Chemiluminescent Enzyme Immunoassay Research Articles

Prognosis of elderly non-valvular atrial fibrillation patients stratified by B-type natriuretic peptide: ELDERCARE-AF subanalysis

B-type natriuretic peptide (BNP) is a risk factor for stroke and cardiac death in patients with atrial fibrillation. We hypothesized the prognostic outcomes of very elderly non-valvular atrial fibrillation patients ineligible for standard anticoagulation treatment would vary according to BNP stratification. In this subanalysis of the ELDERCARE-AF trial, patients were stratified by BNP levels at enrollment, and clinical outcomes compared among BNP subgroups. Hazard ratios were adjusted for age, atrial fibrillation type, body mass index, creatine clearance, congestive heart failure, and D-dimer. BNP levels were measured using chemiluminescence enzyme immunoassays. In total, 984 patients (average age: 86.6 years) not considered eligible for oral anticoagulant therapy at approved doses for stroke prevention were included. The BNP levels at enrollment were <200 (low), 200 to <400 (moderate), and ≥400 (high) pg/mL in 428, 300, and 256 patients, respectively. The number (%) of patients with stroke or systemic embolism (SSE) was 7 (1.2%), 24 (5.9%), and 28 (8.6%) in the low, moderate, and high BNP subgroups, respectively (adjusted hazard ratio 3.82, P=.0025 for low vs moderate BNP and 4.76, P=.0007 for low vs high BNP). There was no significant difference in major bleeding incidence between the BNP subgroups. Edoxaban 15 mg was associated with a consistent reduction in SSE vs placebo in all BNP subgroups. Stratification by BNP level was associated with the incidence of SSE for very elderly non-valvular atrial fibrillation patients ineligible for standard anticoagulation treatment, and the effect of edoxaban 15 mg was consistent across BNP levels.

A new detection system for serum fragmented cytokeratin 18 as a biomarker reflecting histologic activities of human nonalcoholic steatohepatitis.

Caspase‐generated fragmented cytokeratin 18 (fCK18) is recognized as a useful noninvasive biomarker in the diagnosis of nonalcoholic fatty liver disease (NAFLD), particularly nonalcoholic steatohepatitis (NASH). However, fCK18 measurement is not applied clinically due to widely variable cut‐off values under the current enzyme‐linked immunosorbent assay platform. Therefore, we developed a highly sensitive chemiluminescent enzyme immunoassay using newly developed monoclonal antibodies against fCK18 and investigated its relevance in NASH diagnosis. Serum fCK18 levels were measured in the derivation and validation cohort. The correlation between serum fCK18 levels and NAFLD activity score (NAS), fibrosis stage, and liver function was examined. Serum fCK18 levels were significantly correlated with alanine aminotransferase (ALT), aspartate aminotransferase (AST), and gamma‐glutamyl transpeptidase. Serum fCK18 levels were significantly associated with NAS, Brunt's grade/stage, Matteoni's classification, portal inflammation, and fat accumulation in the liver. Notably, hepatocyte ballooning was the only independent variable significantly associated with serum fCK18 in the multivariate linear regression analysis. Serum fCK18 levels were significantly elevated in patients with NAFLD and nonalcoholic fatty liver (NAFL) compared to healthy individuals. They were also significantly elevated in patients with NAFL compared to NASH defined by NAS or Matteoni's classification, with area under the curve values being 0.961 (NAFLD vs. healthy), 0.913 (NAFL vs. healthy), 0.763 (NASH vs. NAFL), and 0.796 (NASH type 3–4 vs. NAFL type 1–2). These results were confirmed by a validation cohort. Notably, changes over time in serum fCK18 levels were significantly correlated with changes in ALT, AST, and the fibrosis‐4 index in 25 patients who underwent lifestyle modification. Serum fCK18 levels were significantly correlated with liver damage associated with NASH pathology. Serum fCK18 levels are accurate in distinguishing patients with NAFL or NASH from healthy individuals and may be useful to monitor NASH over time.

