scholarly journals High-Specificity Targets in SARS-CoV-2 N Protein for Serological Antibody Detection

2020 ◽  
Author(s):  
Jianhai Yu ◽  
Zhiran Qin ◽  
Xiaoen He ◽  
Xuling Liu ◽  
Jinxiu Yao ◽  
...  
Keyword(s):  
Amino Acid Level ◽  
Cell Epitope ◽  
High Specificity ◽  
Cross Reactivity ◽  
Serological Diagnosis ◽  
Detection Methods ◽  
Antibody Detection ◽  
Nucleic Acid Detection ◽  
N Protein ◽  
Antibody Levels

Abstract Background The current coronavirus disease (COVID-19) pandemic has created a pressing need to diagnose and screen a large number of close contacts of confirmed and suspected cases. Numerous nucleic acid detection kits are being rapidly developed and approved for viral etiological diagnosis; however, these are limited by the number of false negatives produced in clinical practice. Therefore, there is an urgent need to establish serological detection methods to serve as supplementary diagnostics. Methods We (1) performed a conservation and specificity analysis of the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid (N) protein, which is the main target of serological diagnosis; (2) integrated various B-cell epitope prediction methods to obtain possible dominant epitope regions for the N protein; (3) applied ELISA to analyze differences in the serological antibody levels for different epitopes; and (4) identified N protein epitopes for IgG and IgM with high specificity. Results SARS-CoV-2 strains showed low mutation rates for the N protein, and the construction of a phylogeny was a good characterization of its molecular evolutionary lineage in relation to other coronaviruses. SARS-CoV-2 showed the closest genetic relationship with SARS-CoV, which showed multiple consecutive long conserved regions at the amino acid level, but differed substantially from other coronaviruses. Tests targeting the SARS-CoV-2 N protein produced strong positive results in SARS-CoV patients in recovery. Of the five epitope dominant regions, using N18-39 and N183-197 for IgG and IgM detection, respectively, can effectively overcome the limitations of cross-reactivity. Conclusions The patients infected with both SARS viruses may exhibit cross-reactivity when using the N protein for antibody detection. However, there are regions of the N protein that can be used for antibody detection and some of these regions showed good specificity even between SARS-CoV-2 and SARS-CoV, and the antibody levels detected were consistent with those detected by the complete N protein. These findings provide a basis for serological diagnosis of SARS-CoV-2 patients, and research ideas for developing vaccines.

C1q-Binding HLA Antibodies Do Not Predict Platelet Transfusion Failure in TRAP Study Participant

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1562-1562
Author(s):  
Rachael P. Jackman ◽  
Douglas Bolgiano ◽  
Mila Lebedeva ◽  
Sherrill J. Slichter ◽  
Philip J. Norris
Keyword(s):  
Conflicts Of Interest ◽  
Fluorescent Antibody ◽  
Study Participant ◽  
Class I ◽  
Detection Methods ◽  
Antibody Detection ◽  
Hla Antibodies ◽  
Luminex Assay ◽  
Single Antigen ◽  
Antibody Levels

