Chemiluminescent Enzyme Immunoassay System Research Articles

Effect of the third dose of BNT162b2 COVID-19 mRNA vaccine on anti-SARS-CoV-2 antibody levels in healthcare workers.

Administration of three doses of Pfizer-BioNTech BNT162b2 COVID-19 mRNA vaccine was completed in Japan in the spring of 2022. This study aimed to evaluate the antibody responses, and kinetics of three doses of vaccine in healthcare workers (HCWs). We conducted a longitudinal cohort study with HCWs, who had no history of COVID-19 or serologic evidence of SARS-CoV-2 infection, from a single hospital. Immunoglobulin G (IgG) titers of anti-SARS-CoV-2 spike protein (SP) and nucleocapsid protein (NP) titers were measured using an automated chemiluminescent enzyme immunoassay system. A total of 636 HCWs participated in the study. The anti-SP IgG titers decreased slowly after the second dose of the BNT162b2 vaccine in all participants, and robust antibody response was observed after the third dose of the vaccine. The peak anti-SP IgG titer after the third dose was approximately 4.1-fold higher than that after the first and second doses, and the rate of decrease in the anti-SP IgG titer after the third dose was significantly more gradual, than that after the second dose. After the second dose of vaccine, the antibody response was weaker in older participants than in younger participants, and in males than in females respectively, whereas the response to the third dose of vaccine did not differ significantly by sex or age. Adverse events following immunization were generally mild to moderate. The third dose of the BNT162b2 vaccine induced a significant and sustained increase in anti-SP IgG titers, and was generally safe and well-tolerated.

Antibody titers against the Alpha, Beta, Gamma, and Delta variants of SARS-CoV-2 induced by BNT162b2 vaccination measured using automated chemiluminescent enzyme immunoassay

BackgroundLevels of 50% neutralizing titer (NT50) reflect the a vaccine-induced humoral immunity after the vaccination against the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Measurements of NT50 are difficult to implement in large quantities. A high-throughput laboratory test is expected for determining the level of herd immunity against SARS-CoV-2. MethodsWe analyzed samples from 168 Japanese healthcare workers who had completed two doses of the BNT162b2 vaccine. We analyzed immunoglobulin G (IgG) index values against spike protein (SP) using automated chemiluminescent enzyme immunoassay system AIA-CL and analyzed the background factors affecting antibody titer. SP IgG index was compared with 50% neutralization titers. ResultsThe median SP IgG index values of the subjects (mean age = 43 years; 75% female) were 0.1, 1.35, 60.80, and 97.35 before and at 2, 4, and 6 weeks after the first dose, respectively. At 4 and 6 weeks after the first dose, SP IgG titers were found to have positive correlation with NT50 titer (r = 0.7535 in 4 weeks; r = 0.4376 in 6 weeks). Proportions of the SP IgG index values against the Alpha, Beta, Gamma, and Delta variants compared with the original strain were 2.029, 0.544, 1.017, and 0.6096 respectively. Older age was associated with lower SP IgG titer index 6 weeks after the first dose. ConclusionsSP IgG index values were rised at 3 weeks after two doses of BNT162b2 vaccination and have positive correlation with NT50. SP IgG index values were lower in the older individuals and against Beta and Delta strain.

A Novel Thyroglobulin Immunoassay Using the Specimen-Pretreatment Process Improves the Accuracy of Thyroglobulin Measurements in Anti-Thyroglobulin Positive Specimens.

Recently, second-generation thyroglobulin (Tg) sandwich immunoassays have been used in clinical laboratories to measure the serum Tg levels, which is a tumor marker used to monitor postoperative patients with differentiated thyroid cancers. However, these immunoassays are often subject to Tg autoantibody (TgAb) interference. TgAb interference is inevitable for almost all Tg immunoassays, resulting in unreliable Tg measurement values of TgAb-positive samples. To address TgAb interference, we have developed a novel immunoassay based on a fully automated chemiluminescent enzyme immunoassay system using the effective specimen-pretreatment process to inactivate TgAb in blood and evaluated its assay performance. The developed assay was traceable to BCR457 IRMM reference material with a limit of quantification of 0.03 ng/mL. The pretreatment process inactivated almost all TgAb in specimens and allowed accurate Tg measurements in TgAb-positive samples in which TgAb interference was observed using the immunoassays. Size-exclusion chromatography analysis of immunoreactive Tg molecule in a TgAb-positive serum verified disruption of the Tg-TgAb immune complex by the pretreatment process. Good correlation of Tg values in TgAb-negative specimens was observed between the new Tg immunoassay and the second-generation sandwich immunoassays. However, there were numerous discrepant samples on bias plots between the new Tg immunoassay and the second-generation sandwich immunoassays for TgAb-positive specimens. This study indicates the new Tg immunoassay with the specimen-pretreatment process is both robust and free from interference by TgAb. Thus, this novel assay is superior to second-generation sandwich immunoassays and gives accurate Tg concentrations even for TgAb-positive cases.

