Serodiagnostic Methods Research Articles

Increased production of orthoflavivirus single-round infectious particles produced in mammalian cells at a suboptimal culture temperature of 28°C

In the employment of serodiagnostic methods for the detection of orthoflavivirus infections, neutralization tests are known to be more accurate than measurements of antibody binding properties employing enzyme-linked immunosorbent assays. However, neutralization tests require infectious virus and laboratories with an appropriate level of biosafety. Single-round infectious particles (SRIPs), which encode a reporter gene instead of the viral structural protein genes, are replication incompetent and represent a safe and reliable alternative to the diagnosis of pathogenic viruses in neutralization tests. The orthoflavivirus SRIPs are produced by co-transfection of plasmids expressing virus-like particles and replicons into mammalian cell lines preferably with high transfection efficacy, such as HEK293T cells. However, certain orthoflavivirus SRIPs have limitations in their efficient expression at 37°C, which is the optimal temperature for mammalian cell growth, resulting in insufficient yields for neutralization tests. Here, we demonstrate that the production of orthoflavivirus SRIPs increases at the lower temperature of 28°C compared to 37°C. Moreover, infections with 28°C-cultured SRIPs in microneutralization tests were specifically inhibited in the presence of serum from mice infected with homologous viruses, suggesting that these SRIPs preserved their neutralizing epitopes for antibodies. Our method to produce high titer SRIPs is anticipated to promote efficient and safe SRIPs neutralization tests as a general serodiagnostic method for detecting virus-specific neutralizing antibodies against orthoflaviviruses.

Adaptive Immune Response Investigation in Lyme Borreliosis.

To diagnose Lyme Borreliosis, it is advised to use an enzyme-linked immunosorbent test to check for serum antibodies specific for Lyme and all tests with positive or ambiguous enzyme-linked immunosorbent assay (ELISA) results being confirmed by immunoblot. This method of measuring the humoral immunity in human fluids (e.g., by ELISA) has provided robust and reproducible results for decades and similar assays have been validated for monitoring of B cell immunity. These immunological tests that detect antibodies to Borrelia burgdorferi are useful in the diagnosis of Borreliosis on a routine basis. The variety of different Borrelia species and their different geographic distributions are the main reasons why standards and recommendations are not identical across all geographic regions of the world. In contrast to humoral immunity, the T cell reaction or cellular immunity to the Borrelia infection has not been well elucidated, but over time with more studies a novel T cell-based assay (EliSpot) has been developed and validated for the sensitive detection of antigen-specific T cell responses to B. burgdorferi. The EliSpot Lyme assay can be used to study the T cell response elicited by Borrelia infections, which bridges the gap between the ability to detect humoral immunity and cellular immunity in Lyme disease. In addition, detecting cellular immunity may be a helpful laboratory diagnostic test for Lyme disease, especially for seronegative Lyme patients. Since serodiagnostic methods of the Borrelia infection frequently provide false positive and negativeresults, this T cell-based diagnostic test (cellular assay) may help in confirming a Lyme diagnosis. Many clinical laboratories are convinced that the cellular assay is superior to the Western Blot assay in terms of sensitivity for detecting the underlying Borrelia infection. Research also suggests that there is a dissociation between the magnitude of the humoral and the T cell-mediated cellular immune responses in the Borrelia infection. Lastly, the data implies that the EliSpot Lyme assay may be helpful to identify Borrelia infected individuals when the serology-based diagnostic fails to do so. Here in this chapter the pairing of humoral and cellular immunity is employed to evaluate the adaptive response in patients.

Atypical chronic active herpesvirus infections: Etiological structure, frequency of occurrence, clinical syndromes associated with them

