Detection Of Antibodies Research Articles

Approaching onchocerciasis elimination in Equatorial Guinea: Near zero transmission and public health implication

Abstract Background Onchocerciasis and lymphatic filariasis (LF) are endemic in Equatorial Guinea with notable variations in disease incidence between island and mainland regions. Historically, efforts to control and map these diseases were concentrated in Bioko Island, where loiasis is absent, allowing for targeted onchocerciasis interruption strategies. With the cessation of onchocerciasis transmission on Bioko and no reported cases on Annobon island, assessing the transmission status in the previously unaddressed mainland region has become imperative. Mapping efforts in mainland Equatorial Guinea have proven low to moderate level of transmission for LF and onchocerciasis, although the results so far have not been very conclusive. The current study aims to update the prevalence estimates for onchocerciasis and LF in mainland Equatorial Guinea using various diagnostic techniques. Methods This is the first cross-sectional study carried out to estimate the prevalence of onchocerciasis and LF in the mainland area of Equatorial Guinea, from September to December 2019, based on the combination of skin snip biopsies, thick blood smears, laboratory serological tests (ELISA tests for the detection of IgG4 antibodies against Onchocerca volvulus recombinant antigen Ov16 and Wuchereria bancrofti recombinant antigen Wb123) and molecular laboratory tests. Frequencies and prevalence rates, along with 95% confidence intervals for interval estimation of a binomial proportion, were computed. Results The overall onchocerciasis seroprevalence calculated for the study was 0.3% (95% CI: 0.1 to 0.5%). Microscopic examination of skin biopsies from the eight individuals seropositive for Ov16, out of the 3951 individuals initially tested, revealed no O. volvulus microfilariae. However, DNA extracted from one skin snip was successfully amplified, with subsequent sequencing confirming the presence of O. volvulus. Among the 3951 individuals, 182 were found to have anti-Wb123 antibodies, suggesting exposure to W. bancrofti, with an estimated seroprevalence of 4.6% (95% CI: 4.0 to 5.3%). Microscopy and Filaria-real time-PCR (F-RT-PCR) analysis for W. bancrofti were negative across all samples. Conclusions The findings indicate that onchocerciasis may no longer constitutes a public health problem in Equatorial Guinea, positioning the country on the verge of achieving elimination. Additionally, the mapped prevalence of LF will facilitate the formulation of national strategies aimed at eradicating filariases countrywide. Graphical Abstract

Abstract A074: Early detection of breast implant associated anaplastic large cell lymphoma by multiplex lateral flow assay of CD30 and IL-10

Abstract Introduction: More than 1300 women have developed a unique breast implant associated anaplastic large cell lymphoma (BIA-ALCL) around breast implants. BIA-ALCL presents as an accumulation of fluid (seroma) a median of 9 years after implant placement. When detected early, survival with surgery alone is nearly 100%. Remaining patients present with masses and/or lymph node metastases requiring radiation, chemotherapy and/or immunotherapy and fatalities have occurred. Methods: Preliminary studies identified CD30 and IL-10 as the most sensitive and specific biomarkers for BIA-ALCL in seroma fluids. Therefore, we constructed a multiplex LFA to detect both CD30 and IL-10 in 30 mL seroma fluid within 20 minutes. LFA is a point of care immunoassay using capture and detection antibodies that detect separate epitopes of the target molecule. The detection antibody is linked to a colored particle; in this multiplex assay CD30 antibody was linked to blue and IL-10 to red particles. Separate positive test lines (blue and red) indicate presence of CD30 or IL-10. A control line confirms migration of the seroma fluid past the test lines. To determine the dynamic range of sensitivity we spiked recombinant CD30 and IL-10 into benign seromas. Test and control lines were quantitated with NIH Image J software and ratios of test line to control line calculate for each sample. Forty-eight hour supernatants of supernatants of BIA-ALCL lines TLBR1 and TLBR2 were tested for IL-10 and CD30. Fifteen patients, 8 with BIA-ALCL and 7 with benign seromas provided consent for testing of their seroma fluids. Results: Multiplex CD30/IL10 LFA produced positive test lines for neat and 1:10 dilutions of 48 hour supernatnats of BIA-ALCL lines TLBR1 and TLBR2. Both IL-10 and CD-30 could be detected on the multiplex LFA strip using a 1:3 dilution of seroma fluids. The multiplex LFA shows both IL-10 and CD30 are significantly higher in malignant samples (IL-10 TL/CL: malignant, 0.2±0.16 n=8 vs benign, 0.004±0.007 n=7, p<0.05; CD30 TL/CL: malignant, 0.38±0.2 vs benign, 0.02±0.02, p<0.001, two-way ANOVA). Importantly, when either IL-10 or CD30 yields a faint positive test line, the other is not detected reducing the possibility of false positives. Conclusions: The CD30/IL-10 multiplex LFA had 100% positive and negative predictive value in this pilot study. The assay was able to distinguish lymphoma from recurrent breast cancer and more common benign seromas. The use of this multiplex assay mitigates positive false positive results from more sensitive CD30 LFA which has been detected in some benign seromas. This LFA will potentially allow early pre-operative detection of ALCL allowing surgeons to plan their surgical approach. The LFA can replace more costly time-consuming flow cytometry, immunohistochemistry, cyto- and histopathologic studies which require at least 10 ml of fluid. The LFA can be performed outside of medical centers in rural and underserved areas. A larger prospective multicenter study including intra-operative sampling is planned to validate these results. Citation Format: Peng Xu, Katerina Kourentzi, Richard Willson, Marshall Kadin. Early detection of breast implant associated anaplastic large cell lymphoma by multiplex lateral flow assay of CD30 and IL-10 [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr A074.

