Enzyme-linked Immunosorbent Assay Antibody Titers Research Articles

Correlation of desmoglein 1 and 3 immunohistochemistry with autoantibody levels and clinical severity in pemphigus.

Pemphigus is a chronic potentially fatal autoimmune bullous disorder. Pemphigus vulgaris (PV) and pemphigus foliaceus (PF) are the two common subtypes. PV is the most common and aggressive type characterized by oral mucosal erosions and cutaneous lesions. PF presents with blisters on the scalp, face, and upper trunk, and spares the mucosae. Direct immunofluorescence (DIF) is the gold standard for diagnosis. Immunohistochemistry (IHC) is an emerging alternate diagnostic tool. In this study, our objectives were to identify the staining patterns of desmoglein 1 (dsg 1) and desmoglein 3 (dsg 3) IHC and to correlate the same with autoantibody levels and clinical severity in patients with PV and PF. Forty-nine clinically, histologically, and DIF-confirmed cases of pemphigus were included in the study. The IHC patterns were scored from 0 to 3+ with 3+ dsg 1 IHC exhibiting intense membranous staining in the upper layers of the epidermis and 3+ dsg 3 IHC showing intense basal layer staining. Enzyme-linked immunosorbent assay (ELISA) for anti-dsg 1 and 3 antibodies was performed in 38 cases where serum samples were available. The pemphigus disease activity index system was utilized for clinical scoring. A 0 to 1+ score was observed for dsg 1 IHC in 100% of PF cases. A score of 0 to 1+ was observed for dsg 3 IHC in 97.3% of PV cases. One hundred percent of cases with PF and 83.9% of patients with PV tested positive for ELISA anti-dsg 1 and 3 antibody titers, respectively. Anti-dsg 1 and 3 ELISA titers significantly correlated with the dsg 1 and dsg 3 IHC scores. The mucosal scores showed a significant association with both dsg 1 and 3 IHC (p < 0.001). The cutaneous scores showed a significant association with the dsg 3 IHC (p < 0.001). The IHC patterns for dsg 1 and 3 proved reliable in giving concordant results with the ELISA antibody titers and clinical severity.

Serological responses and protection levels in chickens administered with Newcastle disease vaccines

Vaccination against Newcastle disease (ND) is the most effective means of controlling the disease, and these vaccines are commercialized only after their safety and effectiveness have been verified through tests that comply with Korean Standards of National Lot Release for Veterinary Biologics. This study investigated whether a relatively convenient and safe serological test can be used in place of the challenge test using highly virulent ND virus. Hemagglutination inhibition (HI) assay and enzyme-linked immunosorbent assay (ELISA) were considered positive of log2 2 or more and cutoff value of 200 or more, respectively, in both live and inactivated vaccines. However, when the antibody levels of the live and inactivated vaccines induced using the Ulster 2C, KBNP-C4152R2L, and K148/08 strains were compared, the antibody titers for inactivated vaccines were significantly higher than those for live vaccines in both the HI assay and ELISA. A strong positive correlation was observed between HI and ELISA antibody titers. The live vaccines corresponded to a survival rates of ≥ 80% and the inactivated vaccines corresponded to 100% survival rates. This study confirmed that standard efficacy tests can serve as serological tests, and can replace the challenge test and that the vaccine approval process can be improved.

CASTING: A Potent Supramolecular Strategy to Cytosolically Deliver STING Agonist for Cancer Immunotherapy and SARS-CoV-2 Vaccination

CASTING: A Potent Supramolecular Strategy to Cytosolically Deliver STING Agonist for Cancer Immunotherapy and SARS-CoV-2 Vaccination

Trials for preparation and evaluation of a combined inactivated reassorted H5N1 and Escherichia coli O157 vaccine in poultry.