BACKGROUND: Every year, a steady, progressive increase in the number of atypical chronic, active forms of infections caused by herpesviruses is recorded. Diagnosing and selecting adequate therapeutic strategies for treating these infections present significant difficulties for physicians due to the polysyndromicity and many clinical manifestations.
 AIMS: This study determines the prevalence of atypical chronic, active infections caused by herpesviruses among patients infected with herpesvirus infections and studies the etiological structural features and clinical manifestations/criteria and signs of atypically occurring chronic forms of herpes viral infections.
 MATERIALS AND METHODS: Under our supervision at the Clinical and Diagnostic Center Medsi in Belorusskaya (Moscow), 98 patients of both sexes aged 23 to 60 years suffering from atypical chronic, active forms of infections caused by herpesviruses comprised the herpesviruses group. The comparison group consisted of 30 conditionally healthy subjects comparable in sex and age to patients. In addition to traditional methods (history collection, physical examination, general blood test, and others), serodiagnostic methods with ELISA were used to detect herpesvirus infections. ELISA was also used for detecting the genome of viruses in biomaterials. The study was approved by the ethics committee, and all patients received informed consent to participate in the study. Statistical analysis was performed using adequate methods.
 RESULTS: The study of the etiological structure of herpesvirus infections in patients with atypical chronic, active forms of infections caused by herpesviruses, mixed herpesvirus infections were shown to occur in 83.4% of patients, and mono herpesvirus infections in 16.6% of cases. It was shown that the EpsteinBarr virus was the dominant virus among patients with mono and mixed herpesvirus infections. A high rate of EpsteinBarr virus DNA detection was demonstrated in saliva (84.2%), posterior pharyngeal wall scrapings (73.5%), tonsils (42.9%), urine (12.6%), and blood (8.3%) is a marker of the high replicative activity of the virus. The primary clinical syndromes associated with mono and mixed atypical chronic and active forms of infections caused by herpesviruses were identified.
 CONCLUSIONS: This study identified and quantitatively assessed the viral load associated with the severity of the course and clinical manifestations of atypical chronic, active forms of infections caused by herpesviruses. Clarifying the features of clinical manifestations and syndromes in patients suffering from various mono and mixed herpesvirus infections will allow us to outline the goals for the further development of an adequate diagnostic algorithm for these atypical forms of herpesvirus infections and the concept of targeted, personalized etio- and immunopathogenetic therapy.

Comparative evaluation of four commercial analyzers for the serological screening of hepatitis A, B, C and HIV.

Independent evaluations that deploy clinical patient samples are important in assessing the performance of commercial tests used for serological screening of viral hepatitis and HIV in clinical laboratories. We compared the analytical performance of Abbott Architect i2000SR, Abbott Alinity i, DiaSorin Liaison XL, and Siemens Atellica for the following analytes: anti-HAV IgG/anti-HAV total, anti-HAV IgM, HBsAg, anti-HBc IgM, Anti-HBc, HBeAg, anti-HBe, anti-HBs, anti-HCV, and HIV Ag/Ab. In addition, anti-HBc IgM, HBeAg, and anti-HBe were evaluated for Abbott Architect, Abbott Alinity and DiaSorin Liaison. Pseudonymized clinical serum specimens (N=98-200 for each analyte) were selected for the analysis according to their reactivity on the Abbott Architect. The results were compared against Abbott Architect and against consensus. A generally high agreement was observed between the tests. Abbott Alinity had the lowest anti-HAV IgG/total specificity (75.9% against Abbott Architect and 83.0% against consensus). The comparatively low sensitivity of Siemens Atellica (78.2%), Abbott Alinity (87.5%) and DiaSorin Liaison (89.3%) for anti-HAV IgM against Abbott Architect may reflect a higher false-positive rate of Abbott Architect. Particular variation was observed in the sensitivity values of anti-HBc, HBsAg and HIV Ag/Ab between the test methods. DiaSorin Liaison anti-HBs gave consistently higher values as compared to the other tests. The serodiagnostic methods for HIV and viral hepatitis of Abbott Architect, Abbott Alinity, DiaSorin Liaison, and Siemens Atellica performed well in comparison with each other. The observed differences between the tests will provide useful information for clinical laboratories in planning their workflows for screening and confirmation.

On the question of the practical value of the Stamm antigen according to Kalinin and Ginsburg

Soon after the Wassermann reaction was introduced for the diagnosis of syphilis, aspirations appeared that had the goal, on the one hand, to improve the method of serodiagnosis of syphilis in the sense of obtaining more accurate results, since the Wassermann reaction, even with the most careful formulation, is still sometimes gave unsatisfactory results, on the other hand, a simplification of this technique, in order thereby to be able to apply serodiagnostic methods in primitive laboratory conditions, without laboratory animals.

To serodiagnosis of echinococcal diseases. Z. V. Ermolaeva. (Tr. I Congress of Khir. North-Caucasian Territories)

Z. V. Ermolaeva. (Tr. I Congress of Hir. North Caucasus. Territory), making an assessment of various sero-diagnostic methods of echinococcosis, highlights the intradermal test, due to its immediacy and demonstrativeness.

