Indirect Enzyme-linked Immunosorbent Assay Method Research Articles

Examination of the cross-reactivity between vaccinia virus Tiantan strain and monkeypox virus

AimTo investigate the cross-reactivity between the sera collected from Vaccinia Virus Tiantan Strain vaccinated rabbits and viral antigens of monkeypox virus. MethodsVaccinia viruses were prepared on chicken embryo fibroblasts (CEF) and Vero cells respectively named as CEF-VTT NVSI-1 and Vero-VTT NVSI-1. Rabbits were inoculated with a total of three doses of adjuvanted 1.3 × 108 PFU CEF-VTT NVSI-1 each dose or adjuvanted 3.9 × 107 PFU Vero-VTT NVSI-1 (Freunds complete adjuvant) via the subcutaneous route. We then performed the enzyme-linked immunosorbent assay (ELISA) and bio-layer interferometry (BLI) for determination of the binding activity and affinity of immune sera to five crucial surface antigens on monkeypox virus including A35, B6R, H3 and to corresponding homologous antigens A33R, B5 and L1R of vaccinia virus. For comparison, plaque reduction neutralizing tests were used to evaluate the neutralization of immune sera against vaccinia virus. ResultsBoth CEF-VTT NVSI-1 and Vero-VTT NVSI-1 vaccinations following planned schedule could induce neutralizing antibody titers greater than 1:2048 in rabbit sera. Binding antibodies targeting monkeypox viral antigens were confirmed by both indirect ELISA and BLI methods. Indirect ELISA for rabbit sera revealed 1:51200 binding antibody titers to A35/B6R/H3 monkeypox virus antigens while BLI tests yielded affinities at 2 × 10−6 to 8 × 10−7 between the sera and the three antigens. Similarly, such sera showed binding strength to vaccinia virus antigens A33R/B5/L1R consistent with that to three preceding monkeypox virus antigens. These results demonstrated the cross-reactivity between the sera of vaccinia virus vaccinated animals and monkeypox virus antigens. Traditional ELISA test and BLI method displayed a high consistency in antigen screening and they were further proved to correlate to the results of plaque reduction neutralizing test, which indicates that BLI could be utilized as an indirect alternative for assessment of neutralizing activity of samples in response to live virus. ConclusionsSera of vaccinia virus-vaccinated rabbits exhibited cross-reactivity with viral antigens of monkeypox virus. Potential in improving the accuracy of antigen discovery while reducing the lengthy work needed for the screening as BLI method possesses, it contributes greatly to the rapid preliminary evaluation of immune response generated by vaccines.

Development of silver nano-based indirect ELISA and Dot-ELISA methods for serological diagnosis of a bacterial fish pathogen Aeromonas veronii

Rapid and accurate detection of bacterial pathogens is critical in controlling disease outbreaks affecting farmed fish. The present study aimed to develop a novel serological diagnostic approach using nano‑silver based Enzyme-linked immunosorbent assay (ELISA) for speedy detection of Aeromonas veronii infections in Nile tilapia. A. veronii isolates used in ELISA assays were recovered from moribund Nile tilapia during a disease outbreak in a private fish farm in Egypt. A. veronii isolates were identified based on alignment analysis of the gyrB and 16S rRNA gene sequences. A. veronii antisera used in ELISA assays were prepared in tilapia, and the bacterial antigens were formalin-killed. The cut-off values were 0.46 and 0.48 in traditional and nano-based ELISA. There were no cross-reactions with bacterial isolates (Aeromonas hydrophila, Aeromonas caviae, Aeromonas sobria, Pseudomonas fluorescens, and Vibrio vulnificus). The lowest antigen concentration that produced positive results after checkerboard titration in indirect-ELISA (i-ELISA) and dot ELISA was 15 μg and 250 ng of prepared antigen, respectively. Nano-ELISA and nano-based dot-ELISA antigen concentration was 10 μg and 100 ng, respectively. Sera concentration was 1:100 in indirect-ELISA and dot-ELISA, while it was 1:50 in nano-based ELISA and nano dot-ELISA. The i-ELISA successfully detected anti-Aeromonas IgG antibodies with 83.33% sensitivity and 66.67% specificity, while in the dot-ELISA, the sensitivity and specificity were 83.33% and 100%, respectively. Nano dot-ELISA had 100% sensitivity, specificity, and accuracy. Nano dot-ELISA assays have higher specificity, sensitivity, and accuracy than traditional ELISAs in detecting A. veronii. Further studies are needed to develop a rapid test kit for on-site field diagnosis.

