Detection of EHDV Antibodies Using Enzyme-Linked Immunosorbent Assay (ELISA).

Enzyme-linked immunosorbent assay (ELISA) is a relatively inexpensive, rapid, and high-throughput diagnostic tool to detect antibodies raised against epizootic hemorrhagic disease virus (EHDV) in ruminant serum. While the presence of EHDV antibodies only confirms prior exposure to the virus, it does not conclusively determine infection status. The c-ELISA can be used in conjunction with other diagnostic tests (e.g., real-time PCR) to reinforce diagnosis of infection or as a surveillance tool to support disease control. The EHDV competition ELISA (c-ELISA) described here is a commercial diagnostic assay, recommended by the World Organisation for Animal Health (WOAH), that detects ruminant antibodies against the highly conserved EHDV structural protein, VP7.

Diagnostic Accuracy of IgA Anti-Transglutaminase Assessed by Chemiluminescence: A Systematic Review and Meta-Analysis.

A new chemiluminescence immunoassay method (CLIA) for detecting IgA anti-transglutaminase (atTG IgA) in celiac disease (CD) has prompted inquiries into its diagnostic performance. We conducted a systematic review and meta-analysis comparing CLIA with traditional enzyme-linked immunosorbent assay (ELISA) and fluorescence enzyme immunoassay (FEIA). We searched PubMed, Medline, and Embase databases up to March 2024. The diagnostic references were intestinal biopsy and ESPGHAN guidelines. We calculated the sensitivity and specificity of atTG IgA assessed by CLIA and the odds ratio (OR) between the assays. Eleven articles were eligible for the systematic review and seven for the meta-analysis. Sensitivity and specificity of atTG IgA CLIA-assay were 0.98 (95% CI, 0.95-0.99) and 0.97 (95% CI, 0.94-0.99), respectively. The sensitivity of atTG IgA antibody detection did not significantly vary across the three assay modalities examined (CLIA vs. ELISA OR: 1.08 (95% CI, 0.56-2.11; p = 0.8); CLIA vs. FEIA OR: 6.97 (95% CI, 0.60-81.03; p = 0.1). The specificity of atTG IgA assessed by FEIA was higher than for CLIA (OR 0.17 (95% CI, 0.05-0.62); p < 0.007). According to the systematic review, normalization of atTG IgA levels in CD patients following a gluten-free diet was delayed when using CLIA compared to ELISA and FEIA methods. Conflicting findings were reported on the antibody threshold to use in order to avoid biopsy confirmation.

Methods to Analyze the Surface Composition of Bacterial Membrane Vesicles.

Bacterial membrane vesicles (BMVs) are small, spherical structures released by Gram-positive and Gram-negative bacteria that play essential roles in intercellular communication, nutrient acquisition, and antibiotic resistance. BMVs typically range from 40 to 400 nm in diameter and contain a single membrane derived from the bacterial membrane, comprising proteins, lipids, nucleic acids, and other biomolecules. Notably, the molecules located on the surface of BMVs facilitate interactions with neighboring cells, including the transfer of functional genes, coordination of bacterial growth through quorum sensing, and delivery of toxins during infections. In addition, BMVs exhibit heterogeneity in their surface composition, which influences their interactions with host and bacterial cells. It is therefore essential to understand not just the composition of BMVs, but the localization of the molecules of interest, particularly those on the surface. In this chapter, we describe several methods that can be used to quantify and characterize the protein and nucleic acid composition, particularly on the surface of BMVs. We describe quantitative immunoblot and ELISA protocols that enable quantification of the concentration of a particular protein of interest. We also describe an enzymatic digestion protocol to determine whether the protein of interest is located on the surface or within the lumen of the BMV, as well as a nucleic acid staining procedure that enables quantification of dsDNA specifically located on the surface of the BMVs. Together, these tools provide a detailed analysis of the protein and nucleic acid composition of BMVs that can be further combined with various separation techniques to study variations within different populations.

Performance evaluation of microfluidic microplate-based fluorescent ELISA for qualitative detection of SARS-CoV-2-specific IgG and IgM.

