Comparing the performance of dengue virus IgG and IgG-capture enzyme-linked immunosorbent assays in seroprevalence study.

BACKGROUND: Dengue virus (DENV) is the leading cause of arboviral diseases in humans worldwide. Currently Dengvaxia, the first dengue vaccine licensed in 20 countries, was recommended for DENV seropositive individuals aged 9-45 years. Studying dengue seroprevalence can improve our understanding of the epidemiology and transmission dynamics of DENV, and facilitate future intervention strategies and assessment of vaccine efficacy. Several DENV envelope protein-based serological tests including IgG and IgG-capture enzyme-linked immunosorbent assays (ELISAs) have been employed in seroprevalence studies. Previously DENV IgG-capture ELISA was reported to distinguish primary and secondary DENV infections during early convalescence, however, its performance over time and in seroprevalence study remains understudied. METHODS: In this study, we used well-documented neutralization test- or reverse-transcription-polymerase-chain reaction-confirmed serum/plasma samples including DENV-naïve, primary and secondary DENV, primary West Nile virus, primary Zika virus, and Zika with previous DENV infection panels to compare the performance of three ELISAs. RESULTS: The sensitivity of the InBios IgG ELISA was higher than that of InBios IgG-capture and SD IgG-capture ELISAs. The sensitivity of IgG-capture ELISAs was higher for secondary than primary DENV infection panel. Within the secondary DENV infection panel, the sensitivity of InBios IgG-capture ELISA decreased from 77.8% at < 6 months to 41.7% at 1-1.5 years, 28.6% at 2-15 years and 0% at > 20 years (p < 0.001, Cochran-Armitage test for trend), whereas that of IgG ELISA remains 100%. A similar trend was observed for SD IgG-capture ELISA. CONCLUSIONS: Our findings demonstrate higher sensitivity of DENV IgG ELISA than IgG-capture ELISA in seroprevalence study and interpretation of DENV IgG-capture ELISA should take sampling time and primary or secondary DENV infection into consideration.

Zika Virus IgM 25 Months after Symptom Onset, Miami-Dade County, Florida, USA.

We assessed IgM detection in Zika patients from the 2016 outbreak in Miami-Dade County, Florida, USA. Of those with positive or equivocal IgM after 12-19 months, 87% (26/30) had IgM 6 months later. In a survival analysis, ≈76% had IgM at 25 months. Zika virus IgM persists for years, complicating serologic diagnosis.

Establishment of the cross-clade antigen detection system for H5 subtype influenza viruses using peptide monoclonal antibodies specific for influenza virus H5 hemagglutinin.

The H5 subtype of highly pathogenic avian influenza (H5 HPAI) viruses is a threat to both animal and human public health and has the potential to cause a serious future pandemic in humans. Thus, specific and rapid detection of H5 HPAI viruses is required for infection control in humans. To develop a simple and rapid diagnostic system to detect H5 HPAI viruses with high specificity and sensitivity, we attempted to prepare monoclonal antibodies (mAbs) that specifically recognize linear epitopes in hemagglutinin (HA) of H5 subtype viruses. Nine mAb clones were obtained from mice immunized with a synthetic partial peptide of H5 HA molecules conserved among various H5 HPAI viruses. The antigen-capture enzyme-linked immunosorbent assay using the most suitable combination of these mAbs, which bound specifically to lysed H5 HA under an optimized detergent condition, was specific for H5 viruses and could broadly detect H5 viruses in multiple different clades. Taken together, these peptide mAbs, which recognize linear epitopes in a highly conserved region of H5 HA, may be useful for specific and highly sensitive detection of H5 HPAI viruses and can help in the rapid diagnosis of human, avian, and animal H5 virus infections.

Comparison of enzyme-linked immunosorbent assay systems using rift valley fever virus nucleocapsid protein and inactivated virus as antigens.