BSA Interference in Immunoassays in Individuals with Egg Allergy

Aim: The aim of current study was to determine interference by bovine serum albümine (BSA) as blocking agent in enzyme-linked immunosorbent assay (ELISA) carried out in individuals with egg allergy. Material and Methods: 14 people diagnosed with egg allergy and 7 people without allergy were included. The sample were studied with an indirect ELISA method for egg-white IgG antibody developed in our laboratory. Effect BSA on interference was studied by manipulating antigene coating (none vs. egg white extract), blocking (1% BSA vs. Tween 80), and sample diluent (PBS vs. PBS + 0.5% BSA). Results: In wells that were blocked with 1% BSA without being coated with antigen, positive samples cross-reacted with BSA to give an optical density (OD) of 0.99 ± 0.16, while negative samples gave an OD of 0.08±0.01 (p<0.05). However, when the same samples were diluted with 0.5% BSA, the OD of positive samples decreased (from 0.99±0.16 to 0.08±0.01), and the statistical difference with negative samples disappeared. It was observed that tween, which was used as a blocking and diluting agent, did not cross-react with the samples. Positive samples gave an OD of 0.66±0.07 in antigen (egg white extract) coated and tween-blocked wells, and 1.01±0.11 OD in BSA blocking (p<0.05). When the samples were diluted with 0.5% BSA, positive samples gave 0.18±0.01 OD on the antigen coated plate, while negative samples gave 0.12±0.04 OD (p<0.05). Conclusion: Ovalbumin, which is found in high levels in eggs, has a similar molecular structure to BSA, and some antibodies produced against ovalbumin in people with egg allergy may also cross-react against BSA. Therefore, it was concluded that the use of BSA in both dilution and blocking solution should be avoided if the samples of individuals with egg sensitivity are to be analyzed by ELISA method. It has been observed that Tween can be easily applied as an alternative blocking agent in allergy ELISA tests.

Improvements in skills and knowledge after a comprehensive ELISA teaching course for biotechnology undergraduates.

As a universal and extensively adopted technique, enzyme-linked immunosorbent assay (ELISA) can be used to detect and quantify small molecules in many applications both clinical and analytical. However, generally, students experiment mechanically using commercial ELISA kits according to the instructions and eventually produce a standard curve to calculate the concentration of the sample to be measured, cannot understand the critical factors and process of method establishment. This study systematically introduced undergraduates to using the pathogen-specific antigen and establishing an indirect ELISA method to detect the diagnostic target pathogen Burkholderia pseudomallei. This course aimed to develop the experimental skills of the students and improve their scientific research knowledge, which fully embody the organic combination of scientific research and teaching. Students independently selected the diagnostic antigen target of interest, obtained the antigen proteins using genetic engineering techniques, and established an ELISA method through a series of conditional optimization experiments. In addition, typical student-generated data, experimental methods, and a student feedback interpretation are presented in this study. Overall, the students were able to combine abstract knowledge with practice and understand the principles and applications of antigen-antibody interactions, thus enabling them to gain practical experience in molecular biology techniques, and learn how to use this principle to establish an ELISA method for detecting infectious diseases.