We evaluated the diagnostic performance of newly developed microfluidic microplate-based fluorescent ELISA for anti-SARS-CoV-2 antibody detection: the Veri-Q opti COVID-19 IgG and IgM ELISAs (hereafter, "Opti IgG/M"; MiCo BioMed, Gyeonggi-do, Republic of Korea), in comparison with conventional ELISAs. A total of 270 serum samples were analyzed, among which 90 samples were serially obtained from 25 COVID-19 patients. Another 180 samples were collected from 180 SARS-CoV-2-negative individuals. As comparative assays, we used SCoV-2 Detect IgG/M ELISA (hereafter, "InBios IgG/M"; InBios, Seattle, WA, USA) and Veri-Q COVID-19 IgG/IgM ELISA (hereafter, "Veri-Q IgG/M"; MiCo BioMed). Compared with conventional ELISAs, the Opti IgG yielded 97.1-100.0% positive percent agreement, 95.2-98.0% negative percent agreement, 96.3-97.8% total percent agreement, and kappa values of 0.90-0.94. Between the Opti IgM and the InBios IgM, the values were 93.7%, 96.6%, 95.9%, and 0.89, respectively. For the Opti IgG, sensitivities for the samples collected from 0-7, 8-14, 15-21, and ≥ 22 days after symptom onset were 40.0, 58.3, 94.1, and 100.0%, respectively. The values for the Opti IgM were 30.0, 54.2, 88.2, and 80%, respectively. The diagnostic specificities of the Opti IgG and IgM were 99.4 and 97.2%, respectively. The microfluidic microplate-based fluorescent ELISAs showed comparable diagnostic performance to conventional ELISAs for detecting anti-SARS-CoV-2 antibodies. With the combination of high throughput, a simplified workflow, and the ability to analyze reduced volumes, this new technology has great potential for improving SARS-CoV-2 serologic testing.

Baculovirus expression and purification of virion core and envelope proteins of goatpox virus to evaluate their diagnostic potential.

Goatpox and sheeppox are highly contagious and economically important viral diseases of small ruminants. Due to the risk they pose to animal health, livestock production, and international trade, capripoxviruses are a considerable threat to the livestock economy. In this study, we expressed two core proteins (A4L and A12L) and one extracellular enveloped virion protein (A33R) of goatpox virus in a baculovirus expression vector system and evaluated their use as diagnostic antigens in ELISA. Full-length A4L, A12L, and A33R genes of the GTPV Uttarkashi strain were amplified, cloned into the pFastBac HT A donor vector, and introduced into DH10Bac cells containing a baculovirus shuttle vector plasmid to generate recombinant bacmids. The recombinant baculoviruses were produced in Sf-21 cells by transfection, and proteins were expressed in TN5 insect cells. The recombinant proteins were analysed by SDS-PAGE and confirmed by western blot, with expected sizes of ~30 kDa, ~31 kDa, and ~32 kDa for A4L, A12L, and A33R, respectively. The recombinant proteins were purified, and the immunoreactivity of the purified proteins was confirmed by western blot using anti-GTPV serum. The antigenic specificity of the expressed proteins as diagnostic antigens was evaluated by testing their reactivity with infected, vaccinated, and negative GTPV/SPPV serum in indirect ELISA, and the A33R-based indirect ELISA was optimized. The diagnostic sensitivity and specificity of the A33R-based indirect ELISA were found to be of 89% and 94% for goats and 98% and 91%, for sheep, respectively. No cross-reactivity was observed with other related viruses. The recombinant-A33R-based indirect ELISA developed in the present study shows that it has potential for the detection of antibodies in GTPV and SPPV infected/vaccinated animals.

A reporter virus particle seroneutralization assay for tick-borne encephalitis virus overcomes ELISA limitations.