Background and Aim:Avian influenza (AI), which is one of the major respiratory diseases of poultry, and Escherichia coli (E. coli) have caused major economic losses around the world, including in Egypt. Therefore, in this study, we aimed to produce a vaccine from E. coli O157 and AI H5N1 formulated with Montanide ISA70 for the protection of poultry against both diseases.Materials and Methods:We divided one hundred 3-week-old chicks into four groups: Group 1 was vaccinated with prepared inactivated AI H5N1formulated with Montanide ISA70, Group 2 was vaccinated with inactivated E. coli formulated with Montanide ISA70, Group 3 was vaccinated with combined E. coli and AI H5N1 formulated with Montanide ISA70, and Group 4 was an unvaccinated control group. We measured the immune response using the HI (hemagglutination inhibition) test, enzyme-linked immunosorbent assay (ELISA), and the challenge test.Results:We found the three vaccines to be safe and sterile during all periods of examination and observation. The HI test showed that Group 1 exhibited specific antibody titers of 2.3 log2, 4.3 log2, 7.5 log2, 7.8 log2, 8 log2, and 8.1 log2 from week 2 to week 7, respectively, post-vaccination. Group 3 exhibited antibody titers of 3.3 log2, 5.8 log2, 7.8 log2, 8 log2, 8.3 log2, and 8.3 log2 from week 2 to week 7, respectively, post-vaccination. The immune response in both groups reached a high titer at week 6. The combined inactivated E. coli and AI H5N1 vaccine generated a higher immune response than the inactivated AI H5N1 vaccine, and a significant difference exists between the two groups. For Groups 2 and 3, the ELISA antibody titer exhibited its lowest value, 1996.5 and 2036.7, respectively, at week 1 post-vaccination; whereas, both groups exhibited the highest titers, 2227.7 (for Group 2) and 2287.3 (for Group 3), in week 3 post-booster. The ELISA for the combined inactivated E. coli and AI H5N1 vaccine had a higher titer than did the inactivated E. coli vaccine, and a significant difference exists between the two groups. Moreover, the protection rate was higher in Group 3, with 100% for E. coli and 90% for the AI H5N1 vaccine.Conclusion:Our findings demonstrate that producing a combined vaccine using E. coli and AI H5N1 formulated with Montanide ISA70 is recommended for protection against both diseases.

Correlation of Automated Chemiluminescent Method with Enzyme-Linked Immunosorbent Assay (ELISA) Antibody Titers in Convalescent COVID-19 Plasma Samples: Development of Rapid, Cost-Effective Semi-Quantitative Diagnostic Methods.

BackgroundWe investigated the utility of an automated chemiluminescent SARS-CoV-2 IgG antibody assay platform in quantifying the amount of binding antibodies present in donated convalescent plasma.MethodsA total of 179 convalescent plasma units were analyzed for the presence of SARS-CoV-2 IgG antibodies using the Beckman-Coulter chemiluminescent immunoassay (CLIA) platform. The equipment-derived numerical values (S/Co ratio) were recorded. Aliquots from the same units were subjected to enzyme-linked immunosorbent assay (ELISA) that detects IgG antibodies against the receptor-binding domain (RBD) of the SARS-CoV-2 S1 protein. The relationship between ELISA titers and CLIA S/Co values was analyzed using linear regression and receiver operating characteristics (ROC) curve.ResultsTwenty-one samples (11.7%) had S/Co values of less than 1.0 and were deemed negative for antibodies and convalescent plasma had S/Co values between >1.0 and 5.0 (70/179, 39.1%). Fifteen units (8.4%) had negative ELISA titer. The majority of the units (95/179. 53.1%) had titers ≥1:1024. The sensitivities of ELISA to CLIA were comparable (90.5% vs 88.3%, respectively; p=0.18). There was positive linear correlation between CLIA S/Co values and ELISA IgG titer (Rho = 0.75; Spearman’s rank = 0.82, p-value = <0.0001). The agreement between the two methods was fair, with a κ index of 0.2741. Using the ROC analysis, we identified a CLIA S/Co cutoff value of 8.2, which gives a sensitivity of 90% and a specificity of 82% in predicting a titer dilution of ≥1:1024.ConclusionThe utility of automated antibody detection systems can be extended from simply a screening method to a semi-quantitative and quantitative functional antibody analysis. CLIA S/Co values can be used to reliably estimate the ELISA antibody titer. Incorporation of chemiluminescent-based methods can provide rapid, cost-effective means of identifying anti-SARS-CoV-2 antibody titers in donated plasma for use in the treatment of COVID-19 infection.

