Serum prolactin, Preptin, CCL 18 and genetic polymorphisms in Iraqi women with polycystic ovary syndrome.

The polycystic ovary syndrome is an endocrine condition. One of the leading causes of female infertility and the most common disorder among women. The work was being carried out on 100 Iraqi women (50 cases confirmed with PCOS and 50 controls). Between October 2019 and March 2020, blood samples were collected from the Advanced Institute of Infertility Diagnosis and Assisted Reproductive Technology at AL-Nahrain University and a private laboratory. ELISA was used to evaluate the biochemical parameters of preptin, FSH, insulin, LH, and CCL 18 in serum samples from the AFIAS-6 (AFIAS Automated Immunoassay System). The findings of the analysis indicate that, as opposed to the control group, values of prolactin (ng/ml), LH (mIU/ml), Preptin (pg/ml) and CCL 18 (ng/ml) Quite higher in PCOS sickness (p < 0.001) Compared with the patient group, the values of testosterone (ng/ml) and FSH (mIU/ml) was noticeably higher (p <0.05), and PRLR gene expression levels in PCOS patients were significantly increased by 3.6 times. I n summary, the levels of Preptin and CCL18 can be regarded as PCOS markers.

Analytical evaluation of the Nittobo Medical tartrate resistant acid phosphatase isoform 5b (TRACP-5b) EIA and comparison with IDS iSYS in different clinically defined populations

Objectives Tartrate-resistant acid phosphatase, isoform 5b (TRACP-5b) is a bone resorption marker not influenced by renal function or food intake. TRACP-5b can be measured with Nittobo Medical enzymatic-immunoassay and IDS-iSYS automated immunoassay. We evaluated the Nittobo assay and established reference ranges for a Western-European population. We compared Nittobo and IDS results in different well-defined clinical populations. Methods We established the limits of detection and quantification (LOD-LOQ), linearity, imprecision and the reference ranges in 119 males, 50 women (<45 years) and 120 women (>60 years) for TRACP-5b with the Nittobo assay. We compared both assays in 30 hemodialyzed (HD), and 40 stage 3–5 patients suffering from chronic kidney disease (CKD), 40 patients suffering from rheumatoid arthritis and osteoporosis and 80 post-menopausal women. We measured TRACP-5b, β-crosslaps (β-CTX), bone alkaline phosphatase (B-ALP) and PTH in 20 hemodialyzed (HD) and 40 CKD patients. Results LOD and LOQ were 0.02 and 0.35 U/L. CV ranged from 8.3 to 4.3% (2/5 samples presenting CV > desirable CV). Method was linear up to of 11.3 U/L. Upper and lower limits of normality were 0.8–7.6 U/L in men, 0.9–4.7 U/L in women <45 and 0.9–7.1 U/L in women >60. The regression equation between the 2 methods was Nittobo = 1.13 (95% CI: 1.09–1.16) × iSYS − 0.4 (95% CI: −0.5; −0.3). TRACP-5b and b-ALP were in their respective reference ranges for most of CKD and HD patients. That was not the case for β-CTX, which increased with decreasing eGFR. Conclusions Nittobo TRACP-5b presents interesting analytical features and a good concordance with IDS iSYS. These methods could thus potentially be harmonized.

Performance and usefulness of a novel automated immunoassay HISCL SARS-CoV-2 Antigen assay kit for the diagnosis of COVID-19

Here, we aimed to evaluate the clinical performance of a novel automated immunoassay HISCL SARS-CoV-2 Antigen assay kit designed to detect the nucleocapsid (N) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This kit comprises automated chemiluminescence detection systems. Western blot analysis confirmed that anti-SARS-CoV antibodies detected SARS-CoV-2N proteins. The best cut-off index was determined, and clinical performance was tested using 115 serum samples obtained from 46 patients with coronavirus disease 2019 (COVID-19) and 69 individuals who tested negative for COVID-19 through reverse transcription quantitative polymerase chain reaction (RT-qPCR). The HISCL Antigen assay kit showed a sensitivity of 95.4% and 16.6% in samples with copy numbers > 100 and < 99, respectively. The kit did not cross-react with human coronaviruses causing seasonal common cold and influenza, and none of the 69 individuals without COVID-19 were diagnosed with positive results. Importantly, 81.8% of the samples with low virus load (< 50 copy numbers) were diagnosed as negative. Thus, using HISCL antigen assay kits may reduce overdiagnosis compared with RT-qPCR tests. The rapid and high-throughput HISCL SARS-CoV-2 Antigen assay kit developed here proved suitable for screening infectious COVID-19 and may help control the pandemic.