Development of in-house ELISA for detection of antibodies against lumpy skin disease virus in cattle and assessment of its performance using a bayesian approach

Lumpy skin disease (LSD) is a contagious disease among cattle and buffalo worldwide. Currently, an enzyme-linked immunosorbent assay (ELISA) has been recognized as an efficient diagnostic tool that is less time-consuming and easier than the viral neutralization test to measure the antibody levels. In the present study, an in-house method of indirect ELISA was developed to detect the bovine antibodies against Lumpy skin disease virus (LSDV) and its performance was assessed using field samples. This in-house method has been compared with the commercial ELISA test kit for detection of bovine antibodies against LSDV. The sensitivity (Se) and the specificity (Sp) of the test were estimated using a Bayesian latent class model. Checkerboard titration was performed using the naturally LSDV-infected bovine sera and colostrum-deprived calf sera. The LSDV antigen concentrations (1 TCID50/mL), the sample serum (1:500), and goat anti-bovine immunoglobulin G (IgG) labeled with horseradish peroxidase (HRP) (1:10,000) were determined to be optimal for this assay. The calculated cut-off value was 0.067, and there were no differences in the results of tests that utilized positive and negative sera (p < 0.05). The characteristics of two diagnostic tests were evaluated using a conditional dependent and one-population Bayesian model. The Se value of an in-house indirect ELISA were almost similar to ELISA test kit. On the other hand, the Sp value of the in-house ELISA test was lower than that of the commercial ELISA test with the median values of 89% (95% PPI = 75.9–99.3%) and 91.4% (95% PPI = 85.3–95.5%), respectively. A posterior estimate for the prevalence was 66.9% (95% PPI = 60.8–83.3%) and higher than initially expected.

A highly efficient indirect ELISA and monoclonal antibody established against African swine fever virus pK205R.

African swine fever (ASF) is a contagious infectious disease with high lethality which continuously threatens the global pig industry causing huge economic losses. Currently, there are no commercially available vaccines or antiviral drugs that can effectively control ASF. The pathogen of ASF, ASF virus (ASFV) is a double-stranded DNA virus with a genome ranging from 170 to 193 kb and 151 to 167 open reading frames in various strains, which encodes 150-200 proteins. An effective method of monitoring ASFV antibodies, and specific antibodies against ASFV to promote the development of prevention techniques are urgently needed. In the present study, pK205R of ASFV was successfully expressed in mammalian cells using a suspension culture system. An indirect enzyme-linked immunosorbent assay (ELISA) based on the purified pK205R was established and optimized. The monoclonal antibody (mAb) against pK205R recognized a conservative linear epitope (2VEPREQFFQDLLSAV16) and exhibited specific reactivity, which was conducive to the identification of the recombinant porcine reproductive and respiratory syndrome virus (PRRSV) expressing pK205R. The ELISA method efficiently detected clinical ASFV infection and revealed good application prospects in monitoring the antibody level in vivo for recombinant PRRSV live vector virus expressing the ASFV antigen protein. The determination of the conserved linear epitope of pK205R would contribute to further research on the structural biology and function of pK205R. The indirect ELISA method and mAb against ASFV pK205R revealed efficient detection and promising application prospects, making them ideal for epidemiological surveillance and vaccine research on ASF.

The Indirect ELISA and Monoclonal Antibody against African Swine Fever Virus p17 Revealed Efficient Detection and Application Prospects.

Since 2018, the outbreak and prevalence of the African swine fever virus (ASFV) in China have caused huge economic losses. Less virulent ASFVs emerged in 2020, which led to difficulties and challenges for early diagnosis and control of African swine fever (ASF) in China. An effective method of monitoring ASFV antibodies and specific antibodies against ASFV to promote the development of prevention techniques are urgently needed. In the present study, ASFV p17 was successfully expressed in CHO cells using a suspension culture system. An indirect enzyme-linked immunosorbent assay (ELISA) based on purified p17 was established and optimized. The monoclonal antibody (mAb) against p17 recognized a conservative linear epitope (3TETSPLLSH11) and exhibited specific reactivity, which was conducive to the identification of recombinant porcine reproductive and respiratory syndrome virus (PRRSV) expressing p17. The ELISA method efficiently detected clinical ASFV infection and effectively monitored the antibody levels in vivo after recombinant PRRSV live vector virus expressing p17 vaccination. Overall, the determination of the conserved linear epitope of p17 would contribute to the in-depth exploration of the biological function of the ASFV antigen protein. The indirect ELISA method and mAb against ASFV p17 revealed efficient detection and promising application prospects, making them ideal for epidemiological surveillance and vaccine research on ASF.

