Inter-assay Coefficients Of Variation Research Articles

Development of Simultaneous Drug Concentration Measurement Method Using an Automated Pretreatment Liquid Chromatography/Tandem Mass Spectrometry System for Therapeutic Drug Monitoring

Therapeutic drug monitoring (TDM) is a personalized treatment approach that involves optimizing drug dosages based on patient-specific factors, such as drug plasma concentrations, therapeutic efficacy, or adverse reactions. The plasma concentration of drugs is determined using liquid chromatography/tandem mass spectrometry (LC-MS/MS) or various immunoassays. Compared with immunoassays, LC-MS/MS requires more pretreatment time as the number of samples increases. Recently, fully automated pretreatment LC-MS/MS systems have been developed to automatically perform whole-sample pretreatment for LC-MS/MS analysis. In this study, we developed a method for simultaneous concentration determination of five analytes (clozapine, mycophenolic acid, sunitinib, N-desethylsunitinib, and voriconazole) using LC-MS/MS for clinical TDM using a fully automated LC-MS/MS pretreatment system. In the developed method, the intra- and inter-assay relative error (RE) values ranged between −14.8% and 11.3%; the intra- and inter-assay coefficient of variation (CV) values were <8.8% and <10.5%, respectively. The analytes showed good stability, with RE values ranging between −13.6% and 10.9% and CV values <8.9%. Furthermore, the plasma concentrations in clinical samples using this method and the conventional manual pretreatment method showed similar results. Therefore, the method developed in this study could be considered a useful pretreatment method for routine TDM in patients.

Multiplex qPCR development for the simultaneous and rapid detection of largemouth bass virus and infectious spleen and kidney necrosis virus in aquaculture

Largemouth bass virus (LMBV) and infectious spleen and kidney necrosis virus (ISKNV) are both belong to Iridoviridae that cause considerable economic losses in the fish industry. There is no reported literature that can detect these two viruses simultaneously. In this study, we established a multiplex quantitative polymerase chain reaction (qPCR) assay that can specifically and simultaneously detect both LMBV and ISKNV in fish samples. The specificity experiment showed that the method only amplified LMBV and ISKNV but not the other 10 common fish viruses. The slope (m), efficiency (E) and linearity (R2) determined from the generated standard curve were all within the optimal range of qPCR values. The detection limit of the multiplex qPCR assay was as low as 4 copies/μL for LMBV DNA and 7 copies/μL for ISKNV DNA, respectively. The established method exhibited adequate repeatability and reproducibility, and the intra- and inter-assay coefficients of variation were both less than 3%. The accuracy of the multiplex qPCR method was validated using 229 fish samples and was more precise than that of the conventional PCR assay. In summary, the established multiplex qPCR assay can simultaneously detect LMBV and ISKNV to monitor the risk of infection LMBV and ISKNV and control the disease early.

Simultaneous detection of 10 cancer-associated amino acids and polyamines by high-performance liquid chromatography-high resolution mass spectrometry in pleural effusion cells obtained from lung adenocarcinoma patients

The deregulation of amino acid and polyamine metabolism is a hallmark of malignancy that regulates cancer cell proliferation, angiogenesis, and invasion. A sensitive mass spectrometry method was developed to simultaneously quantify 10 cancer-associated metabolites in pleural effusion cells for the diagnosis of malignancy and to complement conventional pleural cytology. Analytes were detected by high-performance liquid chromatography-high resolution mass spectrometry (HPLC-HRMS) using C8-reversed-phase HPLC for separation and sequential window acquisition of all theoretical fragment ion spectra (SWATH) acquisition for obtaining high-resolution quantitative MS/MS chromatograms. This method was validated and applied to pleural effusion cells from patients with lung adenocarcinoma (LUAD, n = 48) and those from benign controls (n = 23). The range of the above metabolites was 2–200 ng/mL for proline, aspartate, ornithine, creatine, glutamine, glutamate, arginine, citrulline, and spermine and 10–1000 ng/mL for putrescine. The intra-assay and inter-assay coefficient of variation was below 13.70 % for all analytes. The joint detection of these metabolites in pleural effusion cells achieved a clinical sensitivity of 75.0 % and specificity of 95.7 % differentiating LUAD patients from benign controls. This assay enabled the detection of 10 cancer-associated metabolites in pleural effusion cells, and the increased concentration of these metabolites was correlated with the presence of LUAD.