Tick-borne encephalitis (TBE) virus is the most prevalent tick-transmitted orthoflavivirus in Europe. Due to the nonspecific nature of its symptoms, TBE is primarily diagnosed by ELISA-based detection of specific antibodies in the patient serum. However, cross-reactivity between orthoflaviviruses complicates the diagnosis. Specificity issues may be mitigated by serum neutralization assays (SNT), although the handling of clinically relevant orthoflaviviruses requires biosafety level (BSL) 3 conditions and they have highly divergent viral kinetics and cell tropisms. In the present study, we established a reporter virus particle (RVP)-based SNT in which the infectivity is measured by luminescence and that can be performed under BSL-2 conditions. The RVP-based SNT for TBEV exhibited a highly significant correlation with the traditional virus-based SNT (R2 = 0.8637, p < 0.0001). The RVP-based assay demonstrated a sensitivity of 92.3% (95% CI: 79.7%-97.4%) and specificity of 100% (95% CI: 81.6%-100%). We also tested the cross-reactivity of serum samples in RVP-based assays against other orthoflaviviruses (yellow fever virus, dengue virus type 2, Zika virus, West Nile virus and Japanese encephalitis virus). Interestingly, all serum samples which had tested TBEV-positive by ELISA but negative by RVP-based SNT were reactive for antibodies against other orthoflaviviruses. Thus, the RVP-based seroneutralization assay provides an added value in clinical diagnostics as well as in epidemiological studies.

Establishment and application of PDCoV antigen-specific DAS-ELISA detection method.

BACKGROUND: Porcine deltacoronavirus (PDCoV) is a swine enteropathogenic coronavirus that affects young pigs, causing vomiting, acute diarrhea, dehydration, and even death. There is growing evidence that PDCoV can undergo cross-species as well as zoonotic transmissions. Due to the frequent outbreaks of this deadly virus, early detection is essential for effective prevention and control. Therefore, developing a more convenient and reliable method for PDCoV detection is the need of the hour. RESULTS: This study utilized a high-affinity monoclonal antibody as the capture antibody and a horseradish peroxidase labeled polyclonal antibody as the detection antibody to develop an enzyme-linked immunosorbent assay (DAS-ELSA) for PDCoV detection.Both antibodies target the PDCoV nucleocapsid (N) protein. The findings of this study revealed that DAS-ELISA was highly specific to PDCoV and did not cross-react with other viruses to cause swine diarrhea. The limit of detection of the virus titer using this method was 103 TCID50/mL of PDCoV particles. The results of a parallel analysis of 239 known pig samples revealed a coincidence rate of 97.07% (κ = 0.922) using DAS-ELISA and reverse transcriptase PCR (RT-PCR). The DAS-ELISA was used to measure the one-step growth curve of PDCoV in LLC-PK cells and the tissue distribution of PDCoV in infected piglets. The study found that the DAS-ELISA was comparable in accuracy to the TCID50 method while measuring the one-step growth curve. Furthermore, the tissue distribution measured by DAS-ELISA was also consistent with the qRT-PCR method. CONCLUSION: The developed DAS-ELISA method can be conveniently used for the early clinical detection of PDCoV infection in pigs, and it may also serve as an alternative method for laboratory testing of PDCoV.

Identification and serological responses to a novel Plasmodium vivax merozoite surface protein 1 (PvMSP-1) derived synthetic peptide: a putative biomarker for malaria exposure.

Background: The integration of diagnostic methods holds promise for advancing the surveillance of malaria transmission in both endemic and non-endemic regions. Serological assays emerge as valuable tools to identify and delimit malaria transmission, serving as a complementary method to rapid diagnostic tests (RDT) and thick smear microscopy. Here, we evaluate the potential of antibodies directed against peptides encompassing the entire amino acid sequence of the PvMSP-1 Sal-I strain as viable serological biomarkers for P. vivax exposure. Methods: We screened peptides encompassing the complete amino acid sequence of the Plasmodium vivax Merozoite Surface Protein 1 (PvMSP-1) Sal-I strain as potential biomarkers for P. vivax exposure. Here, immunodominant peptides specifically recognized by antibodies from individuals infected with P. vivax were identified using the SPOT-synthesis technique followed by immunoblotting. Two 15-mer peptides were selected based on their higher and specific reactivity in immunoblotting assays. Subsequently, peptides p70 and p314 were synthesized in soluble form using SPPS (Solid Phase Peptide Synthesis) and tested by ELISA (IgG, and subclasses). Results: This study unveils the presence of IgG antibodies against the peptide p314 in most P. vivax-infected individuals from the Brazilian Amazon region. In silico B-cell epitope prediction further supports the utilization of p314 as a potential biomarker for evaluating malaria transmission, strengthened by its amino acid sequence being part of a conserved block of PvMSP-1. Indeed, compared to patients infected with P. falciparum and uninfected individuals never exposed to malaria, P. vivax-infected patients have a notably higher recognition of p314 by IgG1 and IgG3.

