Commercial Chemiluminescent Immunoassay Research Articles

Prognostic value of serum 25-hydroxyvitamin D levels and malnutrition status on postoperative complications in patients following laryngectomy with neck dissection.

Postoperative complications (PCs) following total laryngectomy remain a significant challenge, with recent investigations directed toward the impact of nutrition status and vitamin D deficiency. To elucidate the association between preoperative vitamin D level status, malnutrition risk score, and surgical and survival outcomes in patients with advanced laryngeal cancer following total laryngectomy. Prospective cohort study. Sixty-four patients with advanced laryngeal carcinoma treated with total laryngectomy were included in the study. Serum levels of 25(OH) D3 were measured employing a commercial chemiluminescent immunoassay kit, while nutrition status was evaluated using the nutrition risk index (NRI) and Malnutrition universal screening tool (MUST). The mean serum 25(OH) D level was 37.1 ± 19.4 nmol/L (range 11.0-100.6 nmol/L), with 47% of patients exhibiting vitamin D deficiency and 31% displaying insufficiency. Medium/high MUST score had 53% of patients, and moderate/severe NRI was verified in 48% of patients. Univariate logistic regression analysis identified MUST score, GPS score, neutrophil-to-lymphocyte ratio, and circulating 25(OH) D levels as predictive for the occurrence of PCs. In multivariate analysis, MUST score and circulating 25(OH) D levels remained significantly associated with PCs. Patients with high nutrition risk had significantly lower two-year OS rates compared to the medium and low nutrition risk groups, respectively (30% vs. 62% and 83%, p = 0.010). Early identification of malnourished or patients with vitamin D deficiency and those who would benefit from specific nutritional support could be beneficial for minimizing the risk of development of surgical complications and help improve our clinical outcomes.

Detection of intrathecal IgG antibody for varicella and measles diagnosis by evaluation and comparison of a commercial IgG chemiluminescent immunoassay with two ELISAs.

Analyse alternative methods of intrathecal antibody detection by comparing chemiluminescent immunoassay (CLIA) and enzyme-linked immunosorbent assay (ELISA) techniques to determine if CLIA can replace ELISA in the diagnosis of CNS infections. A panel of 280 paired samples-cerebrospinal fluid (CSF) and serum-with known antibody reactivities (Varicella, n = 60; Measles, n = 120) and negative samples (n = 100) were used to evaluate the performance of six serological test kits (Enzygnost, VirClia®, and Serion ELISA (Measles and Variella). For Measles virus IgG, the VirClia® IgG monotest revealed 97% and 94% positive and negative agreement to the Enzygnost as reference test,respectively. In contrast, Serion ELISA kits yielded values of 18% and 90%. For theVaricella Zoster virus (VZV) IgG, the VirClia® IgG monotest showed 97% and 90% positive and negative agreement compared to Enzygnost. The Serion ELISA kits showed values of 55% and 86%, respectively. ROC analysis revealed that the areas under the curve for Measles and VZV IgGs were 0.7 and 0.852, respectively, using the Serion kit, and 0.963 and 0.955, for Vircell S.L CLIA technique. VirClia® monotest values were calculated using an antibody index cut-off of 1.3. The findings indicate that CLIA testing can improve antibody detection in CSF samples, aiding the diagnosis of infectious neurological impairments.

Exploring cell-free assays for COVID-19 serosurvey

Serosurveys to monitor immunity toward COVID-19 in the population are primarily performed using an ELISA to screen samples for SARS-CoV-2 antibodies, followed by confirmation by a virus neutralization test, which is considered the Gold Standard. However, virus neutralization test may not be feasible for some laboratories because of the requirement for specific facilities and trained personnel. In an attempt to address this limitation, we evaluated three cell-free methods as potential alternatives for assessing SARS-CoV-2 seroprevalence in human population from plasma. We report the establishment of two inhibition ELISAs designed to detect anti-Spike RBD IgG antibodies and a microsphere quantitative suspension array technology assay, based on the Luminex xMAP platform, to measure the presence of antibodies against various SARS-CoV-2 antigens, including anti-RBD. These methods were also compared to a commercial chemiluminescent immunoassay designed for anti-RBD antibodies detection and to the combined ELISA + virus neutralization test strategy. These cell-free assays performed equally to estimate the percentage of positive and negative samples and could be used to determine the prevalence of SARS-CoV-2 antibodies in human population, at least in cohort with high-expected prevalence, without the use of seroneutralization assay.

