Intra-assay Coefficient Of Variation Research Articles

Development of a time-resolved fluoroimmunoassay for rituximab and its application to therapeutic drug monitoring.

Rituximab pharmacokinetics in patients with membranous nephropathy (MN) exhibit significant interindividual variability. Accurate measurement of serum rituximab concentrations is essential for effective therapeutic monitoring. This study develops a highly sensitive time-resolved fluoroimmunoassay (TRFIA) for rituximab (rituximab-TRFIA) with a wide detection range, aimed at enhancing therapeutic drug monitoring in MN treatment. A capture -type rituximab-TRFIA was developed using streptavidin-coated microplates, biotinylated anti-rituximab idiotypic antibodies, and Eu3+-labeled mouse anti-human IgG targeting the Fc fragment of rituximab. The assay was used to measure rituximab serum concentrations in MN patients treated with rituximab. The linear range of rituximab-TRFIA is 0.50 to 2500ng/mL, with a limit of detection (LOD) of 0.062ng/mL. The intra-assay coefficient of variation (CV) ranges from 1.97% to 9.50%, while the inter-assay CV ranges from 7.44% to 9.99%. The recovery rate ranges from 99.14% to 107.75%. No cross-reactivity was observed with other monoclonal antibody drugs (mAbs). The detection range is two orders of magnitude higher, and the sensitivity is six times greater than that of the enzyme-linked immunosorbent assay (ELISA). Rituximab-TRFIA showed strong consistency within the same measurement range compared to ELISA (P < 0.0001). The serum rituximab levels in MN patients were significantly higher than those in the control group (P < 0.0001). Among patients who did not achieve remission, 60% to 70% had insufficient drug concentrations, and rituximab pharmacokinetics followed the expected trends. The rituximab-TRFIA provides high sensitivity, a wide detection range, and reliable performance for monitoring serum rituximab concentrations in MN patients, supporting personalized treatment strategies.

Quantification and clinical performance of serum parathyroid hormone 1-84 via immunocapture coupled to LC-MS/MS in chronic renal failure.

Accurate measurement of serum parathyroid hormone (PTH) is crucial for diagnosing and managing endocrine and osteological diseases. Conventional immunoassay methods struggle with cross reactivity issues between full-length PTH and truncated fragments or post-translationally modified forms. Both the standardization of PTH assays and the peptide's stability are concerning. This study addresses these issues by establishing an immunocapture coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to precisely quantify PTH1-84. PTH1-84 was isolated from one mL serum samples by immunocapture on a polystyrene bead and eluted from matrix, then quantitated by LC-MS/MS. The results from 268 serum samples were compared to an intact PTH immunoassay. The assay's linear range was 5.0-1000.0 pg/mL. The intra-assay coefficients of variation (CVs) ranged from 3.2 % to 6.8 %, and the inter-assay CVs ranged from 4.6 % to 9.5 %. The extraction efficiencies were 98.0 %-100.5 %, with no significant matrix effects observed after internal standard correction. The correlation coefficient between LC-MS/MS and immunoassay was 0.989, but the bias between the methods was substantial. Nevertheless, the immunocapture purification coupled LC-MS/MS method offers a promising approach for accurate PTH measurement.

Development and application of a quadruple RT-qPCR assay for the simultaneous detection of NoV GI, NoV GII, and HAV in bivalve shellfish.

To achieve rapid and simultaneous detection of NoV GI, NoV GII, and HAV, a quadruple real-time fluorescence quantitative PCR (RT-qPCR) assay was developed using MS2 bacteriophage as a process control virus. The quadruple RT-qPCR assay effectively detected NoV GI, NoV GII, HAV, and MS2 RNA with detection limits of 102 copies/μL, 103 copies/μL, 102 copies/μL, and 103 copies/μL, respectively, within 1 hour 50 minutes. The quadruple RT-qPCR assay could specifically detect NoV GI, NoV GII, HAV, and MS2 without cross-reactions with other common pathogens, demonstrating good reproducibility with intra-assay and inter-assay coefficients of variation all below 2.11%. In this study, 337 bivalve shellfish samples collected from various regions of Hebei Province were pretreated using the proteinase K-PEG 8000 precipitation-chloroform method, and viral nucleic acids were enriched and extracted from a volume of viral solution that was doubled. The developed quadruple RT-qPCR assay was used to detect NoV GI, NoV GII, and HAV in bivalve shellfish samples, and the positive rates were 19.88% (67/337), 20.47% (69/337), and 4.75% (16/337), respectively. In addition, mixed infections of NoV GI and NoV GII (10.68%, 36/337) and NoV GI and HAV (0.89%, 3/337) were observed. In all, 200 bivalve shellfish samples were randomly selected for the assay, and it was found that the total, positive, negative coincidence rates, and Kappa values of the quadruple RT-qPCR assay were 98.3%, 99.1%, 98.2%, and 0.945, respectively, compared with the single RT-qPCR assay. These results show that the developed quadruple RT-qPCR assay has comparable performance to the single RT-qPCR assay.IMPORTANCEFood-borne diseases caused by viral contamination have become a growing concern, and bivalve shellfish is a crucial source of infection, with many outbreaks of non-bacterial acute gastroenteritis associated with raw food or the use of undercooked shellfish such as oysters. As food contamination problems caused by NoV and HAV become more severe, it is important to study and establish a sensitive and efficient assay to simultaneously detect NoV and HAV by applying the MS2 process control virus for the protection of bivalve shellfish food safety and the monitoring of the above food-borne viral contamination. In addition, bivalve shellfish samples contain a large number of PCR inhibitors such as polysaccharides, lipids, and proteins, so optimization of the virus enrichment and extraction method is essential and is expected to provide a research basis for subsequent related experiments.