Performance of an enzyme-linked immunosorbent-based serological assay for Entamoeba histolytica: Comparison with an indirect immunofluorescence assay using stored frozen samples

IntroductionEntamoeba histolytica infections are increasingly diagnosed as sexually transmitted infections in Japan. However, the stool ova–parasite examination (O&P) test has been the only approved diagnostic method used in Japan since production of the indirect immunofluorescence assay (IFA) serum antibody test was discontinued at the end of 2017. Herein, we assessed whether an enzyme-linked immunosorbent assay (ELISA)-based serological test could substitute for IFA. MethodsThis cross-sectional study used stored frozen serum samples from the Biobank of the National Center for Global Health and Medicine. A serological ELISA-based test was performed on these samples and their titers were compared with those previously measured by IFA based on the medical record data. ResultsSixty seven stored frozen serum samples with differing recorded IFA antibody titers (16 samples with titers < ×100, 13 samples × 100, 16 samples × 200, 11 samples × 400, and 11 samples ≥ × 800) were analyzed. The sensitivity and specificity values for ELISA vs. IFA were 92.2% [95% confidential interval: 81.5–96.9] and 87.5% [64.0–97.8], respectively. A strong correlation between the antibody titers was confirmed by a one-way ANOVA (R square 0.83, p value < 0.0001) for the two diagnostic methods. ConclusionThe ELISA and IFA antibody titers for E. histolytica were well correlated, and results from these methods were highly concordant. Introduction of an ELISA-based serological test for E. histolytica should be considered to improve E. histolytica infection diagnosis in Japan.

Clinical significance of positive anti-neutrophil cytoplasmic antibodies without evidence of anti-neutrophil cytoplasmic antibodies-associated vasculitis.

Anti-neutrophil cytoplasmic antibodies (ANCA) have a role in the diagnostic workup of ANCA-associated vasculitis. However, the clinical significance of positive ANCA in the absence of vasculitis is yet to be determined. Therefore, we sought to investigate the clinical spectrum and rate of patients with a positive ANCA without evidence of vasculitis. Retrospective analysis of patients positive for cytoplasmic ANCA (C-ANCA) and proteinase 3 (PR3) or P-ANCA and myeloperoxidase (MPO) between 2007 and 2016 in the Chaim Sheba Medical Center, Israel. The proportion of patients who had no evidence of vasculitis among all patients with a positive C-ANCA/PR3 or P-ANCA/MPO was calculated according to tertiles of enzyme-linked immunosorbent assay (ELISA) antibody levels. Among 113 patients who tested positive for C-ANCA/PR3 or P-ANCA/MPO, 68 (60.1%) had no evidence of vasculitis. ELISA antibody titers were significantly higher among patients with vasculitis than those without (6.2 vs 3.2, for C-ANCA/PR3 and 5.4 vs 2.6 for P-ANCA/MPO, P<0.05). The proportion of patients without vasculitis among all patients with a positive C-ANCA/PR3 and among all patients with a positive P-ANCA/MPO declined in parallel to the increases in ELISA antibody level tertiles (96%, 57% and 22% in the 1st, 2nd and highest tertiles, respectively, for patients with C-ANCA/PR3 patients and 100%, 66% and 20% in the 1st, 2nd and highest tertiles, respectively, for patients with P-ANCA/MPO). A significant proportion of patients with a positive C-ANCA/PR3 or P-ANCA/MPO do not have evidence of vasculitis, particularly those with low-medium ELISA antibody titers. Using a higher threshold of ANCA titers may be required to improve specificity.

High Prevalence of MERS-CoV Infection in Camel Workers in Saudi Arabia.

Middle East respiratory syndrome (MERS), a highly lethal respiratory disease caused by a novel coronavirus (MERS-CoV), is an emerging disease with high potential for epidemic spread. It has been listed by the WHO and the Coalition for Epidemic Preparedness Innovations (CEPI) as an important target for vaccine development. While initially the majority of MERS cases were hospital acquired, continued emergence of MERS is attributed to community acquisition, with camels likely being the direct or indirect source. However, the majority of patients do not describe camel exposure, making the route of transmission unclear. Here, using sensitive immunological assays and a cohort of camel workers (CWs) with well-documented camel exposure, we show that approximately 50% of camel workers (CWs) in the Kingdom of Saudi Arabia (KSA) and 0% of controls were previously infected. We obtained blood samples from 30 camel herders, truck drivers, and handlers with well-documented camel exposure and from healthy donors, and measured MERS-CoV-specific enzyme-linked immunosorbent assay (ELISA), immunofluorescence assay (IFA), and neutralizing antibody titers, as well as T cell responses. Totals of 16/30 CWs and 0/30 healthy control donors were seropositive by MERS-CoV-specific ELISA and/or neutralizing antibody titer, and an additional four CWs were seronegative but contained virus-specific T cells in their blood. Although virus transmission from CWs has not been formally demonstrated, a possible explanation for repeated MERS outbreaks is that CWs develop mild disease and then transmit the virus to uninfected individuals. Infection of some of these individuals, such as those with comorbidities, results in severe disease and in the episodic appearance of patients with MERS.IMPORTANCE The Middle East respiratory syndrome (MERS) is a coronavirus (CoV)-mediated respiratory disease. Virus transmission occurs within health care settings, but cases also appear sporadically in the community. Camels are believed to be the source for community-acquired cases, but most patients do not have camel exposure. Here, we assessed whether camel workers (CWs) with high rates of exposure to camel nasal and oral secretions had evidence of MERS-CoV infection. The results indicate that a high percentage of CWs were positive for virus-specific immune responses but had no history of significant respiratory disease. Thus, a possible explanation for repeated MERS outbreaks is that CWs develop mild or subclinical disease. These CWs then transmit the virus to uninfected individuals, some of whom are highly susceptible, develop severe disease, and are detected as primary MERS cases in the community.