Antibody Sustainability in SARS-CoV-2 Healthcare Professionals' Patient Cohort

In this study, it was aimed to evaluate one-year follow-up of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) specific antibodies formed against the virus binding site, in a coronavirus disease-2019 (COVID-19) positive case cohort (n= 413) between the period March 2020 to December 2020 in Manisa Celal Bayar University Hospital, until July 2021. SARS-CoV-2 antibodies were determined by the chemiluminescent enzyme immunoassay (CLIA) method. Values of 1.0 and above were considered positive. Chi-square tests and Joinpoint regression analysis (version 4.7.0) were used in the statistical analyses. The mean age of the participants was 34.9 ± 9.3 and 60.2% of them were women. Between 21-30 days after the diagnosis of COVID-19, total antibody level was above the threshold value in 72.2% (n= 126) of the participants, while this rate increased to 79.1% (n= 240) in 31-60 day interval. In the following period, this rate decreased to 38.8% (n= 108) in days 211st to 240th. Antibody response could not be detected in 76 (20.7%) of 367 employees who have initially been followed up. The percentage of total antibody positivity prevalence ranged from 98.9% to 96.1% in the 31-210th day after diagnosis, in the follow-up of 291 employees whose total antibody positivity was detected after diagnosis. According to the results of the Joinpoint regression analysis, after the diagnosis of COVID-19, the curve showing the percentage of antibody positivity was broken at two points: The first breaking point was observed in 181-210th days (6-7 months) (p= 0.069), and the second breaking point was in 271-300th days (9-10 months) (p< 0.001). As a result, the highest antibody positivity rates were detected after the 30th day of the disease onset and antibody positivity was maintained in the first seven months after diagnosis; the antibody positivity rate decreased to 25% at the end of the first year.

IDF21-0105 Serum Vitamin B12 In Relation To Anemia And Neuropathy By MNSI Among Type 2 Diabetes Mellitus Adults On Metformin

Background: Type 2 diabetes mellitus (T2DM) patients on metformin (the first line therapy to treat T2DM) are prone to have low serum B12 as evident from western literature. There is limited evidence from India regarding the same. Symptoms of B12 deficiency can manifest as hematological and neurological manifestations. The study was planned to understand the association of B12 deficiency, anemia and neuropathy among T2DM patients on metformin. Aim: To assess the magnitude of B12 deficiency and study it in relation to anemia and peripheral neuropathy (PN) among T2DM patients on metformin. Method: A hospital based cross sectional survey of 245 T2DM adults on metformin from a tertiary health care hospital of Delhi was conducted for PN assessment by Michigan Neuropathy Screening Instrument (MNSI). After meeting the inclusion and exclusion criteria, 155 subjects gave fasting blood samples for estimation of Vitamin B12, HbA1c,Hemoglobin and cell morphology by chemiluminescent enzyme immunoassay (C.L.I.A), HPLC and C.B.C respectively. Results: The prevalence of B12 deficiency (B12≤200pg/ml) was 52% among T2DM adults on metformin. Odds Ratio of B12 deficiency and vegetarian diet was 2.33(C.I. 1.22-4.47) (p<0.05). Anemia was present in 40% but it was not associated with B12 deficiency as there were no cases of macrocytic anemia. The prevalence of PN was 38% by PN score ≥2.5 and the mean PN scores was 2.14 ± 1.98.There was 39.2% low grade PN (PN score > 0 ≤ 2.5) followed by high grade PN (34.3%) (PN score >2.5).By Receiver Operating Curve (ROC) analysis serum B12 has shown fair test performance in predicting PN scores of 2.25 (p<0.001). Discussion: The prevalence of vitamin B12 deficiency in T2DM adults on metformin in our study is higher than that reported in other countries. The higher prevalence of B12 deficiency in our study (52 %) is not stunning owing to the higher prevalence of vitamin B12 deficiency in general population in India. The prevalence of B12 deficiency in T2DM patients in our study is more than other Indian studies on metformin. This study showed that though metformin lowers serum B12 but this had no impact on hematological parameter (hemoglobin and cell morphology) assessed in our study. Similar results have been reported by an Indian study and study from the other country. In our study the vitamin B12 deficiency was associated with PN in T2DM adults on metformin which is similar to the finding of a multi centric study from an Asian country Pakistan and several studies from other countries It can be said from our study that vitamin B12 test has fair chance of predicting PN among T2DM patients on metformin and taking a decision regarding which patients should be referred to a neurologist for electrophysiological studies. Conclusions: Metformin use was associated with biochemical B12 deficiency but not with clinical B12 deficiency as none had macrocytic anemia. Those who were B12 deficient were probable to have PN scores of 2.5 by MNSI physical assessment score. Serum B12 screening and use of MNSI for PN is recommended in T2DM patients on metformin.