Validation of a focus reduction neutralization test (FRNT) to rapidly titrate human SARS-CoV-2 neutralizing antibodies by using the CTL Immunospot S6 universal analyzer

In this paper we describe the validation of a focus reduction neutralization test (FRNT) to quantitate human SARS-CoV-2 neutralizing antibodies by using the CTL Immunospot S6 Universal Analyzer. We employed a previously published protocol and compared its performances to a well-established and traditional serum-neutralization assay (SN). To assess diagnostic sensitivity, a total number of 201 human sera positive by SN for SARS-CoV-2 NAbs were processed: 196/201 tested positive by FRNT50 (97.51 %). A diagnostic specificity of 100 % was obtained by evaluating 206 negative serum samples. Repeatability of the test was evaluated by determining the intra and inter-assay coefficient of variation (CV). A standard deviation of 0.83 and a CV of 13 % were evidenced demonstrating an acceptable reproducibility of the assay. Moreover, a Cohen's Kappa of 0.975 was obtained proving an extremely high level of agreement between the FRNT protocol and the SN. Despite an acceptable correlation between methods (p < 0.05), FRNT demonstrated a statistically significant increase in NAbs titres compared to SN as well as higher data variability and asymmetry. These discrepancies could be attributed to FRNT sensitivity or most probably to the subjective interpretation of SN, although this aspect needs to be further investigated with a more representative number of samples.Basing on our results, it is reasonable to replace the SN with the FRNT assay as, with this, fast processing time (less than 2 days) and operator bias-free results registrations are guaranteed.

Serum anti-Müllerian hormone is an indirect predictor of ovarian reserve in domestic cats

Anti-Müllerian hormone (AMH) serves as an indirect marker for predicting primordial follicles that are representative of ovarian reserve. In this study the possibility of using AMH and age to predict the ovarian reserve in domestic cats. Ovaries and blood were collected from 30 cats undergoing routine ovariohysterectomy. The animals were divided into three age groups: prepubertal (<4 mo, n = 10), adult (1–5 y, n = 10), and senior (>5 y, n = 10). Blood was collected at surgery for serum AMH measurements using the AMH Gen II ELISA kit. The intra-assay coefficient of variation (CV) and inter-assay CV were 3.56 % and 7.68 %, respectively. One side of the ovary was processed to determine AMH localization using immunohistochemistry and for a histological count of follicles, which is the gold standard. The expression of AMH protein was quantified from the contralateral ovary by Western blot analysis. Primordial follicles exhibited the most pronounced inverse relationship with age (rho = −0.779, P < 0.05), followed by a positive association with serum AMH concentration (rho = 0.490, P < 0.05), indicating that both age and AMH are potential markers indicative of primordial follicles. Furthermore, secondary (rho = 0.651, P < 0.05) and small antral follicles (rho = 0.648, P < 0.05) were identified as the major sources of circulating AMH, as indicated by the stronger correlation with serum AMH concentrations compared with primary follicles. However, there was no significant correlation between the expression of AMH protein and other factors, including age, primordial follicles, primary follicles, secondary follicles, small antral follicles, and serum AMH concentration. A model for predicting primordial follicle number using serum AMH concentration (AIC = 672.66, P < 0.05) and age (AIC = 668.93, P < 0.05) was established. In conclusion, both serum AMH concentration and age may serve as comparable markers of ovarian reserve in domestic cats. Moreover, AMH is particularly useful in situations where age information is not available.

Quantification of multiple steroid hormones in serum and human breast cancer tissue by liquid chromatography-tandem mass spectrometry analysis.