Anti-nuclear valosin-containing protein-like autoantibody is associated with calcinosis and higher risk of cancer in systemic sclerosis.

Systemic sclerosis (SSc)-specific autoantibodies allow the diagnosis and predict the prognosis of SSc patients with different clinical characteristics. The aim of this study was to describe new SSc-related autoantibodies by a novel protein immunoprecipitation (IP) assay. Serum samples and clinical data were collected from 307 SSc patients. Antinuclear autoantibodies were tested in all patients by indirect immunofluorescence (IIF) on HEp-2 cells. SSc-specific autoantibodies were evaluated with a commercial immunoblot and chemiluminescence immunoassay, and traditional RNA-IP. Patients negative for all these autoantibodies (n = 51) were further tested with a non-radioactive protein IP assay. Protein bands detected on SDS-PAGE were then analysed by mass spectrometry (MS) and confirmed by western blot (WB). Additional 56 patients with nucleolar pattern by IIF were tested by protein IP-WB. Five patients who underwent protein IP testing showed a 110-115kDa molecular weight band on SDS-PAGE and a homogeneous nucleolar pattern by IIF. MS identified the bands as nuclear valosin-containing protein-like (NVL). An additional positive patient was detected by IP-WB. As compared with the remaining 101 negative patients, anti-NVL positive patients showed a greater prevalence of calcinosis (100% vs 18.9%, P < 0.001), and cancer (66.7% vs 8.9%, P = 0.002), with a particular association with synchronous cancer (OR = 16.3; P = 0.024). We identified NVL as a new autoantibody target by a novel protein IP assay in SSc patients with a homogeneous nucleolar IIF pattern, testing negative for all known SSc-specific autoantibodies by commercial assays and RNA IP. Anti-NVL identifies a new clinical phenotype, characterized by calcinosis and cancer.

Relationship between galectin-3 level and disease activity in ankylosing spondylitis patients

Background and aimsAnkylosing spondylitis (AS) is a chronic inflammatory disease that chiefly affects the sacroiliac joints and the spine. Galectin-3, a chimera-type member of the galectin family, binds glycoconjugates containing N-acetyllactosamine. Galectins play a role in regulation of embryogenesis, angiogenesis, neurogenesis, and immunity. The aim of the present study was to evaluate the serum galectin-3 level and its possible association with disease activity in AS patients. Forty five AS patients and 35 healthy controls enrolled in this study. All participants with a history of hyperlipidemia, liver, renal, hematological, familial thyroid, neoplastic, autoimmune infectious diseases and using anti-inflammatory drugs were excluded from the study. Serum galectin-3 levels concentration was measured using a commercial chemiluminescent microparticle immunoassay. Erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) levels were measured. The Bath Ankylosing Spondylitis Disease Activity Index (BASDAI) and Ankylosing Spondylitis Disease Activity Score–CRP (ASDAS-CRP) were used to evaluate disease activity in AS patients.ResultsSerum galectin-3 levels were significantly higher in AS patients compared to the control group (p = 0.04). A correlation was determined between the serum galectin-3 levels and BASDAI and ASDAS-CRP scores in the AS patients (r = 0.49, p < 0.001; r = 0.56, p < 0.001, respectively). In AS patients, serum galectin-3 levels were significantly related with CRP levels but were not related with ESR (r = 0.57, p < 0.001; r = 0.25, p = 0.09, respectively).ConclusionsThe serum galectin-3 levels were higher in AS patients and were correlated with disease activity. This study may be useful to reveal the role of galectin-3 in inflammation and to evaluate disease activity in AS patients.

Investigation of SARS-CoV-2 seroprevalence in relation to natural infection and vaccination between October 2020 and September 2021 in the Czech Republic: a prospective national cohort study

ObjectiveExamine changes in SARS-CoV-2 seropositivity before and during the national vaccination campaign in the Czech Republic.DesignProspective national population-based cohort study.SettingMasaryk University, RECETOX, Brno.Participants22 130 persons provided blood samples at two...