Establishment of an indirect ELISA method for antibody detection of porcine pseudorabies by recombinant gB, gC, and gD proteins.

Pseudorabies virus (PRV), as a neuroherpes virus, leads to heavy economic losses in the pig industry worldwide.This study was designed to establish recombinant PRV glycoprotein B (gB), C, and D proteins as PRV diagnostic antigens. The gB/C, gC/D, and gB/C/D fusion sequences were synthesized and inserted into pET-28a+ vector to generate the recombinant plasmids. The identified positive recombinant plasmids were transformed into BL21 Escherichiacoli.The results of thepolymerase chain reactionand enzyme digestion showed that the gB/C, gC/D, and gB/C/D fusion proteins were successfully expressed. An indirect sandwich ELISA was developed with the gB/C, gC/D, and gB/C/D as coating antigens. The results of indirect enzyme-linked immunosorbent assay(ELISA) analysis of 184 PRV-positive porcine sera showed that the positive coincidence rates of three recombinant proteins ELISAs relative to IDEXX kit were 98.25%, 95.32%, and 98.83%, and the negative coincidence rates were 85.71%, 75% and 100%, respectively. The inter and intra batch repeatability tests showed that the coefficient of variations of our kits were all less than 5%. Especially, the gB/C/D-ELISAhas the highest specificity and sensitivity among the ELISA methods developed in this study.We established a series expression system of gB/C, gC/D, and gB/C/D antigen epitope genes and Recombinant protein-based indirect ELISA, providing new ideas for PV diagnosis and vaccine development.

Bacterial coloration immunofluorescence strip for ultrasensitive rapid detection of bacterial antibodies and targeted antibody-secreting hybridomas

The indirect enzyme-linked immunosorbent assay (ELISA) is the gold standard method for monoclonal antibody (McAb) detection and plays a unique role in the preparation of bacterial antibodies. To solve the laborious issues associated with indirect ELISA, a novel bacterial coloration immunofluorescence strip (BCIFS) for antibody detection using colored bacteria instead of a labeled antibody as the antigen and tracer simultaneously and goat anti-mouse IgG as the test line was developed. The affinity range survey of BCIFS indicated that hybridoma cell cultures of E. coli O157:H7 (D3, E7) and Vibrio parahemolyticus (H7, C9) were detected, which complied with the results of indirect ELISA. Compared with the traditional indirect ELISA, the BCIFS sensitivity for E7 cell cultures, ascites, and purified antibodies was at least 4-fold more sensitive, and the BCIFS cross-reactivity for E7 cell cultures was almost consistent with that of indirect ELISA. In addition, the BCIFS isotypes for E. coli O157:H7 cell cultures and Vibrio parahemolyticus were IgG2a and IgG1, respectively, which were identical to the indirect ELISA. Furthermore, the BCIFS method was confirmed by McAb preparation, effective antibody use, and targeted antibody-secreted hybridoma preparation and screening, which showed excellent performance and substitution of the indirect ELISA method. Combined with methylcellulose semisolid medium, BCIFS offers a novel, easy to operate, rapid preparation method for antigen-specific hybridomas. This is the first report using BCIFS instead of indirect ELISA for bacterial antibody detection and application in different samples, which demonstrates a rapid and powerful tool for antibody engineering.

Serological responses to a soluble recombinant circumsporozoite protein-VK210 of Plasmodium vivax (rPvCSP-VK210) among Iranian malaria patients