Systemic and local steroid hormone levels may function as novel prognostic and predictive biomarkers in breast cancer patients. We aimed at developing a novel liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous measurement of multiple, biologically pivotal steroid hormones in human serum and breast cancer tissue. The quantitative method consisted of liquid-liquid extraction, Sephadex LH-20 chromatography for tissue extracts, and analysis of steroid hormones by liquid-chromatography-tandem mass spectrometry. We analyzed serum and tissue steroid hormone levels in 16 and 40 breast cancer patients, respectively, and assessed their correlations with clinical parameters. The method included quantification of nine steroid hormones in serum [including cortisol, cortisone, corticosterone, estrone (E1), 17β-estradiol (E2), 17α-hydroxyprogesterone, androstenedione (A4), testosterone and progesterone) and six (including cortisone, corticosterone, E1, E2, A4, and testosterone) in cancer tissue. The lower limits of quantification were between 0.003-10 ng/ml for serum (250 µl) and 0.038-125 pg/mg for tissue (20 mg), respectively. Accuracy was between 98%-126%, intra-assay coefficient of variations (CV) was below 15%, and inter-assay CV were below 11%. The analytical recoveries for tissue were between 76%-110%. Tissue levels of E1 were positively correlated with tissue E2 levels (p<0.001), and with serum levels of E1, E2 and A4 (p<0.01). Tissue E2 levels were positively associated with serum E1 levels (p=0.02), but not with serum E2 levels (p=0.12). The levels of tissue E2 and ratios of E1 to A4 levels (an index for aromatase activity) were significantly higher in patients with larger tumors (p=0.03 and p=0.02, respectively). The method was convenient and suitable for a specific and accurate profiling of clinically important steroid hormones in serum. However, the sensitivity of the profile method in steroid analysis in tissue samples is limited, but it can be used for the analysis of steroids in breast cancer tissues if the size of the sample or its steroid content is sufficient.

A multiplex digital PCR assay for detection and quantitation of porcine circovirus type 2 and type 3.

Porcine circovirus (PCV) has become a major pathogen, causing major economic losses in the global pig industry, and PCV type 2 (PCV2) and 3 (PCV3) are distributed worldwide.We designed specific primer and probe sequences targeting PCV2 Cap and PCV3 Rap anddeveloped a multiplex crystal digital PCR (cdPCR) method after optimizing the primerconcentration, probe concentration, and annealing temperature.The multiplex cdPCR assay permits precise and differential detection of PCV2 and PCV3, with a limit of detection of 1.39 × 101 and 1.27 × 101 copies/reaction, respectively, and no cross-reaction with other porcine viruses was observed. The intra-assay and interassay coefficients of variation (CVs) were less than 8.75%, indicating good repeatability and reproducibility. To evaluate the practical value of this assay, 40 tissue samples and 70 feed samples were tested for both PCV2 and PCV3 by cdPCR and quantitative PCR (qPCR). Using multiplex cdPCR, the rates of PCV2 infection, PCV3 infection, and coinfection were 28.45%, 1.72%, and 12.93%, respectively, and using multiplex qPCR, they were 25.00%, 0.86%, and 4.31%, respectively This highly specific and sensitive multiplex cdPCR thus allows accurate simultaneous detection of PCV2 and PCV3, and it is particularly well suited for applications that require the detection of small amounts of input nucleic acid or samples with intensive processing and complex matrices.

Development of a triplex quantitative reverse transcription-polymerase chain reaction for the detection of porcine epidemic diarrhea virus, porcine transmissible gastroenteritis virus, and porcine rotavirus A.

Porcine viral diarrhea is caused by many pathogens and can result in watery diarrhea, dehydration and death. Various detection methods, such as polymerase chain reaction (PCR) and real-time quantitative PCR (qPCR), have been widely used for molecular diagnosis. We developed a triplex real-time quantitative reverse transcription PCR (qRT-PCR) for the simultaneous detection of three RNA viruses potentially associated with porcine viral diarrhea: porcine epidemic diarrhea virus (PEDV), porcine transmissible gastroenteritis virus (TGEV), and porcine rotavirus A (PoRVA). The triplex qRT-PCR had R2 values of 0.999 for the standard curves of PEDV, TGEV and PoRVA. Importantly, the limits of detection for PEDV, TGEV and PoRVA were 10 copies/μL. The specificity test showed that the triplex qRT-PCR detected these three pathogens specifically, without cross-reaction with other pathogens. In addition, the approach had good repeatability and reproducibility, with intra-and inter-assay coefficients of variation <1%. Finally, this approach was evaluated for its practicality in the field using 256 anal swab samples. The positive rates of PEDV, TGEV and PoRVA were 2.73% (7/256), 3.91% (10/256) and 19.14% (49/256), respectively. The co-infection rate of two or more pathogens was 2.73% (7/256). The new triplex qRT-PCR was compared with the triplex RT-PCR recommended by the Chinese national standard (GB/T 36871-2018) and showed 100% agreement for PEDV and TGEV and 95.70% for PoRVA. Therefore, the triplex qRT-PCR provided an accurate and sensitive method for identifying three potential RNA viruses for porcine viral diarrhea that could be applied to diagnosis, surveillance and epidemiological investigation.