Evaluation of the MAGLUMI HIV Ab/Ag combi test for the detection of HIV infection

Abstract Background Human immunodeficiency virus (HIV) infection screening and diagnosis are critical to control the HIV epidemic. Testing for anti-HIV antibodies (Ab) and antigens (Ag) in blood samples is the first step to screen people who have been potentially exposed to the virus. This study aimed to evaluate the performance of the MAGLUMI HIV Ab/Ag Combi for detection of HIV antibodies and antigens. Methods We used residual samples to assess the diagnostic specificity and sensitivity of the MAGLUMI HIV Ab/Ag Combi retrospectively. All samples that met the test criteria were tested with the MAGLUMI HIV Ab/Ag Combi according to manufacturer’s instruction. Results of the MAGLUMI HIV Ab/Ag Combi were compared with the Architect HIV Ag/Ab Combo test. Results The specificity of the MAGLUMI HIV Ab/Ag Combi was 99.85% in 5,057 unselected blood donors and 100.00% in 213 hospitalized patient samples, respectively. The sensitivity of the Test in 614 HIV-1 Ab, HIV-1 Ag or HIV-2 Ab positive samples was 100.00%. Seroconversion sensitivity from results of 30 panels was comparable between the MAGLUMI HIV Ab/Ag Combi and the Architect assay. Conclusions The reactivity of the MAGLUMI HIV Ab/Ag Combi test is comparable to the Architect HIV Ag/Ab Combo assay.

Abstract A030: Rapid extraction and detection of extracellular vesicle-derived PD-L1 in a microfluidic platform

Abstract Background. Extracellular vesicles (EV) are emerging as new biomarkers for cancer diagnostics and therapeutic monitoring1. For example, in patients that receive PD1/PD-L1 immune checkpoint inhibitors (a key pillar of cancer immunotherapy), circulating EV-derived PD-L1 is gaining attention as a non-invasive biomarker for therapeutic efficacy. Standard methodologies to measure EV-derived PD-L1 requires ultracentrifugation to first separate EVs and then quantify those that express PD-L1 using nano flow cytometry. These are tedious processes that require trained technologists in centralized facilities, which is a key barrier to frequently monitoring EV-based biomarkers. Yet, frequent monitoring is crucial to quickly identify and stop an ineffective therapy while more fruitful options are still available. Method. To bridge the gap, we develop a proof-of-concept digital microfluidic (DMF) device that can separate EVs directly from biofluids and quantify PD-L1+ EVs automatedly. Briefly, DMF device supports automatedly droplet handling, including droplet transport, splitting and mixing, based on electrowetting principles2. Immunomagnetic beads were used to capture EVs from biofluids, and EVs were then detected by electrochemical sensors inserted into the device. These tips of the electrochemical sensors were modified with gold nanoparticle self-assembled layers to enhance target binding, and CD9 antibodies were immobilized on the tips to bind to EVs. We then used anti-PD-L1 as secondary antibodies for target detection. Differential Pulse Voltammetry (DPV)3 were performed to obtain the results. Results. We used the DMF device to extract 109- 1011 #/mL EVs secreted by a human breast cancer cell line (MDA-MB-231). Briefly, 10 mL of cell culture media was loaded into the DMF device, and the sample was mixed and incubated with a 10 mL droplet of immunomagnetic beads to capture EVs. After 5 min, EVs were eluted in 20 mL of elution buffer, and the beads were discarded. We estimated the number of EVs using Nanoparticle Tracking Analysis; the number of EVs extracted on-chip was comparable with those that were extracted using standard in-tube method. We also verified the purity of the EVs extracted on-chip using Western Blot. Our results showed that the electrochemical sensors can detect as low as 104 #/mL PD-L1+ EVs, with a linear range of 104 – 107 #/mL (R2=0.9701). The entire process can be completed within 1 hour. Conclusion. We integrated automated EV extraction and their surface marker detection in a single DMF device. Our next step is to extract and detect EV-based biomarkers from plasma samples. This platform holds promise for the regular monitoring of EV-based biomarkers that are indicators of therapeutic responses in patients that receive cancer immunotherapies.