BackgroundCircumsporozoite protein (CSP) has a central immune domain that includes short regions of repeating amino acid sequences. This immunodynamic region is an epitope of B cells that can elicit an immune response in human and laboratory animals. The aim of the present study was to express the recombinant PvCSP-VK210 antigen and evaluate it for assaying antibodies obtained during human P. vivax infection by Western blotting and indirect ELISA (enzyme-linked immunosorbent assay).MethodGenomic DNA of P. vivax was isolated from a blood sample of an Iranian person with vivax malaria, and by PCR, the fragment of the PvCSP-VK210 gene was amplified. The gene fragment was cut after gel purification by BamHI and HindIII enzymes and then cloned into pET28a expression vector. Finally, the recombinant pET28a was transformed into the E.coli BL21 (DE3) as the expression host. In order to produce His-tagged protein, the expression host was cultured in LB medium. The protein was purified by Ni–NTA columns and immobilized metal affinity chromatography, and after confirmation by Western blotting technique, was used as the antigen in the indirect ELISA test.ResultsThe recombinant protein was expressed and purified as a 32-kDa protein. The sensitivity and specificity of the indirect ELISA test with the recombinant PvCSP-VK210 antigen were 61.42% and 97.14%, respectively, based on OD = 0.313. Between the results of the microscopic test and the indirect ELISA test with the recombinant PvCSP-VK210 antigen there was a Kappa coefficient of 0.586. The positive and negative predictive value and validity of the ELISA test with the recombinant PvCSP-VK210 antigen were 95.55%, 71.57%, 79.28%, respectively.ConclusionThe sensitivity of the indirect ELISA method with the recombinant PvCSP-VK210 antigen was 61.42%, which is the first report from Iran.

A novel antigen immunochromatography fluorometric strip for rapid detection and application of pathogenic bacterial high-quality antibody

Unlike traditional immunoassay strips, a novel antigen immunechromatography fluorometric strip (AICFS) using inactivated bacterial antigen instead of an antibody as a test line and goat anti-mouse IgG-FITC as a tracer was developed. The applicability survey of AICFS indicated that E. coli O157:H7 (D3) and Acidovorax citrulli (6F) hybridoma cell cultures could be detected, but Vibrio parahemolyticus (H7, C9) hybridoma cell cultures were missed compared with the indirect enzyme-linked immunosorbent assay (ELISA). The four antibody affinity constants (Ka) were measured and compared, and AICFS could be suitable for high-affinity antibody detection. Compared with the traditional indirect ELISA, the AICFS sensitivity for D3 cell cultures, ascites, and purified antibodies was at least 2-fold more sensitive, the AICFS specific for D3 cell cultures by comparative interpretation was compliant except for the strain ATCC 43895, and the indirect ELISA missed it. More importantly, the AICFS method was confirmed by various real samples that it could be used in different scenarios regarding the antibody, including McAb preparation, the effective antibody use, and high-affinity antibody-secreted hybridoma auxiliary preparation and screening. It could be an excellent alternative method with less than 5% corresponding processing time for indirect ELISA method for pathogenic bacterial high-quality antibody detection. This is the first report of using AICFS for bacterial high-quality antibody detection and application in different samples, which demonstrates a rapid auxiliary tool for high-affinity antibody secreted-hybridoma screening and an excellent alternative method for high-quality antibody application.

Development and evaluation of an indirect ELISA based on recombinant structural protein VP2 to detect antibodies against duck hepatitis A virus

An indirect enzyme-linked immunosorbent assay (I-ELISA) based on the VP2 protein of duck hepatitis A virus type 3 (DHAV-3) was established in this study. The optimal dilutions of antigen, serum and goat anti-duck IgG conjugate were 1:1600 (2.23 μg/mL), 1:160 and 1:2000, respectively. The optimal blocking buffer was 1% skim milk. The cut-off value for the method was 0.25, and the analytical sensitivity of the method was 1:5120. The results of specific evaluation showed that except for DHAV-1, DHAV-3 antisera did not cross-react with any other common duck-sensitive pathogens, indicating that this method can be used to detect DHAV-3 and DHAV-1 antibodies. The coefficients of variation (CVs) were lower than 10 %. The coincidence rate between the VP2-DHAV-3-ELISA and the neutralization test was 93.3 %. In summary, the I-ELISA method based on VP2 protein has high sensitivity, specificity, and coincidence rate compared with the neutralization test and has advantages in serum monitoring. The I-ELISA method based on VP2 protein provides a simple and rapid method for the detection of anti-DHAV antibodies and the epidemiological monitoring of DHAV.