Clinical validation of quantitative SARS-CoV-2 antigen assays to estimate SARS-CoV-2 viral loads in nasopharyngeal swabs

BackgroundExpansion of the testing capacity for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an important issue to mitigate the pandemic of coronavirus disease-2019 (COVID-19) caused by this virus. Recently, a sensitive quantitative antigen test (SQT), Lumipulse® SARS-CoV-2 Ag, was developed. It is a fully automated chemiluminescent enzyme immunoassay system for SARS-CoV-2. MethodsIn this study, the analytical performance of SQT was examined using clinical specimens from nasopharyngeal swabs using reverse transcription polymerase chain reaction (RT-PCR) as a control. ResultsReceiver operating characteristic analysis of 24 SARS-CoV-2-positive and 524 -negative patients showed an area under the curve of 0.957 ± 0.063. Using a cut-off value of 1.34 pg/ml, the sensitivity was 91.7%, the specificity was 98.5%, and the overall rate of agreement was 98.2%. In the distribution of negative cases, the 99.5 percentile value was 1.03 pg/ml. There was a high correlation between the viral load calculated using the cycle threshold value of RT-PCR and the concentration of antigen. The tendency for the antigen concentration to decrease with time after disease onset correlated with that of the viral load. ConclusionsPresented results indicate that SQT is highly concordant with RT-PCR and should be useful for the diagnosis of COVID-19 in any clinical setting. Therefore, this fully automated kit will contribute to the expansion of the testing capability for SARS-CoV-2.

Analytical and clinical validation of rapid chemiluminescence enzyme immunoassay for urinary thioredoxin, an oxidative stress-dependent early biomarker of acute kidney injury

BackgroundOxidative stress is now recognized to be an important therapeutic target in kidney diseases. However, there are currently no biomarkers that can be used clinically to diagnose renal oxidative stress. MethodsA rapid assay system for urinary thioredoxin 1, an oxidative stress-dependent biomarker of acute kidney injury (AKI), was developed as a chemiluminescence enzyme immunoassay and validated analytically and clinically. ResultsAnalytic evaluation revealed that hemolytic hemoglobin caused measurements to be abnormally high, above the detectable range. However, urine sediment containing red blood cells did not affect the measurements. Assays using our proposed chemiluminescence enzyme immunoassay were completed within as little as 6 min, whereas a conventional ELISA > 4 h. Aciduria <pH 6.0 led to a significant underestimation of thioredoxin 1 concentrations. However, the effects of aciduria were completely reversible with use of a buffer developed for pH adjustment. Urinary thioredoxin 1 was increased in patients with AKI, but was unaffected by extrarenal oxidative stress diseases, including hypoxemia and myocardial infarction, or by chronic kidney disease in which serum creatinine concentrations were comparable. ConclusionsThese results suggest that the chemiluminescent enzyme immunoassay system for urinary thioredoxin 1 enables rapid and specific diagnosis of AKI associated with oxidative stress.

Clinical evaluation of a novel and highly sensitive immunoassay for anti-hepatitis B core antigen using a fully automated immunochemical analyzer.

Recently, the measurement of hepatitis B surface antigen and anti-hepatitis B core antigen (HBcAb) and/or anti-hepatitis B surface antigen has been recommended before various therapies to identify patients at risk of hepatitis B virus (HBV) reactivation. However, a recent study reported that HBV reactivation occurred in HBcAb-negative patients, indicating that it is challenging to identify patients with a history of HBV infection using conventional HBcAb reagent. We developed a highly sensitive HBcAb (HBcAb-HS) assay for reducing the risk of HBV reactivation. The HBcAb-HS assay is an automated chemiluminescent enzyme immunoassay system, which is suitable for clinical use. The cut-off was set at 0.020IU/mL from the distribution patterns of HBcAb-negative specimens, and we evaluated the performance of this assay compared with conventional reagents. This new assay showed a 27-81-fold greater sensitivity than conventional HBcAb reagents; the quantified measurement range was from 0.005IU/mL to 1.500IU/mL, and it showed excellent quantitative performance and correlated well with two conventional assays, using the HBcAb-positive specimens. Moreover, it showed 100% specificity for the 469 purchased HBcAb-negative specimens. Notably, this newly developed HBcAb-HS assay showed positivity in the preserved specimens before HBV reactivation, for which conventional HBcAb reagents gave negative results, and the HBcAb-HS assay could detect the lower HBcAb levels even after intensive immunosuppressive therapies, including autologous hematopoietic stem cell transplantation. The clinical efficacy of the newly developed, highly sensitive HBcAb assay would enable the identification of patients at risk of HBV reactivation more accurately.