Glial Fibrillary Acidic Protein’s Usefulness as an Astrocyte Biomarker Using the Fully Automated LUMIPULSE® System

Background: Glial fibrillary acidic protein (GFAP) is an important biomarker for neuroinflammatory conditions. Recently, advancements in the treatment of neurological diseases have highlighted the increasing importance of biomarkers, creating a demand for accurate and simple measurement systems for GFAP levels, which are essential for both research and clinical applications. This study presents the development and validation of a novel fully automated immunoassay for the quantitative determination of GFAP levels in biological samples. Methods: We examined the analytical performance of the GFAP assay on the LUMIPULSE platform. The assay’s parameters, including antibody concentrations, incubation times, and detection methods, were optimized to enhance sensitivity and specificity. GFAP levels were measured in 396 serum or plasma samples, comprising both healthy controls and patients with neurodegenerative diseases. Results: In the analytical performance studies, intra- and inter-assay coefficients of variation (CV) were below 5%, indicating high reproducibility. Additionally, the assay demonstrated good linearity over the measurement range. The limit of quantification (LoQ) for this assay was 6.0 pg/mL, which is sufficient for measuring specimens from healthy controls. In clinical validation studies, GFAP levels were significantly elevated in patients with neurodegenerative diseases compared to healthy controls. Conclusions: This automated GFAP assay provides a robust and reliable tool for GFAP measurement, facilitating further research into GFAP’s role in neurological disorders and potentially aiding in the diagnosis and monitoring of these conditions.

Circulation of West Nile virus and Usutu virus in birds in Germany, 2021 and 2022

Background Usutu virus (USUV) and West Nile virus (WNV) are zoonotic arthropod-borne orthoflaviviruses. The enzootic transmission cycles of both include Culex mosquitoes as vectors and birds as amplifying hosts. For more than 10 years, these viruses have been monitored in birds in Germany by a multidisciplinary network. While USUV is present nationwide, WNV used to be restricted to the central-east. Methods In 2021 and 2022, over 2300 live bird blood samples and organs from over 3000 deceased birds were subjected to molecular and serological analysis regarding the presence of WNV and USUV. The samples were collected at sites all over Germany. Results Circulation of both viruses increased in 2022. For USUV, the nationwide presence of lineages Africa 3 and Europe 3 reported in previous years was confirmed. Lineage Europe 2, formerly restricted to the German east, was able to expand westward. Nonetheless, USUV neutralizing antibody (nAb) detection rates remained low (< 9%). Years 2021 and 2022 were characterized by stable enzootic circulation of WNV lineage 2, dominated by one previously identified subcluster (95% of generated sequences). In 2022, >20% of birds in the endemic region in eastern Germany carried nAb against WNV. Serological data also indicate expanding WNV circulation west and south of the known hotspots in Germany. Conclusions USUV circulates enzootically nationwide. Emergence of WNV at several new locations in Germany with a potential increase in human infections may be imminent. In this context, wild bird monitoring serves as a capable early warning system in a One Health setting.

