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)

Abstract

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.

Keywords: Creutzfeldt–Jakob disease; cerebrospinal fluid; chemiluminescent enzyme immunoassay; diagnostic test; human prion disease; prion protein.

Figure 1

(a–d) Storage stability of CSF samples. The 500 uL CSF samples from all the patients are divided into five 100 uL aliquots. T-tau protein levels decline after 2 days of storage at room temperature, with an approximate deactivation rate of 6–9% within a week under the same conditions. T-tau protein concentrations remain relatively stable in samples stored at 4 °C, showing only around a 3% decrease compared to those stored at 14 °C. T-tau protein levels measured in samples stored at −20 °C and −80 °C remain nearly unchanged over a period of 14 days but demonstrate around a 5% decrease after storage for up to 29 days. Considering the results obtained from multiple samples with concentrations exceeding 1000 pg/mL, we infer that any variations in the T-tau protein measurements between samples stored at −20 °C and −80 °C fall within the margin of error. CSF, cerebrospinal fluid. Every line of the same shape is from the same random sample. A total of 3 samples were randomly selected, and the three random samples were divided into 4 equal parts, with each abcd having one part. The abcd diagram represents the experimental results at room temperature, the experimental results at 4 degrees, the experimental results at minus 20 degrees, and the experimental results at minus 80 degrees.

Figure 2

Relationship between T-tau protein levels measured using ELISA and CLEA. ROC analysis is performed using Lumipulse to investigate the diagnostic performance between CLEA and ELISA and evaluate the ROC analysis of the new automated method. According to the detection results, there is a strong correlation between CLEA and ELISA, indicating that both detection methods can effectively detect T-tau protein. CLEA, chemiluminescent enzyme immunoassay; ELISA, enzyme-linked immunosorbent assay; ROC, receiver operating characteristic.