BACKGROUND: Rift Valley Fever (RVF) is a mosquito-borne viral zoonosis. To detect RVF virus (RVFV) infection, indirect immunoglobulin G (IgG) and immunoglobulin M (IgM) enzyme linked immunosorbent assays (ELISAs) which utilize recombinant RVFV nucleocapsid (RVFV-N) protein as assay antigen, have reportedly been used, however, there is still a need to develop more sensitive and specific methods of detection. METHODS: RVFV-N protein was expressed in Escherichia coli (E. coli) and purified by histidine-tag based affinity chromatography. This recombinant RVFV-N (rRVFV-N) protein was then used as antigen to develop an IgG sandwich ELISA and IgM capture ELISAs for human sera. Ninety six serum samples collected from healthy volunteers during the RVF surveillance programme in Kenya in 2013, and 93 serum samples collected from RVF-suspected patients during the 2006-2007 RVF outbreak in Kenya were used respectively, to evaluate the newly established rRVFV-N protein-based IgG sandwich ELISA and IgM capture ELISA systems in comparison with the inactivated virus-based ELISA systems. RESULTS: rRVFV-N protein-based-IgG sandwich ELISA and IgM capture ELISA for human sera were established. Both the new ELISA systems were in 100% concordance with the inactivated virus-based ELISA systems, with a sensitivity and specificity of 100%. CONCLUSIONS: Recombinant RVFV-N is a safe and affordable antigen for RVF diagnosis. Our rRVFV-N-based ELISA systems are safe and reliable tools for diagnosis of RVFV infection in humans and especially useful in large-scale epidemiological investigation and for application in developing countries.

Development of a rapid method for the detection of Yersinia enterocolitica serotype O:8 from food.

In this study, a new and alternative method based on monoclonal antibodies (MAbs) for the rapid detection of Yersinia enterocolitica O:8 was developed. This microorganism is an emerging foodborne pathogen causing gastrointestinal disease in humans. The transmission can occur through contaminated food such as raw or undercooked meat, milk and dairy products, water and fresh vegetables. Nine MAbs (46F7, 54B11, 54C11, 62D10, 64C7, 64C10, 72E8, 72E10, 72G6) were characterized and selected versus Y. enterocolitica O:8, and only 2 of them showed also a weak cross-reaction with Campylobacter jejuni. The MAb 54B11 was used for the development of Y. enterocolitica capture-ELISA in food matrices, i.e. meat and dairy products (n = 132). The method was validated by ISO 16140:2003 and compared with the official method for the detection of presumptive pathogenic Y. enterocolitica (ISO 10273:2003). Relative accuracy, sensitivity and specificity corresponded to 100%. The selectivity was evaluated on other food samples (n = 126) showing a lower confidence limit of 90.3% and an upper confidence limit of 100%. The results from this study demonstrated that the developed method was rapid and cheap, specific and sensitive for the screening of the pathogen in food.

Evaluation of 5 Commercially Available Zika Virus Immunoassays.

Because of the global spread of Zika virus, accurate and high-throughput diagnostic immunoassays are needed. We compared the sensitivity and specificity of 5 commercially available Zika virus serologic assays to the recommended protocol of Zika virus IgM-capture ELISA and plaque-reduction neutralization tests. Most commercial immunoassays showed low sensitivity, which can be increased.

Anti-Taenia solium monoclonal antibodies for the detection of parasite antigens in body fluids from patients with neurocysticercosis.

Neurocysticercosis (NCC), an infection of the brain by Taenia solium (Ts) cysts, is the most common cause of adult-onset epilepsy in developing countries. Serological testing consists primarily of varying methods to detect antibodies in body fluids and more recently antigen (Ag) detection assays to identify individuals or animals with viable parasites. Antigen assays currently in use employ monoclonal antibodies (mAbs) raised against T. saginata, which have known cross reactivity to animal cestodes but are highly specific in human samples. We produced, characterized and tested 21 mAbs raised against T. solium whole cyst antigens, vesicular fluid or excretory secretory products. Reactivity of the TsmAbs against specific cyst structures was determined using immunofluorescence and immunohistochemistry on histological sections of Ts muscle cysts. Four TsmAbs reacted to vesicular space alone, 9 to the neck and cyst wall, one to the neck and vesicular space and 7 to the neck, cyst wall and vesicular space. An in-house ELISA assay to detect circulating Ts antigen, using the TsmAbs as capture antibodies and a rabbit polyclonal anti-Ts whole cyst antibody as a detector antibody demonstrated that eight of the 21 TsmAbs detected antigens in known NCC-positive human sera and three of these also in urine samples. Reactivity was expressed as normalized ratios of optical densities (OD positive control/OD negative control). Three TsmAbs had ratios >10 and five between 2 and 10. The TsmAbs have potential utility for the diagnosis and post-treatment monitoring of patients with viable NCC infections.

