Chemiluminescent Enzyme Immunoassay Research Articles

Concordance between APOE genotyping and proteotyping using the novel Lumipulse G Pan‐ApoE and Lumipulse G ApoE4 RUO assays

AbstractBackgroundThe APOE gene has three different alleles that encode for three different ApoE protein isoforms (APOE2, APOE3, and APOE4), resulting in 6 different phenotypes. Nucleic acid amplification test remains the gold standard for APOE genotyping, however quantification of the ApoE proteins using immunoassays could provide another piece of information, related to the expression levels of the proteins. In this regard, ApoE as a protein biomarker can be used in research to determine its value alone or as part of a panel of plasma biomarkers.MethodThe LUMIPULSE G System is a chemiluminescent enzyme immunoassay platform enabling fully automated processing of samples using ready‐to‐use immunoreaction cartridges. Time to result in these single use test cartridges takes about 30 minutes. The analytical performance of the newly developed Lumipulse G Pan‐ApoE and Lumipulse G ApoE4 RUO assays was determined. Parameters analyzed were precision, sensitivity, linearity, prozone and potential impact of interfering substances. Proof of concept of these novel assays was verified with a set of 230 genotyped plasma samples.ResultThe total within‐lab variability of both assays was ≤8% CV confirming high precision of the LUMIPULSE G system. Using low ApoE concentrated plasma samples, the observed LoD and LoQ (@20% CV) was 0.072 µg/mL (Pan‐ApoE) and 0.152 µg/mL (ApoE4). Linearity was shown across the assay range (0 – 100 µg/mL) for both assays. Neither prozone effect nor significant impact (recovery within 100±10%) of commonly tested endogenous substances (bilirubin, haemoglobin, low and high Protein content, and human anti‐mouse antibodies (HAMA)) was observed. Potential interference was observed for triglycerides and rheumatoid factor (RF) with observed recovery within 100±20%. ApoE proteotyping based on the novel assays resulted in a 99.8% concordance with APOE genotyping (1 discordant result). Moreover, applying the ratio ApoE4/Pan‐ApoE with the measurement values of respective assays, classified correctly between hetero‐ and homozygous E4 carrier status.ConclusionThe analytical performance studies demonstrate low variability and high sensitivity enabling measurement of pan‐ApoE and ApoE4 in plasma. Pan‐apoE and apoE4 quantification can offer relevant insights into the assessment of subjects at risk for AD or adverse events following anti‐amyloid treatments and should be studied further.

Preliminary performance of the Lumipulse G pTau 217 Plasma prototype assay on plasma samples

AbstractBackgroundMultiple publications demonstrated that plasma pTau species (e.g., 181, 217, and 231) have the potential to be a good predictor of amyloid status by either PET or cerebrospinal fluid (CSF). Such blood‐based biomarker testing could be used as a simple, accessible, and scalable approach to help support the diagnosis of Alzheimer’s disease (AD), avoiding the complexity of CSF sampling, and the high costs and limited availability of PET imaging. Multiple plasma pTau assays, established on different immunoassay platforms, are currently being evaluated in research settings with variable outcomes in terms of analytical and clinical performance. At Fujirebio, an assay for the measurement of plasma pTau217 was developed on the LUMIPULSE G platform to complement the commercially available plasma pTau181 RUO assay. The aim of this study was to determine the preliminary performance of the Lumipulse G pTau 217 Plasma prototype assay.MethodThe LUMIPULSE G System is a fully automated chemiluminescent enzyme immunoassay platform processing samples in ready‐to‐use immunoreaction cartridges in about 30 minutes. The assay uses an in‐house developed monoclonal antibody for capturing of pTau217. Analytical assay characteristics such as sensitivity, precision and dilutional linearity were determined. The analytical studies included a series of buffer and K2EDTA plasma samples, each tested in several replicates. Preliminary clinical performance was explored on a small set of patient and control samples.ResultAll analytical parameters tested were within the acceptance criteria. All clinical samples tested had a result above the limit of quantification (LoQ). The LoQ was obtained based on the evaluation of low concentrated plasma pTau217 samples. Imprecision was less than 15% CV on buffer‐based as well as plasma samples. Dilutional linearity on spiked samples was within ±20%. The Lumipulse G pTau 217 Plasma prototype assay outperformed the Lumipulse G pTau 181 Plasma RUO assay in a ROC curve analysis of clinically typed patients and controls.ConclusionThis preliminary performance evaluation of the Lumipulse G pTau 217 Plasma prototype assay indicated good results in terms of high sensitivity, low variability, dilutional linearity and clinical capability. More studies on well‐characterised and diverse cohorts are needed to confirm the assay’s clinical performance.

