Diluted Serum Samples Research Articles

Electrochemical Determination of B-Type Natriuretic Peptide with an Epitope-Imprinted Polymer-Based Sensor

B-type natriuretic peptides (BNP) are produced and secreted by the myocardium to reduce blood pressure and cardiac load. They cause vasodilation, natriuresis, growth suppression, and inhibition of the sympathetic nervous system and the renin–angiotensin–aldosterone system. The measurement of plasma BNP levels provides clinically useful information concerning the diagnosis and management of left ventricular dysfunction and heart failure, complementing other diagnostic testing procedures. In this work, three epitopes from the N-terminal (BNPnt), C-terminal (BNPct), and the cystine-bridged cyclic peptides (BNPr) of B-type natriuretic peptides were synthesized as templates for molecular imprinting. These peptides were doped into aniline (AN) and m-aminobenzenesulfonic acid (MSAN) for electropolymerization, thus forming epitope-imprinted poly(AN-co-MSAN) conductive films (EIPs). The monomer ratio was optimized using the electrochemical signals during polymerization. The optimized films were then characterized using a scanning electron microscope (SEM), atomic force microscope (AFM), and AC impedance. The electrochemical response of the films to the target peptides and to BNP was then measured. The sensing range of the EIPs-coated electrodes was from 0.001 to 1000 pg/mL for BNP. Finally, the BNP concentration in diluted serum samples was measured with the BNPrIP-coated electrode, giving 3.15 ± 0.07 pg/mL. By spiking the sample with known BNP concentrations, the accuracy was determined to be better than ±5%.

Synthesis and characterization of core–shell magnetic molecularly imprinted polymer nanocomposites for the detection of interleukin-6

Interleukin-6 (IL-6) belongs to the cytokine family and plays a vital role in regulating immune response, bone maintenance, body temperature adjustment, and cell growth. The overexpression of IL-6 can indicate various health complications, such as anastomotic leakage, cancer, and chronic diseases. Therefore, the availability of highly sensitive and specific biosensing platforms for IL-6 detection is critical. In this study, for the first time, epitope-mediated IL-6-specific magnetic molecularly imprinted core–shell structures with fluorescent properties were synthesized using a three-step protocol, namely, magnetic nanoparticle functionalization, polymerization, and template removal following thorough optimization studies. The magnetic molecularly imprinted polymers (MMIPs) were characterized using dynamic and electrophoretic light scattering (DLS and ELS), revealing a hydrodynamic size of 169.9 nm and zeta potential of +17.1 mV, while Fourier transform infrared (FTIR) spectroscopy and fluorescence spectroscopy techniques showed characteristic peaks of the polymer and fluorescent tag, respectively. Scanning electron microscopy (SEM) and high-resolution transmission electron microscopy (HRTEM) investigations confirmed the successful encapsulation of the magnetic core within the ca. 5-nm-thick polymeric shell. The MMIP-based electrochemical sensing platform achieved a limit of detection of 0.38 pM within a linear detection range of 0.38–380 pM, indicating high affinity (dissociation constant KD = 1.6 pM) for IL-6 protein in 50% diluted serum samples. Moreover, comparative investigations with the non-imprinted control polymer demonstrated an imprinting factor of 4, confirming high selectivity. With multifunctional features, including fluorescence, magnetic properties, and target responsiveness, the synthesized MMIPs hold significant potential for application in various sensor techniques as well as imaging.

B-336 Analytical verification of the EXENT® solution for the identification an quantitation of monoclonal immunoglobulins by mass spectrometry

