Electrochemiluminescence Immunoassay Research Articles

Complement proteins and complement regulatory proteins are associated with age-related macular degeneration stage and treatment response

BackgroundDysregulation of the complement system is involved in development of age-related macular degeneration (AMD). The complement cascade is regulated by membrane bound complement regulatory proteins (Cregs) on mononuclear leukocytes among others. This study aims to investigate systemic complement proteins and Cregs in AMD stages and their association with treatment response in neovascular AMD (nAMD).MethodsIn this clinical prospective study, treatment-naïve patients with nAMD, intermediate AMD (iAMD) and healthy controls were recruited and systemic complement proteins C3, C3a and C5a were investigated with electrochemiluminescence immunoassays, and Creg expression (CD35, CD46 and CD59) on T cells (CD4 + and CD8+) and monocytes (classical, intermediate and non-classical) investigated with flow cytometry. Treatment response in nAMD patients was evaluated after loading dose and after one year, and categorized as good, partial or poor. Complement proteins and Creg expression levels were compared between healthy controls, iAMD and nAMD, as well as between good, partial and poor nAMD treatment response groups. Polymorphisms in the CFH and ARMS2 genes were analyzed and compared to complement proteins and Creg expression levels in nAMD patients.ResultsOne hundred patients with nAMD, 34 patients with iAMD and 61 healthy controls were included. 94 nAMD patients completed the 1-year follow-up. Distribution of treatment response in nAMD was 61 (65%) good, 26 (28%) partial, and 7 (7%) poor responders. The distribution of 1-year treatment response was 50 (53%) good, 33 (36%) partial, and 11 (11%) poor responders. The concentrations of systemic C3, C3a, and the C3a/C3-ratio were significantly increased in patients with nAMD compared to healthy controls (P < 0.001, P = 0.002, and P = 0.035, respectively). Systemic C3 was also increased in iAMD compared to healthy controls (P = 0.031). The proportion of CD46 + CD4 + T cells and CD59 + intermediate monocytes were significantly decreased in patients with nAMD compared to healthy controls (P = 0.018 and P = 0.042, respectively). The post-loading dose partial treatment response group had significantly lower concentrations of C3a and C5a compared to the good response group (P = 0.005 and P = 0.042, respectively). The proportion of CD35 + monocytes was significantly lower in the 1-year partial response group compared to the 1-year good response group (P = 0.039). High-risk CFH genotypes in nAMD patients was associated with increased C3a, C3a/C3-ratio, and expression levels of CD35 + CD8 + T cells and CD46 + classical monocytes, while expression level of CD46 + non-classical monocytes was decreased.ConclusionElevated concentrations of systemic complement proteins were found in patients with iAMD and nAMD. Decreased Creg expression levels were found in patients with nAMD. Partially responding nAMD patients had a dysregulated complement system and Cregs compared to good responders.

Photoinduced Electrochemiluminescence Immunoassays.

Optimization of electrochemiluminescence (ECL) immunoassays is highly beneficial for enhancing clinical diagnostics. A major challenge is the improvement of the operation conditions required for the bead-based immunoassays using the typical [Ru(bpy)3]2+/tri-n-propylamine (TPrA) system. In this study, we report a heterogeneous immunoassay based on near-infrared photoinduced ECL, which facilitates the imaging and quantitative analysis of [Ru(bpy)3]2+-modified immunobeads at low anodic potential. The photovoltage generated by the photoanode under near-infrared light promotes oxidation processes at the electrode/electrolyte interface, thus considerably lowering the onset potential for both TPrA oxidation and ECL emission. The anti-Stokes shift between the excitation light (invisible to the human eyes) and the visible emitted light results in a clear and stable signal from the immunobeads. In addition, it offers the possibility of site-selective photoexcitation of the ECL process. This approach not only meets the performance of traditional ECL immunoassays in accuracy but also offers the additional benefits of lower potential requirements and enhanced stability, providing a new perspective for the optimization of commercial immunoassays.

Prevalence and risk factors for blood-borne viruses among multi-transfused patients in Sana'a City, Yemen

Background: Blood-borne viruses (BBVs), like hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus (HIV) among multi-transfused patients (MTPs), remain a major public health problem in Yemen. This study aimed to determine the seroprevalence of BBVs and associated risk factors among MTPs. Methods: A cross-sectional study was conducted on MTPs at the Yemeni Society for Thalassemia, National Oncology Center, and Al Kuwait University Hospital in Sana’a City. Data were collected through face-to-face interviews using a predesigned questionnaire. Blood samples were drawn and tested for HBsAg, anti-HCV, and HIV-1,2 by using an electrochemiluminescence immunoassay technique. Results: Among 361 MTPs, the overall seroprevalence of BBVs was 6.8%; seropositivity of HBsAg, anti-HCV, and anti-HIV-1,2 was 3.9%, 2.9%, and 0.0%, respectively. An increase in family size and low income were significantly associated factors with HBV infection. In addition, increases in age and blood transfusion units were found to be significant predictors for HCV (p&lt;0.05). Conclusion: Although the seroprevalence of BBVs is low, it remains a major problem among MTPs. The most frequent infection was HBV, followed by HCV, with a significantly higher rate among leukemia patients. Regular mentoring for viral markers and mandatory implementation of HCV antigen-antibodies testing as well as advanced technology in blood screening are highly recommended.

