Detection Of Serum Antibodies Research Articles

#2239 Identification of non-HLA antibodies associated with the development of antibody-mediated damage after kidney transplantation

Abstract Background and Aims Non-HLA antibodies may play a role in the development of antibody-mediated rejection (ABMR) in the presence of donor-specific anti-HLA antibodies (HLA-DSA) or microvascular inflammation (MVI) without HLA-DSA in kidney transplantation (KT). The development of multiplex panels allowed the analysis of multiple antibodies simultaneously and new antibodies potentially related to ABMR/MVI have been identified. Method Retrospective study of a cohort of 169 KT recipients with: a) pre- and post-KT sera; b) follow-up HLA antibody monitoring; and c) follow-up biopsies. We determined the presence of 60 non-HLA antibodies in sera using a multiplex test: the Single Non-HLA Beads kit (LIFECODES®) suitable for the SAB assay performed using Luminex® technology. Results Pre-KT, we detected non-HLA antibodies in 85% of the recipients, with a median of 3 (2-5) positive antibodies. During follow-up, 73 patients developed ABMR/MVI. Pre-KT, in a univariate analysis including the presence of HLA-DSA (8.3% in the cohort) we detected 7 antibodies individually associated with ABMR/MVI. In multivariate analysis (Figure) GSTT1, NCL, PLA2R1 and Thyroglobulin antibodies together with HLA DSA were significantly associated with the development of ABMR/MVI, although only GSST1 and Thyroglobulin appear in a relevant number of patients (>10 patients) involved in 32 cases of ABMR and 6 cases of MVI. Post-KT, only the detection of anti-GSTT1 antibodies in sera 1- and 3-years post-TR was associated with ABMR/MVI in 3-year biopsies. Conclusion Pre-KT the detection of antibodies against GSST1 and Thyroglobulin using a multiplex non-HLA antibody detection panel is independently associated with the development of ABMR/MVI during follow-up. Post-KT detection of GSST1 is also associated with ABMR/MVI in 3-year biopsies.

Human and camel cystic echinococcosis - a polyclonal antibody-based sandwich ELISA for its serodiagnosis with molecular identification.

Cystic echinococcosis (CE) is an emergent neglected disease affecting human and animals in Egypt with a wide distribution and incidence. This study aimed to evaluate the use of a polyclonal antibody-based sandwich ELISA in the detection of Echinococcus granulosus antigen in human and camel sera. Hydatid cyst protoscoleces antigen (PsAg) was isolated from hydatid cysts collected from naturally infected camel livers and lungs. PsAg was used for immunization of rabbits to raise IgG polyclonal antibodies (IgG PsAb). IgG PsAb were then precipitated, purified using Protein-A Sepharose gel and labeled with horseradish peroxidase enzyme. We assayed the purity of the IgG PsAb, and the two prepared E. granulosus antigens CPsAg from camel cysts and HPsAg from human cysts by Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The resulted protein bands of the prepared CPsAg appeared at different molecular weights: 180, 90, 68, 54, 42 and 22kDa while, HPsAg shared with it in 4 common bands at 68, 54, 42, and 22kDa. The purified IgG PsAb had been resolved at two bands at 52kDa and at 32kDa. Sandwich ELISA were performed for the detection of circulating E. granulosus antigens in sera of human (n = 183) and camels (n = 190). The purified IgG PsAb showed strong reactivity against E. granulosus infected human and camel samples and no cross reactivity neither with free-healthy negative sera nor with others parasitic diseases (Schistosomiasis, Fascioliasis, Toxoplasmosis, Ancylostomiasis for human samples and Fascioliasis, ticks' infestation, Eimeriosis, Cryptosporidiosis, Nasal myiasis, Toxoplasmosis for camel samples). The sensitivity of the assay was 98.25% (56/57) and 96.9% (31/32) against human and camel samples, respectively. Specificity was 100% in both human and camel samples. Sandwich ELISA detected CE in 33.3% (24/72) and 55.6% (50/90) random human and camel samples, respectively. Indirect ELISA, using CPsAg, was used for detection of antibodies in positive human and camels' sera and detected 96.5% (55/57) and 93.8% (30/32) of human and camel samples, respectively. In our study, Genomic DNA was extracted from protoscoleces fluid of human liver hydatid cysts to identify the Echinococcus sp. isolate based on NADH dehydrogenase subunit 1 (NAD1) gene by Polymerase Chain Reaction (PCR) and the isolate (GenBank: OP785689.1) were identified as E. granulosus sensu lato genotype. In conclusion, Sandwich ELISA technique was found to be a potent and sensitive assay for detection of hydatid antigen in both human and camel samples.