Suppression of Highly Pathogenic Avian Influenza A/H5N1 Infection Using Migratory Antibody Passed from Mother to Chick

Avian influenza is the most contagious disease not only in poultry, but also in humans. Avian influenza in humans occurs mainly in Southeast Asia, but no human-to-human pandemic has occurred. Meanwhile, outbreaks of avian influenza in poultry occur on a global scale and cause a large economic loss. Migration antibodies passed from mother birds via eggs are said to be an important component of the immune system that protects birds from infection. Thus, the immunity status of mother birds can determine the ability of offspring to defend against infection. In this study, we investigated the presence of anti-avian influenza virus antibody in chickens hatched on a poultry farm in Indonesia and examined the involvement of migratory antibodies in protecting against virus infection by infectious experiments of highly pathogenic avian influenza in chickens. Blood was collected from randomly selected chicks, and antibodies against avian influenza virus were evaluated in all birds. Since these young birds had no history of vaccination, the antibodies were deemed to have been transferred from the mother birds. The enzyme-linked immunosorbent assay antibody titer in each bird varied. Infection of these birds with highly pathogenic avian influenza virus A/H5N1 intra-nasally resulted in a high mortality rate in chicks with low antibody titers but a low mortality rate in chicks with high antibody titers. These findings indicate that migratory antibody prevented highly pathogenic avian influenza A/H5N1 infection in chicks, suggesting that such a preventive effect could also be expected with outdoor natural infection.

Discrepant serological assays forPneumococcusin renal transplant recipients - a prospective study

Vaccine immunoprotection for Streptococcus pneumoniae is mediated by opsonizing antibodies targeting serotype-specific capsular polysaccharides. Quantitative antibody levels enzyme-linked immunosorbent assay (ELISA) and antibody-mediated opsonophagocytic assays (OPA) measure vaccine-induced protection; correlation of these assays in transplantation requires investigation. This study examines the laboratory assessment of antibody titers in vaccinated renal recipients. Streptococcus pneumoniae 19A is common in immunocompromised hosts and is represented in protein-conjugate vaccines (PCV) and polysaccharide vaccines (PSV). Antibodies to 19A in serial sera from 30 vaccinated renal transplant recipients were compared using ELISA and OPA assays. Subject titers were classified as protected or not by ELISA (>0.35 μg/ml) and OPA titer (>1:8). Antibody titers analyzed using McNemar's test indicate that protection measured by the two assays are not the same (P = 0.0078); simple linear regression of within-subject geometric means of 19A enzyme-linked immunosorbent assay (ELISA) antibody levels versus 19A opsonophagocytic assays (OPA) titers demonstrates significant correlation between the two assays (P < 0.001). Vaccination is increasingly important given increasing antimicrobial resistance worldwide. OPA and ELISA antibody assays do not correlate well using current values for protective immunity against the Pneumococcus in immunosuppressed transplant recipients. Future studies of vaccination in transplant recipients should evaluate protective antibody levels using both functional antibody assays and standard ELISA antibody titers. (ClinicalTrials.gov: NCT00307125).

Human polyclonal immunoglobulin G from transchromosomic bovines inhibits MERS-CoV in vivo.