Validation of the LUMIPULSE automated immunoassay for the measurement of core AD biomarkers in cerebrospinal fluid

Objectives The core cerebrospinal fluid (CSF) biomarkers; total tau (tTau), phospho-tau (pTau), amyloid β 1-42 (Aβ 1-42), and the Aβ 1-42/Aβ 1-40 ratio have transformed Alzheimer’s disease (AD) research and are today increasingly used in clinical routine laboratories as diagnostic tools. Fully automated immunoassay instruments with ready-to-use assay kits and calibrators has simplified their analysis and improved reproducibility of measurements. We evaluated the analytical performance of the fully automated immunoassay instrument LUMIPULSE G (Fujirebio) for measurement of the four core AD CSF biomarkers and determined cutpoints for AD diagnosis. Methods Comparison of the LUMIPULSE G assays was performed with the established INNOTEST ELISAs (Fujirebio) for hTau Ag, pTau 181, β-amyloid 1-42, and with V-PLEX Plus Aβ Peptide Panel 1 (6E10) (Meso Scale Discovery) for Aβ 1-42/Aβ 1-40, as well as with a LC-MS reference method for Aβ 1-42. Intra- and inter-laboratory reproducibility was evaluated for all assays. Clinical cutpoints for Aβ 1-42, tTau, and pTau was determined by analysis of three cohorts of clinically diagnosed patients, comprising 651 CSF samples. For the Aβ 1-42/Aβ 1-40 ratio, the cutpoint was determined by mixture model analysis of 2,782 CSF samples. Results The LUMIPULSE G assays showed strong correlation to all other immunoassays (r>0.93 for all assays). The repeatability (intra-laboratory) CVs ranged between 2.0 and 5.6%, with the highest variation observed for β-amyloid 1-40. The reproducibility (inter-laboratory) CVs ranged between 2.1 and 6.5%, with the highest variation observed for β-amyloid 1-42. The clinical cutpoints for AD were determined to be 409 ng/L for total tau, 50.2 ng/L for pTau 181, 526 ng/L for β-amyloid 1-42, and 0.072 for the Aβ 1-42/Aβ 1-40 ratio. Conclusions Our results suggest that the LUMIPULSE G assays for the CSF AD biomarkers are fit for purpose in clinical laboratory practice. Further, they corroborate earlier presented reference limits for the biomarkers.

Seroprevalence of the SARS-CoV-2 antibody in healthcare workers: a multicentre cross-sectional study in 10 Colombian cities

BackgroundHealthcare workers are at increased risk of infection due to occupational exposure to SARS-CoV-2-infected patients. The objective of this study was to determine the seroprevalence of SARS-CoV-2 in healthcare workers in Colombia.MethodsThis study is a cross-sectional study focused on estimating the seroprevalence of SARS-CoV-2 antibodies in healthcare workers from 65 hospitals in 10 cities in Colombia during the second semester of 2020. The seroprevalence was determined using an automated immunoassay (Abbott SARS-CoV-2 CLIA IgG). The study included a survey to establish the sociodemographic variables and the risk of infection. A multivariate model was used to evaluate the association between the results of seroprevalence and risk factors.ResultsThe global seroprevalence of antibodies against SARS-CoV-2 was 35% (95% Bayesian CI 33% to 37%). All the personnel reported the use of protective equipment. General services personnel and nurses presented the highest ratios of seroprevalence among the healthcare workers. Low socioeconomic strata have shown a strong association with seropositivity.ConclusionThis study estimates the prevalence of SARS-CoV-2 infection among healthcare workers. Even though all the personnel reported the use of protective equipment, the seroprevalence in the general services personnel and nurses was high. Also, a significant difference by cities was observed.