Simultaneous Detection of Porcine Respiratory Coronavirus, Porcine Reproductive and Respiratory Syndrome Virus, Swine Influenza Virus, and Pseudorabies Virus via Quadruplex One-Step RT-qPCR.

Porcine respiratory coronavirus (PRCoV), porcine reproductive and respiratory syndrome virus (PRRSV), swine influenza virus (SIV), and pseudorabies virus (PRV) are significant viruses causing respiratory diseases in pigs. Sick pigs exhibit similar clinical symptoms such as fever, cough, runny nose, and dyspnea, making it very difficult to accurately differentially diagnose these diseases on site. In this study, a quadruplex one-step reverse-transcription real-time quantitative PCR (RT-qPCR) for the detection of PRCoV, PRRSV, SIV, and PRV was established. The assay showed strong specificity, high sensitivity, and good repeatability. It could detect only PRCoV, PRRSV, SIV, and PRV, without cross-reactions with TGEV, PEDV, PRoV, ASFV, FMDV, PCV2, PDCoV, and CSFV. The limits of detection (LODs) for PRCoV, PRRSV, SIV, and PRV were 129.594, 133.205, 139.791, and 136.600 copies/reaction, respectively. The intra-assay and inter-assay coefficients of variation (CVs) ranged from 0.29% to 1.89%. The established quadruplex RT-qPCR was used to test 4909 clinical specimens, which were collected in Guangxi Province, China, from July 2022 to September 2023. PRCoV, PRRSV, SIV, and PRV showed positivity rates of 1.36%, 10.17%, 4.87%, and 0.84%, respectively. In addition, the previously reported RT-qPCR was also used to test these specimens, and the agreement between these methods was higher than 99.43%. The established quadruplex RT-qPCR can accurately detect these four porcine respiratory viruses simultaneously, providing an accurate and reliable detection technique for clinical diagnosis.

Advancing cortisol measurements in zebrafish: Analytical validation of a commercial ELISA kit for skin mucus cortisol analysis

Cortisol is the main stress biomarker used for zebrafish. However, zebrafish small size made it challenging to extract cortisol without harming or killing the fish. Thus, researchers adopted a terminal method, the trunk cortisol, as standard practice. Here, we developed and validated an alternative and minimally invasive technique for measuring cortisol in the skin mucus of adult zebrafish, using a commercial enzyme-linked immunosorbent assay (ELISA). For this, AB zebrafish were randomly assigned to a precision, accuracy, and specificity test. Each sample contained the skin mucus of five to ten fish or one fish trunk. The cortisol was extracted using methanol as organic solvent. The results obtained showed an adequate precision (intra-assay coefficient of variation (CV) <15%; inter-assay CV = 26%), accuracy (CV <120%), and specificity (r2 =0.96–0.98) for skin mucus cortisol levels, as well as for trunk cortisol.•A commercial ELISA was analytically validated to measure cortisol in the skin mucus of zebrafish.•Skin mucus cortisol is a non-terminal method that reduce the number of animals used and allows longitudinal studies.

The development, evaluation, performance, and validation of micro-PCR and extractor for the quantification of HIV-1 &-2 RNA

HIV infection has been a global public health threat and overall reported ~ 40 million deaths. Acquired immunodeficiency syndrome (AIDS) is attributed to the retroviruses (HIV-1/2), disseminated through various body fluids. The temporal progression of AIDS is in context to the rate of HIV-1 infection, which is twice as protracted in HIV-2 transmission. Q-PCR is the only available method that requires a well-developed lab infrastructure and trained personnel. Micro-PCR, a portable Q-PCR device, was developed by Bigtec Labs, Bangalore, India. It is simple, accurate, fast, and operationalised in remote places where diagnostic services are inaccessible in developing countries. This novel micro-PCR determines HIV-1 and HIV-2 viral load using a TruePrep™ extractor device for RNA isolation. Five ml blood samples were collected at the blood collection centre at AIIMS, New Delhi, India. Samples were screened for serology, and a comparison of HIV-1/2 RNA was done between qPCR and micro-PCR in the samples. The micro-PCR assay of HIV-RNA has compared well with those from real-time PCR (r = 0.99, i < 0.002). Micro-PCR has good inter and intra-assay reproducibility over a wide dynamic range (1.0 × 102–1.0 × 108 IU/ml). The linear dynamic range was 102–108 IU/ml. The clinical and analytical specificity of the assay was comparable, i.e., 100%. Intra-assay and inter-assay coefficients of variation ranged from 1.17% to 3.15% and from 0.02% to 0.46%, respectively. Moreover, due to the robust, simple, and empirical method, the Probit analysis has also been done for qPCR LODs to avoid uncertainties in target recoveries. The micro-PCR is reliable, accurate, and reproducible for early detection of HIV-1 and HIV-2 viral loads simultaneously. Thus, it can easily be used in the field and in remote places where quantification of both HIV-1/2 is not reachable.