Abstract Background Recently a new mass spectrometry (MS) technology, EXENT® solution (The Binding Site, part of Thermo Fisher Scientific), for the identification and quantification of IgG, IgA and IgM intact monoclonal immunoglobulins (M-Ig) has been launched in the European markets. In this study we present the results of onsite verification study performed in our hospital. Methods Study design, using patients serum samples, including data analysis were based on the Clinical & Laboratory Standards Institute (CLSI). Relevant standards used were: Precision-CLSI EP15-A3:2014, Linearity-EP06 ED2:2020 and Comparison - EP09C ED3:2018. Samples were automatically prepared on the EXENT-iP®500 liquid handler, sample spots were analysed using the EXENT-iX®500 MALDI-TOF mass spectrometer. Light chain spectral peaks were identified by EXENT-iQ® software and then quantified indirectly in combination with total immunoglobulins concentrations by immunoturbidimetry in the Optilite® platform (The Binding Site, part of Thermo Fisher Scientific). We compared the results of MS with capillary electrophoresis (CZE) (IgG: n=82; IgA n=19; IgM n=19) and immunofixation (sIFE) (n=541). Results Precision was, for IgG with concentrations between 50.77 g/L and 3.51 g/L of 4.14% and 4.47%, for IgA between 61.88g/L and 5.02 g/L of 1.42% and 7.17%, and for IgM between 68.32 g/L and 7.30 g/L of 5.32% and 8.44%, respectively. Deviation from linearity of this assay, using serially diluted serum samples over the following ranges for IgG (65.18 g/L - 0.23 g/L), IgA (48.91 g/L - 0.26 g/L) and IgM (49.65 g/L -0.23 g/L) was <10% for all isotypes. M-Ig concentration comparison for specimens with detectable IgG, IgA or IgM M-Ig with CZE, assessed by Passing Bablok regression was IgG g/L MS = -0.9756 + 1.523x CZE g/L (slope -2.058 to -0.3322 and intercept 1.331 to 1.679), IgA g/L MS = -2.114 + 1.646x CZE g/L (slope -12.50 to -0.5059 and intercept 1.105 to 3.296), IgM g/L MS = -1.886 + 1.935x CZE g/L (slope -5.205 to -0.3571 and intercept 1.239 to 2.462). All samples MS g/L = -1.182 + 1.556x CZE g/L (n=120). Agreement between EXENT/IFE was of 99.1% for IgGK(209/211), 97.2% IgGL(105/108), 100% AK(97/97), 100% AL(51/51), 100% MK(6/6), 100% ML(3/3), 89.2% free K(33/37), 89.3% free L(25/28), 66,7% non-secretory(4/6). Discordances intact immunoglobulin was due to samples with two M-Ig, where IFE wasn’t able to identify the major clone, for free K, free L was due to EXENT’s ability to identify the heavy chain that matched with the light chain, and for non-secretory patients due to EXENT was able to identify one M-Ig in 2/6 samples. Conclusions These on-site verification results demonstrate that the EXENT solution has a wide measuring interval, to detect M proteins with very low concentrations and to provide stable and reproducible performance with a high level of accuracy for the detection and typing of monoclonal immunoglobulins. However, the bias observed against CZE to quantify M-Ig would be justified for the lowest CZE sensitivity, CZE variability and also due to the CZE quantification uses the total protein concentration to quantify M-Ig.

B-021 Impact of Assay Conditions on FT4 Measurement Accuracy of Equilibrium Dialysis LC/MS/MS Clinical Assay

Abstract Background Reliable FT4 measurements are vital for accurate diagnosis and effective management of thyroid disorders. However, currently used FT4 immunoassays (IAs) lack standardization, leading to significant variations between platforms. Equilibrium dialysis (ED) coupled with LC-MS/MS analysis, considered the gold standard for FT4 measurement, has been adopted as a reference measurement procedure (RMP) for IA standardization. This study aimed to establish a high-throughput version of the routine clinical FT4 assay based on ED-LC/MS/MS technology and assess the impact of altered assay conditions on measurement accuracy. Methods A commercially available micro-ED plate was employed for ED-LC/MS/MS analysis. FT4 extraction from the dialysate was performed by utilizing a 96-well C18 SPE plate, followed by FT4 quantitation via LC-MS/MS in positive ion mode. Certified primary reference material was used to calibrate the assay. To evaluate assay robustness, the effects of minor method modifications were studied, including incubation time of ED, serum/buffer ratios in ED inserts, ED incubation temperature, shaker speed during ED, serum sample dilution ratio, and dialysate sample dilution ratio. Additionally, short-term and long-term stability of serum FT4 were assessed. Results The developed ED-LC/MS/MS assay successfully resolved T4, T3, reverse triiodothyronine (rT3), and other interferences within 4 minutes using C18 chromatography. The assay's linear range (0.5-100 pg/mL) encompassed clinically relevant FT4 concentrations across hypo- and hyperthyroid patient samples. Excellent agreement was observed between the routine assay based on ED-LC/MS/MS and CDC RMP with a Deming regression slope near 1 (95%CI: 0.87-1.06). Furthermore, serum/buffer ratios from 1:1 to 1:2.5 in ED inserts, shaker speeds from 75 to 150 rpm, and ED incubation times from 16 to 20 hours did not significantly affect FT4 measurement. However, elevated ED temperature (38°C) led to a more than 10% increase in FT4 values. Serum FT4 remained stable when exposed to room temperature for 12 hours, five freeze-thaw cycles, and -70°C storage for 1.5 years. Additionally, dilution of sera by up to 3-fold before ED did not alter FT4 measurement results. Diluting dialysate samples at least 3 times prior to SPE extraction proved accurate for samples exceeding the calibration curve range. Conclusions In conclusion, we successfully developed a routine clinical FT4 assay based on ED-LC/MS/MS that generates data comparable to the RMP. The assay exhibits robustness, with several altered conditions not significantly impacting measurement accuracy. This high-throughput and accurate assay demonstrates suitability for both clinical laboratories and large-scale epidemiological studies. Disclaimer: The findings and conclusions in this report are those of the author(s) and do not necessarily represent the official position of the Centers for Disease Control and Prevention/the Agency for Toxic Substances and Disease Registry. Use of trade names is for identification only and does not imply endorsement by the Centers for Disease Control and Prevention, the Public Health Service, and the US Department of Health and Human Services.