Türkiye’de bir üçüncü basamak hastanede sağlık çalışanlarında SARS-CoV-2 seroprevalansı

Objective: The aim of this study is to investigate the previous four months (March-July 2020) SARS-CoV-2 infection rate, seroprevalence and the variables affecting these in HCWs in a university hospital. Methods: The present study is a SARS-CoV-2 seroprevalence study on HCWs working in a tertiary hospital during the first stage (March-July 2020) of the outbreak in Turkey. The presence of IgM and IgG antibodies against the spike structure of the virus was investigated by the chemiluminescent enzyme immunoassay (CLIA) method using the commercial antibody kit (COV2T, Siemens®, Tarrytown, NY, US). Participants’ socio-demographic characteristics, health status, lifestyle, risky occupational and social and personal protective equipment (PPE) usage were independent variables of the study. Chi-square test and Fisher’s exact test were used in univariate analyzes, and accepted type 1 error value was 0.05. The analyzes were made using the SPSS 23.0 package program. Results: 1177 out of a total of 1702 health workers participated in the study. Participation rate was 69.1% . The mean age of the study group was 35.3 ± 9.8 and 62.7% were females. SARS-CoV-2 infection rate detected by nucleic acid amplification test (NAAT-PCR) or antibody test (Elisa) was (18/1177) 1.5%; The seroprevalence of SARS-CoV-2 was 1.01%. 17% of the entire SARS-CoV-2 cases were asymptomatic. The highest infection prevalence was significantly higher in auxiliary health workers (3.7%) compared to other groups. The presence of symptoms HCW’s and their family members that did not exist before in the last 15 days, being overweight or obese and consulting as contacted person in survelliance unit were significantly related to having SARS-CoV-2 infection (p

Conformity of cleia and clia HBsAg quantitative assay with qPCR gold standard in hepatitis B patients

Quantitative HBsAg assay is an alternative marker of potential viremia and monitoring response to antiviral treatment. Various methods have been developed to assess HBsAg quantification. This cross-sectional observational research aims to understand the performance of quantitative HBsAg assay with CLEIA (Chemiluminescence Enzyme Immunoassay) and CLIA (Chemiluminescence Immunoassay) methods and their compliance with qPCR. Serum samples were obtained from 69 Hepatitis B patients at the Outpatient Clinic. Quantitative HBsAg assay was performed with Sysmex HISCL-5000 and Mindray CL-900i, HBV DNA by using GeneXpert® HBV Viral Load. The results showed that the HBsAg value of the CLEIA method ranged from 9.77 to &gt;2,500 IU/mL, while the CLIA method ranged from 19.97 to &gt;50,000 IU/mL. Analysis of the CLEIA and CLIA methods showed agreement ((LoA: -5014.01 – 1815.33) with the relationship between the two methods showed a positive correlation (p &lt;0.05). There is a positive correlation between HBsAg results using CLEIA method and HBV DNA (p &lt;0.05) and also between the HBsAg CLIA method and HBV DNA (p &lt;0.05).

BP180NC16a autoantibody positivity may predict low-risk mucous membrane pemphigoid.