PATHFAST, a novel chemiluminescent enzyme immunoassay for measuring estradiol in equine whole blood and serum.

A novel chemiluminescent enzyme immunoassay system, PATHFAST, for the measurement of estradiol in horses was evaluated. The concentrations of estradiol in the whole blood and serum of mares were measured using PATHFAST and the estradiol concentrations measured by PATHFAST were compared with those measured by a time-resolved fluoro-immunoassay (FIA). To monitor physiological changes, serum estradiol concentrations in mares were measured using PATHFAST throughout the gestation period. The serum estradiol concentrations correlated highly with those in whole blood samples. The serum concentrations of estradiol measured by PATHFAST also correlated well with FIA. Circulating estradiol increased during mid-gestation and high levels of serum estradiol were maintained in late gestation, followed by an abrupt decline to term. These results demonstrate the utility of PATHFAST in equine clinics as an accurate diagnostic tool for the rapid assay of estradiol within 26 min using unextracted whole blood.

AB0071 A Possible Role of Serum IL-6 Quick Measure System in RA Patients

BackgroundInterleukin 6 (IL-6) has a crucial role in the pathogenesis of rheumatoid arthritis (RA) [1]. Measurement of serum and plasma IL-6 level has been reported that is useful for monitoring...

A New Chemiluminescent Enzyme Immunoassay for Plasma Tissue Factor Detection

High concentrations of tissue factor (TF) in the plasma may have a major role in the elevated risk of thrombosis in patients with malignant diseases. The quantification of plasma TF concentrations in cancer patients may enable accurate assessments of the risk of thrombosis during the process of cancer progression. Whether the plasma TF concentration is associated with the tumor volume in pancreatic cancer in the field of cancer biology remains uncertain. Rat anti-human TF monoclonal antibodies were prepared in our laboratory and were used in a chemiluminescent enzyme immunoassay (CLEIA) for TF detection. In an in vitro experiment, the antibodies for human TF did not crossreact with mouse TF. Therefore, tumor-derived TF was specifically detected using a novel sensitive human TF detection system developed for this study. The plasma concentration of tumor-derived TF was positively correlated with the tumor volume in mice bearing the human pancreatic cancer cell line BxPC-3. Thus, the plasma TF concentration may be a useful tumor marker. These data warrant further preclinical and clinical investigations of our CLEIA system.

Evaluation of a CLEIA automated assay system for the detection of a panel of tumor markers

Tumor markers are commonly used to detect a relapse of disease in oncologic patients during follow-up. It is important to evaluate new assay systems for a better and more precise assessment, as a standardized method is currently lacking. The aim of this study was to assess the concordance between an automated chemiluminescent enzyme immunoassay system (LUMIPULSE® G1200) and our reference methods using seven tumor markers. Serum samples from 787 subjects representing a variety of diagnoses, including oncologic, were analyzed using LUMIPULSE® G1200 and our reference methods. Serum values were measured for the following analytes: prostate-specific antigen (PSA), alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), cancer antigen 125 (CA125), carbohydrate antigen 15-3 (CA15-3), carbohydrate antigen 19-9 (CA19-9), and cytokeratin 19 fragment (CYFRA 21-1). For the determination of CEA, AFP, and PSA, an automatic analyzer based on chemiluminescence was applied as reference method. To assess CYFRA 21-1, CA125, CA19-9, and CA15-3, an immunoradiometric manual system was employed. Method comparison by Passing-Bablok analysis resulted in slopes ranging from 0.9728 to 1.9089 and correlation coefficients from 0.9977 to 0.9335. The precision of each assay was assessed by testing six serum samples. Each sample was analyzed for all tumor biomarkers in duplicate and in three different runs. The coefficients of variation were less than 6.3 and 6.2 % for within-run and between-run variation, respectively. Our data suggest an overall good interassay agreement for all markers. The comparison with our reference methods showed good precision and reliability, highlighting its usefulness in clinical laboratory's routine.