Use of blood matrices and alternative biological fluids for antibody detection in animal tuberculosis

Bovine tuberculosis (bTB) control programs can be improved by implementation of advanced ante-mortem testing algorithms. Serodiagnostic methods using traditional blood or blood-derived specimens may benefit from the use of less invasive alternative biological fluids, provided those mirror systemic antibody responses. In the present study, we used Dual Path Platform (DPP) and Multiantigen Print Immunoassay (MAPIA) to compare antibody levels in ten sample types including whole blood (fresh and hemolyzed), plasma (fresh and leftover from Bovigam testing), serum, saliva, broncho-alveolar lavage, urine, diaphragm extract, and bile collected from cattle aerosol-infected with Mycobacterium bovis. High correlation (r = 0.97−0.99) in measurements of IgG antibodies to MPB70/MPB83 fusion antigen by DPP assay was found between all blood-derived specimens, supporting matrix equivalency. Broncho-alveolar lavage and diaphragm extract yielded positive results in all the infected animals tested, showing high correlation with matching serum data (r = 0.94 and r = 0.95, respectively) and suggesting their potential use in antibody assays. Characterized by MAPIA, the antigen reactivity patterns obtained with paired sera and alternative specimens were nearly identical, with slight differences in intensity. Antibodies were also found by DPP assay in saliva, urine, and bile from some of the infected animals, but the titers were relatively low, thus reducing the diagnostic value of such specimens. The proposed approach was evaluated in a pilot field study on warthogs diagnosed with M. bovis infection. Relative levels of antibody in tissue fluid obtained from lymph nodes or lungs were consistent with those detected in sera and detectable in all infected warthogs. The findings support the diagnostic utility of non-traditional biological fluids and tissue samples when used as alternative test specimens in serologic assays for bTB.

Borrelia burgdorferi BmpA-BBK32 and BmpA-BBA64: New Recombinant Chimeric Proteins with Potential Diagnostic Value.

Currently, the diagnosis of Lyme disease is based mostly on two-tiered serologic testing. In the new generation of immunoenzymatic assays, antigens comprise whole-cell lysates of members of the Borrelia burgdorferi sensu lato (s.l.) species complex, with the addition of selected recombinant proteins. Due to the high diversity of members of the B. burgdorferi s.l. genospecies and the low degree of conservation among the amino acid sequences of their proteins, serodiagnostic methods currently in use are not sufficient for the correct diagnosis of borreliosis. Two divalent chimeric proteins (BmpA-BBK32 and BmpA-BBA64) were expressed in Escherichia coli. Following purification by one-step metal-affinity chromatography, preparations were obtained containing milligram levels of chimeric protein exhibiting electrophoretic purity in excess of 98%. Reactivity of the new chimeric proteins with specific human IgG antibodies was preliminarily determined by Western blot. For this purpose, 20 negative sera and 20 positive sera was used. The new chimeric proteins were highly reactive with IgG antibodies contained in the serum of patients suffering from borreliosis. Moreover, no immunoreactivity of chimeric proteins was observed with antibodies in the sera of healthy people. These promising results suggest that new chimeric proteins have the potential to discriminate between positive and negative sera.

Assessment of an In-House Enzyme-Linked Immunosorbent Assay and IgG Avidity Test Based on SAG1 and GRA7 Proteins for Discriminating between Acute and Chronic Toxoplasmosis in Humans.

To improve serodiagnostic methods for diagnosis of acute from chronic toxoplasmosis, an economical in-house enzyme-linked immunosorbent assay (ELISA) for measuring Toxoplasma-specific IgG, IgM, and IgG avidity has been developed and assessed based on use of various Toxoplasma gondii antigens, including SAG1, GRA7, and a combination of SAG1 and GRA7 (SAG1+GRA7), as well as Toxoplasma lysate antigens (TLAs). Performances of in-house IgM, IgG, and IgG avidity assays were compared to those of ELISA commercial kits and VIDAS Toxo IgG avidity. A set of 138 sera from patients with acquired T. gondii infection and seronegative people were assessed. Receiver operating characteristic (ROC) analysis revealed an area under curve (AUC) of 0.98, 0.97, 0.99, and 0.99 for IgM-TLAs, IgM-SAG1, IgM-GRA7, and IgM-SAG1+GRA7, respectively. Furthermore, AUC was calculated as 0.99, 0.99, 0.98, and 0.99 for IgG-TLAs, IgG-SAG1, IgG-GRA7, and IgG-SAG1+GRA7, respectively. The current study showed that GRA7 included 100% sensitivity for the detection of Toxo IgM, while SAG1 included 89.7% sensitivity. Furthermore, the highest specificity (97.2%) to detect Toxo IgM was achieved using SAG1+GRA7 antigen. For the detection of Toxo IgG, the highest sensitivity (100%) was recorded for SAG1+GRA7, followed by TLAs (97.9%). The SAG1+GRA7 showed the greatest potential for assessing avidity of IgG antibodies, with 97.1% sensitivity and 96.6% specificity compared to those of VIDAS Toxo IgG avidity. The preliminary results have promised better discriminations between acute and chronic infections using a combination of SAG1 and GRA7 recombinant antigens compared to those using TLAs.