Target Antigens in Western and Line Immunoblots for Supporting the Diagnosis of Lyme Disease. Comment on Porwancher et al. Immunoblot Criteria for Diagnosis of Lyme Disease: A Comparison of CDC Criteria to Alternative Interpretive Approaches. Pathogens 2023, 12, 1282.

An article was recently published in Pathogens on using different target antigens from Borrelia species that cause Lyme disease for detecting serum antibodies to support a clinical diagnosis of Lyme disease (LD) [...].

Immune response stability to the SARS-CoV-2 mRNA vaccine booster is influenced by differential splicing of HLA genes

Many molecular mechanisms that lead to the host antibody response to COVID-19 vaccines remain largely unknown. In this study, we used serum antibody detection combined with whole blood RNA-based transcriptome analysis to investigate variability in vaccine response in healthy recipients of a booster (third) dose schedule of the mRNA BNT162b2 vaccine against COVID-19. The cohort was divided into two groups: (1) low-stable individuals, with antibody concentration anti-SARS-CoV IgG S1 below 0.4 percentile at 180 days after boosting vaccination; and (2) high-stable individuals, with antibody values greater than 0.6 percentile of the range in the same period (median 9525 [185–80,000] AU/mL). Differential gene expression, expressed single nucleotide variants and insertions/deletions, differential splicing events, and allelic imbalance were explored to broaden our understanding of the immune response sustenance. Our analysis revealed a differential expression of genes with immunological functions in individuals with low antibody titers, compared to those with higher antibody titers, underscoring the fundamental importance of the innate immune response for boosting immunity. Our findings also provide new insights into the determinants of the immune response variability to the SARS-CoV-2 mRNA vaccine booster, highlighting the significance of differential splicing regulatory mechanisms, mainly concerning HLA alleles, in delineating vaccine immunogenicity.

A candidate competitive ELISA based on monoclonal antibody 3A8 for diagnosis of contagious bovine pleuropneumonia

For the development of a competitive ELISA (cELISA) to detect serum antibodies against the Mycoplasma mycoides subsp. Mycoides (Mmm) (strain PG1), the causative agent of contagious bovine pleuropneumonia (CBPP), all the proteins of this pathogen were analyzed. Then, a specific extracellular region of a transmembrane protein with the potential for diagnosis was identified. After that, a monoclonal antibody (Mab) named 3A8 was obtained using this extracellular region as an immunogen. Finally, a cELISA was established with the extracellular domain of this transmembrane protein as the coating antigen, Mab 3A8 as the competitive antibody, and HRP-labeled goat anti-mouse IgG as the enzyme-labeled antibody. This established method was used to detect the antibody dynamic regularity of goats which are artificially immunized Mmm and was also compared with a commercial ELISA kit. Further, the sera of 1011 different cattle from border provinces of China were monitored using a candidate Mab 3A8 cELISA. The detection results of known background sera used in this study indicate that a candidate diagnostic marker was successfully identified by analyzing all the coding proteins of Mmm in this research, and the cELISA established based on the Mab 3A8 against this protein can detect CBPP-positive serum with specificity and has no cross-reaction with other related epidemic disease-positive sera. In addition, we tested the sera collected from the border areas of China using the established ELISA, and no positive sample was detected. The research protocol of the CBPP cELISA established in this study is different from the traditional method, which can greatly reduce the investment of manpower and capital and save development time. We believe that this study’s protocol could serve as a reference for the development of detection methods for mycoplasma and other complex pathogens.Key points• A Mmm-specific diagnostic marker was obtained based on protein characteristics.• A cELISA was established for CBPP serum antibody detection.• The serological investigation was conducted for CBPP in the border areas of China.

Generation of porcine circovirus type 4 virus-like particles and their use to detect serum antibodies.