Japanese encephalitis associated acute encephalitis syndrome cases in West Bengal, India: A sero-molecular evaluation in relation to clinico-pathological spectrum.

Japanese encephalitis (JE) is a major public health problem in Asia and worldwide and it is responsible mainly for viral acute encephalitis syndrome (AES). The sole etiologic agent of JE is Japanese encephalitis virus (JEV). Although JE/AES cases have been regarded traditionally as a disease of children, a growing number of patients with JE/AES cases are also seen in the adult age group every year in the state of West Bengal, India in spite of vaccination. Therefore, a systematic study was performed to differentiate and characterize the clinico-pathological parameters and viral diversity among the patients of different age groups. Viral diversity was also evaluated from the JE/AES cases, depending on their disease severity. A total of 441 JE/AES cases were included in this study. By MAC-ELISA, 111 samples were found JEV IgM positive and among the IgM negative cases, 26 samples were found RT-PCR positive against JEV infection. Neck rigidity, abnormal behavior, convulsion, protein in CSF, WBC in CSF, and aspartate transaminase in blood differed significantly among the patients of pediatric-adolescent and adult group in both IgM positive and RT-PCR positive cases. Viral diversity was increased significantly in the pediatric-adolescent group compared to adult patients. Interestingly, with the rise in disease severity the viral diversity was found to be increased among the patients, irrespective of their age distribution. Based on clinico-pathological parameters and analysis of viral diversity, it can be concluded that viral diversity which occurs naturally is likely to affect disease severity, especially in the patients of pediatric-adolescent group.

Differential detection of chicken heterodimeric cytokines, interleukin 12 and 23 using their subunit-specific mouse monoclonal antibodies.

Interleukin-23 (IL-23) is a recently identified member of the IL-12 family of heterodimeric cytokines that play a critical role in regulating T helper cell function. IL-12 and IL-23 share a common p40 subunit, but differ in their p35 and p19 subunits, respectively. This difference in subunit composition results in distinct signaling pathways and biological functions for IL-12 and IL-23. Here, we report the functional characterization and immunomodulatory properties of chicken IL-12 and IL-23 using the panels of newly developed mouse anti-IL-12p40, IL-12p35-α and IL-23p19 monoclonal antibodies (mAbs). Western blot and indirect ELISA analysis demonstrated that the anti-chicken IL-12p40 mAbs (chIL-12p40; #10G10F4 and #10D8G2) bound to both recombinant proteins (IL-12 and IL-23), the anti-chicken IL-12p35 mAb (chIL-12p35; #2F1) specifically recognized recombinant IL-12, and the anti-chicken IL-23p19 mAb (chIL-23p19; #15A3) exhibited specificity for recombinant IL-23, without any cross-reactivity. Two ELISAs detecting specific chicken IL-12 (#10G10F4 and #2F1) or IL-23 (#10D8G2 and #15A3) were developed using newly developed mAb combinations, #10G10F4/ #2F1 and #10D8G2/#15A3 for IL-12 and IL-23, respectively, identified through a pairing assay. The levels of IL-12 and IL-23 in Resiquimod-848 stimulated-HD11 chicken macrophage cells were monitored over time using antigen-capture sandwich ELISA developed in this study. Furthermore, the levels of chicken IL-12 and IL-23 in the circulation of Eimeria maxima (E. maxima) and Eimeria tenella (E. tenella)-infected chickens were determined. Notably, the anti-chIL-12p40 mAbs (#10G10F4 and #10D8G2) neutralized the function of both chIL-12 and chIL-23 proteins, which share the p40 subunit, while the anti-chIL-23p19 mAb (#15A3) specifically neutralized chIL-23 protein in HD11 cells in vitro. The anti-chIL-12p35 mAb (#2F1), which is specific to the p35 subunit of IL-12, showed a partial neutralizing effect on chIL-12 protein. Collectively, our study validates the specificity and significance of 2 newly developed antigen-capture immunoassays for chIL-12 and chIL-23 which will expand our understanding of the functional characteristics of IL-12 and IL-23 and their association in normal and diseased chickens. These mAbs for each subunit, anti-chIL-12p35, anti-chIL-12p40 and anti-chIL-23p19, will serve as valuable immune reagents to elucidate host immune responses against disease pathogenesis in both fundamental and applied studies of avian species.