Diagnostic Potential of an IgE-ELISA in Detecting Strongyloidiasis.

Strongyloides stercoralis infection is prevalent worldwide and can cause lifelong infection in immunocompetent individuals, and potentially death in immunosuppressed patients. The diagnosis is hindered by the low sensitivity of microscopic examination, thus making serology an important complementary test to improve the detection rate. However, there were reports that some Strongyloides-infected individuals were negative with specific IgG and IgG4 assays, and other helminth infections were positive with commercial Strongyloides IgG-ELISAs. Thus, there is a need to develop better serodiagnostic methods for strongyloidiasis. We investigated the diagnostic potential of IgE-ELISAs using Strongyloides larval lysate. Sera from two groups infected with Strongyloides served as the positive reference, that is, 1) positive by commercial IgG-ELISAs and IgG4 rapid test, and stool samples positive by microscopy and/or PCR (group IA; n = 20); and 2) negative by IgG-ELISAs and IgG4 rapid test, but stool samples were PCR positive (group IB sera; n = 11). Sera from another two groups served as negative reference (controls), that is, 1) infected with other parasites (group II; n = 73) and 2) healthy donors (group III; n = 22). Results showed a 100% diagnostic sensitivity in detecting sera from groups IA and IB. The latter group of individuals probably had early infection because their IgG and IgG4 assays were negative. The optical density values of group IB sera were also significantly lower than those of group IA (P < 0.003). The IgE-ELISA was 100% specific when tested against sera from groups II and III. This study highlights the diagnostic potential of IgE-ELISA using larval lysate to detect strongyloidiasis, especially those with probable early infection.

Seroprevalence of Toxocara spp. infection in Southeast Asia and Taiwan.

Human toxocariasis is a worldwide neglected zoonotic parasitic disease and caused mainly by Toxocara canis, and to a lesser event, by T. cati. There are only 16 epidemiological studies and 5 clinical toxocariasis case reports in 11 Southeast Asia countries and Taiwan (SEAT) that were found by searching data from PubMed in the period from January 1992 to August 2019. The overall seroprevalence in SEAT varied from 3.9% to 84.6% chiefly detected by using T. canis larval excretory-secretory antigen (TcES)-based ELISA. Playing with dogs or contacting Toxocara eggs from the contaminated soil or vegetables or eating raw meats/viscera containing encapsulated larvae seem likely the major risk factors leading to human toxocariasis in SEAT. Nevertheless, undertaking comprehensive seroepidemiological studies to establish the baseline data and beware of clinical toxocariasis cases by physicians as well as establishing adequate serodiagnostic methods in detection of Toxocara infection, e.g., TcES-based immunoblotting method in helminth endemic SEA are strongly required.

Glycopeptides and -Mimetics to Detect, Monitor and Inhibit Bacterial and Viral Infections: Recent Advances and Perspectives.

The initial contact of pathogens with host cells is usually mediated by their adhesion to glycan structures present on the cell surface in order to enable infection. Furthermore, glycans play important roles in the modulation of the host immune responses to infection. Understanding the carbohydrate-pathogen interactions are of importance for the development of novel and efficient strategies to either prevent, or interfere with pathogenic infection. Synthetic glycopeptides and mimetics thereof are capable of imitating the multivalent display of carbohydrates at the cell surface, which have become an important objective of research over the last decade. Glycopeptide based constructs may function as vaccines or anti-adhesive agents that interfere with the ability of pathogens to adhere to the host cell glycans and thus possess the potential to improve or replace treatments that suffer from resistance. Additionally, synthetic glycopeptides are used as tools for epitope mapping of antibodies directed against structures present on various pathogens and have become important to improve serodiagnostic methods and to develop novel epitope-based vaccines. This review will provide an overview of the most recent advances in the synthesis and application of glycopeptides and glycopeptide mimetics exhibiting a peptide-like backbone in glycobiology.