The objective of the present study was to explore the clinical characteristics of BP180NC16a autoantibody-positive mucous membrane pemphigoid. Data from 22 patients were collected by retrospective chart review. For the detection of BP180NC16a autoantibody, a commercially available enzyme-linked immunosorbent assay or chemiluminescent enzyme immunoassay kit was used. The patients were classified into the following clinical phenotypes: low-risk patients who had lesions only in the oral mucosa or in the oral mucosa and skin and high-risk patients who had lesions in the oral mucosa and any of the following sites: the eyes, upper respiratory tract, or esophagus. Eleven of the 22 patients (50%) were BP180NC16a-positive at the time of diagnosis. All the positive patients were in the low-risk group. There was a significant difference in the incidence of low-risk mucous membrane pemphigoid between BP180NC16a autoantibody-positive and BP180NC16a autoantibody-negative patients (p=0.004). Patients' age, disease duration, and oral disease activity scores were not significantly different between the two groups. BP180NC16a reactivity was associated with the clinical phenotype characterized by only oral mucosal lesions or combined oral mucosal and skin lesions. BP180NC16a autoantibody might be useful as a serum marker to predict low-risk mucous membrane pemphigoid.

Comparison of CLEIA and ELISA for SARS-CoV-2 Virus Antibodies after First and Second Dose Vaccinations with the BNT162b2 mRNA Vaccine.

The global severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has required rapid action to control its spread and vaccines are a fundamental solution to this pandemic. The development of rapid and reliable serological tests to monitor the antibody response to coronavirus disease vaccines is necessary for post-vaccination immune responses. Therefore, in this study, anti-SARS-CoV-2 antibody titers after the first and second doses were monitored using two different measurement systems, a highly sensitive analytical platform of chemiluminescent enzyme immunoassay (CLEIA) and an enzyme-linked immunosorbent assay (ELISA). Our study included 121 participants who received two doses of the BNT162b2 vaccine. Both methods show significant increase in anti-spike protein IgG antibody levels one week after the first vaccination, and then reached at a plateau at week five (week two after the second dose), with a 3.8 × 103-fold rise in CLEIA and a 22-fold rise in ELISA. CLEIA and ELISA showed a good correlation in the high titer range, >10 binding antibody unit (BAU)/mL. Both methods detected higher IgG antibody levels in females compared with male participants after the second vaccination, while CLEIA exhibits the sex difference after the first dose. Thus, our study showed better performance of CLEIA over ELISA in sensitivity, especially in the low concentration range, however ELISA was also useful in the high titer range (>10 BAU/mL) corresponding to the level seen several weeks after the first vaccination.

Re-Assessment of the Oral Salt Loading Test Using a New Chemiluminescent Enzyme Immunoassay Based on a Two-Step Sandwich Method to Measure 24-Hour Urine Aldosterone Excretion.

Since April 2021, the plasma aldosterone concentration has been measured by chemiluminescent enzyme immunoassay (CLEIA) in Japan. In the present study, we developed a new CLEIA using a two-step sandwich method to measure the 24-hour urine aldosterone level. We collected 115 urine samples and measured 24-hour urine aldosterone levels employing radioimmunoassay (RIA), CLEIA, and liquid chromatography–tandem mass spectrometry (LC-MS/MS). The results showed that the 24-hour urine aldosterone levels measured using CLEIA and LC-MS/MS were significantly correlated (ρ = 0.992, P < 0.0001). Based on the results of Passing–Bablok regression analysis, the slope was 0.992 and the intercept –19.3. The 24-hour urine aldosterone levels measured using CLEIA and RIA were also significantly correlated (ρ = 0.905, P < 0.0001). However, the aldosterone level measured by CLEIA was lower than that measured by RIA (slope, 0.729; intercept, 120.9). In Japan, a new guideline for primary aldosteronism has been announced, with changes in the aldosterone measurement method. The cutoff values for oral sodium loading test (OSLT) were changed, but clinical verification using real-world urine samples has not been performed. Therefore, we examined the cut-off value of the 24-hour urine aldosterone level after the OSLT. Receiver operating characteristic analysis revealed a cut-off value for primary aldosteronism of 3 μg/day.