A Quadruplex RT-qPCR for the Detection of African Swine Fever Virus, Classical Swine Fever Virus, Porcine Reproductive and Respiratory Syndrome Virus, and Porcine Pseudorabies Virus.

African swine fever virus (ASFV), classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), and porcine pseudorabies virus (PRV) induce similar clinical signs in infected pigs, including hyperthermia, anorexia, hemorrhage, respiratory distress, neurological symptoms, and/or abortions in pregnant sows. The differential diagnosis of these diseases relies on laboratory examinations. In this study, a quadruplex RT-qPCR was established using four pairs of specific primers and probes aimed at the B646L (p72) gene of ASFV, the 5' untranslated region (5'UTR) of CSFV, the ORF6 gene of PRRSV, and the gB gene of PRV for the detection and differentiation of ASFV, CSFV, PRRSV, and PRV. The assay exhibited great sensitivity with limits of detection (LODs) of 134.585, 139.831, 147.076, and 142.331 copies/reaction for ASFV, CSFV, PRRSV, and PRV, respectively. The assay exclusively identified ASFV, CSFV, PRRSV, and PRV, yielding negative results for the other control swine viruses used in this study. The intra-assay and inter-assay coefficients of variation (CVs) were not higher than 1.12%, indicating good reproducibility of the assay. The quadruplex RT-qPCR assay was used to analyze 3116 clinical tissue samples from pigs in Guangxi province, China, from April 2023 to September 2024. ASFV, CSFV, PRRSV, and PRV had positivity rates of 10.84% (338/3116), 0.80% (25/3116), 14.92% (465/3116), and 1.38% (43/3116), respectively, demonstrating a coincidence rate of ≥99.45% with the previously described RT-qPCR assays, which were also used to test these same samples. The established assay was rapid, sensitive, and accurate in detecting and differentiating ASFV, CSFV, PRRSV, and PRV.

Carbohydrate-binding module could integrate with ELISA and serve the simple and specific quantification of hyaluronic acid

Quantification is essential in the research and development of polysaccharides. However, achieving simplicity and specificity in polysaccharide quantification remains a challenging task. Enzyme-linked immunosorbent assay (ELISA) based on antibodies provides a straightforward and specific quantification strategy. Nevertheless, acquiring antibodies for polysaccharides is complicated. Carbohydrate-binding modules (CBMs), which can be efficiently obtained, exhibit the capability to specifically recognize and bind carbohydrates. In this study, we verified the feasibility of a CBM-based ELISA for the quantification of hyaluronic acid (HA) by replacing an antibody with a CBM. The CBM-based ELISA, which employed a HA-specific CBM, exhibited a linear detection range spanning from 10 to 100 μg/mL. Both intra-assay and inter-assay coefficients of variation remained below 15 % and recoveries ranged from 96.26 % to 98.22 %, indicating favorable precision and accuracy. The method exhibited specificity exclusively to HA, suggesting its reliance on CBM binding specificity. The effectiveness of the method in analyzing commercial products was confirmed. Additionally, a comparison with the carbazole assay revealed a highly significant correlation (r = 0.994). By integrating CBMs with ELISA, the study presented a novel and easily implementable solution for simple and specific quantification of HA.

Development and application of a quadruplex TaqMan real-time fluorescence quantitative PCR assay for four porcine digestive pathogens.

Salmonella, Escherichia coli, Lawsonella intracellularis, and Brachyspira hyodysenteriae are the primary pathogens responsible for gastrointestinal diseases in pigs, posing a significant threat to the health and productivity of pig production systems. Pathogen detection is a crucial tool for monitoring and managing these infections. We designed primers and probes targeting the invA gene of Salmonella, the 23S rRNA gene of Escherichia coli, the aspA gene of Lawsonella intracellularis, and the nox gene of Brachyspira hyodysenteriae. We developed a quadruplex TaqMan real-time quantitative PCR assay capable of simultaneously detecting these four pathogens. This assay demonstrated high sensitivity, with detection limits of 100 copies/μL for the recombinant plasmid standards pEASY-23S rRNA, pEASY-aspA, and pEASY-nox, and 10 copies/μL for pEASY-invA. The standard curves exhibited excellent linearity (R2 values of 0.999, 0.999, 1, and 0.998, respectively) and high amplification efficiencies (93.57%, 94.84%, 85.15%, and 81.81%, respectively). The assay showed high specificity, with no cross-reactivity detected against nucleic acids from Streptococcus suis, porcine epidemic diarrhoea virus (PEDV), porcine transmissible gastroenteritis virus (TGEV), Pasteurella multocida, Clostridium perfringens, Gracilaria parapsilosis, porcine delta coronavirus (PDCoV), porcine group A rotavirus (GARV), and porcine teschovirus (PTV). The assay also exhibited excellent repeatability, with inter- and intra-assay coefficient of variation (CV) ranging from 0.15% to 1.12%. High concentrations of nucleic acids did not interfere with the detection of low concentrations, ensuring robust performance in complex samples. Among 263 diarrhoeic samples, the assay detected Salmonella in 23.95%, Escherichia coli in 26.24%, Lawsonella intracellularis in 33.84%, and Brachyspira hyodysenteriae in 22.43%. This quadruplex TaqMan qPCR assay offers a rapid, sensitive, and specific tool for the simultaneous detection of Salmonella, Escherichia coli, Lawsonella intracellularis, and Brachyspira hyodysenteriae in pigs.