Target-responsive triplex aptamer nanoswitch enables label-free and ultrasensitive detection of antibody in human serum via lighting-up RNA aptamer transcriptions

Accurate and sensitive monitoring of the concentration change of anti-digoxigenin (Anti-Dig) antibody is of great importance for diagnosing infectious and immunological diseases. Combining a novel triplex aptamer nanoswitch and the high signal-to-noise ratio of lighting-up RNA aptamer signal amplification, a label-free and ultrasensitive fluorescent sensing approach for detecting Anti-Dig antibodies is described. The target Anti-Dig antibodies recognize and bind with the nanoswitch to open its triplex helix stem structure to release Taq DNA polymerase and short ssDNA primer simultaneously, which activates the Taq DNA polymerase to initiate downstream strand extension of ssDNA primer to yield specific dsDNA containing RNA promoter sequence. T7 RNA polymerase recognizes and binds to these promoter sequences to initiate RNA transcription reaction to produce many RNA aptamer sequences. These aptamers can recognize and bind with Malachite Green (MG) dye specifically and produce highly amplified fluorescent signal for monitoring Anti-Dig antibodies from 50 pM to 50 nM with a detection limit down to 33 pM. The method also exhibits high selectivity for Anti-Dig antibodies and can be used to discriminate trace Anti-Dig antibodies in diluted serum samples. Our method is superior to many immunization-based Anti-Dig antibody detection methods and thus holds great potential for monitoring disease progression and efficacy.

Highly catalytic sulfur-doped and bimetal-coordinated CoFe(CN)5NO nanoparticles coupled with PER/HCR amplification cascades for sensitive electrochemical aptamer luteinizing hormone assay

Sensitive monitoring of luteinizing hormone (LH), a glycoprotein that regulates the synthesis of regulatory steroid hormones, can facilitate the diagnosis of various reproductive diseases. In this work, a new and highly catalytic Sulfur-doped and bimetal-coordinated CoFe(CN)5NO (denoted as S–CoFe(CN)5NO) nanoparticles are synthesized. Such material is further used to construct high performance sensing interface and coupled with primer exchange reaction (PER) and hybridization chain reaction (HCR) amplification cascades for sensitive electrochemical aptamer-based LH assay. Target LH molecules bind aptamer sequences in DNA duplex probes to liberate ssDNA strands, which initiate subsequent PER/HCR amplification cascades for the capture of many ferrocene (Fc)-tagged DNAs on sensing interface. S–CoFe(CN)5NO subsequently leads to catalytic oxidation of these Fc tags for yielding substantially magnified currents for realizing ultrasensitive assay of LH with the detection limit of 0.69 pM in range from 5 pM to 10 nM. Owing to the high specificity of aptamer, such sensor has high selectivity and can achieve low levels of LH assay in diluted serum samples. With the successful demonstration for detecting trace LH, such sensor can be easily extended as a universal aptamer-based electrochemical sensing method for monitoring various target analytes in the biomedical and biological fields.

