Evaluation and Limitations of the Novel Chemiluminescent Enzyme Immunoassay Technique for Measuring Total Tau Protein in the Cerebrospinal Fluid of Patients with Human Prion Disease: A 10-Year Prospective Study (2011-2020).

BACKGROUND: Recently, the investigation of cerebrospinal fluid (CSF) biomarkers for diagnosing human prion diseases (HPD) has garnered significant attention. Reproducibility and accuracy are paramount in biomarker research, particularly in the measurement of total tau (T-tau) protein, which is a crucial diagnostic marker. Given the global impact of the coronavirus disease pandemic, the frequency of measuring this protein using one of the world's fully automated assays, chemiluminescent enzyme immunoassay (CLEA), has increased. At present, the diagnosis and monitoring of neurological diseases mainly rely on traditional methods, but their accuracy and responsiveness are limited. There is limited knowledge of the accuracy of CLEA in tau measurements. We aimed to measure T-tau protein using CLEA and to elucidate its merits and limitations. METHODS: We randomly selected 60 patients with rapidly progressive dementia, using ELISA and CLEA analysis of cerebrospinal fluid specimens. Additionally, we used Western blotting to detect the presence of 14-3-3 protein and employed real-time quaking-induced conversion (RT-QuIC) assays to analyze the same set of samples. Furthermore, we examined the correlation coefficient between ELISA and CLEA results in a subset of 60 samples. Moreover, using CLEA, we evaluated the diurnal reproducibility, storage stability, dilutability, and freeze-thaw effects in three selected samples. RESULTS: In 172 patients, 172 samples were extracted, with each patient providing only one sample, and a total of 88 (35 men and 53 women) tested positive for HPD in the RT-QuIC assay. In contrast, all CSF samples from the remaining 84 patients without HPD (50 men and 34 women) tested negative in the RT-QuIC assay. Both ELISA and CLEA showed perfect sensitivity and specificity (100%) in measuring T-tau protein levels. In addition, ELISA and CLEA are similar in terms of measurement sensitivity and marginal effect of detection extrema. CLEA analysis exhibited instability for certain samples with T-tau protein levels exceeding 2000 pg/mL, leading to low reproducibility during dilution analysis. CONCLUSIONS: Our findings indicate that CLEA outperforms ELISA in terms of diurnal reproducibility, storage stability, and freeze-thaw effects. However, ELISA demonstrated superior performance in the dilution assay. Therefore, it is imperative to develop innovative approaches for the dilution of biomarker samples for CLEA measurements during clinical trials.

Impact of the transition from radioimmunoassay (RIA) to chemiluminescent enzyme immunoassay (CLEIA) for the measurement of plasma aldosterone concentration (PAC) on the diagnosis of primary aldosteronism (PA) via retrospective analyses in Okinawa, Japan.

In Japan, the traditional method for measuring plasma aldosterone concentration (PAC) was radioimmunoassay (RIA), which had several challenges, including poor traceability of certified reference materials and reduced detection sensitivity at low concentrations. To overcome these issues, a chemiluminescent enzyme immunoassay (CLEIA) for PAC measurement was introduced in April 2021 and the Japan Endocrine Society published new guidelines for primary aldosteronism (PA). This study aimed to evaluate the impact of the transition from RIA to CLEIA for PAC measurement on PA diagnosis. Data from 190 patients admitted to the Second Department of Internal Medicine, University of the Ryukyus Hospital, between April 2012 and March 2021 were analyzed. Patients who were diagnosed with PA underwent adrenal venous sampling. The PAC measured by RIA (PAC(RIA)) was converted to the estimated PAC measured by CLEIA (ePAC(CLEIA)) using a conversion formula. The present study evaluated the discordance rates in diagnoses based on screening (SC), captopril challenge test (CCT), saline infusion test (SIT), and diagnosis of PA between results judged by PAC(RIA) according to the previous guidelines and those judged by ePAC(CLEIA) according to the new guidelines. The results revealed discordant diagnosis rates of 6.4% for SC and 10.1% for CCT, with no discordance for SIT. The discordant diagnosis rate for PA was 3.7%. Our study reveals the challenges in establishing appropriate diagnostic criteria for PA using PAC(CLEIA) and highlights the demand for further research on provisionally positive categories.

Relationship Between Cerebrospinal Fluid Alzheimer's Disease Biomarker Values Measured via Lumipulse Assays and Conventional ELISA: Single-Center Experience and Systematic Review.

Background: Although Lumipulse assays and conventional ELISA are strongly correlated, the precise relationship between their measured values remains undetermined. Objective: To determine the relationship between Lumipulse and ELISA measurement values. Methods: Patients who underwent cerebrospinal fluid (CSF) Alzheimer's disease (AD) biomarker measurements and consented to biobanking between December 2021 and June 2023 were included. The relationship between values measured via Lumipulse assays and conventional ELISA were evaluated by Passing-Bablok analyses for amyloid-ß 1-42 (Aß42), total tau (t-tau), and phospho-tau 181 (p-tau 181). Studies using both assays were systematically searched for in PubMed and summarized after quality assessment. Results: Regression line slopes and intercepts were 1.41 (1.23 to 1.60) and -77.8 (-198.4 to 44.5) for Aß42, 0.94 (0.88 to 1.01) and 98.2 (76.9 to 114.4) for t-tau, and 1.60 (1.43 to 1.75) and -21.1 (-26.9 to -15.6) for p-tau181. Spearman's correlation coefficients were 0.90, 0.95, and 0.95 for Aß42, t-tau, and p-tau181, respectively. We identified 13 other studies that included 2,117 patients in total. Aß42 slope varied among studies, suggesting inter-lab difference of ELISA. The slope and intercept of t-tau were approximately 1 and 0, respectively, suggesting small proportional and systematic differences. Conversely, the p-tau181 slope was significantly higher than 1, distributed between 1.5-2 in most studies, with intercepts significantly lower than 0, suggesting proportional and systematic differences. Conclusions: We characterized different relationship between measurement values for each biomarker, which may be useful for understanding the differences in CSF biomarker measurement values on different platforms and for future global harmonization.

