Immunoassay System For Detection Research Articles

910. Evaluation of an Enzymatic Immunoassay System for Detection and Differentiation of Rat Hepatitis E Virus Infection in Humans

BackgroundPreviously, hepatitis E in humans was believed to be caused exclusively by species A variants of the Orthohepevirus genus (HEV-A) of the family Hepeviridae. However, we have previously demonstrated that Orthohepevirus species C (HEV-C), also known as rat hepatitis E virus, also causes hepatitis in humans. Due to high sequence divergence between HEV-A and HEV-C, serological tests based on HEV-A are often insensitive for HEV-C diagnosis. Therefore, we developed an enzymatic immunoassay (EIA) system for differentiating HEV-A and HEV-C antibody signatures in patient sera. MethodsHEV-A and HEV-C peptide homologs spanning the entire immunogenic E2s region of HEV ORF2 capsid protein were expressed in E. coli. These peptides, HEV-A4 p239 and HEV-C p241, form virus-like particles (figure 1). Both peptides were coated in separate 96-well plates. Sera obtained from patients with RT-PCR confirmed acute HEV-A infection (n = 54), HEV-C infection (n = 15), and uninfected HEV seronegative controls (n = 126) were tested in parallel in HEV-A4 p239 and HEV-C p241 EIAs for respective IgG antibodies. Sample optical densities (ODs) were divided by mean OD + 3SD of the seronegative controls to generate signal/noise (S/N) ratios. S/Ns marking positivity in either assay were determined by ROC analysis. An algorithm for assay interpretation was developed (table 1) and the performance of this algorithm was measured against the RT-PCR gold standard. HEV-A4 p239 and HEV-C1 p241 virus like particles Transmission electron microscopy images of the two peptides used in this studyDiagnostic testing algorithm interpretation Interpretation of results of testing using parallel enzymatic immunoassaysResultsUsing cutoffs determined by ROC analysis (figure 2), HEV-A4 p239 and HEV-C p241 EIAs detected species-specific antibody responses well (sensitivity: 92.6% and 80% respectively) and were specific (92.9% and 98.3% respectively). The DC was 100% congruent with HEV-C RT-PCR and 88.9% congruent with HEV-A RT-PCR in RT-PCR positive samples. Incorporating all three cutoffs into the algorithm, we derived a 3×3 confusion matrix of RT-PCR sample assignation vs EIA algorithm classification (table 2). The Cohen’s κ value was 0.883 indicating excellent inter-rater reliability. ROC analysis for determining S/N cutoffs Curve for A/A represents analysis for HEV-A4 p239 EIA. Curve for C/¬C represents analysis for HEV-C p241 EIA. Curve for r(C/A) represents analysis for the differentiating ratio.3×3 confusion matrix comparing sample assignations by RT-PCR vs. EIA algorithm ConclusionA parallel EIA system accurately differentiated HEV-A and HEV-C serological signatures in acute patient sera. This method can now be applied to seroprevalence studies to determine seroprevalence of rat hepatitis E in human populations.Disclosures Siddharth Sridhar, FRCPath, Abbott (Other Financial or Material Support, Speaker’s honoraria)

Procalcitonin measurement using antibody-conjugated fluorescent microspheres distinguishes atypical bacterial meningitis from viral encephalitis in children

Examination of cerebrospinal fluid in atypical bacterial meningitis (ABM) is similar to that of viral encephalitis (VE), so ABM can easily be misdiagnosed as VE, which can delay diagnosis and treatment. We developed a simple, rapid hand-held lateral flow immunoassay detection system based on fluorescent microspheres (FMS) for procalcitonin (PCT) detection, which provides an indicator to differentiate between ABM and VE. With this novel method, the antigen-antibody reaction systems involve different species, making the test strips more stable than those utilizing one species. The strips exhibited a wide dynamic range (0.04–50 ng/mL) and good sensitivity (0.03 ng/mL). The function of PCT in the identification of ABM and VE in children was further studied. A significant difference in PCT levels was observed between the ABM and VE groups (P = 0.00) and between the ABM and the normal control groups (P = 0.00). PCT levels were not significantly different between the VE and normal control groups (P = 0.30). The area under the receiver operating characteristic curve of PCT for the diagnosis of ABM was 0.95. These findings collectively indicate the usefulness of the PCT detection method based on FMS for clinically differentiating between ABM and VE.