One-Step Multiplex Real-Time Fluorescent Quantitative Reverse Transcription PCR for Simultaneous Detection of Four Waterfowl Viruses.

Duck Tembusu virus (DTMUV), duck hepatitis virus (DHV), Muscovy duck reovirus (MDRV), and Muscovy duck parvovirus (MDPV) represent four emergent infectious diseases impacting waterfowl, which can be challenging to differentiate due to overlapping clinical signs. In response to this, we have developed a one-step multiplex real-time fluorescence quantitative reverse transcription PCR (qRT-PCR) assay, capable of simultaneously detecting DTMUV, DHV, MDRV, and MDPV. This method exhibits high specificity, avoiding cross-reactivity with other viruses such as Fowl adenoviruses (FADV), infectious bursal disease virus (IBDV), infectious bronchitis virus (IBV), infectious laryngotracheitis virus (ILTV), Haemophilus paragallinarum (Hpg), duck circovirus (DUCV), goose astrovirus (GoAstV), and mycoplasma gallisepticum (MG). The limit of detection (LOD) established for DTMUV, DHV, MDRV, and MDPV was determined to be 27 copies/μL. In the repeatability test, the intra-assay and inter-assay coefficients of variation (CVs) of the recombinant plasmid standard were less than 2%. Utilizing this method, we analyzed 326 clinical specimens sourced from Guangxi over the period spanning October 2021 through December 2023, yielding promising and precise outcomes. The qRT-PCR method established herein exhibits commendable specificity, sensitivity, and repeatability. Furthermore, it boasts a high clinical detection rate, making it a highly effective tool for diagnosing these pathogenic agents in waterfowl.

Comparative study of immunoassays, a microelectromechanical systems-based biosensor, and RT-QuIC for the diagnosis of chronic wasting disease in white-tailed deer

BackgroundChronic wasting disease (CWD) is a fatal transmissible spongiform encephalopathy in cervids. The disease is caused by a pathogenic prion, namely PrPSc. Currently, diagnosis of CWD relies on IHC detection of PrPSc in the obex or retropharyngeal lymph nodes (RPLN) or ELISA screening of obex and RPLN followed by IHC confirmation of positive results. In this study, we assessed the performance characteristics of two immunoassays: CWD Ag-ELISA and TeSeE ELISA, RT-QuIC, and MEMS biosensor via testing 30 CWD + and 30 CWD- white-tailed deer RPLN samples.ResultsBoth CWD Ag-ELISA and TeSeE ELISA correctly identified all CWD + and CWD- samples. A greater intra-assay coefficient of variation (CV) in S/P ratios was observed for the TeSeE ELISA (16.52%), compared to CWD Ag-ELISA (9.49%). However, the high CV did not affect the qualitative results of triplicate assays when the corresponding manufacturer’s cutoff was used. The MEMS biosensor not only correctly identified all CWD + and CWD- RPLN samples, but also demonstrated a 100% detection rate for all CWD + samples at dilutions from 10− 0 to 10− 3. Evaluation of RT-QuIC indicated that the rate of false negative reactions decreased from 21.98% at 10− 2 dilution to 0% at 10− 4 and 10− 5 dilutions; and the rate of false positive reactions reduced from 56.42% at 10− 2 dilution to 8.89% and 2.22% at 10− 4 and 10− 5 dilutions, respectively. Based on a stringent threshold of 2 x the first 10 fluorescent readings of each well and a final cutoff of 2/3 positive reactions for each sample, RT-QuIC correctly identified all positive and negative samples at 10− 4 and 10− 5 dilutions. Both MEMS biosensor and RT-QuIC achieved 100% sensitivity and 100% specificity under the experimental conditions described in this study.ConclusionsThe two immunoassays (CWD Ag-ELISA and TeSeE ELISA) performed comparably on white-tailed deer RPLN samples. MEMS biosensor is a reliable portable tool for CWD diagnosis and RT-QuIC can be used for routine testing of CWD if appropriate testing parameters and interpretive criteria are applied.

Development and application of a quadruplex real-time PCR method for Torque teno sus virus 1, Porcine circovirus type 2, pseudorabies virus, and porcine parvovirus.