Establishment and evaluation of receptor binding domain (RBD)-based ELISA for Middle East respiratory syndrome coronavirus (MERS-CoV) antibody detection

Objective To establish an indirect enzyme-linked immunosorbent assay (ELISA)and to compare the efficiency of receptor binding domain (RBD) proteins in different forms for Middle East respiratory syndrome coronavirus (MERS-CoV) antibody detection. Methods The monomeric and trimeric forms of MERS-CoV RBD were expressed in Bac-insect cells, 293T cells and ExpiCHO-S™ expression system and then purified. The purified RBD proteins were identified with native gel electrophoresis and Western blot. Then, an equal amount of each RBD protein was used as coating antigen to establish an ELISA for detecting MERS-CoV IgG titer. For comparison, the newly developed ELISA and the commercial MERS-CoV IgG antibody detection kit (Euroimmune with S1 as the coating antigen) were used to measure the MERS-CoV antibody reference panel supplied by World Health Organization (WHO). Results The purified monomeric and trimeric MERS-CoV RBD were successfully prepared using 293T cells and ExpiCHO-S™ system. RBD antigens of different forms and from different systems could recognize MERS-CoV specific antibody without having any cross reaction with the sera from healthy adults. The in-house RBD-based ELISA had good detection consistency with the Euroimmune commercial kit. The positive samples showed higher and more concentrated values based on the RBD trimer than the monomer. Conclusions Novel indirect ELISA methods based on the monomeric and trimeric forms of RBD protein were established. The trimetric form-based ELISA achieved higher detection efficiency than the one using the monomer antigen, suggesting that it could be uses as a competent alternative to the commercial kit. Key words: Middle East respiratory syndrome coronavirus (MERS-CoV); Receptor binding domain (RBD); Antibody; Enzyme-linked immunosorbent assay (ELISA)

Development of indirect competitive ELISA for determination of dehydroabietic acid in duck skin and comparison with the HPLC method

Defeathering with rosin results in rosin residue in duck skin, which may present as potential risk to human health. Dehydroabietic acid (DHAA) is a major component of rosin. An indirect competitive enzyme-linked immunosorbent assay (ELISA) was developed for determination of DHAA in duck skin. A set of parameters was optimized, including coating antigen concentration, dilution of antiserum, dilution of HRP-IgG antibody, incubation time, and temperature for antigen reaction with antiserum. The indirect competitive ELISA yielded an excellent specificity against DHAA with low cross-reactivity toward other resin acids. The limit of detection and the working concentration range of DHAA in duck skin were 16.4 ng/g and from 40 to 8,060 ng/g, respectively. The indirect competitive ELISA was applied to the determination of DHAA in duck skin samples spiked with DHAA at different contents, and recoveries were found between 78.2 and 97.2%. Finally, DHAA contents in 32 duck samples were quantified by the indirect competitive ELISA and high performance liquid chromatography-fluorescence detector (HPLC-FLD) method. No significant difference was found between DHAA concentrations from indirect competitive ELISA and HPLC-FLD method for all samples, which indicated the indirect competitive ELISA established in this article was of the same accuracy as the HPLC-FLD method. The indirect competitive ELISA was simple, rapid, and reliable, which could be used to identify the duck carcasses defeathered with rosin in the market.

Development and application of a fiber2 protein-based indirect ELISA for detection of duck adenovirus 3

Duck adenovirus 3 (DAdV-3) is a newly identified duck adenovirus that has recently emerged in China. The incidence of duck infection caused by this virus is very high, with very large economic losses to the poultry industry. Thus, there is an urgent need for a serological assay for the specific detection of DAdV-3. To this end, prokaryotic expression of the fiber2 protein of DAdV-3 was used as a coating antigen to establish an indirect enzyme linked immunosorbent assay (ELISA) method for the specific detection of antibodies against DAdV-3. The method was found to be specific, repeatable and more sensitive than the agarose gel precipitation test (AGP). This indirect ELISA method based on the recombinant fiber2 protein may be used for the clinical detection of DAdV-3 infection and for monitoring antibody levels after vaccine immunization and is of great significance for the effective prevention and control of the disease.