Abstract 3180: Development of a fully automated immunoassay system for detection of serum NY-ESO-1 and XAGE1 antibodies as biomarkers predicting the clinical efficacy of anti-PD-1 therapy

Abstract Background: Programmed cell death-1 (PD-1) and PD-ligand1 (PD-L1) inhibitors have become standard therapies for advanced non-small-cell lung cancer (NSCLC) due to prolonged patient survival. However, extended survival with anti-PD-1/PD-L1 therapy is limited to a small subset of NSCLC patients. Therefore, predictive biomarkers are needed to determine which patients will benefit. Recently, we reported that serum antibodies (Abs) against NY-ESO-1 and/or XAGE1 cancer-testis antigens are useful biomarkers that predict the clinical efficacy of anti-PD-1 monotherapy for NSCLC. The serum Abs were measured with enzyme-linked immunosorbent assay (ELISA); however, the ELISA method has several technical limitations for clinical applications. To overcome limitations in Ab measurement, we have developed a fully automated immunoassay system (HISCLTM) using chemiluminescent magnetic immunoassay technology. HISCLTM is widely used in many clinical fields because it is superior to ELISA due to its rapid reaction (17 minutes), wide dynamic ranges, and high reproducibility. Patients and Methods: Sera of NSCLC patients were obtained before anti-PD-1 monotherapy (nivolumab or pembrolizumab) as a part of standard care. NY-ESO-1/XAGE1 serum Abs were measured by ELISA and HISCLTM and their clinical performance was evaluated. In HISCLTM system, serum Abs were captured by magnetic beads coated with antigens, and the antigen-Ab complex was incubated with anti-human IgG conjugated with alkaline phosphatase. The chemiluminescent intensity acquired was then used for analyses. Results: Twenty-eight NSCLC patients were selected as the initial cohort for this study. Half of the patients responded to anti-PD-1 monotherapy and the remaining half did not. There were strong correlations between HISCLTM chemiluminescence signal intensity and titers of NY-ESO-1 and XAGE1 serum Abs in ELISA (r>0.93 for both). Twelve of thirteen Ab-positive patients in HISCLTM responded to anti-PD-1 therapy, while only two of fifteen Ab-negatives did (92% vs. 13%, p<0.001). Notably, the signal intensities in HISCLTM correlated well with tumor reduction rates after anti-PD-1 therapy. Serum NY-ESO-1/XAGE1 Ab-positivity in HISCLTM predicted efficacy with an AUC of 0.898, resulting in a sensitivity of 86% and specificity of 93%. Conclusions: We have developed a rapid and highly reproducible immunoassay system for detecting the serum NY-ESO-1 and XAGE1 Abs as predictive biomarkers of clinical benefits with anti-PD-1 monotherapy in NSCLC. In the future, our system would be examined in large-scale clinical trials for various other cancers. Citation Format: Yumiko Sakai, Hirotaka Abo, Kanako Sakaeda, Yusuke Atarashi, Nobuyuki Ide, Takaaki Yamaoka, Koji Kurose, Kenta Noda, Toshiyuki Sato, Toru Oga, Mikio Oka. Development of a fully automated immunoassay system for detection of serum NY-ESO-1 and XAGE1 antibodies as biomarkers predicting the clinical efficacy of anti-PD-1 therapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3180.