Real-world performance of the NeuMoDx™ HCV Quant Test for quantification of hepatitis C virus (HCV)-RNA

Quantification of hepatitis C virus (HCV)-RNA in serum or plasma samples is an essential parameter in HCV diagnostics. Here, the NeuMoDx™Molecular System (Qiagen) was tested for the most common HCV genotypes and compared to the cobas c6800 system (Roche).HCV-RNA from 131 plasma/serum samples from chronically infected patients was determined in parallel on the NeuMoDx and c6800 systems. Linearity was analysed using the four most common HCV genotypes (1−4) in our cohort. The coefficient of variation (CV) within (intra-assay) and between (inter-assay) runs was calculated based on HCV-RNA concentration. Quantitative HCV-RNA results were highly correlated on both test systems (R2 = 0.7947; y = 0.94 x + 0.37). On average, the NeuMoDx and c6800 HCV RNA levels showed a mean difference of only 0.05 log10 IU/mL but with a broad distribution (±1.2 2 x SD). The NeuMoDx demonstrated very good linearity across all HCV genotypes tested at concentrations between 1.7 and 6.2 log10 IU/mL (R2 range: 0.9257–0.9991) with the highest mean coefficient of determination for genotype 1 (R2 = 0.9909). The mean intra- and inter-assay CV for both serum and plasma samples was <5 %. The NeuMoDx HCV-RNA Assay demonstrates high subtype-independent comparability, linearity, and reproducibility for the quantification of HCV-RNA in serum and plasma samples from chronically infected patients.

Development and validation of streptavidin-biotin-based double antibody sandwich ELISA for ricin diagnosis

BackgroundRicin is a potential biowarfare agent. It is a phytotoxin isolated from castor seeds. At present there is no antidote available for ricin poisoning, patients only get supportive treatment based on their symptoms. This highlights the importance of early detection to avoid severity of accidents and reduce the risk factor. Considering this, our study aimed to develop a highly sensitive and specific sandwich ELISA for the detection of ricin. MethodsRicin was purified from castor seeds. Anti-ricin polyclonal and monoclonal antibodies were generated from rabbit antisera and hybridoma cell (1H6F1) supernatant using a protein A/G column. Antibody titer estimation was done using Indirect ELISA. A streptavidin–biotin-based sandwich ELISA was developed and the limit of detection (LOD), linear range, intra and inter-assay coefficient of variation (CV), and cross-reactivity with other similar toxins were determined. Interference of human plasma samples spiked with ricin was also checked. ResultsThe LOD of the ELISA was found to be 0.45 ng/ml, with a linear range of 0.90–62 ng/ml, intra and inter-assay CV ranged from 3.34 % to 5 % and 5.17 % to 10.80 % respectively. The assay was not cross-reactive with other similar ribosome-inactivating protein (RIP) toxins. Ricin was detected in spiked plasma samples. ConclusionThe developed assay is highly sensitive and specific for detecting ricin and is not cross-reactive with other similar types of toxins. The assay can detect ricin in spiked plasma samples, so it has the potential to be used for the analysis of clinical samples after ricin poisoning.

A triplex crystal digital PCR for the detection of genotypes I and II African swine fever virus.