COVID-19 mRNA Vaccination in Lactation: Assessment of Adverse Events and Vaccine Related Antibodies in Mother-Infant Dyads

BackgroundData regarding symptoms in the lactating mother-infant dyad and their immune response to COVID-19 mRNA vaccination during lactation are needed to inform vaccination guidelines.MethodsFrom a prospective cohort of 50 lactating individuals who received mRNA-based vaccines for COVID-19 (mRNA-1273 and BNT162b2), blood and milk samples were collected prior to first vaccination dose, immediately prior to 2nd dose, and 4-10 weeks after 2nd dose. Symptoms in mother and infant were assessed by detailed questionnaires. Anti-SARS-CoV-2 antibody levels in blood and milk were measured by Pylon 3D automated immunoassay and ELISA. In addition, vaccine-related PEGylated proteins in milk were measured by ELISA. Blood samples were collected from a subset of infants whose mothers received the vaccine during lactation (4-15 weeks after mothers’ 2nd dose).ResultsNo severe maternal or infant adverse events were reported in this cohort. Two mothers and two infants were diagnosed with COVID-19 during the study period before achieving full immune response. PEGylated proteins were not found at significant levels in milk after vaccination. After vaccination, levels of anti-SARS-CoV-2 IgG and IgM significantly increased in maternal plasma and there was significant transfer of anti-SARS-CoV-2-Receptor Binding Domain (anti-RBD) IgA and IgG antibodies to milk. Milk IgA levels after the 2nd dose were negatively associated with infant age. Anti-SARS-CoV-2 IgG antibodies were not detected in the plasma of infants whose mothers were vaccinated during lactation.ConclusionsCOVID-19 mRNA vaccines generate robust immune responses in plasma and milk of lactating individuals without severe adverse events reported.

Capture the Tag: Mitigation of Biotin Interference in ELISA and Automated Immunoassays by Pre-Conjugating Biotinylated Antibodies to the Streptavidin Surface

Introduction Streptavidin to biotin binding is one of the strongest non-covalent interactions in nature, and is therefore, successfully incorporated into many immunoassays to facilitate antibody capture. Biotin-streptavidin coupling assays are susceptible to interference from free biotin in patient specimens, which may falsely decrease or increase the result depending on assay format. Currently, biotin-stripping methods capture free biotin in patient specimens by pre-incubation with streptavidin-coated microparticles. However, this approach increases turnaround time, test cost, and testing steps, which increases the chance of error. Our objective was to determine if pre-conjugating biotinylated antibodies to the assay’s streptavidin solid surface before adding patient specimen could mitigate biotin interference in ELISA and automated sandwich immunoassays. Methods We performed this study on 3 different ELISAs (CYFRA-21, NSE, S100B) and one automated assay (thyroglobulin); all are a sandwich immunoassay format. Serum pools were spiked with biotin (25 - 1000 µg/L) or PBS control. Manufacturer protocols were followed in both the ELISAs and automated assay to evaluate baseline concentration-dependent biotin interference. Mitigation of biotin interference by pre-incubation was then evaluated in the ELISAs by adding biotinylated antibody to the streptavidin-coated wells 0, 10, 15, or 60 min before adding biotin- or PBS-spiked serum specimens. For the automated assay, streptavidin-coated beads and biotinylated antibody were removed from the reagent cartridge, mixed, and incubated 4.5 hours. The mixture containing biotin-tagged antibody bound to streptavidin beads was added back to the cartridge and placed on the analyzer to evaluate spiked specimens. Lastly, we compared the pre-incubation method to a standard biotin-stripping protocol in the ELISAs to compare the effectiveness of mitigating biotin interference. Results We observed biotin interference across the three ELISAs, where 400 µg/L biotin spiked in serum pools reduced analyte detection to between 10 – 15% of the total activity using the standard assay format. Our time-course studies showed 84 – 95% recovery of the total activity when the biotinylated antibody was pre-incubated in the streptavidin-coated wells for 1 hour prior to addition of serum specimens, compared to 69 – 99% by a standard biotin stripping protocol. We extended this concept to the automated immunoassay where at 1000 µg/L biotin in the specimen, only 9 ±0.01% of the total analyte was measured by the conventional method. However, pre-mixing biotinylated antibody and streptavidin beads resulted in 97 ±0.01% of the total analyte recovery in the presence of 1000 µg/L biotin. Conclusion We have demonstrated that pre-conjugating the biotin antibody to streptavidin is as effective as biotin-stripping methods to avoid biotin interference in sandwich immunoassays that utilize the biotin-streptavidin system, with the additional benefit of optimizing turnaround times, cost, and labor. A simple change in manufacturer assay design could make immunoassays more robust against biotin interference in patient samples.