Dengue Fever in Pregnant Women with a Favorable Outcome

Dengue fever is a viral infectious disease, it manifests itself from mild to severe severity, in some cases to fatal outcomes. The number of cases of dengue fever has been increasing in recent years. The results of many studies have also shown adverse effects on pregnant infected women. This article describes the clinical course of Dengue fever in pregnant women, which occurred in moderate severity and without complications. Dengue fever in a pregnant woman manifested itself in the form of complaints of pain in the epigastric region, bleeding gums and petechial hemorrhages within 1 day. She had a high fever for 5 days before going to the hospital. Upon admission, her body temperature was 37 °C, blood pressure was 110/80 mmHg, pulse rate was 80 beats per minute, respiratory rate was 20 beats per minute. She had mild dehydration, normal breathing and heartbeat. The height of the bottom corresponds to the gestation period of 31 weeks, and the fetal heart rate was 144 beats per minute. Petechiae measuring at least 1-2 m in diameter were found around her face, forearms and both basal areas. In a laboratory study, the results showed changes in the number of platelets in the form of thrombocytopenia, leukocytopenia and in the biochemical analysis of blood by increasing the concentration of C-reactive protein and the detection of antibodies in serum during serological analysis. Although many studies have shown that Dengue during pregnancy increases the risk of preeclampsia, hemorrhagic dengue fever (HLD), fetal distress, premature birth, cesarean section and maternal mortality and intrauterine development delay and stillbirth consequences. This clinical case concludes with favorable outcomes. But the physiological effects of dengue fever during pregnancy (for both mother and child), depending on the trimester, as well as research and treatment methods require further study, especially in the region of endemic zones and India.

Magnetoelastic Immunosensor for the Rapid Detection of SARS-CoV-2 in Bioaerosols.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a protein-coated, single-stranded RNA virus that parasitizes and infects primates, including humans. The current detection methods are mainly carried out using humans as a medium, such as a chest computed tomography (CT) examination, nucleic acid detection, antibody detection, and antigen detection. In addition, SARS-CoV-2 in bioaerosols is an important way of transmission and infection, which has attracted wide attention. In this paper, we simulate a sampling system of SARS-CoV-2 in bioaerosols and detect SARS-CoV-2 in bioaerosols by means of a magnetoelastic (ME) sensing device based on an Android intelligent terminal, which can directly detect SARS-CoV-2 in bioaerosols in a certain unit environment. This approach aims to achieve early detection and prevention. The experimental results show that the sampling system can successfully collect SARS-CoV-2 in bioaerosols. In the linear range of 5-20 ng/mL, the ME sensing detection system results closely match those of the calibration instrument (VNA), with goodness-of-fit values of 0.997 and 0.995, respectively. This work demonstrates that the ME sensing detection system proposed is stable, highly specific, real-time, rapid, and has certain reference values and feasibility.

RVSV-ZEBOV vaccination in people with pre-existing immunity to Ebolavirus: an open-label safety and immunogenicity study in Guinean communities affected by Ebola virus disease (l’essai proches)

BackgroundZaire Ebolavirus disease (EVD) outbreaks can be controlled using rVSV-ZEBOV vaccination and other public health measures. People in high-risk areas may have pre-existing antibodies from asymptomatic Ebolavirus exposure that might affect response to rVSV-ZEBOV. Therefore, we assessed the impact pre-existing immunity had on post-vaccination IgG titre, virus neutralisation, and reactogenicity following vaccination.MethodsIn this prospective cohort study, 2115 consenting close contacts (“proches”) of EVD survivors were recruited. Proches were vaccinated with rVSV-ZEBOV and followed up for 28 days for safety and immunogenicity. Anti-GP IgG titre at baseline and day 28 was assessed by ELISA. Samples from a representative subset were evaluated using live virus neutralisation.ResultsTen percent were seropositive at baseline. At day 28, IgG in baseline seronegative (GMT 0.106 IU/ml, 95% CI: 0.100 to 0.113) and seropositive (GMT 0.237 IU/ml, 0.210 to 0.267) participants significantly increased from baseline (both p < 0.0001). There was strong correlation between antibody titres and virus neutralisation in day 28 samples (Spearman’s rho 0.75). Vaccinees with baseline IgG antibodies against Zaire Ebolavirus had similar safety profiles to those without detectable antibodies (63.6% vs 66.1% adults experienced any adverse event; 49.1% vs 60.9% in children), with almost all adverse events graded as mild. No serious adverse events were attributed to vaccination. No EVD survivors tested positive for Ebolavirus by RT-PCR.ConclusionsThese data add further evidence of rVSV-ZEBOV safety and immunogenicity, including in people with pre-existing antibodies from suspected natural ZEBOV infection whose state does not blunt rVSV-ZEBOV immune response. Pre-vaccination serological screening is not required.