Synthetic peptides-based SPR biosensor evaluation towards canine Visceral leishmaniasis diagnosis: A simple and effective approach

A vital component of surface plasmon resonance (SPR) biosensors is the recognition element. It generates the sensor selectivity towards the target biomarker and plays an important role in defining the sensor’s analytical sensitivity. In serological diagnostics, recombinant proteins (purified antigens) have presented better selectivity compared to soluble crude antigens in SPR immunosensors. However, recombinant DNA technology to obtain proteins is only explored by specialized laboratories, and commercial recombinant proteins still have a high cost. Thus, with the aim of developing a much simpler and lower-cost method, we investigated the prospect of using synthetic peptides as recognition elements for constructing an SPR biosensor. Two synthetic peptides, named PEP13 and PEP16, were tested for the serological diagnosis of Canine Visceral Leishmaniasis (CVL), an endemic neglected disease that affects humans and dogs worldwide. Between the two peptides tested, PEP13 was more sensitive when evaluating its responses against purified antibodies in buffer solution (LOD = 1.05 nmol L-1), and it also discriminated the response better when applied in diluted serum samples of infected dogs compared to diluted healthy dogs’ samples. For this reason, the PEP13 immunosensor was used to analyze canine serum samples, precisely identifying all the positive (n = 7) and negative (n = 7) CVL cases (p = 0.00136) in less than 12 min. In brief, this study explored promising biostructures through a simple and fast methodology for serological diagnosis, addressing the suitability of synthetic peptides for use in biosensors in the urgent field of neglected diseases.

A simple chemiluminescent method for the quantification of exosomes based on horseradish peroxidase adsorbed on two-dimensional nanomaterials

The development of simple methods for the isolation and quantification of exosomes in biological samples is important. By using the typical two-dimensional (2D) nanomaterials, graphene oxide (GO), the present work first studied the interaction of liposomes with the nanocomposites formed by adsorbing HRP on the GO surface and found the presence of liposomes led to the release of HRP from the GO surface to the solution phase triggering the luminol-H2O2 chemiluminescence (CL) reaction to emit light. Benefiting from the similarity of exosomes to liposomes in both composition and morphology aspects, the GO-HRP nanocomposites with a mass ratio of 120:1 and 160:1 were employed for the quantitative detection of exosomes in 100-fold diluted serum samples. The whole detection process took about 15 min and as low as 3.2 × 102 particles μL−1 of exosomes could be sensitively detected. In addition to GO-HRP nanocomposites, the CL responses of other nanocomposites obtained from adsorbing HRP on other 2D nanomaterials such as layered MoS2 for exosomes were also tested. MoS2-HRP exhibited similar behavior and the LODs for the detection of exosomes were 5.8 × 102 particles μL−1. The proposed assays were a biomarker-independent quantitative method that achieved the quantification of exosomes in serum samples directly without an isolation process.

To Estimate and Compare the Level of Serum Immunoglobulin A in Patients of Oral Submucous Fibrosis, Leukoplakia, and Control Group.

To estimate and compare the level of serum immunoglobulin A in patients of oral submucous fibrosis (OSMF), leukoplakia, and control group. OSMF is a well-recognized potentially malignant condition in the oral cavity, and a transformation rate as high as 7.6% over a period of 10 years has been reported from India. Leukoplakia has evolved as a clinico-pathologic concept over many years, with the current clinical designation being accepted worldwide. Quantia IgA is a turbidometric immunoassay for the detection of IgA in human serum and is based on the principle of agglutination reaction. The test specimen is mixed with the activation buffer (R1) and then with antihuman IgA reagent (R2) and allowed to react. For the estimation of serum IgA, 500 μl of Quantia IgA activation buffer R1 is taken in a clean test tube. Serum sample is diluted in the ratio of 1:5 with normal saline. Ten microliters of diluted serum sample are added to R1. After incubation for 10 min, 50 μl of R2 is added, which is Quantia IgA antihuman IgA reagent to the sample, and the reading is recorded at wavelength 340 nm at 37°C. The level of IgA will be estimated by a semi-automatic biochemical analyzer machine and then data analysis will be done. The immunologic alterations observed by us in OSMF and leukoplakia are almost similar or nearer to the alterations observed in oral cancer, so it is reasonable to assume that OSMF and leukoplakia can be an intermediate stage in the transformation process of a normal cell to oral malignancy. Prevention and early detection is the best way to reduce the incidence and mortality of premalignant disorders and oral cancer. There are many ways and methods of early detection of premalignant disorders and oral cancer, and the immunological method is one of them.