In clinical diagnosis of porcine diseases, co-infection with multiple viruses often leads to similar clinical symptoms. Postweaning multisystemic wasting syndrome (PMWS) can be caused by infections with TTSuV or PCV2, while PCV2, PRV, and PPV can cause respiratory and reproductive disorders in pigs. The overlapping clinical and pathological features of these infections necessitate the development of a rapid and specific method for differentiating and detecting these four DNA viruses. In this study, four pairs of primers and TaqMan probes were designed targeting the conserved sequence of TTSuV, the Rep gene of PCV2, the gE gene of PRV, and the VP2 gene of PPV. After optimizing reaction conditions, including annealing temperature, primer concentration, and probe concentration, a quadruplex real-time PCR method was developed. This method can specifically detect TTSuV1, PCV2, PRV, and PPV simultaneously, with no cross-reactivity with ASFV, CSFV, PRRSV, PEDV, PSV, and TGEV. The minimum detection limit for each virus was 10 copies/μl, and the inter-assay and intra-assay coefficients of variation ranged from 0.33% to 1.43%. Subsequently, 150 clinical samples were tested to evaluate the practical applicability of this method. The positive rates for TTSuV1, PCV2, PRV, and PPV were 8.6% (13/150), 10.67% (16/150), 14% (21/150), and 11.33% (17/150), respectively. The results indicate that the established quadruplex real-time PCR method can assist in the accurate and rapid diagnosis of TTSuV1, PCV2, PRV, and PPV in clinical settings, providing robust support for the prevention and control of these infections.

Development of a chemiluminescent immunoassay based on magnetic solid phase for quantification of homocysteine in human serum

BackgroundHomocysteine (HCY) is a sulfur-containing amino acid that is an independent or important risk factor for the occurrence of many chronic diseases and is one of the most important indicators for determining health risks. However, existing HCY detection methods do not meet the requirements of clinical diagnosis. Therefore, there is an urgent need to establish new detection methods to meet the needs of clinical detection.ResultsIn this study, we used the principle of competitive method to establish a new method for the determination of HCY in human serum using a chemiluminescent enzyme immunoassay in conjunction with a chemiluminescent assay instrument that uses magnetic microparticles as the solid phase of the immunoreaction. The established method achieved satisfactory results in terms of minimum detection limit, specificity, accuracy, and clinical application. The limit of detection was 0.03 ng/mL. The intra-assay coefficient of variation (CV) was 1.94–5.05%, the inter-assay CV was 2.29–6.88%, and the recovery rate was 88.60–93.27%. Cross-reactivity with L-cysteine ranged from 0.0100 to 0.0200 μmol/L, and cross-reactivity with glutathione ranged from 0.0100 to 0.200 μmol/L, all of which were less than the limit of detection (LoD) of this method. The linear factor R of this method was greater than 0.99.ConclusionsIn summary, the developed method showed a good correlation with the product from Abbott. A total of 996 clinical patients with cardiovascular diseases were evaluated using the method developed in this study.

A-051 Validation and Implementation of the Stanbio Beta-Hydroxybutyrate Assay on Roche c502 Analyzer

Abstract Background β-hydroxybutyrate (βOHB) serves as a crucial biomarker in assessing, managing, and monitoring ketosis in various clinical scenarios, such as diabetic ketoacidosis and instances of alcohol abuse combined with starvation. This study aimed to validate a βOHB assay (Stanbio Laboratory) conducted on a Roche Cobas c502 analyzer and establish performance specifications. Additionally, we conducted a method comparison for three assays: Randox D-3-Hydroxybutyrate (Ranbut) assay on Roche c501 analyzer, Abbott Freestyle Precision B-Ketone strip point-of-care test, and the Stanbio Laboratory β-Hydroxybutyrate LiquiColor assay on Roche c502 analyzer. Methods Precision, linearity, limit of quantification (LOQ), method comparison, and sample stability were determined according to Clinical and Laboratory Standards Institute (CLSI) guidelines, using commercial quality control materials (Randox Multisera) and residual patient samples (Lithium heparin anti-coagulated plasma). The reference interval was verified by residual samples from 20 healthy individuals (normal blood glucose, HbA1c, liver enzymes, and lipid panel results). Statistical analysis was performed by EP-Evaluator (v12). Results Intra-assay and total coefficient of variation (CV) were determined as 0% and 2.4% at 0.3 and 1.21 mmol/L, respectively. The linearity range of the assay was verified from 0.0 to 9.0 mmol/L. Using CV &amp;lt; 20% as criteria, the functional sensitivity (LOQ) was 0.10 mmol/L at least. Lithium heparin anti-coagulated plasma specimens were stable for 24 hours at room temperature or seven days at 4 - 8 ̊C. The manufacturer’s reference interval was verified as 0.00 - 0.40 mmol/L. The Stanbio βOHB assay on the Roche c502 analyzer demonstrated a strong agreement with the Randox D-3-Hydroxybutyrate assay on the Roche c501 analyzer with a slope of 1.053 (95% CI: 1.037, 1.070) and intercept of -0.005 (95% CI: -0.068, 0.059) (coefficient correlation R = 0.9992). A positive bias was observed compared to the Abbott Freestyle Precision B-Ketone strip POC test (mean bias = + 0.50 mmol/L; slope = 1.630, 95% CI: 1.404, 1.857; R = 0.9184), and this discrepancy occurred particularly at βOHB &amp;gt; 5.5 mmol/L measured by Abbott method. Conclusions The Standbio βOHB assay exhibited excellent precision and linearity across a brand range of concentrations. This assay strongly agrees with the Randox assay run on a similar instrument but discordance with Abbott’s POCT method at high βOHB concentration. Overall, the validated method provides a reliable and efficient means for measuring βOHB levels on a Roche chemistry analyzer, facilitating accurate diagnosis and monitoring of ketosis-related conditions in clinical settings.