African swine fever (ASF) is a highly contagious and lethal viral disease that causes severe hemorrhagic fever in pigs. It keeps spreading around the world, posing a severe socioeconomic risk and endangering biodiversity and domestic food security. ASF first outbroke in China in 2018, and has spread to most provinces nationwide. Genotypes I and II ASF virus (ASFV) as the etiological pathogens have been found in China. In this study, three pairs of specific primers and probes targeting the ASFV B646L gene, F1055L gene, and E183L gene were designed to detect universal, genotype I, and genotype II strains, respectively. A triplex crystal digital PCR (cdPCR) was established on the basis of optimizing various reaction conditions. The assay demonstrated remarkably sensitive with low limits of detection (LODs) of 5.120, 4.218, 4.588 copies/reaction for B646L, F1055L, and E183L gene, respectively; excellent repeatability with 1.24-2.01% intra-assay coefficients of variation (CVs) and 1.32-2.53% inter-assay CVs; good specificity for only detection of genotypes I and II ASFV, without cross-reactivity with PCV2, PRV, SIV, PRRSV, PEDV, FMDV, and CSFV. The triplex cdPCR was used to test 1,275 clinical samples from Guangxi province of China, and the positivity rates were 5.05, 3.22, and 1.02% for genotype I, genotype II, and co-infection of genotypes I and II, respectively. These 1,275 clinical samples were also detected using a reported reference triplex real-time quantitative PCR (qPCR), and the agreements of detection results between these two methods were more than 98.98%. In conclusion, the developed triplex cdPCR could be used as a rapid, sensitive, and accurate method to detect and differentiate genotypes I and II strains of ASFV.

Measurement of Feline Alpha-1 Acid Glycoprotein in Serum and Effusion Using an ELISA Method: Analytical Validation and Diagnostic Role for Feline Infectious Peritonitis.

Alpha-1 acid glycoprotein (AGP) may support a clinical diagnosis of feline infectious peritonitis (FIP). In this study, we assessed the analytical and diagnostic performances of a novel ELISA method to measure feline AGP. AGP was measured in sera and effusions from cats with FIP (n = 20) or with other diseases (n = 15). Precision was calculated based on the coefficient of variation (CV) of repeated testing, and accuracy was calculated by linearity under dilution (LUD). The test is precise (intra-assay CVs: <6.0% in individual samples, <15.0% in pooled samples; inter-assay CVs <11.0% and <15.0%) and accurate (serum LUD r2: 0.995; effusion LUD r2: 0.950) in serum and in effusions. AGP is higher in cats with FIP than in other cats in both serum (median: 1968, I-III interquartile range: 1216-3371 μg/mL and 296, 246-1963 μg/mL; p = 0.009) and effusion (1717, 1011-2379 μg/mL and 233, 165-566 μg/mL; p < 0.001). AGP discriminates FIP from other diseases (area under the receiver operating characteristic curve: serum, 0.760; effusion, 0.877), and its likelihood ratio is high (serum: 8.50 if AGP > 1590 μg/mL; effusion: 3.75 if AGP > 3780 μg/mL). This ELISA method is precise and accurate. AGP in serum and in effusions is a useful diagnostic marker for FIP.

An immunochromatographic strip sensor for marbofloxacin residues.

Marbofloxacin (MBF) was once widely used as a veterinary drug to control diseases in animals. MBF residues in animal food endanger human health. In the present study, an immunochromatographic strip assay (ICSA) utilizing a competitive principle was developed to rapidly detect MBF in beef samples. The 50% inhibitory concentration (IC50) and the limit of detection (LOD) of the ICSAs were 2.5 ng/mL and 0.5 ng/mL, respectively. The cross-reactivity (CR) of the MBF ICSAs to Ofloxacin (OFL), enrofloxacin (ENR), norfloxacin (NOR), and Ciprofloxacin (CIP) were 60.98%, 32.05%, 22.94%, and 23.58%, respectively. The CR for difloxacin (DIF) and sarafloxacin (SAR) was less than 0.1%. The recovery rates of MBF in spiked beef samples ranged from 82.0% to 90.4%. The intra-assay and interassay coefficients of variation (CVs) were below 10%. In addition, when the same authentic beef samples were detected in a side-by-side comparison between the ICSAs and HPLC‒MS, no statistically significant difference was observed. Therefore, the proposed ICSAs can be a useful tool for monitoring MBF residues in beef samples in a qualitative and quantitative manner.