Preparation of monoclonal antibody and establishment of indirect competitive chemiluminescence enzyme immunoassay for sodium pentachlorophenolate

In this study, three haptens of sodium pentachlorophenolate (PCP-Na) were designed and synthesized, and the monoclonal antibody against PCP-Na with high specificity and affinity was successfully prepared. Based on this monoclonal antibody, a highly sensitive indirect competitive chemiluminescence enzyme immunoassay (ic-CLEIA) was developed for the determination of PCP-Na and its metabolite pentachlorophenol (PCP). The ic-CLEIA method based on optimal conditions showed that the half maximal inhibitory concentration (IC50), linear range (IC20-IC80) and the detection limit (LOD, IC10) for PCP-Na were 0.083 ng/mL, 0.0076–1.5111 ng/mL, and 0.0032 ng/mL, respectively, and 0.29 ng/mL, 0.0983–0.7008 ng/mL, and 0.0192 ng/mL for PCP, respectively. The results of cross reaction showed that the method had no obvious cross reaction with other six chlorophenols compounds (phenol, 2-chlorophenol, 4-chlorophenol, 2, 4-dichlorophenol, 2, 4, 5-trichlorophenol, 2, 4, 6-trichlorophenol) except the metabolite PCP. The average recoveries in chicken, pork and fish samples were 81.3–105.8 %, with coefficient of variation (CV) lower than 7.6 %. In this study, a new highly rapid, sensitive and accurate ic-CLEIA method was established, which can be used for the sensitive detection of PCP-Na and its metabolite PCP in animal tissues.

Characteristics of cancer-related fatigue and its correlation with anxiety, depression, and stress-related hormones among Chinese cancer survivors: a cross-sectional study.

Fatigue is a common source of distress for cancer survivors. The severity of cancer-related fatigue varies significantly, which may be due to individual differences in host factors. This cross-sectional study aims to explore how demographic, oncological, sociological, psychological, and stress-related hormones levels interact to influence the distinct experiences of fatigue (Cancer-related fatigue [CRF] occurrence and fatigue degree). A cross-sectional study carried out at the oncology outpatient and ward department of Xiyuan Hospital of China Academy of Chinese Medical Sciences recruited 306 cancer patients between January 2021 to December 2021. General information, fatigue, psychological factors was evaluated by general information questionnaire, the Revised Piper's Fatigue Scale-Chinese Version (RPFS-CV), and the self-report Hospital Anxiety and Depression Scale (HADS). Stress-related hormones were measured with chemiluminescent enzyme immunoassay (Zhengzhou Antobio). 306 patients were included, 229 (74.8%) were diagnosed with CRF, including 94 (41.0%) with mild fatigue, 121 (52.8%) with moderate fatigue, and 14 (6.1%) with severe fatigue. Multivariate regression analysis showed that higher depression scores, aldosterone levels may increase the risk of CRF. Patients who are obese (Body mass index ≥ 28 kg/m2) may help to reduce the risk of CRF. Other contributing factors for increased levels of fatigue (p< 0.05) include being female, having anxiety, depression and high aldosterone levels. The research suggested that CRF was a common symptom in cancer survivors and pay attention to these influencing factors may help to better identify patients susceptible to fatigue and provide long-term, targeted interventions.

Development of high-performance point-of-care aqueous VEGF detection system and proof-of-concept validation in RVO patients.

To develop a sensitive point-of-care testing (POCT) aqueous vascular endothelial growth factor (VEGF) detection system, and assess its role for predicting the response to anti-VEGF treatment in macular edema secondary to retinal vein occlusion (RVO-ME) patients. An automatic point-of-care aqueous humor Magnetic Particle Chemiluminescence Enzyme Immuno-Assay (MPCLEIA) VEGF detection system was developed. The predictive values of aqueous cytokine levels, in combination with imaging parameters, on anatomical treatment response (ATR, the relative central macular thickness change [ΔCMT/bl-CMT]) were analyzed. The automatic MPCLEIA system was able to provide results in 45 min with only 20 μL sample. Among the 57eyes with available pre- and post-treatment evaluation, ATR significantly correlated with levels of interleukin (IL)-6, IL-8, monocyte chemoattractant protein-1 (MCP-1) and VEGF measured by Luminex xMAP platform, and VEGF measured by MPCLEIA. Optimal cut-off values for these biomarkers were 13.26 ng/L, 23.57 ng/L, 1,110.12 ng/L, 105.52 ng/L, and 85.39 ng/L, respectively. Univariate analysis showed significant associations between ATR category (good response if ATR≤-25 % or poor response otherwise) and IL-6, IL-8, MCP-1, VEGF-xMAP, and VEGF-MPCLEIA (p<0.05). Multivariate logistic regression revealed that ATR category was significantly associated with aqueous VEGF-MPCLEIA (p=0.006) and baseline(bl)-CMT (p=0.008). Receiver operating characteristics analysis yielded an AUC of 0.959 for the regression model combining VEGF-MPCLEIA and bl-CMT, for predicting ATR category. Our novel MPCLEIA-based automatic VEGF detection system enables accurate POCT of aqueous VEGF, which shows promise in predicting the treatment response of RVO-ME to anti-VEGF agents when combined with bl-CMT.