A Microsphere Immunoassay for the Quantitative Detection of Antigens in Cell Culture Supernatant.

Quantitative immunoassays, such as the traditional enzyme-linked immunosorbent assay (ELISA), are used to determine concentrations of an antigen in a matrix of unknown antigen concentration. Magnetic immunoassays, such as the Luminex xMAP technology, allow for the simultaneous detection of multiple analytes and offer heightened sensitivity, specificity, low sample volume requirements, and high-throughput capabilities. Here, we describe a quantitative immunoassay using the Luminex MAGPIX® System to determine the antigen concentration from liquid samples with unknown concentrations. In detail, we describe a newly developed assay for determining production yields of Drosophila S2-produced Marburg virus (MARV) glycoprotein in insect-cell-culture-derived supernatant. The potential applications of this assay could extend to the quantification of viral antigens in fluids derived from both in vitro and in vivo models infected with live MARV, thereby providing additional applications for virological research.

Evaluation of Somatic Antigens of Adult Toxocara helminthes for Detection of Human Toxocariasis.

We aimed to develop an indirect enzyme-linked immunosorbent assay (ELISA) to evaluate the presence of specific IgG against Toxocara canis and Toxocara cati somatic antigens on the serum of patients with toxocariasis. The sensitivity, specificity, positive and negative predictive values for indirect-ELISA were calculated by receiver operating characteristic curve (ROC) analysis and Youden's J using Likelihood ratio. All statistics were analysed and graphs are plotted using GraphPad Prism version 8.4.3 (Graph Pad Software, La Jolla, CA, USA), with 95% confidence interval (CI). The sensitivity, specificity, positive and negative predictive values for T. canis were 100%, 82%, 79% and 100%, respectively. The mentioned variables for T. cati were 97%, 82%, 78% and 98%, respectively. Five immune reactive bands of 38, 40, 72, 100 and 250 kDa were common in both species. Toxocara crude antigens were highly immunogenic in human sera. Immunoreactive bands against T. canis compared to T. cati somatic antigen were about two times more. Unlike Toxocara excretory-secretory antigen, that was homologue in two species, somatic antigens of T. canis and T. cati showed different immunoreactive bands in our western blot.

[Enzyme immunoassay system for serological diagnosis of hemorrhagic fever with renal syndrome based on inactivated purified Puumala virus (Hantaviridae: Orthohantavirus)].

INTRODUCTION: Hemorrhagic fever with renal syndrome (HFRS) is the most common zoonotic human viral disease in the Russian Federation. More than 98% of the HFRS cases are caused by Puumala orthohantavirus (PUU). Effective serological tests are required for laboratory diagnosis of HFRS. OBJECTIVE: Construction of an enzyme immunoassay (ELISA) test system for detection of specific antibodies using standard antigen in the form of highly purified inactivated PUU virus as immunosorbent. MATERIALS AND METHODS: Preparation of PUU virus antigen, designing the ELISA for detection of specific antibodies, developing parameters of the ELISA system, parallel titration of HFRS patients sera by fluorescent antibody technique (FAT) and the new ELISA. RESULTS AND DISCUSSION: For the first time, ELISA based on purified inactivated PUU virus as standard antigen directly absorbed onto immunoplate was developed. Parallel titration of 50 samples from HFRS patients blood sera using FAT and the developed ELISA showed high sensitivity and specificity of this ELISA, with 100% concordance of testing results and significant level of correlation between the titers of specific antibodies in the two assays. CONCLUSION: The ELISA based on purified inactivated PUU virus as an immunosorbent can be effectively used for HFRS serological diagnosis and for mass seroepidemiological studies.