As of 13 November 2015, 1618 laboratory-confirmed human cases of Middle East respiratory syndrome coronavirus (MERS-CoV) infection, including 579 deaths, had been reported to the World Health Organization. No specific preventive or therapeutic agent of proven value against MERS-CoV is currently available. Public Health England and the International Severe Acute Respiratory and Emerging Infection Consortium identified passive immunotherapy with neutralizing antibodies as a treatment approach that warrants priority study. Two experimental MERS-CoV vaccines were used to vaccinate two groups of transchromosomic (Tc) bovines that were genetically modified to produce large quantities of fully human polyclonal immunoglobulin G (IgG) antibodies. Vaccination with a clade A γ-irradiated whole killed virion vaccine (Jordan strain) or a clade B spike protein nanoparticle vaccine (Al-Hasa strain) resulted in Tc bovine sera with high enzyme-linked immunosorbent assay (ELISA) and neutralizing antibody titers in vitro. Two purified Tc bovine human IgG immunoglobulins (Tc hIgG), SAB-300 (produced after Jordan strain vaccination) and SAB-301 (produced after Al-Hasa strain vaccination), also had high ELISA and neutralizing antibody titers without antibody-dependent enhancement in vitro. SAB-301 was selected for in vivo and preclinical studies. Administration of single doses of SAB-301 12 hours before or 24 and 48 hours after MERS-CoV infection (Erasmus Medical Center 2012 strain) of Ad5-hDPP4 receptor-transduced mice rapidly resulted in viral lung titers near or below the limit of detection. Tc bovines, combined with the ability to quickly produce Tc hIgG and develop in vitro assays and animal model(s), potentially offer a platform to rapidly produce a therapeutic to prevent and/or treat MERS-CoV infection and/or other emerging infectious diseases.

Reflection of serum immunoglobulin isotypes in the egg yolk of laying hens immunized with enterotoxigenic Escherichia coli

Aim: The aim was to study the seroconversion and development of egg yolk immunoglobulins in adult laying White Leghorn hens immunized against an isolate of enterotoxigenic Escherichia coli (ETEC) bearing K91 and K88ac antigens, obtained from diarrheic piglet. Materials and Methods: Adult laying White Leghorn hens were immunized with inactivated enterotoxic E. coli strain isolated originally from a case of piglet diarrhea following recommended schedule. The development of whole antibodies and isotype-specific antibodies in serum and egg yolk were measured using indirect enzyme-linked immunosorbent assay (ELISA). Piglets suffering from diarrhea with fecal samples positive for ETEC were fed with egg yolk and compared with diarrheic control group. Results: The serum and egg yolk ELISA antibody titer against E. coli strain used in the present study was as high as 2666.66±307.92 and 933.33±203.67 respectively on 50 day-post-vaccination (DPV). The immunoglobulin Y (IgY) was the predominant isotype in serum and egg yolk, which reached the peak titer of 2200±519.61 in serum on 40 DPV and 800±244.94 in egg yolk on 50 DPV. IgM titer in serum and egg yolk was found to be meager, and no IgA could be detected. Diarrheic piglets fed with the egg yolk suspension from immunized hens showed a promising result in controlling diarrhea. Conclusion: Egg yolk antibodies are considered a suitable immunotherapeutic alternative to conventional antibiotic therapy. High titer of egg yolk antibodies raised in the immunized hen against an isolate of ETEC holds the potential to be used for passive protection of diarrheic piglets during their most susceptible period of infection.

Recombinant nucleocapsid protein based single serum dilution ELISA for the detection of antibodies to infectious bronchitis virus in poultry

Avian infectious bronchitis is ubiquitous and highly contagious disease of poultry, with profound effect on commercial poultry production. For effective control of infectious bronchitis virus (IBV), quick and specific diagnosis is of utmost importance. In this study, the virus was isolated from clinical samples from India and the full length nucleocapsid (N) gene was amplified, cloned and expressed in a prokaryotic system. The purified recombinant N protein based single serum dilution enzyme linked immunosorbent assay (ELISA) was developed for IBV to measure specific antibody in the sera of chickens. A total of 310 chicken sera samples were tested using the commercial IDEXX kit along with the assay developed. A linear correlation was obtained between predicted antibody titres at a single working dilution of 1:100 and the corresponding serum titres observed as determined by the standard serial dilution method. Regression analysis was used to construct a standard curve from which an equation was derived which confirmed their correlation. The developed equation was then used to extrapolate predicated ELISA antibody titer from corrected absorbance readings of the single working dilution. The assay proved to be specific (95.8%) and sensitive (96.8%) when compared to the commercial IDEXX ELISA test.