Serum Pepsinogens Combined with New Biomarkers Testing Using Chemiluminescent Enzyme Immunoassay for Non-Invasive Diagnosis of Atrophic Gastritis: A Prospective, Multicenter Study

Background: Analysis of serum biomarkers for the assessment of atrophic gastritis (AG), a gastric precancerous lesion, is of growing interest for identification of patients at increased risk of gastric cancer. The aim was to analyze the diagnostic performance of serum pepsinogen testing using another method, chemiluminescent enzyme immunoassay (CLEIA), as well as of other new potential biomarkers. Material and Methods: The sera of patients considered at increased risk of gastric cancer and undergoing upper endoscopy collected in our previous prospective, multicenter study were tested for pepsinogen I (PGI) and II (PGII), interleukin-6 (IL-6), human epididymal protein 4 (HE-4), adiponectin, ferritin and Krebs von den Lungen (KL-6) using the CLEIA. The diagnostic performance for the detection of AG was calculated by taking histology as the reference. Results: In total, 356 patients (162 men (46%); mean age 58.6 (±14.2) years), including 152 with AG, were included. For the detection of moderate to severe corpus AG, sensitivity and specificity of the pepsinogen I/II ratio were of 75.0% (95%CI 57.8–87.9) and 92.6% (88.2–95.8), respectively. For the detection of moderate to severe antrum AG, sensitivity of IL-6 was of 72.2% (95%CI 46.5–90.3). Combination of pepsinogen I/II ratio or HE-4 showed a sensitivity of 85.2% (95%CI 72.9–93.4) for the detection of moderate to severe AG at any location. Conclusion: This study shows that PG testing by CLEIA represents an accurate assay for the detection of corpus AG. Additionally, IL-6 and HE-4 may be of interest for the detection of antrum AG. Mini-abstract: Pepsinogens testing by chemiluminescent enzyme immunoassay is accurate for the detection of corpus atrophic gastritis. IL-6 and HE-4 maybe of interest for the detection of antrum atrophic gastritis.

Serum Interleukin-18 Provides a Clue to the Diagnosis of Adult-Onset Still's Disease: Findings from 6 Japanese Patients with Adult-Onset Still's Disease.

Adult-onset Still's disease (AOSD) is a systemic autoinflammatory disorder accompanied by skin eruption. However, typical skin eruptions, such as evanescent, salmon-pink erythema, are not specific to AOSD and dermatologists often face difficulty in diagnosing AOSD. In this study, we examined serum IL-18 levels as well as IL-6, ferritin and C-reactive protein in 6 Japanese patients with AOSD. Serum levels of IL-6 and IL-18 were evaluated in the acute phase and at the time of remission. Serum levels of IL-6 were analyzed using a commercial chemiluminescent enzyme immunoassay (CLEIA; SRL, Tokyo, Japan). Serum IL-18 levels were measured using a commercial ELISA kit (Medical & Biological Laboratories Co., LTD. Nagoya, Japan). In active AOSD, serum ferritin levels and CRP levels were above normal range in 6 patients. In remission, serum ferritin levels of 3 patients were slightly above the normal range, while CRP serum levels of 6 patients were all normalized. Serum IL-18 levels were markedly elevated in 5 cases during the acute phase. In remission, serum IL-18 levels remained at higher values than the normal range in 5 cases. Serum IL-6 levels were also highly elevated in 5 patients in active AOSD and became normalized in remission except in case 2. High levels of serum IL-18 will be a clue to the diagnosis of AOSD. CRP is also useful biomarker for monitoring disease activity compared with IL-6 and IL-18.

SARS CoV-2 seroprevalence and diagnostic accuracy during a COVID-19 outbreak in a major penitentiary complex in Brazil, June to July 2020.