Investigating the cut-off values of captopril challenge test for primary aldosteronism using the novel chemiluminescent enzyme immunoassay method: a retrospective cohort study.

The measurement evolution enabled more accurate evaluation of aldosterone production in hypertensive patients. However, the cut-off values for novel assays have been not sufficiently validated. The present study was undertaken to validate the novel chemiluminescent enzyme immunoassay for aldosterone in conjunction with other methods. Moreover, we also aimed to establish a new cut-off value for primary aldosteronism in the captopril challenge test using the novel assay. First, we collected 390 plasma samples, in which aldosterone levels measured using liquid chromatography-mass spectrometry ranged between 0.18 and 1346 ng/dL. The novel chemiluminescent enzyme immunoassay showed identical correlation of plasma aldosterone with liquid chromatography-mass spectrometry, in contrast to conventional radioimmunoassay. Further, we enrolled 299 and 39 patients with primary aldosteronism and essential hypertension, respectively. Plasma aldosterone concentrations measured using the novel assay were lower than those measured by radioimmunoassay, which resulted in decreased aldosterone-to-renin ratios. Subsequently, positive results of the captopril challenge test based on radioimmunoassay turned into "negative" based on the novel assay in 45% patients with primary aldosteronism, using the conventional cut-off value (aldosterone-to-renin activity ratio > 20 ng/dL per ng/mL/h). Receiver operating characteristic curve analysis demonstrated that aldosterone-to-renin activity ratios > 8.2 ng/dL per ng/mL/h in the novel assay was compatible with the conventional diagnosis (sensitivity, 0.874; specificity, 0.980). Our study indicates the great measurement accuracy of the novel chemiluminescent enzyme immunoassay for aldosterone, and the importance of measurement-adjusted cut-offs in the diagnosis of primary aldosteronism.

Adrenal venous sampling criteria for chemiluminescent enzyme immunoassay as a preferable alternative to radioimmunoassay in primary aldosteronism.

Plasma aldosterone concentration (PAC) was routinely measured using radioimmunoassay (RIA); however, the RIA kit was discontinued in March 2021 in Japan. This study examined PAC conversion in adrenal venous sampling (AVS) and AVS criteria when measured using chemiluminescent enzyme immunoassay (CLEIA). PAC of 415 adrenal venous blood samples from AVS (including segmental AVS) of 63 patients with primary aldosteronism was measured using RIA (Spac-S aldosterone kit; Fujirebio Inc.) and CLEIA (Lumipulse Presto Aldosterone; Fujirebio Inc.). PAC of 70 AVS samples was also measured using liquid chromatography-mass spectrometry (LC-MS/MS, ASKA Pharma Medical Co., Ltd.). PAC conversion formulas were determined for each AVS sample assay. PAC measured using CLEIA was significantly correlated with that measured using RIA (correlation coefficient = 0.971). The PAC conversion formula was PAC (CLEIA) = PAC (RIA) × 0.772 - 1,199 pg/mL. The PAC of 14,000 pg/mL in RIA was equivalent to 9,613 pg/mL in CLEIA. PAC measured using CLEIA was also correlated with that measured using LC-MS/MS, and the PAC conversion formula was PAC (CLEIA, pg/mL) = 0.97 × PAC (LC-MS/MS, pg/mL) + 211. The inter-assay coefficient of variability (CV) was 1.1-1.3% and intra-assay CV was 1.0-1.7%, measured using CLEIA. The PAC conversion formula for AVS samples was obtained using CLEIA and RIA, and the conversion formula was different from that for peripheral blood. PAC values measured by CLEIA showed preferable accuracy and high concordance with those measured by LC-MS/MS, even in AVS samples. The study outcomes are useful for interpreting AVS results using non-RIA measurement methods.

Development of a sensitive direct competitive chemiluminescent enzyme immunoassay for gentamicin based on the construction of a specific single-chain variable fragment-alkaline phosphatase fusion protein.

A sensitive chemiluminescent enzyme immunoassay (CLEIA) was established for the determination of gentamicin (GEN) residue levels in animal tissue. This assay is based on a fusion protein of single-chain variable fragment (scFv) and alkaline phosphatase (AP). Initially, VL and VH derived from anti-gentamicin monoclonal antibody were linked by a short peptide to construct a scFv. Subsequently, the constructed scFv sequence was accessed into the pLIP6/GN vector, and a soluble scFv-AP fusion protein was generated. The scFv-AP fusion protein was used to develop a direct competitive CLEIA (dcCLEIA) for the determination of gentamicin. In the dcCLEIA, the half inhibitory concentration (IC50) and limit of detection (LOD) were 1.073 ng/mL and 0.380 ng/mL, respectively. The average recoveries of gentamicin spiked in animal tissue samples ranged from 78% to 96%. These results showed a strong correlation with ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). The above results suggest that the anti-GEN scFv-AP fusion protein is suitable for detecting gentamicin residues in edible animal tissues.