Porcine circovirus type 4 (PCV4), first identified in 2019 as a newly emerging pathogen, has been found in several provinces of China, as well as in Korea and Thailand. Since PCV4 is not included in immunization programs, epidemiological investigations should be conducted for detection of anti-PCV4 antibodies. Virus-like particles (VLPs) are frequently used for serological analysis of pathogen infections. However, there have been no reports on using PCV4 VLPs for serological investigation of PCV4 infection. In this study, we generated self-assembled PCV4 VLPs using an E. coli expression system, purified them using a two-step process, and used them to develop an indirect ELISA. This ELISA method was found to be highly specific, sensitive, and repeatable, making it suitable for PCV4 antibody detection in serum samples. Finally, the ELISA was used to analyze 422 serum samples collected from across several regions in China, 134 of which tested positive. Thus, the PCV4-VLP-based ELISA can effectively detect antibodies against PCV4 in serum samples, making it a useful tool for PCV4 epidemiology.

Retrospective Analysis of the Spread of SARS-CoV-2 in the Mediterranean Part of Bulgaria, During the First Wave of the Pandemic

The SARS-CoV-2 pandemic demonstrated the importance of proper understanding of the epidemiology of viral infections in the modern cosmopolitan world. In this retrospective study, we analyzed different factors associated with the spread of SARS-CoV-2 in the Sandanski municipality, a small and isolated region of Bulgaria, with a classical continental Mediterranean climate. We collected 418 serum samples from people without symptoms of SARS-CoV-2 infection or other respiratory diseases. The samples were tested for COVID-19 markers with qualitative and quantitative ELISA for anti-N and anti-S antibodies, and a rapid test for (IgM/G) antibodies. The methods were applied to complement each other with the different characteristics each of them has, thus allowing us to perform more accurate and more comprehensive detection of serum antibodies and to analyze the virus spread. We compared the results with the official government data for the SARS-CoV-2 distribution in Bulgaria. Based on the obtained results and available literature data, we discuss the importance of popular diagnostic methods, the role of the climate, the travel of people and pets, psychological stress and the individual reaction of subjects related to the spread of the virus.

Immunoinformatics-guided recombinant polypeptide-based enzyme-linked immunosorbent assay for seromonitoring of laboratory animals for minute virus of mice and Kilham rat virus.

Subclinical infection of laboratory animals with one or more of several pathogens affects the results of experiments on animals. Monitoring the health of laboratory animals encompasses routine surveillance for pathogens, including several viruses. This study aimed to explore the development of an alternative assay to the existing ones for detecting infection of mice and rats with the parvoviruses minute virus of mice (MVM) and Kilham rat virus (KRV), respectively. Full-length VP2 and NS1 proteins of these parvoviruses, besides fragments containing multiple predicted epitopes stitched together, were studied for serological detection. The optimal dilution of full-length proteins and antigenic regions containing predicted epitopes for coating, test sera, and conjugate was determined using a checkerboard titration at each step. The assays were evaluated vis-à-vis commercially available ELISA kits. The results showed that an engineered fusion of fragments containing multiple predicted MVM VP2 and NS1 epitopes was better than either of the full-length proteins for detecting antibodies in 90% of the tested sera samples. For KRV ELISA, full-length VP2 was better compared to other individual recombinant protein fragments or combinations thereof for the detection of antibodies in sera. This report is the first description of an ELISA for KRV and an improved assay for MVM. Importantly, our assays could be exploited with small volumes of sera. The results also demonstrate the utility of immunoinformatics-driven polypeptide engineering in the development of diagnostic assays and the potential to develop better tests for monitoring the health status of laboratory animals.

