensitivity, because some epitopes are masked during the coating process; hence, it cannot adequately react with antibodies.33,34 For the detection of central nervous system autoantibodies, CBA has the advantages of high sensitivity, strong specificity, and reliable detection.35 Multiple studies have confirmed the merits of CBAs. In 2012, a multicenter investigation on neuromyelitis optica spectrum disorder reported that the sensitivity of CBA (73.3%) surpassed that of ELISA (60%) in detecting the specific antibody aquaporin-4.36 A CBA also demonstrated greater sensitivity (76.8%) than ELISA (58.5%) in detecting acetylcholine receptor antibodies in patients with myasthenia gravis. Furthermore, all samples that tested positive in the ELISA also tested positive in the CBA. However, it is noteworthy that 15 (44.1%) out of 34 patients with myasthenia gravis who tested negative in the ELISA showed positivity for low-affinity acetylcholine receptor antibodies when the CBA was used.37,38 Similarly, Han et al conducted a study on patients with myasthenia gravis, and the results corroborated the superior sensitivity of the CBA method in detecting muscle-specific kinase antibodies compared with the ELISA method.39 Multiple studies have substantiated the superior specificity of CBA over ELISA in the identification of autoimmune antibodies. However, our results showed significantly elevated anti-TRIB2 titers with ELISA in 2 patients with NTI relative to HCs. A similar approach was employed in a study by Cvetkovic-Lopes et al,19 which also detected heightened TRIB2 antibody titers in a minority of patients with NT1. Consequently, individuals with NT1 showing ELISA positivity for anti-TRIB2 antibodies may represent a distinct subtype or phenotype, highlighting the necessity of further investigation to elucidate the underlying cause.

The study conducted by Kawashima et al20 revealed that there was a correlation between the time to onset of cataplexy symptoms and the levels of anti-TRIB2 antibodies in patients diagnosed with narcolepsy with cataplexy, particularly within 2.3 years after symptom onset. Here, we found that in 28 out of 68 (41%) patients with NT1, the duration of cataplexy was less than 2 years, and no positivity for anti-TRIB2 antibodies was detected in these patients using the CBA method. Furthermore, the ELISA results indicated that the TRIB2 antibody titers in these 28 patients with NT1 were lower than the average titer for the HC group plus 1 SD, suggesting a minimal likelihood of false negatives using the CBA method. Additionally, we did not detect a significant correlation between the duration of cataplexy symptoms and the TRIB2 antibody titer.

The present study had some limitations. First, the study population primarily consisted of adolescents. We hope to enlarge the sample size to verify whether there is an effect of age on TRIB2 antibody contents in later work. Second, owing to the constraints of our experiment, we did not conduct a radioligand binding assay to establish a comparative analysis with our findings. Finally, we did not conduct testing on the cerebrospinal fluid. To further explore the pathogenesis of narcolepsy, future investigations should use new technology to detect more types of autoimmune antibodies in both the serum and cerebrospinal fluid of patients diagnosed with narcolepsy.

DISCLOSURE STATEMENT

All authors have seen and approved this manuscript. Work for this study was performed at the Second Affiliated Hospital of Nanchang University. This study was funded by the National Natural Science Foundation of China (grants no. 81601191 and 32160194) and the Recruitment Program of Experts of Jiangxi Province (jxsp2023102164). The authors report no conflicts of interest.

ACKNOWLEDGMENTS

The authors sincerely thank the Biobank Center of the Second Affiliated Hospital of Nanchang University for their valuable support in collecting, handling, and preserving the clinical samples for this study. Author contributions: X.Z.: analysis of the data and writing the manuscript. Y.Y. and Q.Z.: processing of blood specimens. T.Y., C.K., and Y.L.: collection of clinical data. L.D., Y.D., and F.W.: clinical patient diagnosis and treatment. L.X. and W.W.: review of the manuscript and guidance of all work.

ABBREVIATIONS

CBA

cell-based assay

ELISA

enzyme-linked immunosorbent assay

HC

healthy control

NT1

narcolepsy type 1

PSG

polysomnography

SD

standard deviation

TRIB2

Tribbles homolog 2

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