A predictive and prognostic model for surgical outcome and prognosis in ovarian cancer computed by clinico-pathological and serological parameters (CA125, HE4, mesothelin).

Numerous prognostic models have been proposed for ovarian cancer, extending from single serological factors to complex gene-expression signatures. Nonetheless, these models have not been routinely translated into clinical practice. We constructed a robust and readily calculable model for predicting surgical outcome and prognosis of ovarian cancer patients by exploiting commonly available clinico-pathological factors and three selected serum parameters. Serum CA125, human epididymis protein 4 (HE4) and mesothelin (MSL) were quantified by Lumipulse® G chemiluminescent enzyme immunoassay (Fujirebio) in a total of 342 serum samples from 190 ovarian cancer patients, including 152 paired pre- and post-operative samples. Detection of pre-operative HE4 and CA125 was the optimal marker combination for blood-based prediction of surgical outcome (AUC=0.86). We constructed a prognostic model, computed by serum levels of pre-operative CA125, post-operative HE4, post-operative MSL and surgical outcome. Prognostic performance of our model was superior to any of these parameters alone and was independent from BRCA1/2 mutational status. We subsequently transformed our model into a prognostic risk index, stratifying patients as "lower risk" or "higher risk". In "higher risk" patients, relapse or death was predicted with an AUC of 0.89 and they had a significantly shorter progression free survival (HR: 9.74; 95 % CI: 5.95-15.93; p<0.0001) and overall survival (HR: 5.62; 95 % CI: 3.16-9.99; p<0.0001) compared to "lower risk" patients. We present a robust predictive/prognostic model for ovarian cancer, which could readily be implemented into routine diagnostics in order to identify ovarian cancer patients at high risk of recurrence.

New chemiluminescent enzyme immunoassay for quantitative measurement of Mac-2 binding protein glycosylation isomer in chronic liver disease.

This study aimed to evaluate the quantitative measurement of Mac-2 binding protein glycosylation isomer (M2BPGi) levels using the new chemiluminescent enzyme immunoassay. The data of a total of 347 patients with hepatitis C virus (HCV) infection and 150 health volunteers from 13 locations in Japan were evaluated. The quantitative system for measuring M2BPGi-Qt levels was based on a new chemiluminescent enzyme immunoassay. We evaluated the reproducibility and quantitation range in quantitative M2BPGi-Qt measurement. We also investigated the confidence ratio of M2BPGi-Qt levels measured by the new quantitative system to M2BPGi levels measured by the current semi-quantitative system for validating the clinical utility of the new method. The reproducibility of M2BPGi-Qt in HCV samples with negative, positive 1+, and positive 2+ was 0.77 ± 0.02AU/mL, 2.25 ± 0.03AU/mL, and 6.55 ± 0.21AU/mL, respectively, and the corresponding coefficient of variation (CV)s were 2.1%, 1.3%, and 3.2%, respectively. The range of quantification assessment resulted that all CVs showed less than 5% in investigated range. Sample stability testing found that the mean percentage difference between the pre- and post-storage values of 6 samples ranged between 96.2 and 103.9%. The correlation coefficient between M2BPGi and M2BPGi-Qt in patients with HCV and the healthy volunteers was 0.986 and 0.991, respectively. M2BPGi-Qt could be quantitatively assessed in a patient with over 20 C.O.I. Compared with qualitative methods, the M2BPGi quantitative measurement system could provide a numerical value unaffected by interpretation bias, and measurements are more precise at high M2BPGi levels.

Prognostic value of serum Krebs von den Lungen-6 (KL-6) levels in COVID-19 pneumonia

Background and objectivesKrebs von den Lungen-6 (KL-6), expressed by damaged type II pneumocytes, is useful in the diagnosis and severity assessment of many diffuse interstitial lung diseases. The objective of our study was to determine the prognostic value of the initial KL-6 plasma level in COVID-19 pneumonia. MethodsAll patients hospitalized for a suspected COVID-19 pneumonia between March and May 2020 in our Chest department of a French university hospital were included. KL-6 serum concentrations were measured within 72 h of diagnostic suspicion by chemiluminescence enzyme immunoassay Survival analysis was performed using a Cox regression and modeled by a Kaplan-Meier curve. ResultsSixty-six COVID-19 patients (average age = 64 ± 14 years, 71.2 % males) with KL-6 serum measurement were included. Median KL-6 serum concentration was 409 ± 312 U/mL. KL-6 was significantly higher in men (p = 0.003), elders (p = 0.0001) and in patients with greater Charlson's score (p = 0.002). Higher KL-6 concentration was significantly associated with in-hospital mortality (HR: 8.66; 95 % CI:1.1–69.2, p = 0.014), radiological extension of lesions on chest CT scan (p = 0.004) and higher WHO severity score (p = 0.042), but not with admission in intensive care unit. In 9 (14 %) non-surviving COVID-19 patients, KL-6 serum concentration increased whereas it remained stable or decreased in survivors. At 3 months follow-up (n = 48), DLCO was negatively correlated with the initial KL-6 value (r = 0.47, p = 0.001), while FVC, FEV1 and MRC score were not. ConclusionInitial KL-6 serum concentration is significantly associated with in-hospital mortality, unfavorable outcome, and persistent impairment of DLCO at 3 months. Initial KL-6 plasma determination appears as a prognostic biomarker in COVID-19 pneumonia.