Application of gold immunochromatographic assay strip combined with digital evaluation for early detection of Toxoplasma gondii infection in multiple species

BackgroundTimely diagnosis of Toxoplasma gondii infection is necessary to prevent and control toxoplasmosis transmission. The gold immunochromatographic assay (GICA) is a means of rapidly detecting pathogen in samples. GICA-based diagnostic methods have been developed to accurately detect pathogens with high sensitivity and specificity, and their application in T. gondii diagnosis is expected to yield good results.MethodsColloidal gold test strips were produced using T. gondii C-terminal truncated apical membrane antigen 1 (AMA1C). Colloidal gold-AMA1C and colloidal gold-murine protein conjugate were synthesized under optimal conditions. A nitrocellulose membrane was treated with AMA1C and goat anti-mouse antibody as the test line and control line, respectively. In total, 90 cat serum samples were tested using AMA1C-GICA and a commercial enzyme linked immunosorbent assay (ELISA) kit. The GICA results were digitally displayed using a portable colloidal gold immunochromatographic test strip analyzer (HMREADER). The sensitivity, specificity, and stability of AMA1C-GICA were assessed, and this was then used to examine clinical samples, including 203 human sera, 266 cat sera, and 81 dog sera.ResultsAMA1C-GICA had a detection threshold of 1:32 for T. gondii-positive serum. The GICA strips specifically detected T. gondii antibodies and exhibited no reactivity with Plasmodium vivax, Paragonimus kellicotti, Schistosoma japonicum, Clonorchis sinensis, and Schistosoma mansoni. Consequently, 15 (16.7%) positive samples were detected using the AMA1C-GICA and commercial ELISA kits for each of the assays. The receiver-operating characteristic curve showed that GICA had a relative sensitivity of 85.3% and specificity of 92%, with an area under the curve of 98%. After analyzing clinical samples using HMREADER, 1.2%–23.4% of these samples were found to be positive for T. gondii.ConclusionsThis study presents a novel assay that enables timely and efficient detection of serum antibodies against T. gondii, thereby allowing for its early clinical diagnosis. Furthermore, the integration of digital detection using HMREADER can enhance the implementation of GICA.Graphical

Detection of Toxoplasmosis in Pregnant Women’s In Sabratha City of Using VIDS

Toxoplasmosis is usually asymptomatic, but can have severe consequences if It occurs in immunodeficient subject or fetuses. The diagnosis of toxoplasmosis during pregnancy is often based on maternal serological testing for IgM and IgG Anti- Toxoplasma antibodies. Persistence of IgM for long periods, posses' problems in distinguishing acute from chronic infection. The evaluation of specific IgG avidity enables more accurate dating, since avidity rises progressively during the course of infection. Test that measures immunoglobulin G (IgG) is used to determine if a person has been infected. If it is necessary to try to estimate the time of infection, which is of particular importance for pregnant women, a test which measures immunoglobulin M (IgM) is also used along with other tests such as an avidity test. Toxoplasma-specific IgG antibodies are detectable 1-3 weeks after infection and remain detectable for the life of the individual. Toxoplasma-specific IgM antibodies are also detectable 1-3 weeks after infection but generally decline to nil by one year after infection. The VIDAS® TOXO IgM (TXM) assay is intended for use on the instruments of the VIDAS family (VITEK® Immunodiagnostic Assay System) as an automated enzyme-linked fluorescent immunoassay (ELFA) for the presumptive qualitative detection of anti-Toxoplasma gondii IgM antibodies in human serum. Seventy-six (76) women in the first 16 weeks of pregnancy were tested for VIDAS IgM, IgG antibodies and VIDAS Toxo-IgG avidity. Low avidity antibodies were demonstrated in 2 (33.3%) of 6 sera positive with IgM assay and 4 (12.12%) of sera positive with IgG assay. Low avidity was also detected in 2 (3.27%) of 61 sera negative with IgM. The low avidity suggesting a recent infection, while high avidity in 3 (50%) of the 6 positive IgM and 24 (72.72%) of 33 positive IgG indicating that the Infection acquired in the distant past. These findings highlight the value of VIDAS IgG avidity when used in combination with the VIDAS IgM and IgG assays to provide a confirmatory evidence of an acute infection with a single serum specimen for pregnant women.

Identification of a novel B-cell epitope of the African swine fever virus p34 protein and development of an indirect ELISA for the detection of serum antibodies.