A safe, cost-effective, and high-throughput SARS-CoV-2 antigen capture ELISA suitable for large-scale screening in low-resource settings.

Diagnostics employing multiple modalities have been essential for controlling and managing COVID-19, caused by SARS-CoV-2. However, scaling up Reverse Transcription-Quantitative Polymerase Chain Reaction (RT-qPCR), the gold standard for SARS-CoV-2 detection, remains challenging in low and middle-income countries. Cost-effective and high-throughput alternatives like enzyme-linked immunosorbent assay (ELISA) could address this issue. We developed an in-house SARS-CoV-2 nucleocapsid capture ELISA, and validated on 271 nasopharyngeal swab samples from humans (n = 252), bovines (n = 10), and dogs (n = 9). This ELISA has a detection limit of 195 pg/100 µL of nucleocapsid protein and does not cross-react with related coronaviruses, ensuring high specificity to SARS-CoV-2. Diagnostic performance was evaluated using receiver operating characteristic curve analysis, showing a diagnostic sensitivity of 67.78 % and specificity of 100 %. Sensitivity improved to 74.32 % when excluding positive clinical samples with RT-qPCR Ct values > 25. Furthermore, inter-rater reliability analysis demonstrated substantial agreement (κ values = 0.73-0.80) with the VIRALDTECT II Multiplex RT-qPCR kit and perfect agreement with the CoVeasy™ COVID-19 rapid antigen self-test (κ values = 0.89-0.93). Our findings demonstrated that the in-house nucleocapsid capture ELISA is suitable for SARS-CoV-2 testing in humans and animals, meeting the necessary sensitivity and specificity thresholds for cost-effective, large-scale screening.

Development of a new immunodiagnostic tool for poultry coccidiosis using an antigen-capture sandwich assay based on monoclonal antibodies detecting an immunodominant antigen of Eimeria.

This study was conducted to develop an antigen-capture ELISA that detects an immunodominant antigen of Eimeria, 3-1E which is present in all Eimeria species, using a set of 3-1E-specific mouse monoclonal antibodies (mAbs). Highly sensitive 3-1E-specific antigen-capture ELISA was established using compatible mAb pairs (#318 and #320) selected from 6 mAbs (#312, #317, #318, #319, #320, and #323) with high binding activity against recombinant 3-1E protein. These anti-3-1E mAbs specifically recognized E. tenella sporozoites and a higher level of 3-1E was detected in the lysate of sporozoites than in sporocysts. Immunofluorescence assay (IFA) using 2 mAbs (#318 and #320) showed specific staining around the membrane of E. tenella sporozoites. In order to measure the changes in the 3-1E level during in coccidiosis, serum, feces, jejunal, and cecal contents were individually collected daily for 7-days postinfection (dpi) with E. maxima and E. tenella. The new ELISA was sensitive and specific for 3-1E detection in all samples collected daily from E. maxima- and E. tenella-infected chickens for a week, and the detection sensitivity ranges were 2 to 5 ng/mL and 1 to 5 ng/mL in serum, 4 to 25 ng/mL and 4 to 30 ng/mL in feces, 1 to 3 ng/mL and 1 to 10 ng/mL in cecal contents, and 3 to 65 ng/mL and 4 to 22 ng/mL in jejunal contents. Following coccidiosis, the overall 3-1E levels started to increase from 4 dpi, and the highest production was shown on 5 dpi. Among the samples collected from Eimeria-infected chickens, the highest detection level was found in the jejunal contents of E. maxima-infected chickens. Furthermore, the level of IFN-γ in serum was significantly (P < 0.05) increased from 3 dpi and peaked on 5 dpi post E. maxima infection. Post E. tenella infection, the level of IFN-γ in serum gradually (P < 0.05) increased from 2 to 5 dpi and plateaued at 7 dpi. The level of TNF-α in serum was rapidly (P < 0.05) increased from 4 dpi and those levels were kept until 7 dpi post both Eimeria infections (E. maxima and E. tenella). More importantly, the daily changes in the 3-1E levels in different samples from E. maxima- and E. tenella-infected chickens were effectively monitored with this new antigen-capture ELISA. Therefore, this new immunoassay is a sensitive diagnostic tool to monitor coccidiosis in a large field population in the commercial poultry farms before clinical symptoms develop using serum, feces, and gut samples during the entire period of infection cycle starting from 1 d after infection.