N-terminal prohormone of brain natriuretic peptide predicts recurrent cardiovascular events in patients with acute coronary syndrome both in the acute and post-discharge setting: a TRACER substudy

Abstract Background N-terminal prohormone of brain natriuretic peptide (NT-proBNP) is a circulating peptide released by the heart in response to increased wall tension and stretching, for example, occurring during myocardial and/or atrial dysfunction. NT-proBNP is a diagnostic and prognostic marker in patients with heart failure but might also have prognostic utility in the acute phase of patients with acute coronary syndrome (ACS) and at one month after ACS. Purpose To assess the prognostic value of NT-proBNP over time in patients with ACS both in the acute setting and when re-assessed after one month. Methods The TRACER study randomized 12,944 patients with non-ST-segment elevation ACS (NSTE-ACS) to treatment with vorapaxar vs. placebo. Plasma samples for biomarker analyses were obtained at randomization (median 21 hours after hospital admission) in 6,800 patients and at an outpatient visit after one month in 5,313 patients. NT-proBNP was analysed using the Elecsys electrochemiluminescence immunoassays method. The primary composite endpoint in this substudy was the one-year risk of cardiovascular (CV) mortality, myocardial infarction, or ischaemic stroke. Associations between continuous natural log-transformed NT-proBNP levels and outcomes were assessed using Cox regression models accounting for study treatment and established risk factors. Results The median concentration of NT-proBNP during hospitalization for ACS and after one month was 496 ng/L and 264 ng/L, respectively. Higher age, female sex, a history of heart failure, and lower body mass index were independently associated with higher levels of NT-proBNP among patients in the acute setting, with renal disease also being an important factor after one month. The composite outcome of CV mortality, MI, or ischaemic stroke occurred in 901 patients (incidence rate 10.1 per 100 person-years) included at baseline and in 381 patients (incidence rate 5.4 per 100 person-years) included at one month. Higher circulating levels of NT-proBNP were independently associated with a higher risk of the composite endpoint at baseline (adjusted hazard ratio with 95% confidence interval comparing the third vs. the first sample quartile: 1.25 [1.03 – 1.51]) and at one month (1.56 [1.15 – 2.11]) (Figure 1 and 2). Incorporating NT-proBNP at baseline into a model with other established risk factors increased the concordance index of the multivariable model from 0.656 to 0.660 (p &amp;lt; 0.001), and after one month, from 0.711 to 0.733 (p &amp;lt; 0.001). These findings remained consistent across the various components of the composite endpoint. Conclusions In patients with ACS, the level of NT-proBNP is an indicator of increased risk of CV outcomes both in the acute setting and, even more pronounced, one month after the index ACS. Measuring NT-proBNP provided incremental prognostic information beyond established risk factors, both in the acute and post-discharge settings.Fig 1.NT-proBNP quartile groupsFig 2.NT-proBNP and CV outcome

Troponin T as a risk marker for recurrent cardiovascular events in the post-discharge setting one month after acute coronary syndrome: a TRACER substudy

Abstract Background Troponin T (TnT) is a protein found in cardiac myocytes that is released during myocardial injury, such as myocardial infarction (MI). TnT levels rapidly rise during an MI, remain elevated for several days, and are a key criterion for diagnosing MI. Additionally, circulating TnT levels may offer prognostic information later, particularly during the rehabilitation phase after an acute coronary syndrome (ACS). Purpose To examine the relationship between circulating TnT at one month after hospitalisation for ACS and subsequent cardiovascular (CV) events. Methods The TRACER trial randomly assigned 12,944 patients diagnosed with non-ST-segment elevation ACS (NSTE-ACS) to receive either vorapaxar or placebo. Plasma samples were obtained after one month, and high-sensitivity (hs)-TnT was measured among 5,313 patients using the Elecsys electrochemiluminescence immunoassays. In this sub-study, the primary composite endpoint was the one-year risk of CV mortality, MI, or ischaemic stroke. Associations with outcomes were evaluated using Cox regression models, both unadjusted and adjusted for clinical baseline characteristics. Results The median concentration of hs-TnT was 11 ng/L one month after hospitalisation for ACS. Higher levels of hs-TnT at one month were associated with older age, coronary artery bypass surgery during the index ACS, male sex, a history of heart failure, renal disease, and diabetes mellitus. The composite outcome of CV mortality, MI, or ischaemic stroke occurred in 381 patients (incidence rate 5.4 per 100 person-years). Hs-TnT at one month after ACS was independently associated with the risk of the composite CV endpoint (relative hazard ratio with 95% confidence interval comparing the third vs. the first sample quartile: 2.40 [1.80 – 3.20]) (see Figure 2). Similarly, hs-TnT, when added to a model including established risk factors, increased the concordance index of the multivariable model from 0.711 to 0.740 (p&amp;lt;0.001). The results were consistent across the different components of the composite endpoint. Conclusions The level of hs-TnT provides useful information on the risk of future CV events in patients when measured one month after an ACS. This information might be useful for optimising secondary preventive treatment following an ACS.Figure.Hs-TnT and composite CV outcome

Solid-phase electrochemiluminescence immunosensing platform based on bipolar nanochannel array film for sensitive detection of carbohydrate antigen 125

Development of simple solid-phase electrochemiluminescence (ECL) immunosensor with convenient fabrication for high-performance detection of tumor biomarkers is crucial. Herein, a solid-phase ECL immunoassay was constructed based on a bipolar silica nanochannel film (bp-SNA) modified electrode for highly sensitive detection of carbohydrate antigen 125 (CA 125). Inexpensive and readily available indium tin oxide (ITO) electrode was used as the supporting electrode for the growth of bp-SNA. bp-SNA consists of a bilayer SNA film with different functional groups and charge properties, including negatively charged inner layer SNA (n-SNA) and positively charged outer layer SNA (p-SNA). The nanochannels of bp-SNA were used for the immobilization of ECL emitter tris(bipyridine)ruthenium(II), while the outer surface was utilized for constructing the immunorecognition interface. Due to the dual electrostatic interaction composed of electrostatic attraction from n-SNA and electrostatic repulsion from p-SNA, ECL emitter could be stably confined within bp-SNA, providing stable and high ECL signals to the modified electrode. After amino groups on the outer surface of bp-SNA were derivatized with aldehyde groups, recognition antibodies could be covalently immobilized, and an immunosensor was obtained after blocking nonspecific sites. When CA 125 binds to the antibodies on the recognition interface, the formed complex reduces the diffusion of the co-reactant tripropylamine (TPrA) to the supporting electrode, decreasing the ECL signal. Based on this mechanism, the constructed immunosensor can achieve sensitive ECL detection of CA 125. The linear detection range is from 0.01 to 100 U/mL, with a detection limit of 4.7 mU/mL. CA 125 detection in serum is also achieved. The construction immunosensor has advantages including simple and convenient fabrication, high stability of the immobilized emitter, and high selectivity, making it suitable for CA 125 detection.