This study aims to estimate the overall SARS-CoV-2 seroprevalence and evaluate the accuracy of an antibody rapid test compared to a reference serological assay during a COVID-19 outbreak in a prison complex housing over 13,000 prisoners in Brasília. The authors obtained a randomized, stratified representative sample of each prison unit and conducted a repeated serosurvey among prisoners between June and July 2020, using a lateral-flow immunochromatographic assay (LFIA). Samples were also retested using a chemiluminescence enzyme immunoassay (CLIA) to compare SARS-CoV-2 seroprevalence and 21-days incidence, as well as to estimate the overall infection fatality rate (IFR) and determine the diagnostic accuracy of the LFIA test. This study identified 485 eligible individuals and enrolled 460 participants. Baseline and 21-days follow-up seroprevalence were estimated at 52.0% (95% CI 44.9-59.0) and 56.7% (95% CI 48.2-65.3) with LFIA; and 80.7% (95% CI 74.1-87.3) and 81.1% (95% CI 74.4-87.8) with CLIA, with an overall IFR of 0.02%. There were 78.2% (95% CI 66.7-89.7) symptomatic individuals among the positive cases. Sensitivity and specificity of LFIA were estimated at 43.4% and 83.3% for IgM; 46.5% and 91.5% for IgG; and 59.1% and 77.3% for combined tests. The authors found high seroprevalence of anti-SARS-CoV-2 antibodies within the prison complex. The occurrence of asymptomatic infection highlights the importance of periodic mass testing in addition to case-finding of symptomatic individuals; however, the field performance of LFIA tests should be validated. This study recommends that vaccination strategies consider the inclusion of prisoners and prison staff in priority groups.

Positive Anti-HIV ELISA Results in Pregnancy: Is It Reliable?

Background: Human immunodeficiency virus (HIV) can be transmitted from mothers to their babies during pregnancy, delivery through vaginal fluids or breastfeeding. As false positivity anti-HIV results due to pregnancy could be detected and no relevant study have been reported in Northern Cyprus so far, we aimed to estimate the false anti-HIV positivity rate in pregnant women. Methods: A total of 11.977 women admitted to Near East University Hospital between 2015 and 2018 were involved. The fourth generation anti-HIV-1/2 ELISA test was carried out by chemiluminescence enzyme immunoassay. Positive results were confirmed by real-time polymerase chain reaction (rt-PCR). SPSS (Statistical Package for the Social Sciences) Demo Ver 22 program was used for statistical analysis and significance (p<0.05) was measured by Person Chi-Square and Fisher's Exact tests. Results: Anti-HIV-1/2 ELISA test was positive in 7 (0.3%) of pregnant and 11 (0.1%) of non-pregnant women. HIV RNA was not detected in any pregnant however, was detected in 2 (0.02%) of non pregnant. S/Co titer of pregnant and non pregnant who have positive anti-HIV-test without viral load was x̄=2.68±1.64 (1.34-5.20) and x̄=8.63±7.68 (1.56-20.98) respectively. False positivity was significantly higher in pregnants compared to non-pregnants (p=0.033). Conclusion: False positivity can be encountered during pregnancy therefore, positive anti-HIV-1/2 ELISA results should be confirmed with molecular techniques before initiating antiretroviral treatment.

Krebs von den Lungen-6 as Disease Severity Marker for COVID-19 Patients: Analytical Verification and Quality Assessment of the Tosoh AIA-360 Compared to Lumipulse G600II.

Background: Krebs von den Lungen-6 (KL-6) has been proposed as a disease severity marker of COVID-19. All research articles reported the KL-6 assay detected through Fujirebio reagents by Lumipulse G600/G1200 instrument. In the present study, KL-6 assay was analysed through Tosoh AIA-360 and compared with analytical results by Lumipulse G600 in a population of COVID-19 patients. Materials and methods: Sixty-four patients (median age, IQR 67 (58–76) years), all hospitalized for COVID-19 interstitial pneumonia at Siena COVID Unit. KL-6 was measured by two methods, chemiluminescence enzyme immunoassay (CLEIA) and fluorescent enzyme immunoassay (FEIA) method by Lumipulse G600 II and AIA 360 systems, respectively. Results: KL-6 concentrations evaluated by Lumipulse G600II were significantly higher in severe than those in non-severe patients (p < 0.0001) as well as evaluating by AIA360 (p < 0.0001). Receiver operating curve (ROC) curve analysis showed that KL-6 concentrations, by Lumipuse G600II, distinguished severe from non-severe COVID-19 patients with an area under the curve (AUC) of 99.8% and the best cut-off value was 448 U/mL. AUROC between severe and non-severe COVID-19 patients using T0 KL-6 concentrations by AIA360 was 97.4% and the best cut-off value was 398 U/mL. According to T0 KL-6 concentrations in COVID-19 patients, Bland–Altman difference analysis revealed a mean bias of 78 ± 174.8; while using T1 KL-6 concentrations in COVID-19 patients, Bland–Altman difference analysis revealed a mean bias of 48 ± 126 (95% limits of agreement −199–295) between the Lumipulse G600 II and the AIA360 systems. Conclusions: In conclusion, our study demonstrated that CLEIA and FEIA methods for serum KL-6 detection are comparable and reliable. KL-6 was confirmed as an easily detectable and effective biomarker to identify severe COVID-19 patients.