An Analytical Protocol for Detecting Antibody Titer Levels in Serum/Saliva by Indirect Enzyme-Linked Immunosorbent Assay (ELISA).

Enzyme-linked immunosorbent assay (ELISA) detects qualitatively and quantitatively the presence of antibodies or antigens in a sample. Due to its simplicity, high sensitivity, and user-friendliness, the test is widely used in laboratory research, clinical diagnoses, and food testing. This chapter describes the indirect semiquantitative ELISA protocol used to monitor antibody levels in animals and analyze the titer levels of specific antibodies against a target antigen in serum and saliva.

Specificity of serological screening tests and reference laboratory tests to diagnose gambiense human African trypanosomiasis: a prospective clinical performance study.

BACKGROUND: Serological screening tests play a crucial role to diagnose gambiense human African trypanosomiasis (gHAT). Presently, they preselect individuals for microscopic confirmation, but in future "screen and treat" strategies they will identify individuals for treatment. Variability in reported specificities, the development of new rapid diagnostic tests (RDT) and the hypothesis that malaria infection may decrease RDT specificity led us to evaluate the specificity of 5 gHAT screening tests. METHODS: During active screening, venous blood samples from 1095 individuals from Côte d'Ivoire and Guinea were tested consecutively with commercial (CATT, HAT Sero-K-SeT, Abbott Bioline HAT 2.0) and prototype (DCN HAT RDT, HAT Sero-K-SeT 2.0) gHAT screening tests and with a malaria RDT. Individuals with ≥ 1 positive gHAT screening test underwent microscopy and further immunological (trypanolysis with T.b. gambiense LiTat 1.3, 1.5 and 1.6; indirect ELISA/T.b. gambiense; T.b. gambiense inhibition ELISA with T.b. gambiense LiTat 1.3 and 1.5 VSG) and molecular reference laboratory tests (PCR TBRN3, 18S and TgsGP; SHERLOCK 18S Tids, 7SL Zoon, and TgsGP; Trypanozoon S2-RT-qPCR 18S2, 177T, GPI-PLC and TgsGP in multiplex; RT-qPCR DT8, DT9 and TgsGP in multiplex). Microscopic trypanosome detection confirmed gHAT, while other individuals were considered gHAT free. Differences in fractions between groups were assessed by Chi square and differences in specificity between 2 tests on the same individuals by McNemar. RESULTS: One gHAT case was diagnosed. Overall test specificities (n = 1094) were: CATT 98.9% (95% CI: 98.1-99.4%); HAT Sero-K-SeT 86.7% (95% CI: 84.5-88.5%); Bioline HAT 2.0 82.1% (95% CI: 79.7-84.2%); DCN HAT RDT 78.2% (95% CI: 75.7-80.6%); and HAT Sero-K-SeT 2.0 78.4% (95% CI: 75.9-80.8%). In malaria positives, gHAT screening tests appeared less specific, but the difference was significant only in Guinea for Abbott Bioline HAT 2.0 (P = 0.03) and HAT Sero-K-Set 2.0 (P = 0.0006). The specificities of immunological and molecular laboratory tests in gHAT seropositives were 98.7-100% (n = 399) and 93.0-100% (n = 302), respectively. Among 44 reference laboratory test positives, only the confirmed gHAT patient and one screening test seropositive combined immunological and molecular reference laboratory test positivity. CONCLUSIONS: Although a minor effect of malaria cannot be excluded, gHAT RDT specificities are far below the 95% minimal specificity stipulated by the WHO target product profile for a simple diagnostic tool to identify individuals eligible for treatment. Unless specificity is improved, an RDT-based "screen and treat" strategy would result in massive overtreatment. In view of their inconsistent results, additional comparative evaluations of the diagnostic performance of reference laboratory tests are indicated for better identifying, among screening test positives, those at increased suspicion for gHAT. TRIAL REGISTRATION: The trial was retrospectively registered under NCT05466630 in clinicaltrials.gov on July 15 2022.