Evaluation of a multiplexed immunoassay for assessing long-term humoral immunity Orthopoxviruses

BackgroundThe 2022 Monkeypox virus (MPXV) global outbreak boosted development of multiple serological assays to aid understanding of Mpox immunology. ObjectivesThe study aimed to assess a multiplexed solid-phase electrochemiluminescence immunoassay (Meso Scale Discovery (MSD)) for simultaneous detection of antibodies against MPXV, including A35, E8 and M1 antigens, along with corresponding Vaccina Virus (VACV) homologues and demonstrate its accuracy in assessing antibody titres post-vaccination and infection. MethodsAssay performance was assessed for simultaneous detection of antibodies against MPXV and corresponding VACV antigens. Sensitivity and specificity were evaluated with paediatric negatives (n = 215), pre- and post-IMVANEX vaccinated (n = 80), and MPXV (Clade IIb, n = 39) infected serum samples. ResultsThe assay demonstrated high specificity (75.68 % (CI: 69.01–81.29) - 95.98 % (CI:92.54–97.87)) and sensitivity (62.11 % (CI:52.06–71.21) - 98.59 % (CI:92.44 %–99.93 %)) depending on the Orthopoxvirus antigen. Preferential binding was observed between MPXV-infected individuals and MPXV antigens, while vaccinated individuals exhibited increased binding to VACV antigens. These results highlight differential binding patterns between antigen homologues in related viruses. ConclusionOverall, this assay demonstrates high sensitivities in detecting antibodies for multiple relevant MPXV and VACV antigens post-infection and post-vaccination, indicating its utility in understanding immune responses to Orthopoxviruses in current and future outbreaks and evaluating the immunogenicity of new-generation Mpox-specific vaccinations.

Highly Sensitive Electrochemiluminescence Biosensing Method for SARS-CoV-2 N Protein Incorporating the Micelle Probes of Quantum Dots and Dibenzoyl Peroxide Using the Screen-Printed Carbon Electrode Modified with a Carboxyl-Functionalized Graphene.

Obtaining stable electrochemiluminescence (ECL) emissions from a hydrophobic luminophore in aqueous solutions and designing a method without the use of an exogenous coreactant are promising for ECL biosensing. Here, a highly sensitive signal-on ECL immunoassay for the SARS-CoV-2 N protein was developed using micelles as an ECL tag. The micelles were prepared by coencapsulating the luminophore hydrophobic CdSe/ZnS quantum dots and coreactant dibenzoyl peroxide within the hydrophobic core of micelles. The ECL probe was obtained by covalently bonding a SARS-CoV-2 N protein-binding aptamer onto the micelle surface. The construction of the immunosensor was initiated by the immobilization of the anti-SARS-CoV-2 N protein antibody onto the screen-printed carbon electrode (SPCE) with a -COOH-functionalized surface. The surface functionalization of SPCEs was achieved through paste-exfoliated graphene, which was modified with a -COOH group through supramolecular-covalent scaffolds on SPCE. Upon achieving sandwich complexes on the immunosensor, an efficient ECL signal response at -1.4 V versus Ag/AgCl was obtained in phosphate buffer solution. The ECL assay was used for the sensitive determination of SARS-CoV-2 N protein with the linear range from 0.01 to 50 ng mL-1, and the detection limit was 3.0 pg mL-1. The immunosensor showed good reproducibility and stability, and the ECL immunoassay was used to determine the SARS-CoV-2 N protein in serum samples. The proposed approach to obtain micelles is versatile for the preparation of stable ECL luminophores by using hydrophobic materials, and the strategy provides an alternative for ECL bioassays based on the coreactant route.

A Biomimetic Chip with Dendrimer-Encapsulated Platinum Nanoparticles for Enhanced Electrochemiluminescence Detection of Cardiac Troponin I

The measurement of cardiac troponin I (cTnI) is of vital importance for the early diagnosis of acute myocardial infarction. In this study, an enhanced electrochemiluminescent immunoassay for the highly sensitive and precise determination of cTnI was reported. A biomimetic chip with nepenthes peristome surface microstructures to achieve single-layer microbead arrays and integrated microelectrode arrays (MEAs) for ECL detection was microfabricated. Ru@SiO2 nanoparticles were prepared as signal amplificators labeling immunomagnetic beads. Dendrimer-encapsulated platinum nanoparticles (Pt DENs) were electrochemically modified on ITO MEAs. The resulting Pt DEN-modified ITO MEAs preserved good optical transparency and exhibited an approximately 20-fold ECL signal amplification compared to that obtained from bare ITO. The method made full use of the biomimetic chip with Pt DENs to develop single-layer immunomagnetic bead arrays with increasingly catalyzed electrochemical oxidation of the [Ru(bpy)3]2+–TPA system. Consequently, a limit of detection calculated as 0.38 pg/mL (S/N = 3) was obtained with excellent selectivity, demonstrating significant potential for the detection of cTnI in clinical diagnostics.