Development of an Indirect Enzyme-linked Immunosorbent Assay to Detect Antibodies against Serotype A2013 of Foot and Mouth Disease Virus in Cattle.

Foot and mouth disease (FMD) is a contagious animal disease that causes irreparable damage to the economy of countries, including Iran in which this disease is a native one. Among the ways to combat FMD are vaccination and slaughter. Due to the specific situation of Iran, it is not possible to kill infected animals. Therefore, vaccination is the most important way to fight this disease. Serum neutralization test (SNT) and enzyme-linked immunosorbent assay (ELISA) are two main methods to evaluate the safety and calculate antibody titer. In this study, an indirect ELISA test was developed based on the coating of a complete viral particle (140s) which made it possible to determine antibody. In addition, serotype and viral type were determined without the need for time-consuming and complex molecular tasks, including gene expression. Moreover, in case of a new epidemic, a new epidemic condition can be detected using a serum antibody method. However, the coating of the complete viral particle leads to virus purification as well as the conjugated anti-immunoglobulin antibody testing of the same animal. In this study, the SNT was used as a gold standard test to determine the serum antibody level and compare its results with indirect ELISA method to determine the sensitivity and specificity of the indirect ELISA. To measure the anti-virus antibody rate of FMD (type A2013) through receiver operating characteristic analysis with 100% sensitivity and the specificity of 90%, the routine formulas were utilized using 100 % and 82%sensitivity and specificity, respectively. In this study, the cutoff value for the optical density was obtained at 0.3 and there was a significant difference between the vaccinated animals and the unvaccinated ones in terms of antibody level against the A2013 type. This indicates the correctness of the test and the accurate and proportional antibody detection against the understudy viral types of FMD.

Kinetics of serum antibodies in response to infection with Yersinia enterocolitica.

Information on the kinetics of the serum antibody response to infection with Yersinia enterocolitica is essential to allow the estimation and comparison of seroconversion rates in a diversity of pools of cross-sectional serum antibody measurements. Data from 94 patients with acute enteritis caused by Yersinia infection were used. The follow-up period for the longitudinal study was 36 months, addressed by questionnaire. An indirect enzyme-linked immunosorbent assay method was adapted to determine the concentration of antibodies against Y. enterocolitica in human sera. A mathematical within-host model was used to describe the interaction between pathogen and immune system and the waning of immunity after clearing of the pathogen. All observed antibodies (IgG, IgM, IgA) reached peak levels shortly after infection and then decayed slowly indicating that the median levels decreased only little during the observation period. Estimated maximum peak antibody levels were highest in IgG. Seroresponse curves of all antibodies showed large individual variation between patients. There was no apparent pattern of variation with age, nor any notable difference between genders. Estimated half-times were very long for all antibodies, and their posterior distributions were highly skewed. IgA appeared to have the most persistent antibody response, compared with IgG and IgM. Median peak levels of all three antibodies were similar. There was no significance found between peak antibody levels and severity of symptoms of gastrointestinal infection and severity of joint pain. Our findings allow the use of cross-sectional serum antibody measurements as biomarkers, to estimate seroconversion rates. Such seroincidence estimates include asymptomatic seroconversions, thereby avoiding under-reporting, and allows the comparison of infection pressures among countries, independent of their healthcare and surveillance systems.

Expression of glutathione s-transferase-fusion hepatitis E virus ORF2, 3 antigens in escherichia coli and its application for diagnosis