FRI136 Adrenal Venous Sampling Criteria For Chemiluminescent Enzyme Immunoassay

Abstract Disclosure: K. Nakai: None. Y. Tsurutani: None. K. Irie: None. K. Teruyama: Employee; Self; Fujirebio Inc. J. Saito: None. M. Omura: None. T. Nishikawa: None. Objective: In primary aldosteronism (PA), plasma aldosterone concentration (PAC) was measured by radioimmunoassay (RIA), but the supply of the RIA kit was discontinued in March 2021 in Japan. The Japan Endocrine Society's 2021 Clinical Practice Guideline for the Diagnosis and Management of PA recommends that PAC measured by chemiluminescent enzyme immunoassay (CLEIA), which yields standardized LC-MS/MS compliant measurements, be used for clinical judgment. Because CLEIA shows a lower PAC than RIA, various cutoff values for PA need to be revised. Although conversion formulas for RIA and CLEIA for various kits have been provided, these conversions were studied mainly using samples representing aldosterone concentrations in peripheral blood; thus, it is unclear whether the same conversion formula can be used for samples obtained by adrenal venous sampling (AVS). In this study, we examined PAC conversion in AVS samples and the criteria for AVS when measured using CLEIA. Methods: PAC of 415 adrenal venous blood samples from AVS (including segmental AVS) from 63 patients with PA, were measured using RIA and CLEIA methods. The established conversion formula for PAC in peripheral blood samples was PAC (CLEIA) = 0.852 × PAC (RIA) − 36.0. PAC and aldosterone/cortisol ratio (A/C ratio) conversion formulas were determined between the two assays for AVS samples. Moreover, the CLEIA conversion value, which corresponds to the AVS criteria of conventional RIA, was obtained. Results: The median [interquartile range] PAC (CLEIA) in the samples was 9823.3 [3659.8-20600.5]. PAC measured using CLEIA was significantly correlated with RIA, and the correlation coefficient was 0.971. The PAC conversion formula determined was PAC (CLEIA) = PAC (RIA) × 0.7723 - 1199 pg/mL. PAC at 14,000 pg/mL in RIA was equivalent to 9,613 pg/mL in CLEIA. The correlation coefficient for the A/C ratio was 0.997, and the conversion formula was A/C (CLEIA) = A/C (RIA) × 0.7864 - 2.427. Discussion: Samples obtained from AVS had a much higher level of PAC than those obtained from peripheral blood. The PAC conversion formula for samples in AVS has a very large intercept compared with those in peripheral blood. Thus, the results of AVS tested by CLEIA should be interpreted with care because of the differences in the conversion formula. A different cut-off value may also be needed when evaluating the lateralization ratio because of the large intercept. Conclusion: PAC conversion formulas for AVS samples using CLEIA and RIA methods were obtained. The study outcomes are useful for interpreting AVS results using the CLEIA. Presentation: Friday, June 16, 2023

THU595 New Screening Cutoff Values For Detection Of Aldosterone Producing Adenoma Examined By LC-MS/MS And Newly Developed Non-competitive Cleia