B-296 Assessment of Clozapine Assay on The Automated, High-performance Beckman AU Analyzer

Abstract Background Treatment-resistant schizophrenia (TRS) affects approximately one in three patients with schizophrenia. The timely initiation of clozapine therapy is crucial for achieving remission in TRS patients. However, long turnaround times (TATs) associated with sending clozapine tests to reference laboratories cause significant challenges, leading to delays in treatment initiation and compromising patient outcomes. To address this issue, we implemented in-house clozapine testing on Beckman AU analyzers, aiming to facilitate early clozapine initiation and enhance confidence in prescribing and monitoring clozapine therapy for TRS patients. Methods The Beckman AU clozapine assay is a homogenous competitive nanoparticle agglutination assay. Precision and linearity were assessed for the Beckman AU DxC700 clozapine assay using manufacturer-provided standards. We conducted a correlation study involving 31 samples analyzed on both Beckman AU DxC 700 and LC-MS/MS methods. Data analysis was performed using Deming and Linear regression methods. Additionally, we analyzed 356 clozapine tests performed in-house on Beckman AU DxC700 between March 15th and Sept 10th, 2023, and compared them with 1399 clozapine tests sent to a reference laboratory using the LC-MS/MS method from March 13th, 2020, to March 13th, 2023. Turnaround time was evaluated. Results The Beckman AU DxC700 clozapine assay exhibited excellent performance across the measuring range, with intra-assay and inter-assay coefficients of variation ranging from 1.7-3.6% and 2.2-5.3%, respectively. The assay demonstrated linearity over the five recommended ranges (68-1500 ng/mL) without significant bias observed. The Beckman AU DxC700 and LC-MS/MS clozapine assays displayed excellent correlation (R2=0.948, slope=0.941, intercept=6.3). Implementation of in-house clozapine testing led to a significant reduction in total turnaround time, from 3922 minutes to 116 minutes. The time from sample receipt to results also decreased substantially, from 3894 minutes to 32 minutes. Conclusions The clozapine assay was validated and routinely measured in-house using the Beckman AU DxC700 automated assay. Moreover, we validated the correlation by confirming that the results from Beckman AU DxC had around 6% lower bias comparing with LC-MS/MS. Turnaround time serves as a critical quality indicator for evaluating testing process effectiveness and efficiency, as well as clinician and patient satisfaction. The implementation of in-house clozapine testing significantly improved pre-analytical, analytical, and post-analytical Turnaround times.

B-030 Performance evaluation of Siemens Atellica® IM Anti-Müllerian Hormone Assay

Abstract Background Anti-Müllerian hormone (AMH), a glycoprotein dimer, is produced by ovarian granulosa cells and its levels have been shown to directly correlate to the number of eggs remaining in the ovaries (ovarian reserve). Thus, measurements of AMH are widely used as an aid in the assessment of the ovarian reserve in women presenting to fertility clinics. This study aimed to evaluate the performance of the Siemens Atellica® IM AMH assay and compare it to the Beckman Access AMH assay. Methods The Atellica AMH assay is a fully automated chemiluminescent monoclonal sandwich immunoassay, while the Beckman Access AMH assay is also an automated chemiluminescent monoclonal sandwich immunoassay. Precision and linearity were assessed for the Atellica AMH assay using manufacturer-provided standards. Inter-laboratory correlation studies were completed using deidentified patient samples collected from 120 reproductive-age women undergoing fertility outpatient evaluation at Yale New Haven Health. Blood was drawn on the first office visit for fertility workup. AMH values were initially performed at Yale University using the Beckman Access AMH assay. Serum samples were then frozen (-20°C) and shipped on dry ice to the laboratory at the Ohio State University where they were thawed and analyzed using the Siemens Atellica AMH assay. Linear regression was employed for data analysis. Overall percent agreement using an AMH cut-off value of 1.77 ng/mL was calculated. AMH values &amp;gt;1.77 ng/mL were classified as positive and values ≤1.77 were classified as negative. Results The Siemens Atellica AMH assay exhibited good performance across the measuring range, with intra-assay and inter-assay coefficients of variation (CVs) ranging from 1.4% to 3.2% and 2.5% to 6.0%, respectively. The assay demonstrated linearity over the four recommended ranges (0.043-24.0 ng/mL) without significant bias observed. The Access and Atellica AMH assays demonstrated good overall agreement of 97% and were well-correlated with a R2 value of 0.9721. Conclusions The AMH assay was validated and routinely measured in-house using the Atellica automated assay. The Siemens Atellica AMH assay performs well across its measuring range and shows good agreement with Beckman Access AMH at the AMH cut-off of 1.77 ng/mL.