Context Hepatitis E infection, caused by the hepatitis E virus (HEV), is a common cause of acute hepatitis in developing countries with poor sanitation and hygiene. Aims In this paper, we expressed HEV ORF2 C-terminal 802 bp and ORF3 C-terminal 107 bp fragments and its glutathione S-transferase (GST) fusion genes in Escherichia coli and tested the immunoreactivity of HEV antibody from sample serum with recombinant GST-HEV ORF2, 3 antigens by enzyme-linked immunosorbent assay (ELISA). Settings and design Eight primers designed for gene synthesis. Materials and methods HEV ORF2, 3 and its GST-fusion gene fragments were amplified by PCR. Expression of target genes in E. coli was induced by Isopropyl-β-D-Thio-alactopyranoside (IPTG) (0.1 mmol/l) and temperature (42°C). The detection of anti-HEV antibody with purified GST-HEV ORF2, 3 antigens was performed using the indirect ELISA method. Results When our ELISA was compared with the HEV-ELISA kit, the sensitivity of our assay is 97.9%, specificity is 100%, and consistency is 99.6%. The positive rate of anti-HEV antibody from the serum in acute hepatitis patients with recombinant GST-HEV ORF2, 3 antigens by our ELISA was 29.5 and 26.1%, respectively, and this result corresponded with the HEV-ELISA kit. Conclusion The new HEV ELIA developed in the present study is a highly specific diagnostic assay for the detection of anti-HEV antibody in serum specimens obtained from acute hepatitis patients.

Immunochemical Identification of Immunoreactive Ghrelin in Tea Plant, Camellia sinensis (L) O. Kuntze

Ghrelin was originally described as a growth hormone (GH)-releasing and appetite stimulating hormone in mammals, which is highly conserved and extensively expressed in all eukaryotic organisms investigated. Ghrelin is a natural ligand of the growth hormone secretagogue-receptor, and functions primarily as a GH-releasing hormone and an orexigen, as well as having several other biological actions. Although ghrelin has been well characterized in mammalian systems, the most recent investigations demonstrated that plants have ghrelin immunoreactivity. The aim in this study is to determine ghrelin-like immunoreactive substances in fresh vegetative tissue of tea plant [Camellia sinensis (L.) O. Kuntze (Theaceae)]. Total protein was extracted from the plant samples by using trichloroacetic acid/acetone precipitation. Ghrelin-like immunoreactive substances in the tea plant extracts were investigated with indirect enzyme-linked immunosorbent assay method. From the tea plant samples human-ghrelin-like immunoreactive substances were isolated and characterized using tricine-sodium dodecyl sulfate–polyacrylamide gel electrophoresis and immunoblotting techniques. The ghrelin immunoreactivity of tea extract and human serum were found to be 1.94 ± 0.27 and 0.31 ± 0.012 ng/mg protein, respectively. As a result, in fresh vegetative tissues of tea a single ghrelin-like protein band with a molecular mass of ~13 kDa was observed using tricine-SDS-PAGE and western blotting. This study reflects the importance of ghrelin in living organisms, and speculates the ghrelin-like bioactive peptides derived from plants may play a role in the regulation of appetite and body weight control.

Indirect enzyme-linked immunosorbent assay method based on Streptococcus agalactiae rSip-Pgk-FbsA fusion protein for detection of bovine mastitis.

The aim of this study was to establish a rapid and accurate method for the detection of the Streptococcus agalactiae antibody (SA-Ab) to determine the presence of the bovine mastitis (BM)-causative pathogen. The multi-subunit fusion protein rSip-Pgk-FbsA was prokaryotically expressed and purified. The triple activities of the membrane surface-associated proteins Sip, phosphoglycerate kinase (Pgk), and fibronectin (FbsA) were used as the diagnostic antigens to establish an indirect enzyme-linked immunosorbent assay (ELISA) method for the detection of SA-Ab in BM. The optimal antigen coating concentration was 2 μg/mL, the optimal serum dilution was 1:160, and the optimal dilution of the enzyme-labeled secondary antibody was 1:6000. The sensitivity, specificity, and repeatability tests showed that the method established in this study had no cross-reaction with antibodies to Streptococcus pyogenes, Escherichia coli, Staphylococcus aureus, and Staphylococcus epidermidis in the sera. The results of the sensitivity test showed that a positive result could be obtained even if the serum dilution reached 1:12,800, indicating the high sensitivity and good repeatability of the method. The positive coincidence rate of this method was 98.6%, which is higher than that of previous tests established with the Sip or Pgk mono-antigen fusion protein, respectively, demonstrating the relatively higher sensitivity of this newly established method. The detection rate for 389 clinical samples was 46.53%. The indirect ELISA method established in this study could provide a more accurate and reliable serological method for the rapid detection of S. agalactiae in cases of BM.