Abstract Introduction: In the Trial to Reduce Alloimmunization to Platelets (TRAP) study, 101 of the 530 subjects became clinically refractory (CR) to platelet transfusions in the absence of detectable antibodies against HLA as measured by the lymphocytotoxicity assay (LCA). Using more sensitive bead-based detection methods we have previously demonstrated that while many of these LCA- patients do have anti-HLA antibodies, that these low to moderate level antibodies do not predict refractoriness. In addition to being less sensitive then bead based methods, the LCA screen only detects complement-binding antibodies. As these antibodies could be important for platelet rejection, we assessed if previously undetected complement-binding antibodies could account for some of the refractoriness seen in LCA- patients. Methods: 169 LCA- (69 CR, 100 non-CR) and 20 LCA+ (10 CR, 10 non-CR) subjects were selected from the TRAP study. Anti-class I HLA IgG and C1q binding antibodies were measured in serum or plasma using two different multi-analyte, semi-quantitative, bead-based fluorescent antibody detection assays: the LabScreen mixed Luminex assay, and the LabScreen single antigen class I assay with or without added EDTA (One Lambda). Groups were compared using an unpaired t-test, a=0.05, and correlation between variables was also assessed. Results: New measurements of anti-class I HLA IgG antibodies reliably reproduced earlier data with a strong correlation between the old and new measurements (R2=0.9736, p<0.0001). While some of the LCA- subjects did have detectable C1q-binding anti-class I HLA antibodies, and some LCA+ subjects did not, levels of these antibodies were significantly higher among LCA+ subjects (p<0.0001). Levels of C1q-binding anti-class I HLA antibodies were not significantly different between CR and non-CR among either the LCA- or LCA+ subjects. Conclusions: While complement-binding anti-class I HLA antibody levels were higher in the LCA+ subjects, higher levels of these antibodies were not seen in CR LCA+ patients as compared with non-CR LCA+ patients. Complement-binding anti-class I HLA antibodies do not account for refractoriness seen among the LCA- TRAP subjects. This work confirms that low to mid level anti-class I antibodies do not drive refractoriness to platelets, and suggests that antibody-independent mechanisms cause refractoriness in patients lacking higher levels of anti-HLA antibodies. Disclosures No relevant conflicts of interest to declare.

scholarly journals Persistence of Babesia microti Infection in Humans

Pathogens ◽  
2019 ◽  
Vol 8 (3) ◽  
pp. 102 ◽  
Author(s):  
Evan M. Bloch ◽  
Sanjai Kumar ◽  
Peter J. Krause
Keyword(s):  
Severe Disease ◽  
Immunosuppressive Drugs ◽  
Detection Methods ◽  
Antibody Detection ◽  
Babesia Microti ◽  
Nucleic Acid Detection ◽  
Probability Of Survival ◽  
Immunoglobulin Preparations ◽  
Therapeutic Measures ◽  
Peripheral Blood Smears

Persistent infection is a characteristic feature of babesiosis, a worldwide, emerging tick-borne disease caused by members of the genus Babesia. Persistence of Babesia infection in reservoir hosts increases the probability of survival and transmission of these pathogens. Laboratory tools to detect Babesia in red blood cells include microscopic detection using peripheral blood smears, nucleic acid detection (polymerase chain reaction and transcription mediated amplification), antigen detection, and antibody detection. Babesia microti, the major cause of human babesiosis, can asymptomatically infect immunocompetent individuals for up to two years. Chronically infected blood donors may transmit the pathogen to another person through blood transfusion. Transfusion-transmitted babesiosis causes severe complications and death in about a fifth of cases. Immunocompromised patients, including those with asplenia, HIV/AIDS, malignancy, or on immunosuppressive drugs, often experience severe disease that may relapse up to two years later despite anti-Babesia therapy. Persistent Babesia infection is promoted by Babesia immune evasive strategies and impaired host immune mechanisms. The health burden of persistent and recrudescent babesiosis can be minimized by development of novel therapeutic measures, such as new anti-parasitic drugs or drug combinations, improved anti-parasitic drug duration strategies, or immunoglobulin preparations; and novel preventive approaches, including early detection methods, tick-avoidance, and blood donor screening.

scholarly journals Comparison of different detection methods for Ascaris suum infection on Austrian swine farms

2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Anja Joachim ◽  
Christian Winkler ◽  
Ursula Ruczizka ◽  
Andrea Ladinig ◽  
Michaela Koch ◽  
...  
Keyword(s):  
Ascaris Suum ◽  
Standard Procedure ◽  
High Sensitivity ◽  
Current Method ◽  
Serological Diagnosis ◽  
Detection Methods ◽  
Antibody Detection ◽  
Blood Samples ◽  
Specific Antibodies ◽  
Swine Farms