Abstract Disclosure: F. Satoh: None. Y. Ono: None. K. Omata: None. Y. Tezuka: None. Y. Yamazaki: None. H. Sasano: None. S. Kojima: None. Objective: Thinking of the poor accuracy of conventional CLEIA measurements of aldosterone, liquid chromatography-tandem mass spectrometry (LC-MS/MS) is the ideal method but it is costly, labor intensive and time consuming. A novel non-competitive chemiluminescent enzyme immunoassay (sandwich assay) (NC-CLEIA) for measuring aldosterone was recently developed. We validated the accuracy and diagnostic ability of the NC-CLEIA and compared the results obtained with those by LC-MS/MS and also determined the new screen cutoffs by both measurements, respectively, detecting aldosterone-producing adenoma (APA) in hypertensive patients. Design and Method: We measured plasma renin activity by enzyme assay and plasma aldosterone concentrations (PAC) using LC-MS/MS and NC-CLEIA in 133 APA, 100 bilateral hyperaldosteronism and 111 essential hypertension patients to examine the accuracy of the values of PAC measured by this new NC--CLEIA as compared with those by LC-MS/MS, and determine the new screen cutoff values by both measurements to detect APA, respectively. Results: Passing-Bablok analysis revealed that NC-CLEIA (regression slope, intercept and correlation coefficient were 0.962, -0.043, and 0.994, respectively), was significantly correlated and equivalent to LC-MS/MS than RIA. Bland-Altman plot analysis of NC-CLEIA by LC-MS/MS also demonstrated much smaller systemic errors (a bias of -0.348 ng/dL with limits of agreement of -4.390 and 3.694 and 95% confidence interval). The receiver-operating characteristics analysis demonstrated that cut-off values of aldosterone/renin activity ratio obtained by LC-MS/MS and NC-CLEIA were 31.2 and 31.5 (ng/dL per ng/mL/hr) with a sensitivity of 91.0% and 90.2%, and a specificity of 75.4% and 76.8%, respectively, in terms of discriminating APA from non-APA. Therefore, we can recommend an ARR &amp;gt; 30.0 ng/dL per ng/mL/hr as a screening cut-off by both LC-MS/MS and NC-CLEIA, which would discriminate APA with a sensitivity of 91.7% in both assays, and with a specificity of 73.0% and 73.9 %, respectively. Conclusions: This novel NC-CLEIA assay for PAC could serve as a clinically reliable alternative to LC-MS/MS to detect APA among hypertensive patients with more accuracy than the conventional methods. Presentation: Thursday, June 15, 2023

B-320 Development of Two-step Sandwich CLEIA Kit for Tacrolimus With Automated Sample-Pretreatment Process, LUMIPULSE Presto® iTACT® Tacrolimus

Abstract Background Tacrolimus (The other name: FK506) is an immunosuppressive drug used for preventing rejection of allotransplantation and treating atopic dermatitis or rheumatoid arthritis. The complex consisting of Tacrolimus and FK506 binding protein binds to calcineurin and suppresses expression of Interleukin-2 (IL-2) and IL-2 induced immune response. Although tacrolimus is a useful immunosuppressive drug, this medicine causes serious side effects, such as renal dysfunction, opportunistic infections due to the excessive immunosuppression, and so on. Therefore, the measurement of the tacrolimus concentration in blood is required to obtain the maximum tacrolimus therapeutic effect without side effects. It is known that 95 percent of tacrolimus in blood localizes in the inside of erythrocyte, and this localization imposes cumbersome pretreatment process of whole blood. Almost all diagnostic reagents for tacrolimus need the manual whole blood pretreatment process that defaces the reliability of measurement values. And the existing diagnostic reagents often show the high cross-reactivity against the metabolite products of tacrolimus. To solve these inconveniences, we developed a highly-specific chemiluminescence enzyme immunoassay (CLEIA) for tacrolimus using a new automated sample-pretreatment process on a fully automated LUMIPULSE® L2400 analyzer. In this study, we report the basic performance of this new tacrolimus assay named as LUMIPULSE Presto® iTACT® Tacrolimus. Methods LUMIPULSE Presto® iTACT® Tacrolimus is the two-step sandwich CLEIA with the on-board sample-pretreatment process. All evaluations were executed on LUMIPULSE® L2400 analyzer (FUJIREBIO INC.). The on-board sample-pretreatment process is performed simultaneously with the first immune-reaction in sandwich CLEIA, and the total reaction time is 20 min. Limit of detection (LoD) and Limit of quantification (LoQ), within-run precision, day-to-day reproducibility, linearity, endogenous interferences, anticoagulative interferences, tacrolimus metabolites interferences and the correlation with current tacrolimus assay were evaluated in LUMIPULSE Presto® iTACT® Tacrolimus. Results Both of LoD and LoQ of LUMIPULSE Presto® iTACT® Tacrolimus were 0.12 ng/mL. Within-run precisions (% CVs) using three control samples were 0.4%–2.5%. Day-to-day reproducibility in five days were 0.4%–1.9%. We determined the linearity ranged from 0.29 to 48.80 ng/mL. Up to 19.7 mg/dL of conjugated bilirubin, 18.8 mg/dL of free bilirubin, 1620 FTU of chyle, 2000 mg/dL of triglycerides, 1000 IU/mL of RF, and 1010 ng/mL of HAMA, and 4–12 g/dL of proteins including albumin did not affect tacrolimus measurements. In addition, there was no influence by EDTA 2K and EDTA 2Na up to 10 mg/mL. The cross-reactivities of four tacrolimus metabolites (M-I, M-II, M-III, and M-IV) were respectively 0.4%–1.0%, 0.0%–0.8%, -0.7%–0.2%, and 0.7%–1.6%. Comparing to the existing chemiluminescence immunoassay (CLIA) with 128 specimens, the correlation coefficient was 1.0, and the slope calculated by Passing-Bablok method is 1.04. Comparing to the existing electrochemiluminescence immunoassay (ECLIA) with 137 specimens, the correlation coefficient was 1.0, and the slope calculated by Passing-Bablok method is 1.07. Conclusion The fundamental performance of LUMIPULSE Presto® iTACT® Tacrolimus is satisfactory and the on-board sample-pretreatment process of this assay is expected to contribute to reducing the burden and inconvenience of laboratory personnel.