[Development of a PCR Method to Detect a Mysid Species Causing False Positives in Tests for Crustacean Allergens].

Mysids are small crustaceans that are closely related to shrimp/prawns and crabs but not subject to food allergen labeling requirements for raw materials. In the past, a processed food that contained Japanese smelt (wakasagi) was suspected of producing a false-positive result in shrimp/prawn and crab allergen test because of the presence of consumed mysids. However, there was no reported methods to confirm mysid presence. Therefore, we developed a PCR method to detect mysids. The developed PCR method had high specificity for a mysid species, with no amplification observed from samples of shrimp, crab, krill, mantis shrimp, or the meat of Japanese smelt. In addition, DNA extracted from the internal organs of Japanese smelt was amplified by this PCR method, and sequencing revealed mysid DNA. This confirmed that mysids remained in the internal organs of Japanese smelt following consumption. This PCR method for mysid detection even amplified Japanese smelt-containing processed food samples that were suspected to have produced a false-positive result in shrimp/prawn and crab ELISA. Thus, this PCR method would enable to detect such false positives are caused by mysid contamination.

Evaluation of chimeric recombinant antigens for the serodiagnosis of Trypanosoma cruzi in dogs: a promising tool for Chagas disease surveillance.

BACKGROUND: Chagas disease (CD), a neglected parasitic disease caused by Trypanosoma cruzi, poses a significant health threat in Latin America and has emerged globally because of human migration. Trypanosoma cruzi infects humans and over 100 other mammalian species, including dogs, which are important sentinels for assessing the risk of human infection. Nonetheless, the serodiagnosis of T. cruzi in dogs is still impaired by the absence of commercial tests. In this study, we investigated the diagnostic accuracy of four chimeric recombinant T. cruzi IBMP antigens (IBMP-8.1, IBMP-8.2, IBMP-8.3, and IBMP-8.4) for detecting anti-T. cruzi antibodies in dogs, using latent class analysis (LCA). METHODS: We examined 663 canine serum samples, employing indirect ELISA with the chimeric antigens. LCA was utilized to establish a latent variable as a gold standard for T. cruzi infection, revealing distinct response patterns for each antigen. RESULTS: The IBMP (Portuguese acronym for the Molecular Biology Institute of Paraná) antigens achieved area under the ROC curve (AUC) values ranging from 90.9% to 97.3%. The highest sensitivity was attributed to IBMP-8.2 (89.8%), while IBMP-8.1, IBMP-8.3, and IBMP-8.4 achieved 73.5%, 79.6%, and 85.7%, respectively. The highest specificity was observed for IBMP-8.4 (98.6%), followed by IBMP-8.2, IBMP-8.3, and IBMP-8.1 with specificities of 98.3%, 94.4%, and 92.7%, respectively. Predictive values varied according to prevalence, indicating higher effectiveness in endemic settings. CONCLUSIONS: Our findings underscore the remarkable diagnostic performance of IBMP-8.2 and IBMP-8.4 for the serodiagnosis of Trypanosoma cruzi in dogs, representing a promising tool for the diagnosis of CD in dogs. These chimeric recombinant antigens may not only enhance CD surveillance strategies but also hold broader implications for public health, contributing to the global fight against this neglected tropical disease.

Newly developed peptide-ELISA successfully detected anti-IgG antibodies against Maedi-Visna virus in sheep.