DNA Conformation-Regulated Hemin Switch for Lab-on-Chip Chemiluminescent Detection of an Antibody Secreted from Hybridoma Cells.

This work designed a DNA conformation-regulated hemin switch for rapid chemiluminescent (CL) detection of a monoclonal antibodies. This switch was performed with an affinity probe and an inhibition probe, which were conveniently prepared by hybridizing hemin-labeled DNA1 with KHL peptide-labeled DNA2 and binding biotin-labeled DNA3 to streptavidin, respectively. In the absence of the target antibody, streptavidin-DNA3 could hybridize with hemin-DNA1/KHL-DNA2 to release KHL-DNA2, which led to the loss of hemin activity due to the affinity hindrance of streptavidin-DNA3. After the KHL peptide was recognized by the target antibody, the strand replacement hybridization could be inhibited by the bound antibody, which retained the high catalytic activity of hemin overhung on the antibody-bound affinity probe for a CL reaction, leading to a "signal-on" process for CL antibody detection. Using a KHL-specific antibody, anti-proprotein convertase subtilisin/kexin type 9 antibody (PCSK9-Ab), as a target model and common L012-1,2,4-triazole-H2O2 CL system, the designed switch showed a detection range of 10 ng mL-1 to 1 μg mL-1 with a detection limit of 4.16 ng mL-1 (56.2 pM) and a short analytical time of 6.5 min. The proposed quick method could simply be used for lab-on-chip CL detection of PCSK9-Ab in situ-secreted from PCSK9-6E3 hybridoma cells, which showed an accuracy of 90.2% compared with the statistical results from general fluorescence imaging, providing a potential technique for screening specific hybridoma cells.

Purification of Anti-OSH1 IgG and Immunohistochemical Staining of Rice Shoot Apical Meristem.

Immunohistochemical staining (IHC) enables the visualization of protein localization within tissues and organs. Here, we describe IHC to detect the ORYZA SATIVA HOMEOBOX1 (OSH1) protein in sections of rice shoot apical meristems (SAMs). First, anti-OSH1 IgG is purified from OSH1 polyclonal rabbit antisera. For subsequent IHC, rice shoot apices are fixed and embedded in paraffin and sections are prepared for IHC. In this IHC procedure, two different labeling methods, fluorescein isothiocyanate and alkaline phosphatase, are utilized for secondary antibody detection.

Unlocking the potential of dried blood spot sampling for SARS-CoV-2 antibody detection: A pathway to efficient pandemic surveillance

SummaryThe current SARS-CoV-2 pandemic has exerted unprecedented pressure on global healthcare systems, with an urgent demand for widespread testing to monitor virus spread and contribute to disease control. AimThis study aimed to validate the use of Dried Blood Spot (DBS) sampling for analyzing the immune response to SARS-CoV-2 infection, employing an accessible enzyme-linked immunosorbent assay (ELISA) for detecting IgG antibodies. MethodologyTraditional venous blood sampling presents several challenges, including discomfort to patients, risk of healthcare provider exposure, and the need for specialized equipment, which can limit mass testing capabilities. Our research involved the comparison of DBS and venous blood samples from 170 individuals, all of whom had PCR-confirmed SARS-CoV-2 infection, to assess the efficacy of DBS for seroprevalence studies and vaccine response evaluation. ResultsOur results confirmed that DBS sampling could detect SARS-CoV-2 IgG antibodies with 100 % accuracy, showcasing perfect agreement (Cohen's k value of 1.00) between DBS and venous serum samples in detecting the viral antibodies. However, the analysis indicated no significant correlation between the antibody levels in the two sampling methods. ConclusionThese results suggest that DBS is a viable, rapid alternative for serological studies, offering a solution to current testing limitations and enhancing our public health approach to combating future pandemics.