Magnetic particle-based chemiluminescence immunoassay for serum human heart-type fatty acid binding protein measurement.

Human heart-type fatty acid binding protein (HFABP) is a biomarker for diagnosis, risk assessment, and prognosis of acute myocardial infarction, and we aimed to establish an immunoassay for HFABP quantitation. Human HFABP monoclonal antibodies (mAbs) were developed, evaluated by enzyme-linked immunosorbent assay, and a chemiluminescence enzyme immunoassay (CLEIA) generated. Analytical performance of the CLEIA was evaluated by measuring serum HFABP. The prokaryoticallyexpressed rHFABP was purified and four anti-HFABP mAbs withsuperior detection performance were obtained after immunizing BALB/c mice. MAbs 2B8 and 6B3 were selected as respective capture and detection antibodies for HFABP measurement by CLEIA (detection range, 0.01-128μg/L). Results using the CLEIA showed excellent correlation (r, 0.9622) and the correlation coefficient was 0.9809 (P < 0.05) by the Tukey test statistical analysis with those of latex-enhanced immunoturbidimetry in hospitals. Our mAbs and CLEIA for HFABP detection represent new diagnostic tools for measurement of human serum HFABP.

Impact of anti-squamous cell carcinoma antigen antibodies on serum squamous cell carcinoma antigen levels measured by chemiluminescent immunoassay and chemiluminescent enzyme immunoassay.

The serum squamous cell carcinoma antigen (SCCA) level is a well-known tumor marker for squamous cell carcinoma. In this study, we examined the impact of immunoglobulin (Ig)-bound macromolecular SCCA on serum SCCA levels measured by 2 different methods. Seventy-five serum samples with an SCCA level >5.0 ng/mL as determined by a chemiluminescent immunoassay (CLIA) were also analyzed using a chemiluminescent enzyme immunoassay (CLEIA). The levels of IgG- and IgA-type anti-SCCA antibodies, which form immunoglobulins and macromolecules, respectively, were determined using an enzyme-linked immunosorbent assay. An absorption test was performed to confirm the presence of anti-SCCA antibodies. The correlation coefficient between the values measured by CLEIA and CLIA was 0.768. The ratio of SCCA levels measured by CLEIA to those measured by CLIA in 14 samples with IgG-type anti-SCCA antibodies was significantly lower than that in samples without these antibodies (P < .031). Absorption tests showed that SCCA levels measured by CLIA might be falsely high in samples with IgG-type anti-SCCA antibodies, probably due to reactions with SCCA1. The level of SCCA as measured by CLIA and CLEIA methods correlate well, but the presence of SCCA antibodies can affect the results of the CLIA method.

Analytical Performance of a Novel Latex Turbidimetric Immunoassay, "Nanopia TARC", for TARC/CCL17 Measurement: A Retrospective Observational Study.

Thymus- and activation-regulated chemokine (TARC, also known as CCL17) is used as a biomarker for atopic dermatitis. The methods currently used for its measurement are complex, time-consuming, and require large machinery, warranting the need for a method that is simple, has a quick turnaround time, and requires less complex machinery. We evaluated the analytical performance of a novel latex turbidimetric immunoassay method, "Nanopia TARC", on 174 residual serum samples from patients with skin or allergic diseases. This evaluation included the assessment of the limit of blank/detection/quantification (LOB/D/Q), precision, accuracy, linearity, interference, and commutability between Nanopia TARC and "HISCL TARC", based on the chemiluminescent enzyme immunoassay (CLEIA) method. The LOB/D/Q values were 13, 57, and 141 pg/mL, respectively. The coefficient of variation of the repeatability was 0.9-3.8%, and that of the intermediate precision was 2.1-5.4%. The total error of the accuracy was 1.9-13.4%. The linearity was 141 and 19,804 pg/mL for TARC. The correlation coefficient between Nanopia TARC and HISCL TARC determined using the Passing-Bablok regression analysis was 0.999. Furthermore, the concordance of diagnostic criteria with AD was 92%. Nanopia TARC was confirmed to have the same analytical performance for TARC measurement as the existing CLEIA method.