An Oscillator-Based CMOS Magnetosensitive Microarray Biochip With On-Chip Inductor Optimization Methodology

This paper presents a high sensitivity oscillator-based CMOS magnetosensitive microarray biochip to detect clinical biomarkers in human samples and provide accurate information to diagnose human diseases. To increase the detection sensitivity, dynamic detecting range, and target number, a general methodology of designing an on-chip inductor with high quality factor, moderate size, and uniform surface magnetic field distribution is proposed, which meets all the electrical, magnetic and actual application requirements simultaneously, and also provides useful guidance for other oscillator-based magnetosensitive biochip design. As an implementation example, a microarray biochip with 14 sensor units in an area of $2.3 \times 1.9\,\,\text {mm}^{2}$ is implemented in a 180-nm RF CMOS process to verify the biological sensing scheme and the correctness of the proposed theory and simulation. Finally, experiments using magnetic nanoparticles and cTnI were performed on our designed immunoassay detection system, which integrates the oscillator-based biosensor, microfluidic chip and immunoassay reagent into one platform for the first time, and the experimental results show a limit of detection of 50 pg/mL corresponding to 2.17 pM with a biological dynamic detecting range of 60 dB, proving the suitability of the biochip for quantitative analysis in practical medical diagnostics.

A novel monoclonal antibody suitable for the detection of leukotriene B4

Leukotriene B4 as an inflammatory mediator is an important biomarker for different respiratory diseases like asthma, chronic obstructive pulmonary disease or cystic lung fibrosis. Therefore the detection of LTB4 is helpful in the diagnosis of these pulmonary diseases. However, until now its determination in exhaled breath condensates suffers from problems of accuracy. Reasons for that could be improper sample collection and preparation methods of condensates and the lack of consistently assay specificity and reproducibility of the used immunoassay detection system. In this study we describe the development and the characterization of a specific monoclonal antibody (S27BC6) against LTB4, its use as molecular recognition element for the development of an enzyme-linked immunoassay to detect LTB4 and discuss possible future diagnostic applications.

Improvement of immunoassay detection system by using alternating current magnetic susceptibility.

A major goal with this research was to develop a low-cost and highly sensitive immunoassay detection system by using alternating current (AC) magnetic susceptibility. We fabricated an improved prototype of our previously developed immunoassay detection system and evaluated its performance. The prototype continuously moved sample containers by using a magnetically shielded brushless motor, which passes between two anisotropic magneto resistance (AMR) sensors. These sensors detected the magnetic signal in the direction where each sample container passed them. We used the differential signal obtained from each AMR sensor's output to improve the signal-to-noise ratio (SNR) of the magnetic signal measurement. Biotin-conjugated polymer beads with avidin-coated magnetic particles were prepared to examine the calibration curve, which represents the relation between AC magnetic susceptibility change and polymer-bead concentration. For the calibration curve measurement, we, respectively, measured the magnetic signal caused by the magnetic particles by using each AMR sensor installed near the upper or lower part in the lateral position of the passing sample containers. As a result, the SNR of the prototype was 4.5 times better than that of our previous system. Moreover, the data obtained from each AMR sensor installed near the upper part in the lateral position of the passing sample containers exhibited an accurate calibration curve that represented good correlation between AC magnetic susceptibility change and polymer-bead concentration. The conclusion drawn from these findings is that our improved immunoassay detection system will enable a low-cost and highly sensitive immunoassay.

Detection and Follow Up of BCR/ABL Translocation by Cromogen In Situ Hibridization with ABL Centromeric and Telomeric (T/C) Probe Pair. Molecular, Accurate and Inexpensive Assay.