Using Soluble CD38 to Overcome Daratumumab Interference in Pretransfusion Compatibility Testing

Background: CD38 is a protein highly expressed on myeloma (MM) cells that has been shown to be an effective target antigen for monoclonal antibody therapies, so treatment with anti-CD38 monoclonal antibodies is a first line therapy for patients with MM. Although CD38 is weaker expressed on erythocytes anti-CD38 binds to CD38 on reagent RBCs and cause panreactivity in vitro and subsequent false positive reactions in indirect antiglobulin tests (IAT), antibody detection (screening) tests, antibody identification panels, and anti-human globulin (AHG) crossmatches. These findings suggest that the soluble CD38 method could improve transfusion safety for patients receiving anti-CD38 therapy, particularly in urgent clinical settings This study aims to evaluate the effectiveness of a new soluble CD38-based method in mitigating Daratumumab interference during pretransfusion compatibility testing." Methods: We evaluated the Grifols sCD38 method in 20 patients and compared it with the DTT method Results: In our experience the sCD38 method reduced testing time from 150 to 50 minutes and demonstrated 100% efficacy, compared to 90% with DTT. The sCD38 effectively mitigated the interference caused by anti-CD38 antibodies in 10 (100% efficacy) of patient samples tested while DTT was successful in only 9/10 (90% efficacy); no interference was observed in patients presenting anti erythrocyte antibodies. Moreover, there was no negative influence on DTT sensitive blood group systems such as KEL upon sCD38 treatment. Conclusions: In our Laboratory, in the year 2023, we processed 129 patients treated with anti-CD38. Therefore the reduction from 150 to 50 minutes in the time needed to perform tests for mitigation of anti-CD38 interference appears to be relevant with a recovery of approximately 210 technical-hours per year. Another highly appreciated operational aspect was the possibility of treating the patient's plasma and perform tests using automatic instruments (Erytra Grifols) available in our laboratory. In the evaluation of this new method, we did not observe failures in the mitigation of anti-CD38 interference. Furthermore, the results obtained in the samples that presented allo or auto antibodies were not affected by the treatment with sCD38. In our experience Grifols sCD38 assay is straightforward and quick to perform ant it is superior to DTT treatment in the mitigation of anti-CD38 antibody interference in MM patients treated with Daratumumab.

Review of domestic test systems for detecting current or past SARS‑CoV‑2 infection using enzyme‑linked immunosorbent assay

Introduction. Rapid and reliable assessment of circulating antibody levels to SARS-CoV-2 remains the current challenge for monitoring and preventing the spread of COVID-19. As the risk of emergence and dissemination of new viral strains continues to grow, epidemiologic research is also becomes increasingly important. The enzyme-linked immunosorbent assay (ELISA) is useful to assess the vaccination effectiveness and the need for revaccination, the proportion of post-disease and vaccinated individuals, and to monitor population immunity. This article provides an overview of the local ELISA kits currently available for the detection of antibodies against SARS-CoV-2 viral proteins, with a comparison to the certified kit by Mediagnost Ltd. (Germany).The aim. Laboratory testing of seven domestic reagent kits for the determination of antibodies to SARS-CoV-2 viral proteins by ELISA in comparison with the certified test system “Anti-SARS-CoV-2 ELISA E 111-IVD” (Mediagnost Ltd., Reutlingen, Germany).Materials and methods. The study included 80 serum samples from conditionally healthy donors: experimental group (n=70, serum samples collected after 2019) and control group (n=10, serum samples collected before 2018). The experimental group is comprised of non-infected and post-disease individuals; a proportion of those were vaccinated against COVID-19.Results and conclusions. The data obtained indicate high reproducibility, sensitivity (100 %), and specificity for all domestic ELISA kits in detecting both post-disease (90–95 %) and vaccinated volunteers (100 %). The main quality criteria of the obtained results were comparable to those for the certified test system, “Anti-SARS-CoV-2 ELISA E 111-IVD” (Mediagnost Ltd., Germany). All kits detecting IgG to the receptor-binding domain (RBD) and to the S-glycoprotein were effective for the reliable assessment of antibody production after the vaccination. Kits for the detection of antibodies to the RBD and/or viral nucleocapsid are useful for identifying unvaccinated post-disease individuals.