Comparison of five tacrolimus measurement methods underscore the need for a standardized method to improve clinical outcomes

Abstract Tacrolimus is a widely used drug to prevent post-transplantation organ rejection by suppressing host immune response. While its efficacy is well established, tacrolimus is also known for narrow therapeutic index and debilitating adverse effects, rendering accurate measurement for therapeutic drug monitoring essential, to minimize drug toxicity and graft loss, and maximize organ rejection prevention. Tacrolimus can be quantitated by immunoassay (IA) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). However, previous pairwise studies showed significant inter-assay variabilities between different IA methods, and between IA vs LC-MS/MS, which may be associated with the fact that IA measures parent drug and its metabolites, while LC-MS/MS measures only parent drug. No clear conclusion has been drawn about the optimal method. Transplant patients may also have their tacrolimus levels measured at different laboratories by different methods, causing inter-laboratory and intra-individual variabilities which may affect tacrolimus dose adjustment and monitoring and lead to adverse clinical outcomes. Here we performed a multi-platform comparison of tacrolimus quantification by two IA and 3 LC-MS/MS assays. A total of 216 specimens were analyzed for tacrolimus levels using chemiluminescence immunoassay (ChLIA, ARCHITECT, Abbott Diagnostic) and electrochemiluminescence immunoassay (ECLIA, Elecsys, Roche Diagnostic), and three LC-MS/MS assays (assigned names LC-MS/MS A (Agilent), Q (Waters), and W (Agilent)) in five different laboratories at four institutions. In one laboratory, the same analyses were performed on two different individual instruments (LC-MS/MS W1 and W2). Comparative results using LC-MS/MS A as reference were analyzed using Deming regression analysis, Pearson correlation, Bland Altman plots and bias calculation using GraphPad Prism version 10.1.1. While the correlations between each method with the LC-MS/MS A were good (r value: lowest 0.948 (LC-MS/MS Q), highest 0.988 (LC-MS/MS W2)), the ChLIA and ECLIA showed positive absolute bias of 2.1 and 1.2 ng/mL, respectively, and proportional bias of +26% and +17%, respectively. In comparison, the absolute bias of LC-MS/MS Q, W1 and W2 were -0.7, -0.02, and -0.1 ng/mL, respectively, with proportional bias of -11%, -0.2%, and -0.7%, respectively. The LC-MS/MS platforms showed tight agreement with one another, while IA methods were noticeably different, both from each other and from the LC-MS/MS, with a consistent positive bias, more pronounced with the ChLIA than the ECLIA. Here, our data-driven approach demonstrated that LC-MS/MS is superior to IA, and among IAs, ECLIA showed higher accuracy compared to ChLIA. However, inter-assay differences remain, emphasizing a need for tacrolimus assay standardization as well as prospective clinical trials to identify the most optimal method of tacrolimus measurement and improve patient outcomes.

Combining serum CDK1 with tumor markers for the diagnosis of small cell lung cancer.

An investigation of the diagnostic and clinical value of cell cycle-dependent kinase 1 (CDK1) in small cell lung cancer (SCLC). A large tertiary hospital in Jiangxi Province enrolled 80 SCLC cases, 105 cases of non-small cell lung cancer (NSCLC), 114 cases of pulmonary nodule (PN) and 60 control cases from December 2022 to December 2023. ELISA was used to measure CDK1 levels in serum. The expression levers of neuron-specific enolase (NSE), Pro gastrin-releasing peptide (ProGRP), squamous cell carcinoma antigen (SCCA), carcinoembryonic antigen (CEA), carbohydrate antigen 199 (CA199) and cytokeratin 19 fragment (YFRA21-1) were detected by electrochemiluminescence immunoassay. ①CDK1, ProGRP, NSE, and CA199 expressions were significantly higher in the SCLC group compared to the NSCLC, PN and Control groups (P < 0.01). ②Spearman correlation analysis showed that serum levels of CDK1, NSE, and ProGRP were associated with clinical staging and lymph node metastasis in SCLC patients (P < 0.05). ③The serum levels of CDK1, NSE, and ProGRP in patients with extensive-disease (ED) SCLC were higher than those in patients with limited-disease (LD) SCLC (P < 0.05), and the serum levels of CDK1, NSE, and ProGRP in SCLC patients with lymph node metastasis were higher than those without lymph node metastasis (P < 0.05). ④Compared with the NSCLC group, the AUC of subjects diagnosed with SCLC by CDK1 was the largest and the sensitivity was the highest, 0.831 and 72.50%, the specificity of ProGRP in diagnosing SCLC is the highest, at 95.20% (P < 0.01). Compared with the PN group, CDK1 had the highest AUC, sensitivity, and specificity in diagnosing SCLC, with values of 0.93%, 88.80%, and 94.70%, respectively (P < 0.01). ⑤The combination of CDK1, ProGRP and NSE had the highest AUC and sensitivity of 0.903 and 86.30% for the diagnosis of SCLC (P < 0.01). CDK1 not only plays an important role in assisting the diagnosis of SCLC but also in the differential diagnosis between SCLC and NSCLC. The combination of CDK1 and NSE and ProGRP can significantly improve the diagnostic performance and provide new ideas for the clinical diagnosis of SCLC.

Magnetic microbead-based upconversion immunoassay with laser-induced breakdown spectroscopy readout for the detection of prostate-specific antigen.