B-162 Evaluation of two commercially available stable isotope labeled whole-protein infliximab as internal standard for a clinical infliximab LC-MS/MS assay

Abstract Background Liquid chromatography tandem mass spectrometry (LC-MS/MS) based quantitation of infliximab has been successfully introduced in the clinical laboratory for therapeutic drug monitoring in recent years. Although stable isotope labeled (SIL)-peptide or non-labeled analog protein internal standards have been utilized, SIL-whole protein infliximab is generally considered to be the ideal internal standard for the correction of variations during sample preparation and analysis. In this study, we compared two commercially available SIL infliximab proteins, Sigma SILu™MAb and Promise Proteomics, for use in a clinical infliximab assay. Methods Both labeled infliximab proteins incorporate [13C6, 15N4]-arginine and [13C6, 15N2]-lysine in their sequence and are designed for use as an internal standard for LC-MS/MS based quantitative analysis of infliximab. For the comparison of their performance, a laboratory-developed LC-MS/MS clinical method was utilized. Linearity at 6 levels in triplicate with Remicade and 3 biosimilars (-abda, -axxq, and -dyyb), intra-assay precision at 2 levels (n=10/level), and method comparison (n=15) experiments were performed using each labeled infliximab as the internal standard. For sample preparation, internal standard was mixed with the serum sample, followed by protein precipitation with ammonium sulfate, protein denaturation, and tryptic digestion. The peptides YASESMSGIPSR (YASE) from infliximab and YASESMSGIPSR [13C6, 15N4] (SIL-YASE) from labeled infliximab were monitored by LC-MS/MS (Transcend TLX and TSQ Atlis, Thermo Scientific) and utilized for the quantitation of infliximab. Results For the linearity experiment (1-50 µg/mL), while utilizing the Promise Proteomics internal standard to assay had an accuracy range of 103.4% to 114.1% for Remicade, 100.6% to 108.2% for Infliximab-axxq, 103.0% to 110.9% for Infliximab-abda, 102.7% to 108.3% for Infliximab-dyyb. For the Sigma SILu™Mab SIL infliximab, the accuracy range was 92.1% to 99.6% for Remicade, 94.5% to 107.2% for Infliximab-axxq, 99.0% to 104.2% for Infliximab-abda, and 105.1% to 112.0% for Infliximab-dyyb. The mean overall error for Remicade and biosimilars was 0.203 µg/mL (2.0%) for Sigma SILu™Mab SIL infliximab and 0.193 µg/mL (2.0%) for Promise Proteomics internal standardization. Intra-day precision using each SIL infliximab was evaluated with Remicade at low and high levels (5 and 20 µg/mL). Using Promise Proteomics, the coefficient of variation (CV) was 2.8 and 2.2%, while using Sigma SILu™Mab the precision was 6.8 and 6.5% for the low and high levels, respectively. The regression analysis for the method comparison experiment had a correlation coefficient (r) of 0.9996 with a slope of 1.050 (95% confidence interval, 1.030-1.070) and intercept of -0.318 (95% confidence interval, -0.842 to 0.205). Conclusions The mean overall error for the linearity experiment was the same using Sigma SILu™Mab or Promise Proteomics as the internal standard. The Promise Proteomics product demonstrated a lower intra-assay CV compared to the Sigma SILu™Mab. The 2 products displayed a strong correlation during the method comparison experiment. Both Sigma SILu™Mab and Promise Proteomics labeled standards showed acceptable performance as an internal standard for LC-MS/MS based quantitative analysis of infliximab. Having both products available increases the robustness of LC-MS/MS clinical assays in the event of supply issues.

A-131 An External Laboratory Validation of the Atellica IM Serum Neurofilament Light Chain Assay