Objective: To Identify and to standardize a reliable t(9,22) BCR/ABL Molecular diagnosis method for developing countries.Patients, Materials and method: Nineteen patients: Fourteen newly diagnosed Chronic Myeloid Leukemia confirmed by PCR and Cytogenetic study, one Acute Myeloid Leukemia (AML), one Acute Lymphoblastic leukemia (ALL) Phi positive, two myeloproliferatives Phi negative and a Chronic Lymphocyte Leukemia were studied. Mononuclear cell was obtained by Histopaque separation cell medium (Sigma Aldrich) from Bone Marrow and Blood samples. A translocation probe pair ABL (Chromosome 9 band 9q34) T/C digoxigenin and biotin labeled (Zymed - Invitrogen) were used for hybridization both cellular; blood and bone marrow smear. Immunoassay detection system was applied to localize BCR/ABL under bright field microscope by counting between 200 and 500 cells. We performed fifty assays with these samples.Result: No differences between cell, blood or bone marrow smear were found. We improved the method fixing the samples with acetone. The assay was performed in fresh and frozen samples after six months without relevant differences. We were able to identify translocation, deletion and trisomy. The assay is semi quantitative and we can see a diminished number of BCR/ABL after five months with Tyrosin Kinase inhibitor, Imatinib (Glivec, Novartis) in six patients. (Table 1 and graphic 1). We detected a BCR/ABL LLA Phi positive and AML. All the cases were tested with RT-PCR, and five of them with QRT- PCR. We established an accuracy cutoff higher than 5 to 8% of positive cells for accepting BCR/ABL presence in the sample.Conclusion: This low cost method is useful, accurate, easy and fast to diagnose and follow up CML patients treated with Imatinib. Molecular diagnosis is hardly perform in developing countries or in far away towns or cities; training personal with this method in laboratories with basic equipment would solve this critical problem for many people throughout the world.

Clinical Evaluation of Lumiward Immunoassay System for Detection of Specific IgE Associated with Allergic Rhinitis

The detection of specific IgE is a critical prerequisite for both the definitive diagnosis and the therapeutic strategy of allergic rhinitis and other allergic disorders. The aim of the present study was thus to evaluate the clinical significance of the solid phase capture system (CAP) and the lumiward immunoassay system (LMD) in the diagnosis of allergic rhinitis due to Dermatophagoides farinae ( D. farinae ) and Japanese cedar ( Cryptomeria japonica ) pollens. The specificity of both the CAP and the LMD in the detection of D. farinae -specific IgE and Japanese cedar pollen-specific IgE was 100%. The sensitivity to detect D. farinae -specific IgE was 95.76% in the skin test, 86.53% in the CAP and 88.53% in the LMD, respectively. The combination of the nasal provocation test and the CAP substituted for the skin test resulted in correct diagnoses for 98.25% of the patients, and the combination of the nasal provocation test and the LMD substituted for the skin test resulted in correct diagnoses for 98.00% of the patients. Therefore, the diagnostic significance of the LMD for perennial allergic rhinitis is likely to be equal to that of the CAP. The sensitivity to detect Japanese cedar pollen-specific IgE was 94.50% in the skin test, 84.47% in the CAP, and 96.76% in the LMD, respectively. The sensitivity of the CAP in the detection of Japanese cedar pollen-specific IgE was inferior to that of the skin test, but the sensitivity of the LMD in the detection of pollen-specific IgE was somewhat superior to that of the skin test. In addition, the combination of the nasal provocation test and the CAP substituted for the skin test resulted in correct diagnoses for 98.38% of the patients, whereas the combination of the nasal provocation test and the LMD substituted for the skin test resulted in correct diagnoses for 100% of the patients. Therefore, the diagnostic significance of the LMD for seasonal allergic rhinitis due to Japanese cedar pollens is probably larger than that of the CAP. In conclusion, the LMD may be a better “gold standard” for the detection of Japanese cedar pollen-specific IgE than the skin test, and the combination of the nasal provocation test and the LMD is a better diagnostic tool for the detection of Japanese cedar pollen-induced seasonal allergic rhinitis than the combination of the nasal provocation test and the skin test or the CAP.

5096807 Imaging immunoassay detection system with background compensation and its use: David H Leaback, Radlett, United Kingdom assigned to Murex Corporation

5096807 Imaging immunoassay detection system with background compensation and its use: David H Leaback, Radlett, United Kingdom assigned to Murex Corporation

Encapsulation of europium in liposomes for use in an amplified immunoassay detection system

Encapsulation of europium in liposomes for use in an amplified immunoassay detection system