Calreticulin as a potential diagnostic biomarker for lung cancer.

Calreticulin (CRT) is an endoplasmic reticulum luminal Ca(2+)-binding chaperone protein. By immunizing mice with recombinant fragment (rCRT/39-272), six clones of monoclonal antibodies (mAbs) were generated and characterized. Based on these mAbs, a microplate chemiluminescent enzyme immunoassay (CLEIA) system with a measured limit of detection of 0.09ng/ml was developed. Using this CLEIA system, it was found that soluble CRT (sCRT) level in serum samples from 58 lung cancer patients was significantly higher than that from 40 healthy individuals (only 9 were detectable, P<0.0001). Among them, serum sCRT in the small cell lung cancer was lower than that in adenocarcinoma (P=0.0085), while both were lower than that in the squamous cell carcinoma (P=0.013, P=0.0012, respectively). Moreover, it was found that sCRT in sera from the patients after chemotherapy was higher than that from the patients without chemotherapy (P=0.042). Further study by immunohistochemistry showed that CRT was also highly expressed in the cytoplasm and on the membrane of the lung cancer cells, while there was a trace amount of CRT expression in normal lung cells. Correspondingly, the expression level of CRT on lung cancer cell membrane was associated with the tumor pathological grade. This study demonstrates that sCRT concentration in sera of lung cancer patients is higher than that in sera of healthy individuals, and CRT expression level on lung cancer cell membrane is associated with tumor pathological classification and grade. These findings suggest that CRT may be used as a biomarker in lung cancer prediction and diagnosis.

An illustration of the clinical relevance of detecting human antimouse antibody interference by affinity chromatography

Elevated Cancer antigen 125 (CA 125) serum concentrations (up to 221 kU/l) were measured in a 39 year old woman with a positive family history of breast cancer. The serum determinations were performed with the automated Immulite OM-MA chemiluminescent enzyme immunoassay system (Diagnostic Products). Laparoscopic evaluation of the ovaries did not reveal any abnormalities. CA 125 measurements in the same patient using the automated IMx immunoassay system (Abbott) demonstrated normal serum levels. Using a previously reported chromatography procedure IgG type human antimouse antibody activity was found to be present in the serum samples explaining the falsely elevated levels. To prevent this interference the manufacturer modified the assay system by replacing the monoclonal M11 detection antibody with a rabbit polyclonal antibody. Using the modified OM-MA CA 125 assay results were comparable with the IMx values.

Capsaicin-Induced Calcitonin Gene-Related Peptide Release from Isolated Rat Stomach Measured with a New Chemiluminescent Enzyme Immunoassay

The peripheral capsaicin-sensitive afferent nerve has been reported to play an important role in gastroprotection and to release a calcitonin gene-related peptide (CGRP). We developed a new chemiluminescent enzyme immunoassay (CLEIA) for CGRP and measured capsaicin-induced CGRP release from the isolated and inverted rat stomach. The basal CGRP release from the stomach was 0.40 ±0.02 pg/mg wet weight in a 30-min incubation. Capsaicin (1 × 10−8–1 × 10−5 M) stimulated CGRP release in a concentration-dependent manner. In the stomach from rats with defunctionalization of afferent neurons, the levels of the basal and capsaicin-induced CGRP release were below the limit of detection. On the other hand, the capsaicin-induced CGRP release was not blocked by tetrodotoxin treatment. The gangliosym-pathectomy abolished the increase in the CGRP levels. However, the capsaicin-induced CGRP release was not affected by pretreatment with 6-hydroxydopamine, a neurotoxin that causes a complete degeneration of adrenergic nerve terminals. In conclusion, the CLEIA system may be useful for detecting the released CGRP and studying the activity of capsaicin-sensitive nerves, particulary the CGRP-containing nerves. Our results also confirmed that although the CGRP-containing nerve runs in the sympathetic nerve trunk, the activity of the nerve is not affected by adrenergic nerves, and the capsaicin-induced CGRP release may be attributable to the tetrodotoxin-resistant component.

The development of Luminomaster, a fully automated chemiluminescent enzyme immunoassay system.

We have developed a fully automated discrete chemiluminescent heterogeneous enzyme immunoassay system called Luminomaster. The characteristics of this analyser are: 120 test/h throughput, 14 test menu, wide dynamic range, automated sample dilution, automatic retest, communication with a host central processing unit (CPU) and connection with sample transfer system.