African swine fever (ASF) is a viral disease caused by the African swine fever virus that can be highly transmitted and lethal in domestic pigs. In the absence of a vaccine, effective diagnosis is critical for minimizing the virus's spread. In recent years, with the decline of African swine fever virus (ASFV) virulence, antibody detection has become an important means of detection. ASFV nucleocapsid protein p34 is a mature hydrolytic product of pp220, which is highly conserved and has a high content in the structural protein of the virus. Prokaryotic cells were chosen to generate highly active and high-yield p34 protein, which was then used as an antigen for producing mouse monoclonal antibodies. The B-cell epitope 202QKELDKLQT210, which was highly conserved and found on the surface of the p34 protein, was first identified by an anti-p34 monoclonal antibody utilizing the peptide scanning technique and visualized in helix. This supported the viability of p34 protein detection even further. In addition, we established an indirect ELISA assay based on p34 to detect ASFV antibodies. The coincidence rate of this method with commercially available kits was shown to be 97.83%. Sensitivity analysis revealed that it could be detected in serum dilution as low as 1:6400, and there was no cross-reaction with other prevalent porcine epidemic diseases classical swine fever virus (CSFV), foot-and-mouth disease virus (FMDV), porcine reproductive and respiratory syndrome virus (PRRSV), and porcine circovirus 2 (PCV2). In summary, the established ELISA method and anti-P34 monoclonal antibody have demonstrated that the p34 protein has a promising application prospect for the detection of African swine fever antibodies.

Clinicopathological and prognostic characteristics of idiopathic membranous nephropathy with dual antigen positivity.

Idiopathic membranous nephropathy (IMN) is the most common pathological type in adults with nephrotic syndrome. Many target antigens have been discovered. However, dual antigen-positive IMN patients are very rare, with only a few such cases being briefly described in various studies. There is no specific study on the clinicopathological and prognostic characteristics of dual antigen-positive IMN patients, and the disease characteristics of such patients remain unclear. Immunohistochemical staining of PLA2R, THSD7A, and NELL-1 was conducted on kidney tissue samples obtained from patients diagnosed with IMN. Simultaneously, the presence of corresponding serum antibodies was determined. Patients exhibiting positivity for dual antigens were included in the study, identified either through tissue staining or serum antibody detection. We retrospectively collected their clinical, pathological, and follow-up data and measured their serum antibody levels at multiple time points. Additionally, the same type of dual antigen-positive IMN cases reported in the literature were reviewed to extract clinical, pathological, and prognostic information. We compared the data for all of the above dual antigen-positive and PLA2R single-positive IMN cases at our center. We identified 6 IMN patients with dual antigen positivity at our center, approximately 0.7% of whole MN series; the previous literature reports 43 IMN patients with dual antigen positivity, the proportion ranged from 0.2% to 2.8%. The IgG1 positivity rate in the renal tissue of the dual antigen-positive patients at our center was significantly lower than that of dual antigen-positive patients previously reported (16.7% vs. 100.0%, p=0.015), but there was no significant difference in clinical or prognostic aspects. Patients with dual antigen positivity reported at our center and in the literature were combined and compared with PLA2R single-positive IMN reported at our center. Compared with PLA2R single-positive IMN patients, dual antigen-positive IMN patients had a higher renal tissue IgG1 positivity rate (58.3% vs. 22.3%, p=0.016), and the time required to achieve remission was longer [13.5 (3.3,35.0) vs. 3.0 (1.0,8.0), p=0.052]. Overall, The changes in urine protein were consistent with the changes in serum PLA2R antibody levels in dual antigen-positive IMN patients. For patients with primary membranous nephropathy who did not attain remission following prolonged treatment, multiple target antigen staining should still be actively performed, even with positivity for the PLA2R target antigen.

A novel lateral flow immunochromatographic assay using a recombinant VP2 antigen for total antibody detection of canine parvovirus-2.