A Novel Designed Sandwich ELISA for the Detection of Echinococcus granulosus Antigen in Camels for Diagnosis of Cystic Echinococcosis.

Echinococcus spp. are important cosmopolitan zoonotic parasitic tapeworms that cause a disease called hydatidosis or cystic echinococcosis (CE), which has remarkable economic losses. The objective of our study was to develop a specific IgG polyclonal antigen-based ELISA (Sandwich ELISA; capture ELISA) method for the detection of circulating Echinococcus granulosus (E. granulosus) antigens in camels infected with hydatid cysts before slaughtering and its application in serodiagnosis of CE in animals to assess the positive rate of hydatidosis in camels slaughtered in Giza governorate abattoirs in Egypt. In this study, molecular identification of Echinococcus sp. isolate was performed based on the NADH dehydrogenase subunit 1 (NAD1) gene, revealing the isolate (GenBank: OQ443068.1), which is identical to the G6 E. granulosus sensu lato genotype. The positive rate of hydatid cysts was determined in slaughtered camels' organs (n = 587). The results revealed that hydatid cysts were found in 46.5% (273/587) of the examined camels. Pulmonary echinococcosis was significantly more prevalent in the slaughtered camels (60%, 164/273) than hepatic echinococcosis (39.9%, 109/273), (p = 0.001, Chi Square = 11.081). Cyst fertility rates were higher in hepatic (90.8%, 99/109) than in pulmonary cysts (83.5%, 137/164) and the most viable protoscoleces were recorded from fertile the hepatic cysts (67.85 ± 12.78). In this study, hydatid cyst germinal layer antigen (GlAg) was isolated and used for the immunization of rabbits to raise IgG polyclonal antibodies (anti-Echinococcus GlAb IgG). These IgG polyclonal antibodies were purified by affinity chromatography using a protein A column, then labeled with horseradish peroxidase. Electrophoretic analysis of IgG polyclonal antibodies and crude GlAg was performed in 10% polyacrylamide gels. The SDS-PAGE revealed four bands at molecular weights of 77 kDa, 65 kDa, 55 kDa, and 25 kDa. The Sandwich ELISA was performed to evaluate the sensitivity and specificity and cross-reactivity of the prepared IgG polyclonal antibodies. The circulating hydatid antigen was found in 270 out of the 273 samples with hydatidosis, with a sensitivity of 98.9% (270/273), a specificity of 94.9% (296/312) and a diagnostic efficacy of 96.8%. Regarding the cross reactivity, anti-Echinococcus GlAb IgG showed a low cross-reactivity with Fasciola gigantica infected camel sera (3/8), and Myiasis (Cephalopina titillator larvae; 3/20). No cross-reactivity was recorded with uninfected camel sera (negative sera for E. granulosus), and no cross-reactivity was found with antigens of Eimeria spp., Toxoplasma gondii, Cryptosporidium sp., and Hyalomma dromedarii (ticks' infestation). Then, Sandwich ELISA was conducted again to detect E. granulosus antigen in all the collected camel sera, which resulted in a 48.7% (286/587) positive rate of CE compared to 46.5% (273/587) using a postmortem inspection (PM diagnosis) (p = 0.5, Chi Square = 0.302). In conclusion, the Sandwich ELISA technique introduced in this study appears to be a sufficiently sensitive diagnostic assay for the detection of camels' echinococcosis using anti-Echinococcus GlAb IgG. In addition, it might offer a significant medical and veterinary importance in helping the early detection of hydatidosis, as well as its early treatment.