Development of magnetic particle-based chemiluminescence immunoassay for measurement of SARS-CoV-2 nucleocapsid protein

BackgroundRecently, the Coronavirus Disease 2019 (COVID-19) caused by SARS-CoV-2 infection has spread rapidly around the world, becoming a new global pandemic disease. Nucleic acid detection is the primary method for clinical diagnosis of SARS-CoV-2 infection, with the addition of antibody and antigen detection. Nucleocapsid protein (NP) is a kind of conservative structural protein with abundant expression during SARS-CoV-2 infection, which makes it an ideal target for immunoassay. MethodsThe coding sequence for SARS-CoV-2-NP was obtained by chemical synthesis, and then inserted into pET28a(+). The soluble recombinant NP (rNP) with an estimated molecular weight of 49.4 kDa was expressed in E. coli cells after IPTG induction. Six-week-old BALB/c mice were immunized with rNP, and then their spleen cells were fused with SP2/0 cells, to develop hybridoma cell lines that stably secreted monoclonal antibodies (mAbs) against NP. The mAbs were preliminarily evaluated by enzyme-linked immunosorbent assay (ELISA), and then used to develop a magnetic particle-based chemiluminescence enzyme immunoassay (CLEIA) for measurement of SARS-CoV-2-NP. ResultsmAb 15B1 and mAb 18G10 were selected as capture and detection antibody respectively to develop CLEIA, due to the highest sensitivity for rNP detection. The proposed CLEIA presented a good linearity for rNP detection at a working range from 0.1 to 160 μg/L, with a precision coefficient of variance below 10 %. ConclusionThe newly developed mAbs and CLEIA can serve as potential diagnostic tools for clinical measurement of SARS-CoV-2-NP.

Development of an Automated Chemiluminescent Enzyme Immunoassay for Measuring Thrombopoietin in Human Plasma.

Plasma thrombopoietin (TPO) measurements help distinguish between different types of thrombocytopenia but are not feasible in routine clinical practice. We developed a fully automated quantitative chemiluminescent enzyme immunoassay (CLEIA) for measuring TPO (TPO-CLEIA), which is a one-step sandwich-type assay. This assay utilizes a mouse monoclonal capture antibody, which has the neutralizing epitope of the interaction between TPO and the TPO receptor, and a newly generated rabbit monoclonal detector antibody. In analytical performance studies, this assay showed good linearity over the measuring range and high sensitivity. The limit of quantification (LoQ) of this assay was 3.4 pg/mL; low TPO concentration values of almost all healthy individuals exceeded the LoQ value. In clinical validation studies, TPO levels obtained from patients with aplastic anemia (AA) significantly increased, whereas those of patients with immune thrombocytopenia (ITP) were normal or slightly increased. The cutoff value for TPO-CLEIA corresponding to the previously reported values was useful for distinguishing between ITP and AA. These results suggest that TPO-CLEIA can quantify human plasma TPO levels with high accuracy and sensitivity and has the potential to facilitate routine clinical measurement of TPO in patients with various types of thrombocytopenia.

Antibody Responses to BNT162b2 Vaccination in Japan: Monitoring Vaccine Efficacy by Measuring IgG Antibodies against the Receptor-Binding Domain of SARS-CoV-2.