Laser-induced breakdown spectroscopy (LIBS) is a promising technique for the readout of immunochemical assays utilizing indirect detection of labels (Tag-LIBS), typically based on nanoparticles. We have previously demonstrated that Tag-LIBS immunoassay employing yttrium-based photon-upconversion nanoparticles (UCNPs) can reach sensitivity similar to commonly used enzyme and fluorescence immunoassays. In this study, we report on further increasing the sensitivity of UCNP-based Tag-LIBS immunoassay by employing magnetic microbeads (MBs) as the solid phase in the determinationof cancer biomarker prostate-specific antigen. Due to the possibility of analyte preconcentration, MBs enabled achieving a limit of detection (LOD) of 4.0pg·mL-1, representing two orders of magnitude improvement compared withequivalent microtiter plate-based assay (LOD of 460pg·mL-1). In addition, utilizing MBs opens up the possibility of an internal standardization of the LIBS readout by employing iron spectral lines, which improves the assay robustness by compensating for LIBS signal fluctuations and bead-bound immunocomplexes lost throughout the washing steps. Finally, the practical applicability of the technique was confirmed by the successful analysis of clinical samples, showing a strong correlation with the standard electrochemiluminescence immunoassay. Overall, MB-based Tag-LIBS was confirmed as a promising immunoassay approach, combining fast readout, multiplexing possibilities, and high sensitivity approaching upconversion luminescence scanning while avoiding the requirement of luminescence properties of labels.

6644 Glycosylation Patterns of the IgG Variable Region in Thyroglobulin Antibodies and Their Impact on Antigen Binding Affinity

Abstract Disclosure: S. Wang: None. Y. Cao: None. C. Zhao: None. C. Huang: None. K. Zhao: None. Y. Li: None. Y. Huang: None. Z. Ying: None. Y. Zhang: None. Y. Gao: None. Objective: Thyroglobulin antibodies (TgAb), key serological markers in Hashimoto's thyroiditis (HT) patients, are predominantly in immunoglobulin G (IgG) form. Prior research has highlighted the importance of increased glycosylation in TgAb IgG from HT patients. This study investigates the unique glycosylation patterns of F(ab')₂ fragments in TgAb IgG in HT patients, compared to healthy individuals. It focuses on how these patterns differ and their impact on binding to thyroglobulin (Tg). Exploring the role of N-glycosylation in the F(ab')₂ segment of TgAb IgG is crucial for understanding its effect on immune responses in HT, potentially unveiling key mechanisms of the disease. Methods: To assess serum TgAb levels in HT patients (n≥28), we used an electrochemiluminescence immunoassay, dividing patients into medium-level (mHT, n≥14) and high-level (hHT, n≥14) groups. TgAb IgG was isolated from serum of both HT and control groups (n = 14) using affinity chromatography. TgAb IgG was then cleaved with Ides enzyme to yield TgAb IgG F(ab’)2 and Fc fragments. The F(ab')2 fragment of TgAb IgG underwent MALDI-QIT-TOF-MS mass spectrometry for glycosylation profiling. Additionally, mixed TgAb IgG was separated using columns that recognize mannose and terminal sialic acid, yielding high mannose and high terminal sialic acid TgAb IgG. Different TgAb IgG glycoforms were prepared through enzymatic treatment with peptide-N-glycosidase F, β1-4 galactosidase, and α2-3,6,8 neuraminidase. The binding affinity of these TgAb IgG to Tg antigen was measured using Tg specific ELISA and surface plasmon resonance (SPR). Results: Using MALDI-TOF MS, we identified 22 IgG glycan types in both HT and control (Con) groups. In the hHT group, six glycan types (G0F, G1F, G2F, M5A1, A3G1F, A3F) were upregulated compared to Con, with statistical significance (p &amp;lt; 0.05). The mHT group showed increased levels of G1F and G2F compared to Con (p &amp;lt; 0.05). Between hHT and mHT, only A3F was significantly elevated (p &amp;lt; 0.05). Removal of N-glycosylation from IgG TgAb F(ab)2 reduced binding to Tg antigen. Presence of both mannose and terminal sialic acid in TgAb enhanced the binding to Tg. Conclusions: Most TgAb IgG in the variable region of HT patients contain N-glycans. The glycosylation patterns of TgAb IgG F(ab)2 in HT differ significantly from healthy controls, affecting antibody binding affinity. Our findings reveal the complex role of glycosylation patterns in TgAb IgG, providing new insights into HT pathogenesis and potential research directions. Presentation: 6/1/2024

9303 Evaluation of The Consistency of Thyroid Indicators Tests in Minimally Invasive Peripheral Blood and Venous Blood

Abstract Disclosure: Z. Zhu: None. Y. Huang: None. Z. Cao: None. L. Zhang: None. D. Wang: None. S. Wang: None. J. Zhang: None. Y. Gao: None. Y. Zhang: None. Background The detection of thyroid indicators is of high demand in clinical and epidemiological screenings. Compared with existing venous blood tests, peripheral fingertip blood tests are less invasive and have a lower blood collection volume, which is the basis for point-of-care tests of thyroid indicators. Objective To explore the feasibility of minimally invasive and micro blood collection tests for thyroid disease (TD) patients by evaluating the consistency of thyroid indicators and TD diagnostic efficiency between fingertip blood tests and venous blood tests. Method 63 subjects with or without thyroid dysfunction were consecutively recruited. All subjects underwent serum, plasma and fingertip blood (same day collection) measurements for TSH and TT4 detection via the chemiluminescence method and for TPOAb detection via the electrochemiluminescence immunoassay method. The fingertip blood was centrifuged and diluted after EDTA anticoagulation. Bland–Altman plot, Passing–Bablok regression, and weighted kappa test were used for consistency test; AUC of ROC curve, sensitivity and specificity were used for authenticity evaluation among the three collection and testing methods. Results 43 subjects with euthyroidism and 20 subjects with thyroid dysfunction were included; including 29 (46.03%) females, the average age was 50.98±14.76 years. Bland-Altman plots showed more than 90% of the subjects had TSH and TT4 differences within the 95% limits of agreement between peripheral blood (PB) and venous blood (including serum and plasma). Passing–Bablok regression analyses revealed strong correlations between TSH levels in PB and serum (r=0.888) and plasma (r=0.894) (p&amp;lt;0.05); for TT4, there was a strong correlation between PB and serum (r=0.746), and there was also a moderate correlation between PB and plasma (r=0.664) (p&amp;lt;0.05 for both). Weighted kappa tests were performed for low, normal and high TSH values, indicating strong consistency between PB and serum and between PB and plasma, with coefficients of 0.901 and 0.881, respectively (P&amp;lt;0.001); for TT4, the coefficients between PB and serum, PB and plasma were 0.541 and 0.585 (P&amp;lt;0.001), indicating moderate consistency. Regardless of TPOAb positivity or negativity, the consistency among three blood sample sources was 100%. For euthyroidism (n=43), hyperthyroidism (n=6), subclinical hyperthyroidism (n=9), hypothyroidism (n=1), and subclinical hypothyroidism (n=4), the diagnostic sensitivities of PB were 97.06%, 83.33%, 87.50%, 100%, and 75.00%, respectively; the specificities were 94.74%, 97.87%, 95.74%, 98.11%, and 100.00%, respectively. The AUC of PB ROC curve for determining thyroid dysfunction was 0.944 (95% CI 0.867-1.000). Conclusion PB tests of thyroid indicators has high consistency with venous blood tests, offers sufficient diagnostic efficiency for TD, and is a feasible minimally invasive detection method. Presentation: 6/1/2024