Canine parvovirus-2 (CPV-2) is a viral disease of dogs causing acute hemorrhagic gastroenteritis and myocarditis with high morbidity and mortality rates. The infection is still widespread all over the world. Vaccines developed against infection have great importance in preventing infection. However, it is difficult to recommend a practical vaccination program without knowing the antibody level of a puppy. Despite widespread vaccination, difficulties in detecting the maternal antibodies in puppies remain the main cause of vaccination failure. The hemagglutination inhibition (HAI) test is the gold standard to determine the immune status of dogs for canine parvovirus 2, but the HAI test has several disadvantages such as the need for fresh porcine blood, well-equipped laboratory, and long incubation periods. In this study, for the first time we developed a colloidal gold-based competitive lateral flow assay (cLFA) system for the rapid detection of total antibodies in canine serum using CPV-2b-VP2 derived from field isolates. The recombinantly expressed capsid protein of CPV-2 in the prokaryotic expression system was used as a labeled molecule in cLFA. We carried out studies on our cLFA system using the standard antibody solution and the clinical samples from vaccinated puppy serum. We compared the results of the LFAs with the HAI test. Competitive lateral flow assay results showed good correlation with the gold standard method, the HAI test. In the developed platform, the limit of detection of the standard antibody was determined to be 375 ng mL-1, while the cut-off level of antibodies was observed to be 1 : 40 HAI titer in clinical samples. Our reported system will be a strong alternative for CPV-2 antibody-based detection applications.

A Flow Cytometry Study of the Binding and Stimulation Potential of Inactivated Trypanosoma evansi toward Dromedary Camel Leukocytes.

Surra, a wasting disease caused by Trypanosoma evansi, is one of the major animal health burdens in camel-rearing countries, imposing significant economic losses due to reduced fertility and high mortality rates. The present study used inactivated T. evansi (from the Card Agglutination Test for Trypanosomes/Trypanosoma evansi; CATT/T. evansi) and flow cytometry to investigate their binding and activation potential toward camel leukocyte subsets. Labeling T. evansi with propidium iodide (PI) enabled their flow cytometric enumeration and identification with forward scatter (FSC; indicative for cell size) and side scatter (SSC; indicative for cell internal complexity) characteristics that are comparable with values reported for Trypanosoma cruzi. The incubation of PI-labeled non-opsonized T. evansi with camel leukocyte populations revealed that camel monocytes have the highest potential to bind T. evansi, followed by granulocytes and lymphocytes. The identification of pattern recognition receptors (PRRs) on camel immune cells and the pathogen-associated molecular patterns (PAMPs) in T. evansi that are responsible for this different binding capacity requires further studies. Stimulation of camel neutrophils with Trypanosoma evansi induced shape change, reactive oxygen species (ROS) production, and neutrophil extracellular traps (NET)-formation. To ensure that T. evansi, in the parasite concentration used in this study, is not apoptotic or necrotic to camel leukocytes, we evaluated cell apoptosis and necrosis after stimulation with T. evansi. The results revealed no impact of T. evansi stimulation for 2 h on the cell viability of camel leukocytes. Subsequent work may focus on the diagnostic employment of labeled T. evansi and flow cytometry for the detection of anti-Trypanosoma antibodies in camel serum. In addition, more efforts should be deployed to investigate the host-pathogen interaction mechanisms and the escape mechanisms of T. evansi in camels. To complete these data, further studies using the living or freshly killed parasites could also be implemented in camels and/or horses.

Detection of IgG Anti-Giardia duodenalis Antibodies in Sera by Indirect Immunofluorescence and Western Blotting Assays.

Serological assays are alternative laboratory tools for the diagnosis of parasitic infections. The aim of this work was to evaluate the performance of the indirect fluorescent antibody test (IFAT) and Western blotting (WB) for the detection of IgG anti-Giardia antibodies in human sera. Sera from individuals infected with Giardia duodenalis, other parasites or non-parasitized were selected for serological assays. Ninety-seven sera were tested by IFAT at 1:20 and 1:40 dilutions and of these, 40 samples were also analyzed by WB. The sensitivity and specificity of the IFAT was 97% and 46.9% at 1:20 sera dilution, and 39.4% and 59.4% at 1:40 sera dilution. The low molecular weight polypeptides fractions of 25kDa, 27-31kDa and 45-55kDa were the most frequently identified by the sera of individuals infected with G. duodenalis, along with low cross-reactivity, presenting an individual sensitivity of 42.8%, 50.0% and 57.1%, and specificity of 83.3%, 83.3% and 91.7%, respectively. The highest overall sensitivity of WB (85.7%) was based on the immunoreactivity of sera with at least one of those proteins. The concordance between the detection of G. duodenalis in feces by microscopy and the WB results was considered substantial (Kappa = 0.61). Constant exposure to Giardia infection throughout a lifetime can maintain high levels of specific antibodies in serum, even without active infection. Moreover, proteins found in intestinal amoebas may hinder the serological diagnosis of giardiasis in endemic areas due to cross-reactivity, which can be partially solved using Giardia low molecular weight proteins.