Screening and Identification of B-Cell Epitopes in the P61 Protein of Nocardia brasiliensis.

The P61 protein is an immunodominant antigen of Nocardia brasiliensis that is observed in the sera from patients infected with the bacterium. However, the B-cell epitopes of N. brasiliensis are still unresolved. To identify the antigenic determinants of P61, we screened seven monoclonal antibodies (mAbs) against P61 protein that was expressed in the Escherichia coli system. A series of truncated peptides of P61 were then generated and the mAbs were used to screen these peptides by Western blot analyses. Three B-cell epitopes were recognized by the P61 specific mAbs: 461-FEYWTKVDPEIGKRIEEG-478, 427-LVREVFNDAQRDRLVSNVVGGVQEPV. LSRVFEYWTKVDPEIGKRIEEGVRAG-482, and 447-HVLGGVQEPVLSRVFEY WTKVDPEI GKRIEEGVRAGLD-484. The latter two epitopes were further identified by N. brasiliensis-infected mouse serum. These results facilitate future investigations of serodiagnostic methods to identify Nocardia infections.

Design and synthesis of a new peptide derived from Fasciola gigantica cathepsin L1 with potential application in serodiagnosis of fascioliasis

Fascioliasis is a global parasitic disease that affects domestic animals and causes considerable economic losses in the process of domestic animal breeding in endemic regions. The cause of the disease involves a liver trematode of the genus Fasciola, which secretes materials into a host's body (mainly proteins) in order to protect it from the host's immune system. These materials can be involved in the migration, growth, and nutrition of the parasite. Among the expressive proteins of Fasciola, proteases have been introduced as the appropriate targets for diagnosis, treatment, and vaccination against parasites. Cathepsin L (CL) is a member of cysteine proteases; it is widely expressed in the Fasciola species. The aim of this study was to evaluate two synthetic peptides from F. gigantica CL1 for improving serological diagnosis of the Fasciola infection. Therefore, the potential diagnostic value of the surface epitopes of CL1 was assessed using ELISA. In the current study, bioinformatics tools were applied to select two appropriate epitopes of Fasciola Cathepsin L1 as synthetic antigens. Their diagnostic values were evaluated by two methods of indirect ELISA and dot blot analysis.The findings revealed that the first peptide at a dilution ratio of 1:400 and the second peptide at a dilution ratio of 1:100 had the best results and the best concentration of antigens was introduced at 4 μg/ml. Moreover, 191 sera samples were analyzed by both peptides by using the ELISA method, including fascioliasis sera, other parasitic sera and negative sera. The sensitivity of the peptides 1-ELISA and peptide 2-ELISA for the diagnosis of the various cases was 100%. The specificity of the first peptide was 87.3% and its efficacy was determined to be 93.65%. The specificity and the efficacy of the second peptide were 79% and 89.5%, respectively. The positive predictive values of the first and second peptides were obtained to be 86.27% and 79.27% respectively, and the negative predictive values of both peptides was calculated as 100%. In conclusion, the results of this study indicated that the peptide 1 from CL1 may be used as an appropriate antigen for the diagnosis of fascioliasis if the findings are backed up by using other serodiagnostic methods for checking serological cross-reactivity linked to other parasites.

Comparison of recombinant cathepsins L1, L2, and L5 as ELISA targets for serodiagnosis of bovine and ovine fascioliasis.

Infections caused by Fasciola hepatica are of great importance in the veterinary field, as they cause important economic losses to livestock producers. Serodiagnostic methods, typically ELISA (with either native or recombinant antigens), are often used for early diagnosis. The use of native antigens, as in the MM3-SERO ELISA (commercialized as BIO K 211, BIO-X Diagnostics), continues to be beneficial in terms of sensitivity and specificity; however, there is interest in developing ELISA tests based on recombinant antigens to avoid the need to culture parasites. Of the antigens secreted by adult flukes, recombinant procathepsin L1 (rFhpCL1) is the most commonly tested in ELISA to date. However, although adult flukes produce three different clades of CLs (FhCL1, FhCL2, and FhCL5), to our knowledge, the diagnostic value of recombinant FhCL2 and FhCL5 has not yet been investigated. In the present study, we developed and tested three indirect ELISAs using rFhpCL1, rFhpCL2, and rFhpCL5 and evaluated their recognition by sera from sheep and cattle naturally infected with F. hepatica. Although the overall antibody response to these three rFhpCLs was similar, some animals displayed preferential recognition for particular rFhpCLs. Moreover, for cattle sera, the highest sensitivity was obtained using rFhpCL2 (97%), being equal for both rFhpCL1 and rFhpCL5 (87.9%), after adjusting cut-offs for maximum specificity. By contrast, for sheep sera, the sensitivity was 100% for the three rFhpCLs. Finally, the presence of truncated and/or partially unfolded molecules in antigen preparations is postulated as a possible source of cross-reactivity.