7583 Evaluation of The Consistency of Thyroid Indicators Tests in Minimally Invasive Peripheral Blood and Venous Blood

Abstract Disclosure: Z. Zhu: None. Y. Huang: None. Z. Cao: None. L. Zhang: None. D. Wang: None. S. Wang: None. J. Zhang: None. Y. Gao: None. Y. Zhang: None. Background: The detection of thyroid indicators is of high demand in clinical and epidemiological screenings. Compared with existing venous blood tests, peripheral fingertip blood tests are less invasive and have a lower blood collection volume, which is the basis for point-of-care tests of thyroid indicators. Objective To explore the feasibility of minimally invasive and micro blood collection tests for thyroid disease (TD) patients by evaluating the consistency of thyroid indicators and TD diagnostic efficiency between fingertip blood tests and venous blood tests. Method 63 subjects with or without thyroid dysfunction were consecutively recruited. All subjects underwent serum, plasma and fingertip blood (same day collection) measurements for TSH and TT4 detection via the chemiluminescence method and for TPOAb detection via the electrochemiluminescence immunoassay method. The fingertip blood was centrifuged and diluted after EDTA anticoagulation. Bland–Altman plot, Passing–Bablok regression, and weighted kappa test were used for consistency test; AUC of ROC curve, sensitivity and specificity were used for authenticity evaluation among the three collection and testing methods. Results 43 subjects with euthyroidism and 20 subjects with thyroid dysfunction were included; including 29 (46.03%) females, the average age was 50.98±14.76 years. Bland-Altman plots showed more than 90% of the subjects had TSH and TT4 differences within the 95% limits of agreement between peripheral blood (PB) and venous blood (including serum and plasma). Passing–Bablok regression analyses revealed strong correlations between TSH levels in PB and serum (r=0.888) and plasma (r=0.894) (p&amp;lt;0.05); for TT4, there was a strong correlation between PB and serum (r=0.746), and there was also a moderate correlation between PB and plasma (r=0.664) (p&amp;lt;0.05 for both). Weighted kappa tests were performed for low, normal and high TSH values, indicating strong consistency between PB and serum and between PB and plasma, with coefficients of 0.901 and 0.881, respectively (P&amp;lt;0.001); for TT4, the coefficients between PB and serum, PB and plasma were 0.541 and 0.585 (P&amp;lt;0.001), indicating moderate consistency. Regardless of TPOAb positivity or negativity, the consistency among three blood sample sources was 100%. For euthyroidism (n=43), hyperthyroidism (n=6), subclinical hyperthyroidism (n=9), hypothyroidism (n=1), and subclinical hypothyroidism (n=4), the diagnostic sensitivities of PB were 97.06%, 83.33%, 87.50%, 100%, and 75.00%, respectively; the specificities were 94.74%, 97.87%, 95.74%, 98.11%, and 100.00%, respectively. The AUC of PB ROC curve for determining thyroid dysfunction was 0.944 (95% CI 0.867-1.000). Conclusion PB tests of thyroid indicators has high consistency with venous blood tests, offers sufficient diagnostic efficiency for TD, and is a feasible minimally invasive detection method. Presentation: 6/1/2024

Toward Analytical Performance Specifications for Immunosuppressive Drug Quantification in Transplantation: An Opinion Article.

Analytical methods require performance that meets the clinical needs. Different approaches for setting up permissible analytical imprecision goals (pCVA%) for drug analyses have been reported. The aim of this study was to calculate the pCVA% for cyclosporine, tacrolimus, everolimus, sirolimus, and mycophenolic acid using 4 alternative approaches, to compare the results and to critically discuss advantages and disadvantages of each model. The approaches to evaluate pCVA% were (A) based on biological variation observed in routine measurement results between 2022 and 2023 in the authors' laboratory, (B) derived from the terminal elimination half-life and dosing interval of the drugs, and (C and D) explored from the width of the therapeutic ranges (TR) by the 2 methods. For approach A, routine measurement data for cyclosporine and tacrolimus, obtained through liquid chromatography-tandem mass spectrometry and electrochemiluminescence immunoassays, were evaluated separately. The 4 alternative approaches for deriving pCVA% yielded similar results, for cyclosporine and tacrolimus in an analytical method dependent manner. The average pCVA% was 5.2%, 5.6%, 5.1%, 4.8%, and 7.7% for cyclosporine, tacrolimus, everolimus, sirolimus, and mycophenolic acid, respectively. The most challenging goals were those using TR-related approaches, while those using the biological variation approach were most easily achievable. Approach B resulted in more stringent goals for drugs with longer elimination half-lives (eg, everolimus and sirolimus). There is no single ideal approach for setting goals of drug analysis. However, the pCVA% values derived from the various approaches are similar and confirm that a <6% target proposed by the International Association of Therapeutic Drug Monitoring and Clinical Toxicology is adequate and realistic in combination with state-of-the-art measurement technologies. In the authors' opinion, approaches based on the width of the TR are preferable, as they represent a common basis for clinical decisions and reflect elements of biological variation and analytics used to establish the TR.