Examination of Salmonella Prevalence in Pigs Through Rye-Based Feeding and Coarser Feed Structure Under Field Conditions.

Salmonellosis is the second most commonly occurring bacterial zoonosis in Germany. Rye in pig feeding offers new possibilities for addressing that issue due to its high content of non-starch polysaccharides (NSPs). These are fermented in the intestinal tract to specific fermentation products, which seem to have bacteriolytic effects against Salmonella. A coarse feed structure can display synergistic effects. Seven conventional pig fattening farms increased the rye content (40%-70%) while limiting the amount of fine particles (maximum of 20% ≤0.25mm). Samples from pigs were tested for Salmonella antibodies and compared with samples from 167 farms without any changes to the feed. Rye-based diets had a significant (p<0.05) impact on Salmonella antibody (percentage optical density [OD%]) detection. In this study, it became apparent that significantly fewer positive OD% values could be detected due to the increase in rye compared to farms that did not change the feed (Farm 6 P0: 35.45±36.18; P1: 15.48±16.98; P2: 9.36±8.17). An elimination of Salmonella could not be achieved, but especially farms with high antibody counts were able to strongly reduce those in both phases consecutively (Farm 5 P0: 35.17±35.53; P1: 18.56a±20.96; P2: 13.38a±18.99). That was different on farms without adapted feeding, where an increase in Salmonella antibodies was observed (P0: 17.38±22.21; P1: 20.12±25.39; P2: 18.12±25.44). By increasing the proportion of rye and limiting the proportion of fine particles in the feed, Salmonella antibodies (OD% values) in meat juice and blood can be significantly reduced, especially on farms with an initially high incidence of Salmonella. If that is implemented in feeding across the board on farms, an improvement in food safety and a decreased risk of zoonosis can be expected.

Changes in Donor-Derived Cell-Free DNA Before and After Rejection and De Novo DSA Detection in Primary and Repeat Kidney Transplant Recipients.

Serial monitoring of dd-cfDNA and change from baseline can provide meaningful information beyond absolute thresholds. We describe dd-cfDNA trajectories from the baseline before and after acute rejection (AR) and de novo donor-specific antibodies (dnDSA) detection in kidney transplant recipients (KTRs). We included KTR from 02/2019 to 03/2022 with serial dd-cfDNA. The primary analysis compared the time-varying change in dd-cfDNA from baseline in KTR first AR on biopsy [AR] to patients with no-AR on biopsy [no-AR]. 151 KTR were analyzed (AR=56 KTR, no-AR=95 KTRs). In the AR group, dd-cfDNA rose ahead of diagnosis: median rise from baseline was 75% at -3 months, 32% at -2 months, and 325% at -1 month before biopsy. At the time of biopsy, the median rise in dd-cfDNA from baseline was 291% (IQR [interquartile range] 88%-1081%) in AR and 17% (IQR 0%- 194%) in no-AR (p<0.0001). Following treatment, dd-cfDNA values fell in the AR group with a median change from baseline of 94.7% at +1 month, 10.5% at +2 months, and 0% at +3 months. These trajectories were not observed in the no-AR group. Similarly, there were no significant differences in eGFR (estimated glomerular filtration rate) trajectories between the two groups. The median change from baseline to dnDSA detection was 141% (IQR 112%-574%). In KTRs with persistent rejection, median dd-cfDNA was 0.95% (IQR 0.44-1.8) compared to 0.19% (IQR 0.12-0.31) in subjects with no rejection on follow-up (p<0.001). The significant changes from baseline observed before and after AR show how serial monitoring enhances dd-cfDNA utility and allows for earlier identification of evolving injury and monitoring treatment response.