Maedi Visna Virus (MVV) is a retrovirus that can infect sheep. There is still no effective therapy or vaccine against this virus and timely diagnosis is important to combat the complications of the disease. In this study, we aimed to develop an ELISA using peptides derived from gag protein as antigen. For this purpose, B cell epitopes of gag protein were predicted and a docking analysis with the B cell receptor was performed to select peptides to be used in ELISA. After three soluble epitopes with the highest antigenicity were produced as peptides, the immunogenicity of each peptide was determined by ELISA using sheep serum samples categorized as MVV positive (n=24) and negative (n=13). Subsequently, in house ELISA using above mentioned immunogenic peptides as antigen was used to investigate MVV seroprevalence in sheep (n=88). According to the results, among three peptides, two of them strongly reacted with MVV positive serum samples and the mean absorbance values detected among positive and negative serum samples were statistically significant, indicating that these peptides were immunogenic (P=0.016 and P=0.038). The third peptide also reacted with positive serum samples but the mean absorbance value was not statistically significant and this peptide was considered non-immunogenic (P=0.175). The immunogenic two peptides showed the same high sensitivity and specificity values of 91.60 and 92.80 according to the commercial kit. Moreover, MVV seroprevalence detected by peptide-ELISAs using CKQGSKE and CRPQGKAGHKG peptides as antigen was 3.40 % and 4.5 %, respectively. As a result, it was shown that these peptides can be successfully used for serological diagnosis of MVV.

Estimation of Individual Positive Anti-Islet Autoantibodies from 3 Screen ICA Titer.

The 3 Screen ICA ELISA is a novel assay capable of simultaneously measuring autoantibodies to glutamic acid decarboxylase (GADA), insulinoma-associated antigen-2 (IA-2A), and zinc transporter 8 (ZnT8A), making it a valuable tool for screening type 1 diabetes. Despite its advantages, it cannot specify which individual autoantibodies are positive or negative. This study aimed to estimate individual positive autoantibodies based on the 3 Screen ICA titer. Six hundred seventeen patients with type 1 diabetes, simultaneously measured for 3 Screen ICA and three individual autoantibodies, were divided into five groups based on their 3 Screen ICA titer. The sensitivities and contribution rates of the individual autoantibodies were then examined. The study had a cross-sectional design. Sixty-nine percent (424 of 617) of patients with type 1 diabetes had 3 Screen ICA titers exceeding the 99th percentile cut-off level (20 index). The prevalence of GADA ranged from 80% to 100% in patients with a 3 Screen ICA over 30 index and 97% of patients with a 3 Screen ICA ≥300 index. Furthermore, the prevalence of all individual autoantibodies being positive was 0% for ≤80 index and as high as 92% for ≥300 index. Significant associations were observed in specific titer groups: the 20-29.9 index group when all the individual autoantibodies were negative, the 30-79.9 index group when positive for GADA alone or IA-2A alone, the 30-299.9 index group when positive for ZnT8A alone, the 80-299.9 index group when positive for both IA-2A and ZnT8A, the 300-499.9 index group when positive for both GADA and ZnT8A, and the ≥300 index group when positive for all individual autoantibodies. These results suggest that the 3 Screen ICA titer may be helpful in estimating individual positive autoantibodies.

Design and expression of a chimeric recombinant antigen (SsIR-Ss1a) for the serodiagnosis of human strongyloidiasis: Evaluation of performance, sensitivity, and specificity.

BACKGROUND: The sensitivity of parasitological and molecular methods is unsatisfactory for the diagnosis of strongyloidiasis, and serological techniques are remaining as the most effective diagnostic approach. The present study aimed to design and produce a chimeric recombinant antigen from Strongyloides stercoralis immunoreactive antigen (SsIR) and Ss1a antigens, using immune-informatics approaches, and evaluated its diagnostic performance in an ELISA system for the diagnosis of human strongyloidiasis. METHODOLOGY/PRINCIPAL FINDINGS: The coding sequences for SsIR and Ss1a were selected from GenBank and were gene-optimized. Using bioinformatics analysis, the regions with the highest antigenicity that did not overlap with other parasite antigens were selected. The chimeric recombinant antigen SsIR- Ss1a, was constructed. The solubility and physicochemical properties of the designed construct were analyzed and its tertiary structures were built and evaluated. The construct was expressed into the pET-23a (+) expression vector and the optimized DNA sequences of SsIR-Ss1a (873 bp) were cloned into competent E. coli DH5α cells. Diagnostic performances of the produced recombinant antigen, along with a commercial kit were evaluated in an indirect ELISA system, using a panel of sera from strongyloidiasis patients and controls. The physicochemical and bioinformatics evaluations revealed that the designed chimeric construct is soluble, has a molecular with of 35 KDa, and is antigenic. Western blotting confirmed the immunoreactivity of the produced chimeric recombinant antigen with the sera of strongyloidiasis patients. The sensitivity and specificity of the indirect ELISA system, using the produced SsIR-Ss1a chimeric antigen, were found to be 93.94% (95% CI, 0.803 to 0.989) and 97.22% (95% CI, 0.921 to 0.992) respectively. CONCLUSIONS/SIGNIFICANCE: The preliminary findings of this study suggest that the produced SsIR-Ss1a chimeric antigen shows promise in the diagnosis of human strongyloidiasis. However, these results are based on a limited panel of samples, and further research with a larger sample size is necessary to confirm its accuracy. The construct has potential as an antigen in the ELISA system for the serological diagnosis of this neglected parasitic infection, but additional validation is required.