Abstract Background Ascaris suum, the large roundworm of pigs, is one of the economically most important pig parasites worldwide. In Austria it is commonly diagnosed by monitoring livers for milk spots at the slaughterhouse and intravital diagnosis (flotation for detection of fecal egg shedding). Recently, serological diagnosis based on the detection of specific antibodies with an ELISA (SERASCA®) with high sensitivity has been developed. To introduce and evaluate serology for A. suum screening in Austrian pigs, blood (for serology) (n = 177) and feces (for copromicroscopy) (n = 177) were taken from randomly selected slaughter pig batches from 18 farms at a slaughterhouse in Lower Austria. In addition, livers presented at slaughter (n = 844; max. 70/farm) were evaluated for milk spots. Results Overall, 19% of the livers were milk spot-positive (22% of those with complete diagnostic evaluations). Thirteen percent of the fecal samples contained A. suum eggs, while 69% of the blood samples were serologically positive. Despite we did not determine the sensitivity of the ELISA specifically, results ouf our study confirmed the high sensitivity of the ELISA, which was claimed by the manufacturer prior to our work (sensitivity: liver assessment: 23.5–27.0%; copromicroscopy: 8.5–9.0%; ELISA: 99.5%), and a high percentage of A. suum infections that remained undetected by standard liver assessment. Conclusions This suggests that the current method of roundworm diagnostics is insufficient and antibody detection at the end of the fattening period should be established as the standard procedure.

scholarly journals Selection of New Diagnostic Markers for Dirofilaria Repens Infections With the Use of Phage Display Technology

2021 ◽  
Author(s):  
Mateusz Pękacz ◽  
Katarzyna Basałaj ◽  
Alicja Kalinowska ◽  
Maciej Klockiewicz ◽  
Diana Stopka ◽  
...  
Keyword(s):  
Phage Display ◽  
Disease Transmission ◽  
High Specificity ◽  
Cross Reactivity ◽  
Diagnostic Markers ◽  
Specific Cell ◽  
Phage Display Technology ◽  
Parasite Infections ◽  
Display Technology ◽  
Antibody Levels

Abstract Dirofilaria repens is a parasitic nematode causing vector-borne disease (dirofilariasis), considered an emerging problem in veterinary and human medicine. Although main hosts are carnivores, particularly dogs, D. repens shows high zoonotic potential. The disease spreads uncontrollably, affecting new areas. Since there is no vaccine against dirofilariasis, the only way to limit disease transmission is an early diagnosis. Currently, diagnosis depends on the detection of microfilariae in the host bloodstream using modified Knott's test or multiplex PCR. However, the efficacy of tests relying on microfilariae detection is limited by microfilariae periodic occurrence. Therefore, a new reliable diagnostic test is required. Our study aimed to select new diagnostic markers for dirofilariasis with potential application in diagnostics. We focused on single epitopes to ensure high specificity of diagnosis and avoid cross-reactivity with the other parasite infections common in dogs. Using phage display technology and 12-mer peptides library, we selected epitopes highly reactive with IgG from sera of infected dogs. Additionally, our study presents the possibility of detecting D. repens specific cell-free DNA in dogs with no microfilaria but high IgG and IgM antibody levels against parasite somatic antigen.

Development of an antigen-ELISA and a colloidal gold–based immunochromatographic strip based on monoclonal antibodies for detection of avian influenza A(H5) viruses

2021 ◽  
pp. 104063872110275
Author(s):  
Yixin Xiao ◽  
Fan Yang ◽  
Fumin Liu ◽  
Linfang Cheng ◽  
Hangping Yao ◽  
...  
Keyword(s):  
Avian Influenza ◽  
Disease Virus ◽  
Colloidal Gold ◽  
Rapid Detection ◽  
Influenza A ◽  
Field Investigation ◽  
Cross Reactivity ◽  
Detection Methods ◽  
Immunochromatographic Strip ◽  
Antigen Elisa