Recombinant hexon antigen based single serum dilution ELISA for rapid serological profiling against fowl adenovirus-4 causing hydropericardium syndrome in chickens

A recombinant hexon antigen based single serum dilution enzyme linked immunosorbent assay (ELISA) was developed to measure the specific antibody in sera of chickens against Fowl adenovirus-4 (FAdV) causing Hydropericardium syndrome (HPS). An immunodominant partial hexon gene of 737bp was cloned into pRSET vector and expressed in Escherichia coli strain BL21 DE3 pLys S. Expression was tested by Western Blot test. The purified recombinant protein antigen was used in coating ELISA plate for FAdV-4 serology. A linear relationship was found between the predicted antibody titres at a single working dilution of 1:100 and the corresponding observed serum titres as determined by the standard serial dilution method. Regression analysis was used to determine a standard curve from which an equation was derived that allowed the demonstration of this correlation. The equation was then used to convert the corrected absorbance readings of the single working dilution directly into the predicted ELISA antibody titres. The assay proved to be sensitive, specific and accurate as compared to Q-AGID test. Recombinant antigen was also used in Dot ELISA. In an experimental vaccination of broiler chicken at 10 days old age, the geometric mean (GM) antibody titres as measured by ELISA ranged from 5.006±0.11log10 to 4.526±0.04log10 and by Dot ELISA titre were from 2.240±0.08log10 to 0.180±0.04log10 during 5th–8th weeks of age, results were compared with Q-AGID results. Field samples were collected randomly from breeder flocks, found to have antibody titre by both ELISA and Dot ELISA at 10th and only 75% samples were positive at 14th weeks of age. After revaccination at 16th weeks of age, all sera samples were found have considerably high antibody titre on 24th week but all samples were negative at 32nd weeks. Advantages of recombinant hexon antigen based ELISA and Dot ELISA in FAdV-4 serology have been discussed.

Detection of Mycoplasma agassizii in the Texas Tortoise (Gopherus berlandieri)

Mycoplasma agassizii causes upper respiratory tract disease (URTD) in Texas tortoises (Gopherus berlandieri). To determine exposure to and shedding of M. agassizii, we collected blood samples and nasal swabs from 40 free-ranging Texas tortoises on public and private lands in Texas, USA, from May to October 2009. We used an enzyme-linked immunosorbent assay (ELISA) to detect M. agassizii-specific antibodies. Eleven (28%) tortoises were antibody positive, three (8%) were suspect, and the remaining 26 (65%) were negative. Nasal lavage samples were collected from 35 of the 40 tortoises for M. agassizii culture and PCR to detect shedding of M. agassizii. Current infection with M. agassizii was confirmed in one tortoise that had mild clinical signs of URTD and was positive by ELISA (antibody titer >512), PCR, and culture. The clinical isolate was confirmed as M. agassizii by restriction fragment length polymorphism and immunobinding.

Establishment of Reverse Direct ELISA and Its Application in Screening High-Affinity Monoclonal Antibodies

Traditionally monoclonal antibody (MAb) titer is determined by indirect enzyme-linked immunosorbent assay (ELISA), which is primarily used to evaluate the quality of MAbs. In this study, the titer and affinity of a group of MAbs against ovalbumin (OVA) were tested by indirect ELISA and the ELISA method reported previously. Data showed that there may be great differences between the indirect ELISA antibody titer and affinity value of MAbs. For the first time, a simple and effective reverse direct ELISA (RD-ELISA) was established for the detection of high-affinity MAbs. Among the group of MAbs to OVA, a certain proportion of antibodies with high affinity but low indirect ELISA titer do exist and can be clearly and efficiently detected by RD-ELISA. This study demonstrates that RD-ELISA is an effective method for high-affinity MAb screening.