Enhanced detection of African swine fever virus in samples with low viral load using digital PCR technology

Detection of low viral load samples has long been a challenge for African swine fever (ASF) prevention and control. This study aimed to compare the detection efficacy of droplet digital PCR(ddPCR) and quantitative PCR(qPCR) for African swine fever virus (ASFV) at different viral loads, with a focus on assessing the accuracy of ddPCR in detecting low viral load samples. The results revealed that ddPCR had a detection limit of 1.97 (95% CI 1.48 – 4.12) copies/reaction and was 18.99 times more sensitive than qPCR (detection limit: 37.42, 95% CI 29.56 – 69.87 copies/reaction). In the quantification of high, medium, and low viral load samples, ddPCR showed superior stability with lower intra- (2.06% – 7.58%) and inter-assay (3.83% – 7.50%) coefficients of variation than those of qPCR (intra-assay: 8.08%–29.86%; inter-assay: 9.27%–34.58%). Bland-Altman analysis indicated acceptable consistency between ddPCR and qPCR for high and medium viral load samples; however, discrepancies were observed for low viral load samples, where two samples (2/24, 8.33%) exhibited deviations beyond the acceptable range (−46.18 copies/reaction). Moreover, ddPCR demonstrated better performance in detecting ASFV in clinical samples from asymptomatic pigs and environmental samples, with qPCR showing false negative rates of 7.69% (2/26) and 27.27% (12/44), respectively. McNemar analysis revealed significant differences between the two methods (P = 0.000) for samples with a viral load <100 copies/reaction. The results of this study demonstrate that ddPCR has better detection limits and adaptability than qPCR, allowing for a more accurate detection of ASFV in early-stage infections and low-concentration environmental samples. These findings highlight the potential of ddPCR in the prevention and control of ASF.

Abstract 81: High-resolution monitoring of B cell isotype switching for biomarker analysis of adenosine pathway inhibition immunotherapy

Abstract Background B cell isotype switching gives rise to IgG-, IgA-, and IgE-expressing B cells and is an essential step in generating effective humoral responses to antigen challenge. Within the tumor microenvironment, extracellular adenosine may contribute to cancer immune evasion by inhibiting isotype switching through interaction with A2aR, a possibility that has driven investigation of adenosine pathway inhibition as an immunotherapeutic modality. In this context, methods to quantify isotype switching may provide insight into the pharmacodynamics of candidate agent activity and serve as a source of predictive biomarkers of response. We recently clinically validated the Oncomine IGH-LR assay for the measurement of CLL somatic hypermutation (SHM). Given that this assay also detects isotype switching events, we retrospectively analyzed our validation dataset to determine the suitability of the assay as a potential tool for biomarker analysis of adenosine pathway inhibition immunotherapy. Methods The Oncomine IGH-LR assay determines B cell SHM and isotype through targeted next-generation sequencing of IGH chains from RNA. The assay software organizes detected IGH chains into B cell clonal lineages, of which a subset may comprise multi-isotype lineages indicative of isotype switching events. The software further reports secondary repertoire features such as clonality/evenness. Total RNA was extracted from peripheral blood mononuclear cells or bone marrow aspirates from patients identified as having &amp;gt;10% monoclonal B-cell population by flow cytometry. Intra-assay precision was evaluated by processing RNA from 7 samples in triplicate and sequencing in the same run. Inter-assay precision was evaluated by having different operators process 10 samples on different days with different sequencing barcodes. Dominant clone isotype and clonal lineage assignment, total isotype abundance, and repertoire clonality were evaluated across replicates. Results Overall, 100% concordance was achieved for dominant clone isotype and clonal lineage calls for both intra- and inter-assay studies. Intra-assay and inter-assay median coefficient of variation (CV) for reported isotype frequencies ranged from 0.10-0.16 and 0.15-0.28, respectively. Intra-assay and inter-assay median CV for clonality (1 - Normalized Shannon Entropy) was 0.06 and 0.04, respectively. Conclusions We observe high reproducibility and repeatability of the IGH-LR assay for B cell isotyping, clonal lineage analysis, and clonality assessment. Multi-isotype clonal lineages are automatically reported by the software and provide a means to directly monitor isotype switching events in the context of antigen challenge or immunomodulation. Taken together, we anticipate this assay to be a useful tool for biomarker analysis of adenosine pathway inhibition immunotherapy. Citation Format: Ramadevi Prathapam, Kristen Champion, Anne Burkhardt, Chelsey L. Smith, Rajesh Singh, Timothy J. Looney. High-resolution monitoring of B cell isotype switching for biomarker analysis of adenosine pathway inhibition immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 81.