Avian influenza A(H5) viruses (avian IAVs) pose a major threat to the economy and public health. We developed an antigen-ELISA (ag-ELISA) and a colloidal gold–based immunochromatographic strip for the rapid detection of avian A(H5) viruses. Both detection methods displayed no cross-reactivity with other viruses (e.g., other avian IAVs, infectious bursal disease virus, Newcastle disease virus, infectious bronchitis virus, avian paramyxovirus). The ag-ELISA was sensitive down to 0.5 hemagglutinin (HA) units/100 µL of avian A(H5) viruses and 7.5 ng/mL of purified H5 HA proteins. The immunochromatographic strip was sensitive down to 1 HA unit/100 µL of avian A(H5) viruses. Both detection methods exhibited good reproducibility with CVs < 10%. For 200 random poultry samples, the sensitivity and specificity of the ag-ELISA were 92.6% and 98.8%, respectively, and for test strips were 88.9% and 98.3%, respectively. Both detection methods displayed high specificity, sensitivity, and stability, making them suitable for rapid detection and field investigation of avian A(H5) viruses.

scholarly journals Long-Term Longitudinal Evaluation of Six Commercial Immunoassays for the Detection of IgM and IgG Antibodies against SARS CoV-2

Viruses ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 1244
Author(s):  
Iulia Nedelcu ◽  
Raluca Jipa ◽  
Roxana Vasilescu ◽  
Cristian Băicuș ◽  
Costin-Ioan Popescu ◽  
...  
Keyword(s):  
Clinical Performance ◽  
Sample Selection ◽  
Early Time ◽  
Humoral Response ◽  
High Specificity ◽  
Data Interpretation ◽  
Serum Samples ◽  
Chemiluminescent Immunoassay ◽  
Chemiluminescent Microparticle Immunoassay ◽  
Antibody Levels

The number of serological assays for SARS-CoV-2 has skyrocketed in the past year. Concerns have been raised regarding their performance characteristics, depending on the disease severity and the time of the analysis post-symptom onset (PSO). Thus, independent validations using an unbiased sample selection are required for meaningful serology data interpretation. We aimed to assess the clinical performance of six commercially available assays, the seroconversion, and the dynamics of the humoral response to SARS-CoV-2 infection. The study included 528 serum samples from 156 patients with follow-up visits up to six months PSO and 161 serum samples from healthy people. The IgG/total antibodies positive percentage increased and remained above 95% after six months when chemiluminescent immunoassay (CLIA) IgG antiS1/S2 and electro-chemiluminescent assay (ECLIA) total antiNP were used. At early time points PSO, chemiluminescent microparticle immunoassay (CMIA) IgM antiS achieved the best sensitivity. IgM and IgG appear simultaneously in most circumstances, and when performed in parallel the sensitivity increases. The severe and the moderate clinical forms were significantly associated with higher seropositivity percentage and antibody levels. High specificity was found in all evaluated assays, but the sensitivity was variable depending on the time PSO, severity of disease, detection method and targeted antigen.

scholarly journals Electrokinetic Formation of “Microbridges” for Protein Biomarkers as Sensors

2007 ◽  
Vol 12 (5) ◽  
pp. 311-317 ◽  
Author(s):  
Vindhya Kunduru ◽  
Shalini Prasad
Keyword(s):  
Electric Fields ◽  
Microelectrode Array ◽  
Protein Detection ◽  
High Specificity ◽  
Detection Methods ◽  
Label Free ◽  
Protein Biomarkers ◽  
Detection Scheme ◽  
Polystyrene Beads ◽  
Conducting Path

We demonstrate a technique to detect protein biomarkers contained in vulnerable coronary plaque using a platform-based microelectrode array (MEA). The detection scheme is based on the property of high specificity binding between antibody and antigen similar to most immunoassay techniques. Rapid clinical diagnosis can be achieved by detecting the amount of protein in blood by analyzing the protein's electrical signature. Polystyrene beads which act as transportation agents for the immobile proteins (antigen) are electrically aligned by application of homogenous electric fields. The principle of electrophoresis is used to produce calculated electrokinetic movement among the anti-C-reactive protein (CRP), or in other words antibody funtionalized polystyrene beads. The electrophoretic movement of antibody-functionalized polystyrene beads results in the formation of “Microbridges” between the two electrodes of interest which aid in the amplification of the antigen—antibody binding event. Sensitive electrical equipment is used for capturing the amplified signal from the “Microbridge” which essentially behaves as a conducting path between the two electrodes. The technique circumvents the disadvantages of conventional protein detection methods by being rapid, noninvasive, label-free, repeatable, and inexpensive. The same principle of detection can be applied for any receptor—ligand-based system because the technique is based only on the volume of the analyte of interest. Detection of the inflammatory coronary disease biomarker CRP is achieved at concentration levels spanning over the lower microgram/milliliter to higher order nanogram/milliliter ranges.

scholarly journals Serological Diagnosis of Chronic Chagas Disease: Is It Time for a Change?