The evaluation of GM6-based ELISA and ICT as diagnostic methods on a Mongolian farm with an outbreak of non-tsetse transmitted horse trypanosomosis

Trypanosoma equiperdum, which is the etiological agent of dourine, spreads through sexual intercourse in equines. Dourine (T. equiperdum) has been reported in Mongolia, where it is considered an economically important disease of horses. T. evansi has also been reported in Mongolian domestic animals. The objective of this study was to evaluate the potential application of recombinant T. evansi GM6 (rTeGM6-4r)-based diagnostic methods on a farm with an outbreak of non-tsetse transmitted horse trypanosomosis.Ninety-seven percent homology was found between the amino acid sequences of T. equiperdum GM6 and the GM6 of another Trypanozoon, which also shared the same cellular localization. This finding suggests the utility of rTeGM6-4r-based serodiagnostic methods for epidemiological studies and the diagnosis of both surra and dourine in Equidae.Fifty blood samples were examined from a herd of horses. The diagnostic value of an rTeGM6-4r-based ELISA and an rTeGM6-4r-based immunochromatographic test (ICT) were measured in comparison to a T. evansi crude antigen-based ELISA, which is a diagnostic method recommended by the OIE. However, this is not a perfect diagnostic method for trypanosomosis. Positive serum samples were detected in 46%, 42% and 28% of the tested horses using an rTeGM6-4r-based ELISA, crude antigen-based ELISA and rTeGM6-4r-based ICT, respectively. The sensitivity of rTeGM6-based ELISA was 81%, the specificity was 79%, and the agreement was moderate. We conclude that rTeGM6-4r-based ELISA and ICT represent alternative options for baseline epidemiological studies and the on-site diagnosis of horse trypanosomoses in the field, respectively.

The Use of Innovative Two-Component Cluster Analysis and Serodiagnostic Cut-Off Methods to Estimate Prevalence of Pertussis Reinfections.

Bordetella pertussis circulates even in highly vaccinated countries affecting all age groups. Insight into the scale of concealed reinfections is important as they may contribute to transmission. We therefore investigated whether current single-point serodiagnostic methods are suitable to estimate the prevalence of pertussis reinfection. Two methods based on IgG-Ptx plasma levels alone were used to evaluate the proportion of renewed seroconversions in the past year in a cohort of retrospective pertussis cases ≥ 24 months after a proven earlier symptomatic infection. A Dutch population database was used as a baseline. Applying a classical 62.5 IU/ml IgG-Ptx cut-off, we calculated a seroprevalence of 15% in retrospective cases, higher than the 10% observed in the population baseline. However, this method could not discriminate between renewed seroconversion and waning of previously infection-enhanced IgG-Ptx levels. Two-component cluster analysis of the IgG-Ptx datasets of both pertussis cases and the general population revealed a continuum of intermediate IgG-Ptx levels, preventing the establishment of a positive population and the comparison of prevalence by this alternative method. Next, we investigated the complementary serodiagnostic value of IgA-Ptx levels. When modelling datasets including both convalescent and retrospective cases we obtained new cut-offs for both IgG-Ptx and IgA-Ptx that were optimized to evaluate renewed seroconversions in the ex-cases target population. Combining these cut-offs two-dimensionally, we calculated 8.0% reinfections in retrospective cases, being below the baseline seroprevalence. Our study for the first time revealed the shortcomings of using only IgG-Ptx data in conventional serodiagnostic methods to determine pertussis reinfections. Improved results can be obtained with two-dimensional serodiagnostic profiling. The proportion of reinfections thus established suggests a relatively increased period of protection to renewed infection after clinical pertussis.