Sweat cortisol determination utilizing MXene and multi-walled carbon nanotube nanocomposite functionalized immunosensor

In this work, a highly selective and sensitive cortisol immunosensor based on MXene and multi-walled carbon nanotubes (MXene-MWCNTs) hybrid composite was proposed. The immunosensor was developed by screen printing technique on a flexible polyethyleneterephthalate (PET) substrate followed by modification of MXene-MWCNTs and anti-cortisol antibodies with EDC/NHS coupling agent. Owing to the large surface area and excellent conductivity of MXene-MWCNTs, the immunosensor exhibited a low detection limit and a wide linear range toward cortisol from 0.1 fg/mL to 1 µg/mL, which covered the normal range of free cortisol in sweat. Additionally, the immunosensor was applied to evaluate the cortisol level in human sweat samples, and the results were validated by a commercial chemiluminescence immunoassay (CLIA) assay. The present platform provides an effective method for non-invasive monitoring of human sweat cortisol levels, which may be well-suited for hormone screenings in healthy monitoring and medical diagnosis.

ANTIBODY RESPONSES AND SAFETY OF THE COMMERCIALLY AVAILABLE VACCINES AGAINST SARS-COV-2 VIRUS IN ALLOGRAFTED PATIENTS: REAL WORLD DATA FROM A SINGLE CENTER

ObjectivePatients (pts) who have undergone allogeneic stem cell transplantation (alloSCT) are at high-risk for life-threating complications post SARS-CoV-2 infection, and the mortality rates has been reported of approximately 30-35%. The currently available vaccines proved their effectiveness in the general population by reducing the severity of the COVID-19 infection however, scant data exist regarding the safety and efficacy of the commercially available vaccines in allografted pts.MethodologyAfter a median of 2,7 (0,3-6,7) ys post alloSCT, 20 pts received within a median of 42 days, 2 vaccines of either Pfizer (n=17) or combinations of Pfizer with Moderna (n=2) or AstraZeneca (n=1). Off immunosuppression without evidence of active GvHD were 14 pts, 1 was only on Cyclosporine (CSP) while 5 were on steroids plus CSP or MMF or Ibrutinib for GvHD treatment. Automated commercial chemiluminescence immunoassay (CLIA) against spike (S1/S2) protein was used for antibody responses detection.ResultsDuring vaccination program no side effect grade ≥3 (including allergy, thrombosis, heart dysfunction or laboratory abnormalities) was reported. The commonest complains were fatigue (20%), bony pain (10%) and fever <38.5 oC (10%). Satisfactory antibody responses were observed in 66% and 95% of pts after the 1st and 2nd dose respectively. Importantly, active GvHD and intensive immunosuppression, did not negatively affect the antibody responses. None of the vaccinated pts developed COVID infectionConclusionOur retrospective study although with small number of patients and with short term follow-up, in agreement with others, confirms that the current commercially available vaccines against SARS-CoV-2 are safe and highly effective in producing effective humoral responses in allografted patients. Prospective studies with longer follow-up are needed to elucidate the proper timing and the number of necessary doses for a safe and effective approach in preventing severe COVID-19 infection

Triple quantitative detection of three inflammatory biomarkers with a biotin-streptavidin-phycoerythrin based lateral flow immunoassay

Quantified inflammatory biomarkers are effective clinical strategy for correct and reasonable drug treatment. In the study, a triple lateral flow immunoassay (triple LFIA) had firstly been developed for specific and simultaneous detection of three pivotal inflammatory biomarkers (PCT, CRP and SAA) via biotin-streptavidin-phycoerythrin signal amplification system in one strip. The developed triple LFIA adopted phycoerythrin (PE) as chromophore to eliminate auto-fluorescence interference from plasma biomolecules and anti-PE mAb as single control line to reduce the nonspecific adsorption, which featured particular advantages in high sensitivity and specificity in a large range of analyte concentrations with the LODs of 0.106 ng/mL for PCT, 0.345 μg/mL for CRP and 3.112 μg/mL SAA, respectively. And the linear quantitative detection ranges were from 0.106 to 100 ng/mL, from 0.345 to 200 μg/mL, and from 3.112 to 200 μg/mL, respectively. Compared to commercial chemiluminescence immunoassay method, the correlations for tested PCT, CRP and SAA in 108 clinical samples were 0.989, 0.987 and 0.988, respectively. In summary, we had proposed a rapid and accurate plasma detection to measure inflammation factors, which facilitated the clinical value to achieve precise treatment.