2016 ◽  
Vol 54 (6) ◽  
pp. 1566-1572 ◽  
Author(s):  
Alba Abras ◽  
Montserrat Gállego ◽  
Teresa Llovet ◽  
Silvia Tebar ◽  
Mercedes Herrero ◽  
...  
Keyword(s):  
Chagas Disease ◽  
False Positive ◽  
Disease Diagnosis ◽  
Cross Reactivity ◽  
Human Migration ◽  
Serological Diagnosis ◽  
Content Type ◽  
New Generation ◽  
Single Technique ◽  
Chronic Chagas Disease

Chagas disease has spread to areas that are nonendemic for the disease with human migration. Since no single reference standard test is available, serological diagnosis of chronic Chagas disease requires at least two tests. New-generation techniques have significantly improved the accuracy of Chagas disease diagnosis by the use of a large mixture of recombinant antigens with different detection systems, such as chemiluminescence. The aim of the present study was to assess the overall accuracy of a new-generation kit, the Architect Chagas (cutoff, ≥1 sample relative light units/cutoff value [S/CO]), as a single technique for the diagnosis of chronic Chagas disease. The Architect Chagas showed a sensitivity of 100% (95% confidence interval [CI], 99.5 to 100%) and a specificity of 97.6% (95% CI, 95.2 to 99.9%). Five out of six false-positive serum samples were a consequence of cross-reactivity withLeishmaniaspp., and all of them achieved results of <5 S/CO. We propose the Architect Chagas as a single technique for screening in blood banks and for routine diagnosis in clinical laboratories. Only gray-zone and positive sera with a result of ≤6 S/CO would need to be confirmed by a second serological assay, thus avoiding false-positive sera and the problem of cross-reactivity withLeishmaniaspecies. The application of this proposal would result in important savings in the cost of Chagas disease diagnosis and therefore in the management and control of the disease.

scholarly journals A 16S rDNA-based nested PCR protocol to detect Campylobacter gracilis in oral infections

2003 ◽  
Vol 17 (2) ◽  
pp. 142-146 ◽  
Author(s):  
José Freitas Siqueira Júnior ◽  
Isabela das Neves Rôças
Keyword(s):  
16S Rdna ◽  
High Sensitivity ◽  
High Specificity ◽  
Cross Reactivity ◽  
Nested Polymerase Chain Reaction ◽  
Oral Infections ◽  
Root Canals ◽  
Infected Root ◽  
Pcr Products ◽  
Species Specific

The aim of this study was to describe a 16S rDNA-based nested polymerase chain reaction (nPCR) assay to investigate the occurrence of Campylobacter gracilis in oral infections. Samples were collected from ten infected root canals, ten cases of acute periradicular abscesses and eight cases of adult marginal periodontitis. DNA extracted from the samples was initially amplified using universal 16S rDNA primers. A second round of amplification used the first PCR products to detect C. gracilis using oligonucleotide primers designed from species-specific 16S rDNA signature sequences. The nPCR assay used in this study showed a detection limit of 10 C. gracilis cells and no cross-reactivity was observed with nontarget bacteria. C. gracilis was detected in the three types of oral infections investigated - 4/10 infected root canals; 2/10 acute periradicular abscesses; and 1/8 subgingival specimens from adult periodontitis. The method proposed in this study showed both high sensitivity and high specificity to directly detect C. gracilis in samples from root canal infections, abscesses, and subgingival plaque. Our findings confirmed that C. gracilis may be a member of the microbiota associated with distinct oral infections, and its specific role in such diseases requires further clarification.

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