Recombinant UL30 antigen-based single serum dilution ELISA for detection of duck viral enteritis

A recombinant UL30 antigen-based single serum dilution enzyme linked immunosorbent assay (ELISA) was developed to measure specific antibody in the sera of ducks against duck enteritis virus (DEV). The partial UL30 gene of DEV was cloned, expressed, purified and tested for its diagnostic use by designing a single serum dilution enzyme linked immuno-sorbent assay (ELISA). A total of 226 duck sera samples were tested using the assay. A linear relationship was found between the predicted antibody titres at a single working dilution of 1:100 and the corresponding serum titres observed as determined by the standard serial dilution method. Regression analysis was used to determine a standard curve from which an equation was derived which demonstrated this correlation. The equation was then used to convert the corrected absorbance readings of the single working dilution directly into the predicted ELISA antibody titres. The assay proved to be specific, sensitive and accurate as compared to the virus neutralization test with a specificity, sensitivity and accuracy being 96%, 95% and 95% respectively.

Cytomegalovirus Neutralization by Hyperimmune and Standard Intravenous Immunoglobulin Preparations

Cytomegalovirus (CMV) remains one of the most important pathogens after transplantation, potentially leading to CMV disease, allograft dysfunction, acute, and chronic rejection and opportunistic infections. Immunoglobulin G (IgG) preparations with high antibody titers against CMV are a valuable adjunctive prevention and treatment option for clinicians and apart from standard intravenous immunoglobulin (IVIG), CMV hyperimmune preparations are available. The CMV antibody titer of these preparations is typically determined by Enzyme-linked immunosorbent assay (ELISA), also used for the selection of high titer plasma donors for the production of the CMV Hyperimmune product. However, CMV ELISA titers do not necessarily correlate with CMV antibody function which is determined by virus neutralization tests. CMV antibody titers were determined by both ELISA and virus neutralization assay and the IgG subclass distribution was compared between a CMV hyperimmune licensed in Europe and standard IVIG preparations. Although the expected high CMV IgG ELISA antibody titers were confirmed for three lots of a CMV hyperimmune preparation, the functionally more relevant CMV neutralizing antibody titers were significantly higher for 31 lots of standard IVIG preparations. Moreover, considerably lower IgG3 levels were found for the CMV hyperimmune preparation compared with standard IVIG preparations. The higher functional CMV neutralization titers of standard IVIG preparations and the better availability of these preparations, suggest that these products could be a valuable alternative to the CMV hyperimmune preparation.

Seroprevalence of Aichi Virus in a Spanish Population from 2007 to 2008

Viruses are among the most common causes of acute gastroenteritis. In recent years, new viruses causing outbreaks of acute gastroenteritis have been described. Among these, Aichi virus was identified in Japan in 1989. Aichi virus belongs to the Kobuvirus genus in the family Picornaviridae. This virus has been detected in outbreaks of gastroenteritis associated with oyster consumption and in pediatric stool samples, but little is known about its epidemiology or pathogenesis. In the present study, the prevalence of antibodies to Aichi virus in a Spanish population was determined between 2007 and 2008 by using an enzyme-linked immunosorbent assay (ELISA). As in previous studies, a high seroprevalence of antibodies to Aichi virus (70%) was observed, with levels differing according to age. We observed significant differences in titers of antibody to Aichi virus among different age groups, grouped by decades. We report high ELISA and neutralizing antibody titers, and both titers fitted a sigmoid curve significantly. However, this virus is seldom detected; therefore, further studies are needed to gain a better understanding of its importance as a pathogenic agent.

Formation of subviral particles of the capsid protein VP2 of infectious bursal disease virus and its application in serological diagnosis

Infectious bursal disease virus (IBDV) is an immunosuppressive disease of young chicken characterized by severe depletion of B-lymphocytes in the bursa of Fabricius. To provide antigen for diagnostic tests, its major structural protein VP2 was expressed in the yeast Saccharomyces cerevisiae. Electron microscopy of purified VP2 protein demonstrated that when expressed from yeast cells VP2 protein forms subviral particles (SVPs) of approximately 20 nm in diameter. A recombinant VP2 antigen-based single serum dilution enzyme linked immunosorbent assay (ELISA) using the SVPs detected IBDV specific antibodies in chickens. A linear relationship was found between the predicted antibody titres at a single working dilution of 1:1000 and the corresponding observed serum titres when determined by the standard serial dilution method. Regression analysis was used to construct a standard curve from which an equation was derived which confirmed their correlation. The equation was then used to convert the corrected absorbance readings of the single working dilution directly into the predicted ELISA antibody titres. The assay proved to be sensitive, specific and accurate as compared to the serum neutralization test and agar gel immunodiffusion test. The recombinant VP2 antigen is a suitable alternative to whole viral antigen.