Cattle, sheep, and goat humoral immune responses against SARS-CoV-2

Following the emergence of SARS-CoV-2 in late 2019, several species of domestic and wild animals have been found to be susceptible to SARS-CoV-2 infection through experimental inoculation and animal surveillance activities. Detection of SARS-CoV-2 specific antibodies in animals is an important surveillance tool since viral shedding in animals can only be detected for a short period of time. In this study, convenience serum samples were collected from 691 cattle, 698 sheep, and 707 goats from several regions in the United States, between 2019 to 2022. The samples were evaluated for the presence of SARS-CoV-2 specific antibodies using two commercial enzyme-linked immunosorbent assays (ELISA); one based on the inhibition of the SARS-CoV-2 receptor-binding domain (sVNT) and the other based on the nucleocapsid protein (N-ELISA) of SARS-CoV-2. Positive samples from the sVNT were additionally evaluated using a conventional virus neutralization test (VNT) employing the Wuhan-like SARS-CoV-2 USA/WA1/2020 isolate. Our results indicate that ∼1% (6/691) of cattle, ∼2% (13/698) of sheep, and ∼2.5% (18/707) of goat serum samples were positive when using the sVNT, whereas ∼4% of cattle (25/691) and sheep (27/698), and 2.5% (18/707) of goat serum samples tested positive with the N-ELISA. None of the sVNT positive cattle, sheep, or goat serum samples had detectable neutralizing antibody activity (<1:8) against the SARS-CoV-2 USA/WA1/2020 isolate by the VNT. Our results indicate low seropositivity in cattle, sheep, and goats in the U.S., indicating the importance to continue monitoring for SARS-CoV-2 prevalence in animal species that are in close contact with humans.

Detection of Antibodies for Mycoplasma gallisepticum by ELISA in Broiler

Detection of Antibodies for Mycoplasma gallisepticum by ELISA in Broiler

A novel multiplex and glycoprotein-based immunochromatographic serologic IgM test for the rapid diagnosis of Escherichia coli O157 and O145 causing bloody diarrhea and hemolytic uremic syndrome.

Shiga toxin-producing Escherichia coli (STEC) are the main etiological agents of hemolytic uremic syndrome (HUS). Good clinical management of STEC infections and HUS depends on early, rapid, and accurate diagnosis. Here, we have developed and evaluated the first multiplex and glycoprotein-based immunochromatographic test for the detection of IgM antibodies against the O-polysaccharide of the lipopolysaccharide of E. coli O157 and O145 in human serum samples. A retrospective study was carried out resulting in a diagnostic sensitivity of the E. coli O157/O145 LFIA (lateral flow immunoassay) of 97.1% and 98.9% for O157 and O145, respectively, and 97.9% for both serogroups. The diagnostic specificity was 98.7% for O157 and O145, and the overall specificity 97.4%. In samples obtained before 3 days after the onset of diarrhea, the detection percentage was 83%, increasing to 100% from 3 days onward. Finally, the association of bloody diarrhea (BD) or HUS cases to an STEC infection increased from 22.8% to 77.2% when stool culture and stx/Stx detection were combined with serology by LFIA. Our results demonstrate that the E. coli O157/O145 LFIA is a highly accurate and serospecific test for the early and rapid diagnosis of E. coli O157 and O145 infections in BD or HUS cases. This test allows the detection of specific IgM antibodies very early in the course of the infection, making it an ideal diagnostic tool to be implemented in pediatric emergencies and, thus, avoid delays in the application of the correct supportive or specific treatment and prevent complications associated with HUS.

Dual-Fluorescent Quantum Dot Nanobead-Based Lateral Flow Immunoassay for Simultaneous Detection of C-Reactive Protein and Procalcitonin.

Simultaneous detection of C-reactive protein (CRP) and procalcitonin (PCT) at the point of care is crucial for the management of infections in patients with inflammation and in critical care settings. The challenge of detecting high concentrations of CRP alongside low concentrations of PCT in plasma from inflammatory patients has limited the clinical application of multiplexed immunoassays. Herein, we developed a lateral flow immunoassay (LFIA) that employs quantum dot nanobeads (QDNBs) of varying sizes and colors to enable the simultaneous quantification of PCT and CRP in human plasma. To extend the dynamic range of CRP detection, we combined QDNBs with smaller particle sizes with the CRP detection antibodies, thereby increasing the assay's dynamic range and reducing the hook effect. At the same time, the stronger fluorescence emitted by these larger QDNBs, in conjugation with the PCT detection antibodies, allows for the detection of PCT at the nanogram level, meeting the demand for high sensitivity. The results show that this method can detect CRP concentrations from 0.1 to 3 mg/L and PCT with a detection limit of 0.09 ng/mL, which is on par with clinically used methods. By employing this dual-color and dual-size QDNB labeling strategy, we successfully achieved simultaneous detection of CRP with a broad dynamic range and PCT with high sensitivity in a one-step point-of-care rapid test.