ABSTRACTTo fight severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes coronavirus disease 2019 (COVID-19), mass vaccination has begun in many countries. To investigate the usefulness of a serological assay to predict vaccine efficacy, we analyzed the levels of IgG, IgM, and IgA against the receptor-binding domain (RBD) of SARS-CoV-2 in the sera from BNT162b2 vaccinated individuals in Japan. This study included 219 individuals who received two doses of BNT162b2. The levels of IgG, IgM, and IgA against RBD were measured by enzyme-linked immunosorbent assay before and after the first and second vaccination, respectively. The relationship between antibody levels and several factors, including age, gender, and hypertension were analyzed. Virus-neutralizing activity in sera was measured to determine the correlation with the levels of antibodies. A chemiluminescent enzyme immunoassay (CLEIA) method to measure IgG against RBD was developed and validated for the clinical setting. The levels of all antibody isotypes were increased after vaccination. Among them, RBD-IgG was dramatically increased after the second vaccination. The IgG levels in females were significantly higher than in males. There was a negative correlation between age and IgG levels in males. The IgG levels significantly correlated with the neutralizing activity. The CLEIA assay measuring IgG against RBD showed a reliable performance and a high correlation with neutralizing activity. Monitoring of IgG against RBD is a powerful tool to predict the efficacy of SARS-CoV-2 vaccination and provides useful information in considering a personalized vaccination strategy for COVID-19.IMPORTANCE Mass vaccination campaigns using mRNA vaccines against SARS-CoV-2 have begun in many countries. Serological assays to detect antibody production may be a useful tool to monitor the efficacy of SARS-CoV-2 vaccination in individuals. Here, we reported the induction of antibody isotype responses after the first and second dose of the BNT162b2 vaccine in a well-defined cohort of employees in Japan. We also reported that age, gender, and hypertension are associated with differences in antibody response after vaccination. This study not only provides valuable information with respect to antibody responses after BNT162b2 vaccination in the Japanese population but also the usefulness of serological assays for monitoring vaccine efficacy in clinical laboratories to determine a personalized vaccination strategy for COVID-19.

A novel high-sensitive indirect competitive chemiluminescence enzyme immunoassay based on monoclonal antibody for tenuazonic acid (TeA) detection

A novel high-sensitive indirect competitive chemiluminescence enzyme immunoassay based on monoclonal antibody for tenuazonic acid (TeA) detection

Comparison and commutability study between standardized liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) and chemiluminescent enzyme immunoassay for aldosterone measurement in blood

A commutability confirmation test for the blood aldosterone measurement was performed on liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) as a designated comparison method (DCM) and four chemiluminescent enzyme immunoassay (CLEIA) measurement procedures based on metrological traceability. A conventional radioimmunoassay (RIA) and two measurement procedures of CLEIA which obtains RIA equivalent values were also compared. The relationship between the DCM value and the CLEIA value with respect to 120 pg/mL of the RIA value, which is the screening criterion of primary aldosteronism (PA) was clarified. For the correlation test, 75 samples of patient serum and plasma were used. Regression analysis revealed that the standardized LC-MS/MS and four CLEIA measurement procedures were in good agreement. This is the effect of measurement specificity and calibration using by certified reference material (CRM). The median of the LC-MS/MS corresponding to 120 pg/mL of RIA was 48.5 pg/mL. In the mean of standardized four CLEIA values corresponding to the 48.5 pg/mL of LC-MS/MS value was 47.51 pg/mL and the standard deviation (SD) was 2.93 pg/mL. However, the correlation between the RIA value and the RIA equivalent of the two measurement procedures by CLEIA differed depending on the measurement procedure. This is due to the influence of RIA measurement performance. Standardized CLEIA measurements are suitable for routine measurement procedure. When converting the LC-MS/MS equivalent value by the standardized CLEIA to the conventional RIA value, it is necessary to use the conversion formula.

Хемилюминесценция функционализированной поверхности графена

To analyze the modification of the functionalized surface of graphene by protein molecules, a chemiluminescent enzyme immunoassay method was proposed. Using the example of functionalized graphene (FG) purification, the possibilities of chemiluminescent control of the state of its surface are shown. Methods for purifying FG from protein molecules with the restoration of the ability to resorb protein molecules are discussed. It has been shown that the FG surface can be cleaned from sorbed proteins, and a biosensor can be designed again based on such purified graphene, including with a different specificity. Therefore, the graphene sensor can be used repeatedly.