Abstract 7295: A novel XNA technology-based assay to detect JAK2 V617F mutation by real-time PCR

Abstract The Janus kinase 2 (JAK2) gene is located on chromosome 9p24.1 and encodes a non-receptor tyrosine kinase that plays a central role in cytokine and growth factor signaling. The JAK2 protein is especially important for controlling the production of blood cells from hematopoietic stem cells. In fact, the JAK2 mutation at amino acid 617 (Valine 617 to Phenylalanine, V617F) is the most frequently detected mutation in myeloproliferative neoplasms (MPN), including essential thrombocythemia (ET), polycythemia vera (PV), and primary myelofibrosis (PMF). This dominant gain-of-function V617F mutation results in constitutive tyrosine phosphorylation activity, leading to uncontrolled blood cell production, including increased numbers of erythrocytes, neutrophils, and platelets. To detect the JAK2 V617F mutation in DNA from patient blood samples, we developed a unique real-time PCR assay using our proprietary XNA technology that enables the assay to selectively amplify the mutant sequence by using a synthetic DNA analog XNA (Xenonucleic Acid). The JAK2 V617F mutation detection assay is designed to detect a valine-to-phenylalanine mutation at amino acid 617 (V617F). PCR primers, TaqMan probes, and XNA were designed for the JAK2 V617F mutation, along with primers and probes for an internal control gene in the duplex setting. The analytical performance of the assay was verified and validated using a variety of control samples. The results revealed that the XNA completely suppressed the amplification of wildtype sequence while only the V617F mutant sequence was amplified. The JAK2 V617F mutation detection assay is highly reproducible with intra- and inter-assay coefficients of variation of less than 10%. Results for the assay’s analytical sensitivity indicated that V617F could be detected with about 0.25% mutant allele frequency in 10 ng DNA input. In addition, no significant cross-amplification between V617F and V617I was observed. Thus, the JAK2 V617F mutation detection assay is specific for the V617F mutation. The JAK2 V617F mutation detection assay, using XNA-based technology, provides high sensitivity and specificity to detect the JAK2 V617F mutation. Thus, this assay would be a useful tool for clinical decision-making in determining the presence of the JAK2 V617F mutation in DNA from patient blood samples. Citation Format: Shuo Shen, Robert Brown, Daniel Kim, Larry Pastor, Andrew Y. Fu, Pushpinder Kaur, Alisha Babu, Wei Liu, Aiguo Zhang, Hiromi Tanaka, Michael Y. Sha. A novel XNA technology-based assay to detect JAK2 V617F mutation by real-time PCR [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7295.

Electrochemical microfluidic immunosensor with graphene-decorated gold nanoporous for T-2 mycotoxin detection

T-2 is one of the most potent cytotoxic food-borne mycotoxins. In this work, we have developed and characterized an electrochemical microfluidic immunosensor for T-2 toxin quantification in wheat germ samples. T-2 toxin detection was carried out using a competitive immunoassay method based on monoclonal anti-T-2 antibodies immobilized on the poly(methyl methacrylate) (PMMA) microfluidic central channel. The platinum wire working electrode at the end of the channel was in situ modified by a single-step electrodeposition procedure with reduced graphene oxide (rGO)-nanoporous gold (NPG). T-2 toxin in the sample was allowed to compete with T-2-horseradish peroxidase (HRP) conjugated for the specific recognizing sites of immobilized anti-T-2 monoclonal antibodies. The HRP, in the presence of hydrogen peroxide (H2O2), catalyzes the oxidation of 4-tert-butylcatechol (4-TBC), whose back electrochemical reduction was detected on the nanostructured electrode at −0.15 V. Thus, at low T-2 concentrations in the sample, more enzymatically conjugated T-2 will bind to the capture antibodies, and, therefore, a higher current is expected. The detection limits found for electrochemical immunosensor, and commercial ELISA procedure were 0.10 μg kg−1 and 10 μg kg−1, and the intra- and inter-assay coefficients of variation were below 5.35% and 6.87%, respectively. Finally, our microfluidic immunosensor to T-2 toxin will significantly contribute to faster, direct, and secure in situ analysis in agricultural samples.