B-266 Therapeutic Drug Monitoring of Natalizumab and Anti-Natalizumab Antibodies

Abstract Background Natalizumab (NTZ) is a monoclonal antibody directed against the α4-integrin subunit of α4β1 and α4β7 integrins. NTZ is used to treat relapsing and remitting forms of multiple sclerosis. Although a majority of patients on standard dosing (intravenous 300 mg, every 4 weeks) reach therapeutic drug concentrations, about 10% of patients will eventually fail NTZ therapy due to the development of anti-NTZ antibodies. NTZ and anti-NTZ antibody concentrations in serum are used together to provide patient-specific pharmacokinetic and immunogenic assessment to aid in patient management. Methods Serum measurements of NTZ and anti-NTZ antibody levels are performed by lab developed electro-chemiluminescent immunoassays where assay design and principle are equivalent to biologic therapeutic drug monitoring described in our previous publication. The natalizumab drug assay is an immunoassay validated to measure free drug (pharmacodynamically active, ADA-unbound) in serum. The anti-natalizumab antibody assay utilizes a solution phase bridging method designed to measure total binding antibodies (including IgM &amp; IgG subtypes). All anti-natalizumab antibody positive samples are confirmed for drug-specificity by an additional signal suppression step. The anti-natalizumab antibody assay is quantitative and drug-tolerant, meaning that circulating drug in patient serum does not interfere with anti-NTZ detection and quantitation. Results Here, we report concomitant measurement of NTZ and anti-NTZ antibody in 160 patient serum samples. Positive anti-NTZ levels from 24 to 235 ng/mL were found in 7.5% (12/160) patients. Anti-NTZ-free samples (148/160) had NTZ drug levels ranging from undetectable (&amp;lt;1.0) to 85 ug/mL. The mean and median drug level in 90 samples (results &amp;gt;1.0) was 10.8 and 2.7, respectively. 61/90 samples had therapeutic drug concentrations (at least 2.0 ug/mL). In the presence of anti-NTZ, 67% (8/12) had undetectable drug level and (4/12) had mean and median drug levels of 1.2 and 1.3, respectively, which is 9- or 2-fold lower than the mean and median drug levels of the anti-NTZ Ab -free group. Conclusions Low serum NTZ and high anti-NTZ are associated with loss of clinical efficacy, while elevated NTZ may increase progressive multifocal leukoencephalopathy risk. Assays to measure biologic drugs and their anti-drug antibodies in patient serum may be useful tools in dose optimization and patient management.

Highly sensitive detection platform-based diagnosis of oesophageal squamous cell carcinoma in China: a multicentre, case–control, diagnostic study

Early detection and screening of oesophageal squamous cell carcinoma rely on upper gastrointestinal endoscopy, which is not feasible for population-wide implementation. Tumour marker-based blood tests offer a potential alternative. However, the sensitivity of current clinical protein detection technologies is inadequate for identifying low-abundance circulating tumour biomarkers, leading to poor discrimination between individuals with and without cancer. We aimed to develop a highly sensitive blood test tool to improve detection of oesophageal squamous cell carcinoma. We designed a detection platform named SENSORS and validated its effectiveness by comparing its performance in detecting the selected serological biomarkers MMP13 and SCC against ELISA and electrochemiluminescence immunoassay (ECLIA). We then developed a SENSORS-based oesophageal squamous cell carcinoma adjunct diagnostic system (with potential applications in screening and triage under clinical supervision) to classify individuals with oesophageal squamous cell carcinoma and healthy controls in a retrospective study including participants (cohort I) from Sun Yat-sen University Cancer Center (SYSUCC; Guangzhou, China), Henan Cancer Hospital (HNCH; Zhengzhou, China), and Cancer Hospital of Shantou University Medical College (CHSUMC; Shantou, China). The inclusion criteria were age 18 years or older, pathologically confirmed primary oesophageal squamous cell carcinoma, and no cancer treatments before serum sample collection. Participants without oesophageal-related diseases were recruited from the health examination department as the control group. The SENSORS-based diagnostic system is based on a multivariable logistic regression model that uses the detection values of SENSORS as the input and outputs a risk score for the predicted likelihood of oesophageal squamous cell carcinoma. We further evaluated the clinical utility of the system in an independent prospective multicentre study with different participants selected from the same three institutions. Patients with newly diagnosed oesophageal-related diseases without previous cancer treatment were enrolled. The inclusion criteria for healthy controls were no obvious abnormalities in routine blood and tumour marker tests, no oesophageal-associated diseases, and no history of cancer. Finally, we assessed whether classification could be improved by integrating machine-learning algorithms with the system, which combined baseline clinical characteristics, epidemiological risk factors, and serological tumour marker concentrations. Retrospective SYSUCC cohort I (randomly assigned [7:3] to a training set and an internal validation set) and three prospective validation sets (SYSUCC cohort II [internal validation], HNCH cohort II [external validation], and CHSUMC cohort II [external validation]) were used in this step. Six machine-learning algorithms were compared (the least absolute shrinkage and selector operator regression, ridge regression, random forest, logistic regression, support vector machine, and neural network), and the best-performing algorithm was chosen as the final prediction model. Performance of SENSORS and the SENSORS-based diagnostic system was primarily assessed using accuracy, sensitivity, specificity, and area under the receiver operating characteristic curve (AUC). Between Oct 1, 2017, and April 30, 2020, 1051 participants were included in the retrospective study. In the prospective diagnostic study, 924 participants were included from April 2, 2022, to Feb 2, 2023. Compared with ELISA (108·90 pg/mL) and ECLIA (41·79 pg/mL), SENSORS (243·03 fg/mL) showed 448 times and 172 times improvements, respectively. In the three retrospective validation sets, the SENSORS-based diagnostic system achieved AUCs of 0·95 (95% CI 0·90-0·99) in the SYSUCC internal validation set, 0·93 (0·89-0·97) in the HNCH external validation set, and 0·98 (0·97-1·00) in the CHSUMC external validation set, sensitivities of 87·1% (79·3-92·3), 98·6% (94·4-99·8), and 93·5% (88·1-96·7), and specificities of 88·9% (75·2-95·8), 74·6% (61·3-84·6), and 92·1% (81·7-97·0), respectively, successfully distinguishing between patients with oesophageal squamous cell carcinoma and healthy controls. Additionally, in three prospective validation cohorts, it yielded sensitivities of 90·9% (95% CI 86·1-94·2) for SYSUCC, 84·8% (76·1-90·8) for HNCH, and 95·2% (85·6-98·7) for CHSUMC. Of the six machine-learning algorithms compared, the random forest model showed the best performance. A feature selection step identified five features to have the highest performance to predictions (SCC, age, MMP13, CEA, and NSE) and a simplified random forest model using these five features further improved classification, achieving sensitivities of 98·2% (95% CI 93·2-99·7) in the internal validation set from retrospective SYSUCC cohort I, 94·1% (89·9-96·7) in SYSUCC prospective cohort II, 88·6% (80·5-93·7) in HNCH prospective cohort II, and 98·4% (90·2-99·9) in CHSUMC prospective cohort II. The SENSORS system facilitates highly sensitive detection of oesophageal squamous cell carcinoma tumour biomarkers, overcoming the limitations of detecting low-abundance circulating proteins, and could substantially improve oesophageal squamous cell carcinoma diagnostics. This method could act as a minimally invasive screening tool, potentially reducing the need for unnecessary endoscopies. The National Key R&D Program of China, the National Natural Science Foundation of China, and the Enterprises Joint Fund-Key Program of Guangdong Province. For the Chinese translation of the abstract see Supplementary Materials section.