Abstract Background Neurofilament light chain (NfL) in serum is a promising biomarker for axonal injury with potential for use in neurodegenerative disease testing, including multiple sclerosis (MS), Alzheimer’s disease, and amyotrophic lateral sclerosis (ALS). This study validated the Siemens Healthineers Atellica® IM Serum Neurofilament Light Chain (sNfL) assay* for research applications. Methods Precision studies were performed using serum and plasma samples. Intra-assay precision comprised three levels of internal assay quality control (QC) and one plasma or serum pool (consisting of serum spiked with CSF). For plasma, one aliquot of each sample was tested in duplicate in two runs per day &amp;gt;2 hours apart for up to &amp;gt;20 days. Serum inter-assay involved three levels of internal assay QC run over 10 days (1 run/day). Sensitivity studies were performed to evaluate the limit of quantitation (LoQ) for serum and plasma using five low concentration samples. Each concentration was tested in 25 replicates for serum, and 50 replicates for plasma. Linearity studies involved five levels, each run three times in duplicate (plasma) or four times (serum). Method comparison studies were performed according to CLSI EP09-A3 using the Atellica® IM NfL laboratory developed test (LDT) offered by Siemens Healthcare Laboratory, LLC. 50 samples within the measuring interval of the LDT for each sample type (plasma and serum) were tested. Results Intra-assay coefficients of variation (%CVs) for internal QC ranged from 1.7-2.5% for concentrations spanning 12.75 to 147.22 pg/mL. Serum and plasma pool intra-assay %CV were 2.5% and 3.4%, respectively. Inter-assay precision of serum %CVs ranged from 2.9-16.1% for 13.4 to 150.8 pg/mL. Plasma inter-assay %CVs demonstrated a slightly improved precision versus serum across runs with %CV ranging from 2.4-12.8% with plasma concentrations ranging from 13.4 to 158.0 pg/mL. Serum and plasma LoQs, defined as the concentration at which CV is &amp;lt;20%, were 2.5 pg/mL (serum/plasma). The assay was linear between 2.5 to 300 pg/mL for serum and plasma. The Atellica IM sNfL assay method comparison to the Atellica IM NfL LDT was analyzed using a Passing-Bablok linear regression model. The assays exhibited acceptable agreement with slopes in the range of 1.12-1.14 across the sample interval tested, 6.4 to 460 pg/mL for serum/plasma. Conclusions This external validation of the Atellica IM sNfL assay demonstrated performance characteristics suitable for research applications. *For Research Use Only. Not for use in diagnostic procedures.

Molecular detection and quantification of canine parvovirus 2 using a fast and sensitive SYBR® green-based quantitative polymerase chain reaction assay in dogs affected with gastroenteritis

Background and Aim: Viral gastroenteritis in canines is primarily caused by the canine parvovirus 2 (CPV-2). Infections by this virus can cause severe consequences in dogs, such as fever, vomiting, diarrhea, septicemia, systemic inflammation, and immunosuppression. Therefore, the mortality rate of persistent infections caused by this virus is significantly high. The capsid protein VP2 genome of canine parvovirus has undergone many changes, resulting in the emergence of different genotypes, including CPV-2a, CPV-2b, and CPV-2c. Diagnostic procedures often lack the necessary specificity for early infection diagnosis. Early detection of the infection enhances the likelihood of canine survival because the canine will receive prompt therapy. Hence, this study aimed to develop a quantitative polymerase chain reaction (qPCR)-based diagnostic technique using SYBR Green for the rapid and accurate detection and quantification of CPV-2. Materials and Methods: The assay was specifically designed to identify a portion of the conserved NS gene using primers that amplify a 125-bp fragment. The qPCR method was executed in the fast mode to expedite the process using Power up SYBR Green Master Mix reagent. A standard curve was constructed using the amplified and purified PCR product of the NS gene. Results: The limit of detection and quantification were determined in the one amplified-DNA copy. The standard curve showed an efficiency of 99.5% and inter- and intra-assay coefficients of variation of 0.387%–0.976% and 0.085%–0.430%, respectively. The assay was specific for the amplification of CPV-2, as no amplification was observed for other viral genomes (canine adenovirus II, canine distemper virus, canine coronavirus, and canine astrovirus) or from the negative controls. Inter- and intra-tests for repeatability showed low test variability around the run time. To validate the present assay, 200 samples of fezzes from canines with gastroenteritis and symptoms associated with enteric infection were tested using the qPCR protocol. From the analyzed samples, 136 were positive for CPV-2 by qPCR assay, of which 110 were before diagnostic positive for the virus by endpoint PCR, showing high sensitivity of the current assay. CPV-2 was detected in dogs over 2 weeks old up to dogs 9 years old, where the highest viral concentration found was 16429595 gene copies in dogs aged 2 weeks. Conclusion: In the present study, a rapid, specific, repeatable, and sensitive assay was developed for the detection and quantification of CPV-2. Furthermore, it was demonstrated that in the population of domestic dogs in Ecuador affected with gastrointestinal disease, the virus is presented in dogs of different ages and not only in young dogs. Keywords: canine parvovirus, gastroenteritis, quantitative polymerase chain reaction, SYBR green.

High-throughput single telomere analysis using DNA microarray and fluorescent in situ hybridization.

The human telomere system is highly dynamic. Both short and long leucocyte average telomere lengths (aTL) are associated with an increased risk of cancer and early death, illustrating the complex relationship between TL and human health and the importance of assessing TL distributions with single TL analysis. A DNA microarray and telomere fluorescent in situ hybridization (DNA-array-FISH) approach was developed to measure the base-pair (bp) lengths of single telomeres. On average 32000 telomeres were measured per DNA sample with one microarray chip assaying 96 test DNA samples. Various telomere parameters, i.e. aTL and the frequency of short/long telomeres, were computed to delineate TL distribution. The intra-assay and inter-assay coefficient of variations of aTL ranged from 1.37% to 3.98%. The correlation coefficient (r) of aTL in repeated measurements ranged from 0.91 to 1.00, demonstrating high measurement precision. aTLs measured by DNA-array-FISH predicted aTLs measured by terminal restriction fragment (TRF) analysis with r ranging 0.87-0.99. A new accurate and high-throughput method has been developed to measure the bp lengths of single telomeres. The large number of single TL data provides an opportunity for an in-depth analysis of telomere dynamics and the complex relationship between telomere and age-related diseases.