Serological diagnosis of strongyloidiasis: An evaluation of three commercial assays.

BACKGROUND: Strongyloidiasis is caused by a neglected nematode, manifesting as chronic intestinal infection with potentially severe manifestations. The disease is an emerging problem in non-endemic countries affecting travelers and migrants. Diagnosis of strongyloidiasis is hampered by the lack of standardization and absence of a gold standard. Since adequate direct methods to detect the motile larvae in stool samples are not widely available, other techniques such as serology have been developed. METHODS: We evaluated three commercial ELISA kits (DRG Instruments, IVD Research, and Bordier Affinity Products) to detect IgG antibodies against Strongyloides stercoralis assays utilizing serum samples from travelers with microscopically confirmed strongyloidiasis (n = 50) and other imported helminthic infections (n = 159) as well as healthy controls (n = 50). RESULTS: The DRG, IVD, and Bordier assays showed sensitivities of 58.0%, 64.0%, and 56.0%, respectively. Specificity values were 96.0%, 96.0%, and 92.0% in healthy controls, and 67.3%, 62.9%, and 76.7% in cases with other helminth infections, respectively. Cross-reactions were mostly observed in cases with other nematodes (37.5%, 42.5%, and 20.0%, respectively), but also in trematode (33.3%, 38.1%, and 19.0%, respectively) and in cestode infections (25.0%, 30.0%, and 32.5%, respectively). CONCLUSION: The study demonstrates the diagnostic limitations of serological assays to detect or exclude cases of strongyloidiasis in returning travelers, who frequently present with recent or acute infections.

Quantification of DNA Methylation by ELISA in Epigenetic Studies in Plant Tissue Culture: A Theoretical-Practical Guide.

This chapter describes a step-by-step protocol for rapid serological quantification of global DNA methylation by enzyme-linked immunosorbent assay (ELISA) in plant tissue culture specimens. As a case study model, we used the coconut palm (Cocos nucifera), from which plumules were subjected to somatic embryogenesis followed by embryogenic calli multiplication. DNA methylation is one of the most common epigenetic markers in the regulation of gene expression. DNA methylation is generally associated with non-expressed genes, that is, gene silencing under certain conditions, and the degree of DNA methylation can be used as a marker of various physiological processes, both in plants and in animal cells. Methylation consists of adding a methyl radical to carbon 5 of the DNA cytosine base. Herein, the global DNA methylation was quantified by ELISA with antibodies against methylated cytosines using a commercial kit (Zymo-Research™). The method allowed the detection of methylation in total DNA extracts from coconut palm embryogenic calli (arising from somatic embryogenesis) cultivated in liquid or solid media by using antibodies against methylated cytosines and enzymatic development with a colorimetric substrate. Control samples of commercially provided Escherichia coli bacterial DNA with previously known methylation percentages were included in the ELISA test to construct an experimental methylation standard curve. The logarithmic regression of this E. coli standard curve allowed methylation quantification in coconut palm samples. The present ELISA methodology, applied to coconut palm tissue culture specimens, is promising for use in other plant species and botanical families. This chapter is presented in a suitable format for use as a step-by-step laboratory procedure manual, with theoretical introduction information, which makes it easy to apply the protocol in samples of any biological nature to evaluate DNA global methylation associated with any physiological process.