Development of an advanced liquid chromatography–tandem mass spectrometry measurement system for simultaneous sphingolipid analysis

Mass spectrometry-based lipidomics approaches offer valuable tools for the detection and quantification of various lipid species, including sphingolipids. The present study aimed to develop a new method to simultaneously detect various sphingolipid species that applies to diverse biological samples. We developed and validated a measurement system by employing a single-column liquid chromatography-mass spectrometry system utilizing a normal-phase separation mode with positive ionization. The measurement system provided precision with a coefficient of variant below 20% for sphingolipids in all types of samples, and we observed good linearity in diluted serum samples. This system can measure the following sphingolipids: sphingosine 1-phosphate (S1P), sphingosine (Sph), dihydroS1P (dhS1P), dihydroSph (dhSph), ceramide 1-phosphate (Cer1P), hexosylceramide (HexCer), lactosylceramide (LacCer), dh-ceramide, deoxy-ceramide, deoxy-dh-ceramide, and sphingomyelin (SM). By measuring these sphingolipids in cell lysates where S1P lyase expression level was modulated, we could observe significant and dynamic modulations of sphingolipids in a comprehensive manner. Our newly established and validated measurement system can simultaneously measure many kinds of sphingolipids in biological samples. It holds great promise as a valuable tool for laboratory testing applications to detect overall modulations of sphingolipids, which have been proposed to be involved in pathogenesis processes in a series of elegant basic research studies.

In vitro SELEX and application of an African swine fever virus (ASFV) p30 protein specific aptamer

The African swine fever virus (ASFV) has caused severe economic losses in the pig industry. To monitor ASFV spread, the p30 protein has been identified as an ideal infection marker due to its early and long-term expression during the ASFV infection period. Timely monitoring of ASFV p30 enables the detection of ASFV infection and assessment of disease progression. Aptamers are an outstanding substitute for antibodies to develop an efficient tool for ASFV p30 protein detection. In this study, a series of aptamer candidates were screened by in vitro magnetic bead-based systematic evolution of ligands by exponential enrichment (MB-SELEX). An aptamer (Atc-20) finally showed high specificity and affinity (Kd = 140 ± 10 pM) against ASFV p30 protein after truncation and affinity assessment. Furthermore, an aptamer/antibody heterogeneous sandwich detection assay was designed based on Atc20, achieving a linear detection of ASFV p30 ranging from 8 to 125 ng/ml and a detection limit (LOD) of 0.61 ng/ml. This assay showed good analytical performances and effectively detected p30 protein in diluted serum samples, presenting promising potential for the development of ASFV biosensors.

Safety Assessment: a Comparative Analysis of Quantitative Content of Bacterial Endotoxins and Evaluation of Pyrogenicity of the Kazakhstan Vaccine QazCovid-in® against COVID-19.

We measured the levels of bacterial endotoxins in the bulk vaccine product (BVP) and finished vaccine QazCovid-in® and evaluated the effect of aluminum hydroxide (adjuvant) on the results of LAL test and pyrogenicity of samples in vivo (in rabbits receiving intravenous injection into the marginal ear vein). Administration of BVP with LPS resulted in a dose-dependent increase in body temperature in rabbits similar to that caused by LPS alone, which suggests that aluminum hydroxide in the vaccine did not affect the pyrogenic response in rabbits. Moreover, the LAL test showed that the aluminum hydroxide did not hinder LPS activity after serial dilution of samples.

Development and application of an indirect ELISA and nested PCR for the epidemiological analysis of Klebsiella pneumoniae among pigs in China.