Exposure to Brucella spp. in Humans and Cows in a High Milk-Producing Area of Bangladesh.

Brucellosis is a zoonotic disease, caused by some species within the Brucella genus. The primary and secondary objectives of this cross-sectional study were to determine the seroprevalence of Brucella antibodies in humans and cows and identify risk factors for exposure to Brucella spp. among people in Shahjadpur sub-district, Bangladesh. Twenty-five villages were randomly selected from the 303 milk-producing villages in the sub-district. We randomly selected 5% of the total households from each village. At each household, we collected demographic information and history of potential exposure to Brucella spp. in humans. In addition, we collected serum from household participants and serum and milk from cattle and tested to detect antibodies to Brucella sp. Univariate analysis was performed to detect associations between seropositivity and demographics, risk factors, and behaviors in households. We enrolled 647 households, 1313 humans, and 698 cows. Brucella antibodies were detected in sera from 27 household participants (2.1%, 95% confidence interval [95%CI]: 1.2-2.9%). Eleven (1.6%, 95%CI 0.6-2.4%) cows had detectable Brucella antibodies in either milk or serum. About half (53%) of the 698 cows exhibited more than one reproductive problem within the past year; of these, seven (2%) had Brucella antibodies. Households with seropositive individuals more frequently reported owning cattle (78% vs. 32%, P < 0.001). Despite a low prevalence of Brucella seropositivity in the study, the public health importance of brucellosis cannot be ruled out. Further studies would help define Brucella prevalence and risk factors in this region and nationally.

Results of Tick-Borne Encephalitis Virus (TBEV) Diagnostics in an Endemic Area in Southern Germany, 2007 to 2022.

Tick-borne encephalitis virus (TBEV) is the most important tick-transmitted neurotropic flavivirus in Europe and Asia. Our analysis aimed to investigate the contribution of TBEV-specific antibody detection by serological assays and TBEV RNA detection by real-time PCR to the diagnosis of tick-borne encephalitis (TBE). We analyzed data from 3713 patients from 16 years of laboratory TBEV diagnostics in an endemic area in Southern Germany. During this period, 126 cases of TBE were diagnosed. TBEV-specific IgM ELISA tests showed a high clinical sensitivity (96.8%) and a very high clinical specificity (99.7%). In immunocompetent patients, TBE was reliably diagnosed by detection of TBEV IgM antibodies in serum. Intrathecal TBEV IgG antibody synthesis was detected in 46 of 84 (55%) cases by analysis of paired serum and cerebrospinal fluid (CSF) samples. None of the 87 immunocompetent TBE patients tested had detectable TBEV RNA in serum or CSF. In contrast, in two TBE patients without TBEV-specific antibodies, diagnosis could only be made by the detection of TBEV RNA in CSF. Both patients had previously been treated with the B cell-depleting antibody rituximab. Therefore, in patients with CNS infection and humoral immunodeficiency, it is necessary to include TBEV PCR in the diagnostic approach.

False Negative Results of First-Tier Treponemal Antibodies in an HIV-Infected Patient