Prescreening with a Rapid Diagnostic Test Followed by a Confirmatory Qualitative Nucleic Acid Test Can Simplify Hepatitis C Diagnosis.

Asymptomatic hepatitis C virus (HCV) infection without treatment is associated with chronic liver diseases including hepatocellular carcinoma. A major obstacle to hepatitis C diagnosis leading to antiviral treatment in some developing countries is the complicated HCV testing required before treatment. To simplify an HCV test-to-treat strategy, which could lead to timely diagnosis and treatment at the point-of-care, we evaluated the performance of four anti-HCV rapid diagnostic tests (RDTs) (Abon, Blue Cross, Healgen, and SD Bioline). They yielded comparable sensitivity (80-83%), specificity (99-100%), and accuracy (90-91.5%). When we field-tested Abon in 4,769 residents of an HCV-endemic province in Thailand, 306 seropositive individuals (6.4%) were identified. In comparison, laboratory test using an automated commercial chemiluminescent microparticle immunoassay (Abbott ARCHITECT) identified slightly more seropositives (327% or 6.9%). Field implementation suggests that Abon was sensitive (88.7%), specific (99.6%), and accurate (98.9%). Furthermore, 82% (250/306) of Abon-positive samples had detectable HCV RNA as determined by nucleic acid test (Roche cobas). The same 250 samples out of 327 reactive in Abbott immunoassay also had detectable HCV RNA (mean RNA level: log 6.28 IU/mL, range: log 3.06- 7.78 IU/mL). The use of RDT followed by qualitative nucleic acid test can cost-effectively identify the majority of HCV seropositive individuals with active infection, which will obviate the need for expensive viral load quantification tests when simplifying HCV diagnosis for the test-to-treat program at the point-of-care.

Glow and Flash Adjustable Chemiluminescence with Tunable Waveband from the Same CuInS2@ZnS Nanocrystal Luminophore.

All commercial chemiluminescence (CL) assays are conducted with either glow or flash CL of eye-visible waveband from chemical luminophores. Herein, glow and flash, as well as waveband adjustable CL from the same nanoparticle luminophore of thiol-capped CuInS2@ZnS nanocrystals (CIS@ZnS-Thiol), are proposed via extensively exploiting the differed redox nature of CL triggering reagents. Taking thiosalicylic acid (TSA) as the model thiol-capping agent, the electron-injection-initiated charge transfer between CIS@ZnS-TSA and reductant can bring out efficient glow CL while the hole-injection-initiated charge transfer between CIS@ZnS-TSA and oxidant can give off obvious flash CL under optimum conditions. The maximum emission wavelength for CL of CIS@ZnS-TSA is adjustable from 730 nm to 823 nm via employing different triggering agents. Promisingly, the coexistent reductant of N2H4·H2O and oxidant of H2O2 can be employed as dual triggering reagents to trigger eye-visible and highly efficient flash CL from CIS@ZnS-TSA. The maximum emission intensity for flash CL of CIS@ZnS-TSA/N2H4-H2O2 is 101-fold greater than the glow CL of CIS@ZnS-TSA/N2H4 and 22-fold greater than the flash CL of CIS@ZnS-TSA/H2O2, respectively. The flash CL from CIS@ZnS-TSA/N2H4-H2O2 is qualified for highly sensitive and selective CL immunoassay in a commercialized typical procedure with the entire operating process manually terminated within 35 min.