Klebsiella pneumoniae (K. pneumoniae) is an important opportunistic and zoonotic pathogen which is associated with many diseases in humans and animals. However, the pathogenicity of K. pneumoniae has been neglected and the prevalence of K. pneumoniae is poorly studied due to the lack of rapid and sensitive diagnosis techniques. In this study, we infected mice and pigs with K. pneumoniae strain from a human patient. An indirect ELISA was established using the KHE protein as the coating protein for the detection of K. pneumoniae specific antibody in clinical samples. A nested PCR method to detect nuclei acids of K. pneumoniae was also developed. We showed that infection with K. pneumoniae strain from a human patient led to mild lung injury of pigs. For the ELISA, the optimal coating concentration of KHE protein was 10 µg/mL. The optimal dilutions of serum samples and secondary antibody were 1:100 and 1:2500, respectively. The analytical sensitivity was 1:800, with no cross-reaction between the coated antigen and porcine serum positive for antibodies against other bacteria. The intra-assay and inter-assay reproducibility coefficients of variation are less than 10%. Detection of 920 clinical porcine serum samples revealed a high K. pneumoniae infection rate by established indirect ELISA (27.28%) and nested PCR (19.13%). Moreover, correlation analysis demonstrated infection rate is positively correlated with gross population, Gross Domestic Product (GDP), and domestic tourists. In conclusion, K. pneumoniae is highly prevalent among pigs in China. Our study highlights the role of K. pneumoniae in pig health, which provides a reference for the prevention and control of diseases associated with K. pneumoniae.

CRISPR/Cas12a linked sandwich aptamer assay for sensitive detection of thrombin

BackgroundThrombin is a serine protease and hemostasis regulator with multiple functions and recognized as an important biomarker for diseases, and sensitive detection of thrombin is of significance for clinical diagnostics and disease monitoring. Recently, the target-triggered nonspecific single-stranded deoxyribonuclease activity of CRISPR/Cas system is discovered, making it become a powerful tool in assay developments due to the ease of signal amplification. In the short period of development, many CRISPR based nucleic acid detection methods have already played a critical role in clinical diagnostics. However, the application of CRISPR/Cas system for protein biomarkers remains limited. ResultsHere we describe a CRISPR/Cas12a linked sandwich aptamer assay for detection of thrombin, which was based on the formation of a sandwich complex of target by using a capture aptamer or antibody coated on the microplate and a well-designed detection DNA strand. The detection DNA strand contained an anti-thrombin aptamer and an active DNA of Cas12a, thus the sandwich complex was labeled with the active DNA. The active DNA triggered activity of Cas12a in indiscriminately cleaving fluorophore and quencher labeled DNA reporters, causing significant fluorescence increase. Our method enabled sensitive detection of thrombin down to 10 pM, and it showed high selectivity for thrombin. The assay exhibited good performance in diluted serum samples, demonstrating the applicability for thrombin analysis in the real media. SignificanceThis assay combines the merits of high affinity of aptamer, trans-cleavage activity of Cas12a, high selectivity of sandwich format analysis, and high-throughput detection of microplate assay, and it shows promise in applications.

Five-Year Analysis of Anti-Nuclear Antibody (ANA) and Extractable Nuclear Antigen Antibody (anti-ENA) Immunoblot Test Results

Autoimmune disorders may cause systemic disease in which multiple organs are affected or may also be localized as single-organ involvement. Various ancillary immunological tests used in cases where systemic involvement is observed, and differential diagnosis becomes difficult or non-specific symptoms are observed. For this purpose, investigation of antinuclear antibodies (ANA) and antibodies against extractable nuclear antigens (ENA) in patient serums using various methods is a guide for clinicians. In this study, we aimed to evaluate the effectiveness of ANA and ENA antibody tests by examining the test results, the patients’ demographic data, and the clinics that ordered the tests. In the study, a retrospective review of the results of 37,791 ANA tests studied on serum samples sent to the medical microbiology laboratory of our hospital from various clinics between January 2018 and December 2022 is presented. ANA tests were resulted semi-quantitatively by evaluating the slides prepared using diluted serum samples at a ratio of 1:100 and HEp-2 cells and monkey liver tissue under a fluorescent microscope with the indirect immunofluorescence (IIF) antibody method. Of the patients who requested ANA test, 11,604 (30.7%) were male and 26,187 (69.3%) were female. The mean ages were 43.46 (0-94) and 46.94 (0-96), respectively. Of the samples tested, 18.8% (7,107/37,791) were evaluated as ANA positive. The ANA positivity rate was significantly higher in women (24.8%) than in men (19.7%) (p<0.001). ANA positive samples were re-tested using the anti-ENA immunoblot method. Of the 7,107 ANA-positive patients, 3,809 (53.6%) were found to be anti-ENA positive for at least one specific antibody, while 3,298 (46.4%) were negative. In the anti-ENA profile, antibodies against 11 different antigen types were screened and the most frequently detected antibody was anti-SS-A (22.7%), and this was followed by anti-centromere (12.8%), anti-RNP (10.2%), and anti-dsDNA (9.7%) antibodies. Immunoblot-based anti-ENA tests provide specific information for the diagnosis of autoimmune diseases. In our study, the fact that anti-ENA tests were negative in a significant part of ANA positive patients indicates the inadequacy of the immunoblot test panel and reveals the need for immunoblot tests with wider antibody diversity or any alternative methods.