Application of recombinant severe fever with thrombocytopenia syndrome virus nucleocapsid protein for the detection of SFTSV-specific human IgG and IgM antibodies by indirect ELISA.

BackgroundSevere fever with thrombocytopenia syndrome (SFTS) is an emerging disease that was first reported in China in 2011. It is caused by SFTS virus (SFTSV) which is a member of the Phlebovirus genus in the Bunyaviridae family. SFTSV has been classified as a BSL3 pathogen. There is a need to develop safe and affordable serodiagnostic methods for proper clinical management of infected patients.MethodsThe full length nucleocapsid (N) gene of SFTSV Yamaguchi strain was amplified by RT-PCR and cloned to an expression vector pQE30. The recombinant (r) SFTSV-N protein was expressed by using Escherichia coli (E. coli) expression system and purified under native conditions. rSFTSV-N protein based indirect IgG and IgM enzyme linked immunosorbent assay (ELISA) systems were established to detect specific human IgG and IgM antibodies, respectively. One hundred fifteen serum samples from clinically suspected-SFTS patients were used to evaluate the newly established systems and the results were compared with the total antibody detecting sandwich ELISA system.ResultsThe native form of recombinant (r) SFTSV-N protein was expressed and purified. Application of the rSFTSV-N protein based indirect IgG ELISA to the 115 serum samples showed results that perfectly matched those of the total antibody sandwich ELISA with a sensitivity and specificity of 100 %. The rSFTSV-N protein based indirect IgM ELISA missed 8 positive samples that were detected by the total antibody sandwich ELISA. The sensitivity and specificity of rSFTSV-N-IgM capture ELISA were 90.59 and 100 %, respectively.ConclusionsThe rSFTSV-N protein is highly immunoreactive and a good target for use as an assay antigen in laboratory diagnosis. Its preparation is simpler in comparison with that used for the total antibody sandwich system. Our rSFTSV-N protein-based IgG and IgM ELISA systems have the advantage of distinguishing two types of antibodies and require small volume of serum sample only. They are safe to use for diagnosis of SFTS virus infection and especially fit in large-scale epidemiological investigations.

A Label-Free Biosensor Based on Nanoscale Porous Silicon Thin Film for Tuberculosis Detection

Though techniques in medicine develop in a very fast pace, tuberculosis still bothers researchers for its extensive existence. It is urgent to find faster, cheaper and more convenient new ways for diagnosis of tuberculosis. In this paper, we demonstrated a novel serodiagnostic method based on porous silicon thin film. Porous silicon has been proven feasible to function as biosensors in a lot of research. While most serodiagnostic methods are labeled detection, our porous silicon biosensor is a label-free technique. This kind of biosensor is manufactured in a simple way with relatively lower cost while providing an excellent sensitivity and specificity. Through the experiment of LAM antigen and anti-LAM antibody interacting on a porous silicon thin film platform, we proved the feasibility of our new detection approach. Furthermore, we also provided some innovation insights for improving our biosensor which may help it be practically applicable.

Evaluation of Human Serological Response to Recombinant TB10.4 Antigen of Mycobacterium tuberculosis

Introduction: Development of effective immunodiagnostic methods for detection of Mycobacterium tuberculosis infection is crucial. Serodiagnostic methods, based on antibody response to specific antigens could provide promising approaches for rapid, economical and easy to perform diagnostic tests which are crucial for tuberculosis (TB) control. In this study, the level of IgG antibody responses against recombinant TB10.4 and Bacille Calmette-Guerin (BCG) in sera from TB patients and vaccinated healthy controls were evaluated. Methods: Indirect ELISA was used to assess the anti-TB10.4 IgG levels. Serum samples were obtained from vaccinated healthy controls, confirmed TB positive patients, TB cases under antibiotic treatment, and cases with atypical mycobacteria infection. The ratio of test sera optical density to the optical density of pooled negative control was taken as cut off value. Results: Using indirect ELISA method, anti-TB10.4 was detected in 64.5%, 93.5%, 85.7% and 100% of the sera from vaccinated healthy controls, confirmed TB positive cases, TB cases under antibiotic treatment and cases with atypical mycobacteria infection, respectively. The relative sensitivity for TB10.4 was calculated as 84.32%. Conclusion: In this study, the use of TB10.4 protein showed high sensitivity, but low specificity in detection of anti-M. tb antibodies in TB patients. These results suggest that further studies should be undertaken to identify the optimal combinations of antigens for the sensitive and specific serodiagnosis of TB.