A high-throughput platform for the rapid screening of vitamin D status by direct infusion-MS/MS

Vitamin D is an important fat-soluble prohormone with pleiotropic effects on human health, such as immunomodulation of the innate and adaptive immune system. There is an unmet clinical need for a rapid screening platform for 25-hydroxyvitamin D (25OH-D) determination without chromatographic separation that offers better precision and accuracy than immunoassays. Here, we introduce a high-throughput method for assessing vitamin D status from blood specimens based on direct infusion-MS/MS (DI-MS/MS) following click derivatization using 2-nitrosopyridine. We developed an optimized liquid-phase extraction protocol to minimize ion suppression when directly infusing serum or plasma extracts via a capillary electrophoresis system for quantitative determination of 25OH-D. Acceptable reproducibility (mean coefficient of variation = 10.9%, n = 412), recovery (mean = 102% at 15, 30, and 45 nmol/l), and linearity (R2 > 0.998) were achieved for 25OH-D with lower detection limits (limit of detection ∼1.2 nmol/l, S/N ∼ 3), greater throughput (∼3 min/sample), and less bias than a commercial chemiluminescence immunoassay prone to batch effects. There was mutual agreement in 25OH-D concentrations from reference blood samples measured by DI-MS/MS as compared with LC-MS/MS (mean bias = 7.8%, n = 18). We also demonstrate that this method could reduce immunoassay misclassification of vitamin D deficiency in a cohort of critically ill children (n = 30). In conclusion, DI-MS/MS offers a viable alternative to LC-MS/MS for assessment of vitamin D status in support of large-scale studies in nutritional epidemiology as well as clinical trials to rapidly screen individual patients who may benefit from vitamin D supplementation.

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.

Antibody Persistence 6 Months Post-Vaccination with BNT162b2 among Health Care Workers.

Background: We present immunogenicity data 6 months after the first dose of BNT162b2 in correlation with age, gender, BMI, comorbidities and previous SARS-CoV-2 infection. Methods: An immunogenicity evaluation was carried out among health care workers (HCW) vaccinated at the Istituti Fisioterapici Ospitalieri (IFO). All HCW were asked to be vaccine by the national vaccine campaign at the beginning of 2021. Serum samples were collected on day 1 just prior to the first dose of the vaccine and on day 21 just prior to the second vaccination dose. Thereafter sera samples were collected 28, 49, 84 and 168 days after the first dose of BNT162b2. Quantitative measurement of IgG antibodies against S1/S2 antigens of SARS-CoV-2 was performed with a commercial chemiluminescent immunoassay. Results: Two hundred seventy-four HWCs were analyzed, 175 women (63.9%) and 99 men (36.1%). The maximum antibody geometric mean concentration (AbGMC) was reached at T2 (299.89 AU/mL; 95% CI: 263.53–339.52) with a significant increase compared to baseline (p < 0.0001). Thereafter, a progressive decrease was observed. At T5, a median decrease of 59.6% in COVID-19 negative, and of 67.8% in COVID-19 positive individuals were identified with respect to the highest antibody response. At T1, age and previous COVID-19 were associated with differences in antibody response, while at T2 and T3 differences in immune response were associated with age, gender and previous COVID-19. At T4 and T5, only COVID-19 positive participants demonstrated a greater antibody response, whereas no other variables seemed to influence antibody levels. Conclusions: Overall our study clearly shows antibody persistence at 6 months, albeit with a certain decline. Thus, the use of this vaccine in addressing the COVID-19 pandemic is supported by our results that in turn open debate about the need for further boosts.

Initial observations on age, gender, BMI and hypertension in antibody responses to SARS-CoV-2 BNT162b2 vaccine.