B-064 Analytical and Diagnostic Performances of Anti-Integrin Antibody System in Inflammatory Bowel Disease

Abstract Background Anti-integrin αvβ6 autoantibodies may have value in the diagnosis of Inflammatory Bowel Disease (IBD) and more specifically Ulcerative Colitis (UC). Our objective was to establish the analytical and diagnostic performance of this novel autoantibody system in distinguishing IBD from a group of Normal Healthy Volunteers (NHV) and UC from Crohn’s Disease (CD). Methods All testing was performed using a Tecan Evo200 equipped with 8-channel and 96-channel arms, plate washer, and plate reader. The ELISA assay used calibration controls and diluted serum samples added to ELISA plates coated with Integrin αvβ6 antigen. Anti-human IgG horseradish peroxidase complexed with Anti-integrin αvβ6 antibodies was detected using colorimetry. The analytical performance was evaluated by using repeatability and reproducibility with coefficient of variation (CV) calculated. The diagnostic performances were evaluated by testing specimens collected from consented subjects available from a biobank in autoimmune gastrointestinal diseases. This consisted of 60 NHV, and 187 IBD (91 CD and 96 UC). Sensitivity, specificity, diagnostic odds ratio (OR), positive and negative predictive values (PPV and NPV) were estimated. Results Intra-assay CVs ranged from 1.5% to 5.2% over dynamic range. Inter-assay CVs were below 10%. Median titers were below 5 U/mL for NHV and CD, and 26 U/mL for UC (Fig. 1). Cutoff of 5 U/mL was 100% specific for UC and CD. Sensitivity for UC and CD was 80% (77/96), and 15% (14/91), respectively. Diagnostic Odds Ratio yielded 22.3-fold (95% CI: 10.5–47.5) higher likelihood of UC than CD in the presence of Anti-integrin αvβ6 titers above cutoff, with PPV and NPV values of 84% (76%-90%) and 81% (74%–87%), respectively (50% pre-test). Conclusion We have established the analytical performance of an Anti-integrin αvβ6 autoantibody ELISA assay. These preliminary data suggests that the Anti-Integrin αvβ6 autoantibody system is highly specific for UC with high sensitivity and specificity.

Improvement of serum sample preparation and chromatographic analysis of nusinersen used for the treatment of spinal muscular atrophy

The present investigation showed that each of the three different liquid chromatography modes may be successfully used for the qualitative analysis of nusinersen metabolites in a patient's serum sample extract. However, the smallest number was detected by the hydrophilic interaction liquid chromatography. Furthermore, the response of the mass spectrometry is several times greater for ion pair chromatography compared to reversed-phase one. Various extraction methods were applied for the extraction of nusinersen metabolites from serum. Silica with bonded capture strand for hybridization was applied, as well as silica modified with amino and carboxyl groups for dispersive solid phase extraction. The hybridization allows selective extraction of nusinersen analogs, however, it fails in extraction of short metabolites. On the contrary, the efficiency of weak ion exchange-based extraction was high, even in the case of the direct extraction of nusinersen metabolites from diluted serum samples without a protein removal step. The new material is a great alternative to liquid-liquid extraction and hybridization for the isolation of nusinersen metabolites from the serum of patients with spinal muscular atrophy (SMA). It is a very simple method that uses a low concentration of organic salt and desorption occurs after changing its pH. Such complex studies were performed for the first time for nusinersen metabolites extracted from the serum of SMA patients treated with Spinraza.

Dot-ELISA based on recombinant Hypodermin C of Przhevalskiana silenus for field diagnosis of goat warble fly infestation.