Abstract Introduction/Objective Syphilis diagnosis relies on detecting Treponema pallidum antibodies in serum or CSF. In the reverse syphilis screening algorithm, an automated enzyme immunoassay first detects serum antibodies against treponemal antigens. A negative result halts further testing; however, a positive result prompts manual testing for antibodies against non-treponemal antigens such as cardiolipin. The sensitivity of first-tier automated assays is typically high. We present a case of an HIV patient who initially tested negative for syphilis in the first-tier assay but was subsequently diagnosed with the infection. Methods/Case Report A 30-year-old HIV-positive female (CD4+ count 24/mm3), previously diagnosed with chlamydia and gonorrhea, experienced blurred vision, eye pain, fever, and a palmoplantar rash. Despite clinical suspicion of syphilis, the initial first-tier chemiluminescence immunoassay (CIA) for treponemal antibodies (LIAISON Treponema, DiaSorin, Stillwater, MN) returned a negative result, precluding any further syphilis testing at that time. However, a skin biopsy revealed interface dermatitis and spirochetes. CSF analysis showed pleocytosis, increased proteins, and borderline low glucose, with both VDRL and IFA assays negative. Based on these findings, we retested for serum antibodies by repeating the CIA test and performing RPR and TP-PA assays outside the algorithm sequence. Despite reconfirmation of the CIA negativity, both RPR (titer 1:64) and TP-PA were positive, leading to a secondary syphilis diagnosis and penicillin G treatment. Subsequent investigations aimed to explain the initial false negative CIA result. Serial dilution of the patient's serum up to 1:1000 did not alter the result, excluding the 'hook effect' caused by an excessive concentration of antibodies. Further serum testing at a different laboratory using another type of first-tier immunoassay yielded a positive result, pointing toward differences in the composition of treponemal antigens utilized across various manufacturer assays as the likely cause of the discrepancy. Results (if a Case Study enter NA) NA Conclusion Given their high sensitivity, automated first-tier treponemal assays rarely yield false negative results, whereas false positives are the most common ‘lab error’ and are typically resolved with second-tier assays. Nevertheless, this case highlights that false negatives can occur and, thus, stresses the need to re-evaluate the approach in diagnosing syphilis, especially in patients with antibody production abnormalities, such as those with HIV.

Detection of anti-SARS CoV-2 antibodies in human serum by means of Bloch surface waves on 1D photonic crystal biochips

This study presents the development and characterization of a disposable biochip for the detection of antibodies against the SARS-CoV-2 spike protein, a well-known target for vaccine and therapeutic development. This biochip is based on a one-dimensional photonic crystal (1DPC) deposited on a plastic substrate and designed to sustain Bloch surface waves (BSW) in the visible range. The experimental phase was carried out using the biochip in conjunction with a custom-made optical read-out platform capable of real-time refractometric detection and fluorescence-based end-point measurements. Our biochip was functionalized by immobilizing the receptor-binding domain of the spike protein onto the surface using a silanization process. Human serum samples, including a negative control and a positive sample from a recovered COVID-19 patient, were tested on the biochip. The experimental results show that the biochip discriminates between positive and negative samples in a label-free refractometric mode down to a 1:10 dilution of the sera and in quantum dot amplified refractometric and fluorescence mode down to 1:100 dilution. The results demonstrate the potential of the disposable biochip for sensitive and specific detection of COVID-19 antibodies.

Bimetallic nickel/cobalt metal-organic framework-based electrochemical sensor for trace level detection of IgG in simulated human blood serum

Electrochemical sensing is a widely used technique for the detection of biomolecules because of its low cost, high sensitivity, and versatility in terms of the various techniques that can be employed. In this study, we report a facile, cost-effective, and highly selective bimetallic Ni/Co-MOF (Nickel/Cobalt-Metal Organic Framework) on Nickel foam (NF) based electrochemical sensor for the trace-level detection of IgG (Human Immunoglobulin) antibodies in the human blood serum. In this direction, we used a simple and efficient solvothermal method to synthesize the Ni/Co-MOF, and the morphological, structural, and chemical states of the elements are studied using different material characterization techniques like TEM, FESEM, XRD, XPS, etc. The Ni/Co-MOF modified NF sensor showed a dynamic range of detection (30 fM to 10 nM) having superior sensitivity of 28 µA cm−2 mol−1 with a lower limit of detection 27.2 fM. Ni/Co-MOF/NF is highly selective over other interfering species (UA, Cl−, Ca2+, Urea, Sulphate, BSA and HAS). Real-time analysis was done using the DPV technique by the standard addition method and the sensor shows an excellent recovery percentage ̴ 97 to 98 %. Also, the addition of Ni2+ to Co enhances the electrochemical properties of a material by raising the valence of Ni2+ in Ni/Co-MOF by transferring partial electrons from Ni2+ to Co2+. The work presented here confirms Ni/Co-MOF/NF as a flexible, high-performance electrode that can be used to detect other biomolecules used in clinical diagnosis and biomedical research.