BackgroundLiterature data suggests that age, gender and body mass index (BMI) could be associated with difference in immune responses to vaccines. The first goal of the study was to analyze the antibody titre seven days after the second dose of BNT162b2 vaccine in a group of 248 healthcare workers (HCWs). The second goal was to analyze how antibody titre changes in correlation with age, gender, BMI and hypertension.MethodsAn immunogenicity evaluation was carried out among HCWs vaccinated at the Istituti Fisioterapici Ospitalieri (IFO), Rome, Italy. All HCWs were asked to be vaccinated by the Italian national vaccine campaign at the beginning of 2021. 260 vaccinated HCWs were enrolled in the study. All eligible participants were assigned to receive the priming dose in two weeks’ time and the booster dose exactly 21 days thereafter. Blood and nasopharyngeal swabs were collected at baseline and 7 days after second dose of vaccine. Quantitative measurements of IgG antibodies against S1/S2 antigens of SARS-CoV-2 were performed with a commercial chemiluminescent immunoassay. Presence of SARS-Cov-2 in nasopharyngeal swab was determined by commercial RT-PCR testing.Findings248 HWCs were analyzed, 158 women (63.7%) and 90 men (36.3%). After the second dose of BNT162b2 vaccine, 99.5% of participants developed a humoral immune response. The geometric mean concentration of antibodies among the vaccinated subjects after booster dose (285.9 AU/mL 95% CI: 249.5–327.7) was higher than that of human convalescent sera (39.4 AU/mL, 95% CI: 33.1–46.9), with p<0.0001. Multivariate linear regression analysis of AU/mL by age, gender and BMI multivariate was performed by the inclusion of covariates. This analysis demonstrated that age (p<0.0001) and gender (p = 0.038) are statistically associated with differences in antibody response after vaccination, whereas BMI and hypertension have no statistically significant association (p = 0.078 and p = 0.52 respectively).Interpretation99.5% of HCW developed a humoral immune response and female and young participants seem to have an increased capacity to mount humoral immune responses. BMI and hypertension seem not associated with difference in immune response to the vaccine.FundingNone.

Maintenance of neutralizing antibodies over ten months in convalescent SARS-CoV-2 afflicted patients.

Knowledge of the level and duration of protective immunity against SARS‐CoV‐2 after primary infection is of crucial importance for preventive approaches. Currently, there is a lack of evidence on the persistence of specific antibodies. We investigated the generation and maintenance of neutralizing antibodies of convalescent SARS‐CoV‐2‐afflicted patients over a ten‐month period post‐primary infection using an immunofluorescence assay, a commercial chemiluminescent immunoassay and an in‐house enzyme‐linked neutralization assay. We present the successful application of an improved version of the plaque‐reduction neutralization assay which can be analysed optometrically to simplify data interpretation. Based on the results of the enzyme‐linked neutralization assay, neutralizing antibodies were maintained in 77.4% of convalescent individuals without relevant decay over ten months. Furthermore, a positive correlation between severity of infection and antibody titre was observed. In conclusion, SARS‐CoV‐2‐afflicted individuals have been proven to be able to develop and maintain neutralizing antibodies over a period of ten months after primary infection. Findings suggest long‐lasting presumably protective humoral immune responses after wild‐type infection.

A Reliable Indirect ELISA Protocol for Detection of Human Antibodies Directed to SARS-CoV-2 NP Protein.

A few months ago, the availability of a reliable and cost-effective testing capacity for COVID-19 was a concern for many countries. With the emergence and circulation of new SARS-CoV-2 variants, another layer of challenge can be added for COVID-19 testing at both molecular and serological levels. This is particularly important for the available tests principally designed to target the S gene/protein where multiple mutations have been reported. Herein, the SARS-CoV-2 NP recombinant protein was utilized to develop a simple and reliable COVID-19 NP human IgG ELISA. The optimized protocol was validated against a micro-neutralization (MN) assay, in-house S-based ELISA, and commercial chemiluminescence immunoassay (CLIA). The developed assay provides 100% sensitivity, 98.9% specificity, 98.9% agreement, and high overall accuracy with an area under curve equal to 0.9998 ± 0.0002 with a 95% confidence interval of 0.99 to 1.00. The optical density values of positive samples significantly correlated with their corresponding MN titers. The assay specifically detects IgG antibodies to the SARS-CoV-2 NP protein and does not cross-detect IgG to the viral S protein. Moreover, it does not cross-react with antibodies related to other coronaviruses (e.g., the Middle East respiratory syndrome coronavirus or human coronavirus HKU1). The availability of this reliable COVID-19 NP IgG ELISA protocol is highly valuable for its diagnostic and epidemiological applications.