Krebs von den Lungen 6 (KL-6) is a novel diagnostic and prognostic biomarker in pleural mesothelioma

ObjectivesPleural mesothelioma (PM) is a rare disease with dismal outcome. Systemic treatment options include chemotherapy and immunotherapy, but biomarkers for treatment personalization are missing. The only FDA-approved diagnostic biomarker is the soluble mesothelin-related protein (SMRP). Krebs von den Lungen-6 (KL-6) is a human mucin 1 (MUC1) glycoprotein, which has shown diagnostic and prognostic value as a biomarker in other malignancies. The present study investigated whether KL-6 can serve as a diagnostic and/or prognostic biomarker in PM. Materials and methodsUsing a fully-automated chemiluminescence enzyme immunoassay (CLEIA) for KL-6 and SMRP, pleural effusion samples from 87 consecutive patients with PM and 25 patients with non-malignant pleural disorders were studied. In addition, KL-6 and SMRP levels were determined in corresponding patient sera, and in an independent validation cohort (n = 122). MUC1 mRNA and protein expression, and KL-6 levels in cell line supernatants were investigated in PM primary cell lines in vitro. ResultsPM patients had significantly higher KL-6 levels in pleural effusion than non-malignant controls (AUC 0.78, p < 0.0001). Among PM patients, levels were highest in those with epithelioid or biphasic histologies. There was a strong positive correlation between pleural effusion levels of KL-6 and SMRP (p < 0.0001). KL-6 levels in sera similarly associated with diagnosis of PM, however, to a lesser extent (AUC 0.71, p = 0.008). PM patients with high pleural effusion KL-6 levels (≥303 IU/mL) had significantly better overall survival (OS) compared to those with low KL-6 levels (HR 0.51, p = 0.004). Congruently, high tumor cell MUC1 mRNA expression in primary cell lines associated with prolonged corresponding patient OS (HR 0.35, p = 0.004). These findings were confirmed in an independent validation cohort. ConclusionThis is the first study demonstrating KL-6 as a potential novel liquid-based diagnostic and prognostic biomarker in PM.

Performance of plasma Aβ42/40, measured using a fully automated immunoassay, across a broad patient population in identifying amyloid status

BackgroundPlasma biomarkers have emerged as promising screening tools for Alzheimer’s disease (AD) because of their potential to detect amyloid β (Aβ) accumulation in the brain. One such candidate is the plasma Aβ42/40 ratio (Aβ42/40). Unlike previous research that used traditional immunoassay, recent studies that measured plasma Aβ42/40 using fully automated platforms reported promising results. However, its utility should be confirmed using a broader patient population, focusing on the potential for early detection.MethodsWe recruited 174 participants, including healthy controls (HC) and patients with clinical diagnoses of AD, frontotemporal lobar degeneration, dementia with Lewy bodies/Parkinson’s disease, mild cognitive impairment (MCI), and others, from a university memory clinic. We examined the performance of plasma Aβ42/40, measured using the fully automated high-sensitivity chemiluminescence enzyme (HISCL) immunoassay, in detecting amyloid-positron emission tomography (PET)-derived Aβ pathology. We also compared its performance with that of Simoa-based plasma phosphorylated tau at residue 181 (p-tau181), glial fibrillary acidic protein (GFAP), and neurofilament light (NfL).ResultsUsing the best cut-off derived from the Youden Index, plasma Aβ42/40 yielded an area under the receiver operating characteristic curve (AUC) of 0.949 in distinguishing visually assessed 18F-Florbetaben amyloid PET positivity. The plasma Aβ42/40 had a significantly superior AUC than p-tau181, GFAP, and NfL in the 167 participants with measurements for all four biomarkers. Next, we analyzed 99 participants, including only the HC and those with MCI, and discovered that plasma Aβ42/40 outperformed the other plasma biomarkers, suggesting its ability to detect early amyloid accumulation. Using the Centiloid scale (CL), Spearman’s rank correlation coefficient between plasma Aβ42/40 and CL was -0.767. Among the 15 participants falling within the CL values indicative of potential future amyloid accumulation (CL between 13.5 and 35.7), plasma Aβ42/40 categorized 61.5% (8/13) as Aβ-positive, whereas visual assessment of amyloid PET identified 20% (3/15) as positive.ConclusionPlasma Aβ42/40 measured using the fully automated HISCL platform showed excellent performance in identifying Aβ accumulation in the brain in a well-characterized cohort. This equipment may be useful for screening amyloid pathology because it has the potential to detect early amyloid pathology and is readily applied in clinical settings.

The value of plasma presepsin as a diagnostic and prognostic biomarker for sepsis in Southern China.