A Novel Electrochemiluminescence (ECL) Immunoassay for Detection of Anti-drug Antibodies (ADAs) to a Monoclonal Antibody (mAb) PYX-106 in Human Serum

Background: PYX-106 is a fully human monoclonal antibody (mAb), targeting sialic acid binding immunoglobulin-like lectin-15 (Siglec-15), an immunosuppressive agent with widespread expression across various tumor types. Methods: To facilitate the evaluation of immunogenicity for PYX-106 in human serum, a validated method was established to enable the detection (screening, confirmatory, and titration) of antibodies to PYX-106 in human serum samples. Results: The Screening Cut-Point Factor (SCPF), Confirmatory Cut-Point (CCP), and Titration Cut-Point Factor (TCPF) were found to be 1.51, 26.9%, and 1.86, respectively. Sensitivity was determined to be 1.81 ng/mL in the screening assay and 1.25 ng/mL in the confirmatory assay. Low Positive Control (LPC) was set at 6.00 ng/mL, and High Positive Control (HPC) was set at 1000 ng/mL. The drug tolerance was up to 500 μg/mL at the HPC level, up to 241 μg/mL at the ADA 100 ng/mL level, and up to 38.9 μg/mL at the LPC level. The intra-assay percent coefficient of variation (%CV) was ≤ 2.1% for Positive Controls (PCs) in the screening assay and ≤ 1.0% for PCs in the confirmatory assay. The inter-assay %CV was ≤ 15.3% for PCs in the screening assay and ≤ 2.1% for PCs in the confirmatory assay. No hook effect, hemolysis effect, lipemia effect, or bilirubin interference was found in this ADA method. Anti-PYX-106 antibodies were found stable in human serum for at least 23 hours 51 minutes at room temperature or after six freeze/thaw cycles. Conclusion: Anti-PYX-106 ADA, bioanalytical assay validation, was reported for the first time in any biological matrix. This ADA method has been successfully applied to human sample analysis to support a clinical study.

Assessing prognosis by quantifying FcγRIIa on fixed platelets.

Introduction: FcγRIIa amplifies platelet activation and higher platelet FcγRIIa identifies patients at greater risk of subsequent cardiovascular events. We report the accuracy and precision of a modified test to quantify FcγRIIa on previously fixed platelets (pFCG test).Methods & results: An antibody clone (5G1) was developed after exposure of mice to formaldehyde treated FcγRIIa. Accuracy and precision of the modified test was evaluated with biologic specimens (platelets) and engineered synthetic cells conjugated with FcγRIIa (Slingshot Biosciences). The modified pFCG test on fixed platelets (using 5G1) consistently identified modestly more (∼300molecules) of FcγRIIa on platelets compared with the pFCG test on nonfixed platelets (using clone FL18.26). With biologic specimens, the intra-assay coefficient of variation (CV) was 2.1±0.1% (standard error of the mean, n=750). The interassay CV was assessed intraday (4.5±1%) and interday (up to 5days after fixation, 6.5±0.4%, n=50). The pFCG test performed on Slingshot Synthetic cells conjugated with FcγRIIa demonstrated accuracy, linearity (R2=0.984) and similar interassay CV both intraday (2%±0.6%) and interday (20nonconsecutive days, 9.9%±2.1%).Conclusion: In summary, modification of the pFCG test to be performed on fixed platelets allows accurate quantification of pFCG with high precision.

Establishment and Clinical Application in Stroke of a Serum Copeptin Time-Resolved Fluorescence Immunoassay.

The serum biomarker copeptin, an innovative and stable substitute biomarker of vasopressin, is associated with stroke. Therefore, establishing a highly sensitive time-resolved fluorescence immunoassay for copeptin (copeptin-TRFIA) is helpful to measure stroke and evaluate its value in clinical applications. Double antibody sandwich was used to establish copeptin-TRFIA. The established method was then assessed. Two coated and Eu3+-labeled copeptin monoclonal specific antibodies targeting different antigen epitopes were employed. The serum fluorescence counts of patients with stroke and healthy volunteers were detected by using thewell-established copeptin-TRFIA. Serum copeptin levels were measured and analyzed statistically. The actual measurement linearity range of the proposed method was 0.13-44.66 ng/mL. Copeptin-TRFIA had the inter-assay coefficient of variation (CV) of 6.49%-9.08% and the intra-assay CV of 4.75%-7.77%. Patients with cerebral infarction (CI) and intracerebral hemorrhage (ICH) had significantly higher serum copeptin levels than healthy subjects. Copeptin concentrations in the serum of patients with stroke were significantly correlated with the scores of the National Institute for Healthy Stroke Scale (NIHSS) and modified Rankin Scale (mRS). A highly sensitive copeptin-TRFIA was successfully established. Serum copeptin has a certain value in the clinical diagnosis and prognosis of stroke.