Diagnostic value of combined detection of serum neuron-specific enolase and homocysteine in patients with coronary atherosclerosis.

The aim of this paper was to investigate the diagnostic significance and severity assessment of serum neuron-specific enolase (NSE) combined with homocysteine (Hcy) for patients with coronary atherosclerosis (coronary artery disease, CAD). Two hundred sixty-three patients with coronary artery disease were selected as the research group, and 400 healthy individuals who underwent physical examination during the same period were taken as the control group. Electrochemiluminescence immunoassay and biochemical analyzer were employed to detect the serum NSE and Hcy levels of all subjects. The diagnostic value of combined and individual serum NSE and Hcy detection for the combined group was analyzed using the ROC curve. The serum NSE (19.91±9.98 vs. 11.17±2.35) and Hcy levels (15.76±5.37 vs. 10.17±3.71) in the research group were significantly higher than those in the control group, with a statistically significant difference (P<0.05). The serum NSE (16.67±4.02 vs. 18.63±5.49 vs. 20.29±5.87) and Hcy levels (13.28±2.49 vs. 15.56±2.67 vs. 16.66±3.94) gradually increased across groups A, B, and C, and inter-group comparisons showed statistically significant differences (P<0.05). The AUC value of combined serum NSE and Hcy detection for CAD patients was higher (0.879 vs. 0.724 vs. 0.827) than individual NSE and Hcy testing. The specificity of Hcy for the diagnosis of CAD was the highest, reaching 90.3%. The sensitivity of combined NSE and Hcy (82.9%) was higher than the individual testing sensitivity of the two groups. The combined detection of serum NSE and Hcy has high diagnostic efficacy for CAD and provides reference value in assessing the severity of the disease.

Interleukin 1β And Interleukin-6 and Some Oxidants in Patients With Stable Angina

ROS play an essential physiological roles in the cellular homoeostasis and cell signaling under normal circumstances. The overexpression of ROS was involved in the pathological events of several diseases, including cardiovascular, because they cause damage to many cell structures.This study aims to evaluate the levels of inflammatory biomarkers, [interleukin-6 (IL-6) and interleukin-1beta (IL-1β)] in stable angina patients and their relationship with oxidative stress. The current study is a case-control one performed on 88 patients with stable angina and 44 people as a control group attending the Nasiriyah Heart Center from August 2023 to March 2024. All cases were diagnosed by cardiologists and confirmed by laboratory investigations. Blood samples (10 ml) were collected from patients. Serum was isolated after centrifugation and stored at -20°C. Serum CK-MB, MYO and Trop I were determined by electro-chemiluminescence immunoassay. While, the ELISA was employed to assess the levels of human IL-6, human IL-1 β, SOD, (GSH-Px/ GPX), total cholesterol, triglycerides, LDL, VLDL and HDL, using the kits from Solarbio, China. The patients with stable angina didn’t show significant variation in the serum level of troponin I and CK-MB in comparison with control, while serum myoglobin level was significantly increased in patients with stable angina compared to control. Although the level of total cholesterol was insignificantly increased, but the levels of triglyceride (TG), VLDL-C and LDL-C were significantly increased, while the serum level of HDL-C was significantly declined in comparison with control. The serum levels IL-6 and IL-1β, in addition to activities of SOD and GPx were increased significantly in patients of angina compared to control. We could conclude that IL-1β, IL-6, SOD and GPx are increased in stable angina. Inflammatory events and oxidative stress could play an important roles in initiation of atherosclerosis and subsequent angina.