Presepsin is a soluble CD14 subtype that has been considered as a novel marker for patients with sepsis.This study explored the clinical value of presepsin for sepsis in Southern China, and further established models for diagnosis and prognosis of sepsis through using machine learning (ML), by combining presepsin and other laboratory parameters. 269 subjects (105 infected patients, 164 sepsis and septic shock) and 198 healthy controls were enrolled. Laboratory parameters (hematological parameters, coagulation parameters, liver function indices, renal function indices, and inflammatory markers) were collected. Plasma presepsin was tested by chemiluminescence enzyme immunoassay. ML of DxAI™ Research platform was used to establish diagnostic and prognostic models. Sensitivity, specificity, and other performance indicators were used to evaluate the performance of each model. The level of presepsin was obviously increased in sepsis and sepsis shock, compared with that of infected and healthy group (all P < 0.0001). Presepsin concentration was positively correlated with positive blood culture and 30-day mortality in sepsis and septic shock patients. Through ROC curve analysis, Hb, UREA, APTT, CRP, PCT, and presepsin were incorporated into machine learning to construct diagnosis models. Ada Boost model had the best diagnostic efficiency (AUC: 0.94 (95% CI 0.919-0.968) in the training set and AUC: 0.86 (95% CI 0.813-0.900) in validation set). Furthermore, AST, APTT, UREA, PCT, and presepsin were included in the prognosis ML models, and the Bernoulli NB model had greater predictive ability for 30-day mortality risk of sepsis (AUC: 0.706), which was higher than that of PCT (AUC: 0.617) and presepsin (AUC: 0.634) alone. Machine-learning model based on presepsin and routinely laboratory parameters showed good performance of diagnostic and prognostic ability for sepsis patients.

Abstract P305: New Screening Cutoff Values For Detection Of Aldosteronoma Examined By Lc-ms/ms And A Newly Developed Non-competitive Chemiluminescent Enzyme Immunoassay

Thinking of the poor accuracy of CLEIA and RIA measurements of plasma aldosterone concentrations (PACs), LC-MS/MS is the ideal method but it is costly, labor intensive and time consuming. A novel non-competitive CLEIA (NC-CLEIA) for measuring PACs was recently developed. We validated the accuracy and diagnostic ability of the NC-CLEIA as compared to PACs measured by LC-MS/MS and also determined the screen cutoffs by both measurements, respectively, detecting aldosterone-producing adenoma (APA) in hypertensive patients. Method: We measured plasma renin activity by enzyme assay and PACs using LC-MS/MS and NC-CLEIA in 133 APA, 100 bilateral hyperaldosteronism and 111 essential hypertension patients to examine the accuracy of PACs by this NC--CLEIA as compared with those by LC-MS/MS, and determine the screen cutoffs to detect APA. Passing-Bablok analysis revealed that NC-CLEIA (the regression slope, the intercept and the correlation coefficient were 0.962, -0.043, and 0.994, respectively), was significantly correlated and very equivalent to LC-MS/MS. Bland-Altman plot analysis of NC-CLEIA by LC-MS/MS also demonstrated much smaller systemic errors (a bias of -0.348 ng/dL with limits of agreement of -4.390 and 3.694 and 95% confidence interval) than those . The receiver-operating characteristics analysis demonstrated that cut-offs of aldosterone/renin activity ratio obtained by LC-MS/MS and NC-CLEIA were 31.2 and 31.5 (ng/dL per ng/mL/hr) with a sensitivity of 91.0% and 90.2%, and a specificity of 75.4% and 76.8%, respectively, to differentiate APA from non-APA. This novel NC-CLEIA for PAC could serve as a clinically reliable alternative to LC-MS/MS.

Soluble C-type lectin-like receptor 2 in stroke (CLECSTRO) study: protocol of a multicentre, prospective cohort of a novel platelet activation marker in acute ischaemic stroke and transient ischaemic attack

IntroductionSoluble C-type lectin-like receptor 2 (sCLEC-2) is a new biomarker for platelet activation, which can be easily measured by usual blood collection. We conducted the CLECSTRO, a prospective, observational cohort...

Phage-Displayed Nanobody as a Sensitive Nanoprobe to Enhance Chemiluminescent Immunoassay for Cronobacter sakazakii Detection in Dairy Products.

The exploitation of stable, high-affinity, and low-cost nanoprobes is essential to develop immunoassays for real-time monitoring of foodborne pathogens, so as to safeguard human health. The possible interaction of the Fc fragment of antibodies with spA protein on Staphylococcus aureus will result in unexpected interference. To address this consideration, we described herein for the first time the development of nanobodies that by definition are devoid of the Fc fraction. These nanobodies directed against Cronobacter sakazakii (C. sakazakii) were retrieved from a dedicated immune phage-displayed nanobody library. The binders showed superiority of low cost, strong stability, high binding affinity, and adequate load capacity. Thereafter, a phage-mediated sandwich enzyme-linked immunosorbent assay (ELISA) was constructed by using Cs-Nb2 as an antigen-capturing antibody and phage-displayed Cs-Nb1 as a detection probe. To further enhance the sensitivity, a chemiluminescent enzyme immunoassay (CISA) was established by replacing the substrate from 3,3',5,5'-tetramethylbenzidine (TMB) to luminol, providing a limit of detection of 1.04 × 104 CFU/mL, with a recovery of 98.15-114.63% for the detection of C. sakazakii in dairy products. The proposed nanobody-based phage-mediated sandwich CLISA shows various advantages, including high sensitivity, cost effectiveness, enhanced loading capacity of the enzyme, and high resistance to the matrix effect, providing a strategy for the design of immunoassays toward foodborne pathogens.