Goat warble fly infestation (GWFI) is an economically important myiasis caused by larvae of Przhevalskiana silenus (Diptera, Oestridae), prevalent in countries of the Mediterranean Basin and Indian subcontinent. GWFI is characterized by the presence of subcutaneous warbles at the lumbar and sacral region of dorsum in the infested animal. The early larval instars (L1 and L2) remain inaccessible to physical detection due to their small size and subcutaneous presence thus causing hindrance in the diagnosis. The objective of present study was to develop a field applicable early diagnostic intervention for GWFI monitoring and prophylactic management for effective control of the disease. Recombinant Hypodermin C (rHyC) antigen of P. silenus was expressed in Escherichia coli. The purified protein was used for optimizing dot-ELISA in a checkerboard titration using goat warble fly infested serum as known positive. The optimized assay was further tested for lower temperature (18°C) and incubation time (30 min). The optimized assay was assessed for inter-rater reliability and field samples. The optimized conditions require 188 ng of protein/dot, 1:800 dilution of serum sample, 1:4000 dilution of anti-goat IgG conjugate and 5% skim milk powder in phosphate buffer saline as blocking buffer. The assay was found to have a diagnostic sensitivity and specificity of 97.3% and 95.8%, respectively. The inter-rater reliability of dot ELISA with rHyC indirect ELISA was found to be almost perfect with a Cohen's kappa index of 0.973. Further testing at ambient temperature (18°C) and shorter incubation steps (30 min) supported suitability of the assay for field diagnosis of GWFI. The present study provides the first report of a sensitive and specific dot-ELISA for early diagnosis of GWFI which is rapid and cost effective. The test may provide an effective field applicable tool for sustainable control of GWFI.

A particle-based microfluidic fluorescent lateral flow assay for rapid and sensitive detection of SARS-CoV-2 antibody

Prompt isolation and treatment of patients are effective measures for preventing the spread of infectious diseases. Therefore, a sensitive and early diagnostic test is crucial for controlling pandemics. Herein, we present a tubeless integrated microfluidic lateral flow immunoassay chip for rapid and sensitive detection of SARS-CoV-2 antibodies. Fully automated chip operation is demonstrated by incorporating a dry reagent storage chamber, staggered herringbone micromixer, mobile particle-based detection zone, and spontaneous liquid transportation mechanism. The effects of the position and number of dry reagent patterns on assay time and detection sensitivity were investigated. Under laminar flow conditions, a staggered herringbone mixer was used to achieve rapid and complete mixing of the sample and reagent. In addition, we found that the volumetric flux of the sample and reagent complex over the surface of the particles is a crucial factor for improving detection sensitivity. The limit of detection of an anti-SARS-CoV-2 spike protein monoclonal antibody was determined to be 10.35 ng/mL in assay buffer with a total assay time of 15 min. In the analyte concentration range of 15.625–1000 ng/mL, the linearity and spike-recovery of the assay on serially diluted serum samples were excellent (R2 ≈ 0.99 and recovery rate of 82.9–112 %). The integrated microfluidic immunoassay chip demonstrates rapid and simple operation, which is promising for the diagnosis and monitoring of SARS-CoV-2 antibodies.

A butterfly-shaped ESIPT dye for pattern recognition of metal ions

Accurate monitoring of versatile metal ions in complicated environmental or biological samples is meaningful for avoiding heavy metal poisoning accidents. To reduce the production cost of a metal ion sensor array, we intended to develop solvent directed fluorescent sensor arrays on the basis of a single dye molecule. Herein, two fluorescent ESIPT dyes named SC1 and SC2 were designed by incorporating different numbers of benzothiazole units as the metal ion binding site. It was discovered that SC1 was highly selective toward Zn2+ in all tested solvents, while a solvent directed cross-responsive sensing behavior was observed for SC2. Therefore a three-component sensor array SC2SA was readily constructed by simply dissolving SC2 in common solvents including ethanol, DMF and DMSO. The sensor array was determined to be accurate for semi-quantitative discrimination and detection of Zn2+, Cd2+, Mg2+, Fe2+ and Fe3+ in diluted serum media. Moreover, sensor array SC2SA was also successfully applied for differentiating Zn2+/Cd2+ and Fe2+/Fe3+ in pure and mixed forms and reliable identification of unknown Zn2+, Cd2+ and Mg2+ in diluted serum samples.