Comparison of LC-MS/MS and chemiluminescent immunoassays for immunosuppressive drugs reveals organ dependent variation in blood cyclosporine a concentrations

IntroductionLife-long monitoring of immunosuppressive drugs (ISDs) in blood is essential after organ transplantation. However, the ISD concentrations vary depending on the assay employed. ISDs are strongly bound to cytoplasmic proteins in erythrocytes in circulation. Therefore, the relatively rapid sedimentation of blood cells in whole blood samples may affect the results when using liquid handling robots. MethodsWe used 1115 blood samples from outpatients and ward patients with kidney (n = 373), liver (n = 101), heart (n = 29) and bone marrow (n = 155) transplant. Whole blood samples were pretreated by protein precipitation. Alternatively, the samples were hemolyzed by freezing prior to precipitation. ISDs were analyzed by a 2-plexing liquid chromatography tandem mass spectrometry (LC-MS/MS) assay and commercial chemiluminescent microparticle immunoassays (CMIA). ResultsThe difference between the two sample preparation practices was negligible (<2%). Overall, the measured ISD concentrations in patient samples were lower by LC-MS/MS than by CMIA. The difference was the largest (20.2%) and the smallest (9.1%) in samples from liver and from heart transplant patients, respectively. ConclusionsCMIA overestimates blood ISD concentrations as compared to LC-MS/MS. The extent of the difference was found to be organ transplant dependent. The ISDs can be quantitatedeither from intact or hemolyzed blood samples.

A sandwich-type ECL immunosensor based on signal amplification using a ZnO nanorods-L-cysteine-luminol nanocomposite for ultrasensitive detection of prostate specific antigen

An ultrasensitive sandwich-type electrochemiluminescence (ECL) immunosensor was developed for detection of prostate specific antigen (PSA) using an amplification strategy based on ZnO nanorods-l-cysteine-luminol nanocomposites and the biotin-streptavidin system. The biotin-streptavidin system served as a capture probe to increase the number of antibodies. ZnO nanorods not only acted as a nanocarrier that increased the number of luminol molecules and secondary antibodies, but also enhanced the ECL signal of luminol-H2O2 system by promoting H2O2 decomposition, which can further increase ECL intensity. Under optimized conditions, the proposed immunosensor demonstrated excellent analytical performance with a wide linear detection range of 0.03 pg mL−1 to 30 ng mL−1 and a detection limit of 0.01 pg mL−1 (the detection limit in real samples was 0.021 pg mL−1). This method exhibited excellent stability, reproducibility, and selectivity. In addition, the results of PSA determinations in human serum samples obtained using the proposed immunosensor were consistent with data collected using the commercial chemiluminescence immunoassay analyzer.

A five-year retrospective audit of anti-myeloperoxidase antibody titres measured using a commercial chemiluminescent immunoassay

Background: In 2015, ACT Pathology transitioned to a commercial chemiluminescent immunoassay (QUANTA-Flash MPO, Inova Diagnostics) to measure anti-MPO IgG antibodies. Monitoring of internal performance check sera identified discordant results following a change in reagent lot. To investigate this further, we conducted a five-year retrospective audit of all anti-MPO titres measured using this assay. Methods: Samples were stratified according to titre (negative <6 IU/mL, low-positive 6–23 IU/mL and high-positive ≥24 IU/mL), clinical information provided on request forms, ANCA pattern as detected by indirect immunofluorescence (IIF), and reagent lot number. Results: A total of 2005 anti-MPO titres were measured between 15 May 2015 and 13 August 2019. The mean number of tests performed each month doubled between 2015–2016 (25 tests/month) to 2017–2019 (50 tests/month). This was associated with increased testing of patients with known AAV. Some variation in the percentage of low-positive (5.8–16.4%) and high-positive (7.7–21.4%) results was observed between the 12 reagent lots used, however this may have been confounded by duration of use (range 2–296 days). The predicted probability of p-ANCA detection was significantly higher in patients with higher anti-MPO IgG titres (OR=17.66, 95% CI 13.72–21.13, p<0.0001). Conclusion: Anti-MPO IgG testing at ACT Pathology has increased over the last five years. While some variation between reagent lots has been observed, this has not significantly affected overall assay performance.