Development of an Anti-Zika and Anti-Dengue IgM ELISA Assay: Evaluation of Cross Reactivity and Validation.

Zika and dengue viruses (ZIKV and DENV) have been considered major global threats to humans in the past decade. The two infections display similar epidemiological and clinical manifestations. They are transmitted by the same primary vector, accounting for the co-circulation of the two viruses in regions where they are endemic. Highly specific and sensitive serological assays that are able to detect ZIKV and DENV antibodies (Abs) during the acute and convalescent phases of infections would help to improve clinical management and disease control. We report the development and characterisation of two monoclonal Abs, the ZIKV 8-8-11 and the DENV 8G2-12-21, which recognise the Zika non-structural protein 1 (NS1) and the dengue virus type 2 envelope protein, respectively. Both mAbs were used to set up enzyme-linked immunosorbent assays (ELISAs) specific for the detection of anti-Zika immunoglobulin M (IgM) and anti-dengue IgM and whose performance was similar to commercially available kits. These kits, intended to be used with the CHORUS Instruments, are rapid and require ≤ 50 µL of human serum. These tests could represent an affordable and reliable option for the rapid diagnosis of both ZIKV and DENV infections in developing countries, where these flaviviruses are endemic.

Establishment of an antigen-capture enzyme-linked immunosorbent assay for detecting avian reticuloendotheliosis virus.

In this study, an antigen-capturing enzyme-linked immunosorbent assay (AC-ELISA) was established for the detection of avian reticuloendotheliosis virus (REV) using monoclonal and polyclonal antibodies against gp90. New Zealand white rabbits were immunized with recombinant REV-gp90 protein, and polyclonal antibodies were obtained after purification and used as the capture antibody. Mice monoclonal antibody 1A12D against REV-gp90 protein previously prepared in our laboratory was used as the detection antibody. The specificity of the AC-ELISA was confirmed with REV, avian leukosis virus subgroup J, Marek's disease virus serotype Ⅰ, avian hepatitis E virus and Fowl adenovirus serotype 4. The results showed that the AC-ELISA had specific binding reaction with REV, and did not react with other viruses. The detection limit of this assay was 195 TCID50 units of REV. Furthermore, commercial vaccine artificially contaminated with REV was detected by three methods: AC-ELISA, the TaqMan probe fluorescence real-time quantitative RT-PCR (RT-qPCR) and indirect immunofluorescence assay (IFA). The results showed that the positive coincidence rate of RT-qPCR and AC-ELISA was 90.63 %, and the positive coincidence rate of RT-qPCR and IFA was 96.88%, indicating that the AC-ELISA established in this study was effective and feasible. This method simplified the detection process for REV contamination in poultry attenuated vaccines, and provide necessary technical tools for high-throughput detection of REV.

Development of specific monoclonal antibodies for the detection of natural chicken tumor necrosis factor-alpha.

Tumor necrosis factor alpha (TNF-α) is an important proinflammatory cytokine and the only known cytokine that can directly kill tumor cells. Unlike mammalian counterparts, chicken TNF-α (chTNF-α) gene has not been identified until very recently due to its high GC content (∼70%) and long GC fragments. The biological functions of this newly-identified cytokine and its detection methods remain to be further investigated. In this study, the extracellular domain of chTNF-α was cloned into prokaryotic vector after codon optimization and recombinant chTNF-α protein was expressed. Subsequently, using recombinant chTNF-ɑ as immunogen, rabbit polyclonal antibody (pAb) and eight clones of mouse anti-chTNF-ɑ monoclonal antibodies (mAbs) were produced, respectively. Both the pAb and mAbs specifically recognized recombinant chTNF-ɑ expressed in E.coli and transfected COS-7 cells. Further mapping the antigenic region showed that all the mAbs recognized a region of amino acid residues 195-285 of chTNF-ɑ. Furthermore, an antigen-capture enzyme-linked immunosorbent assay for the detection of chTNF-ɑ was established using one mAb and the pAb. This assay showed no cross-reactivity with irrelevant Trx-fused antigens and could detect natural chTNF-ɑ expressed by mitogen-activated chicken splenocytes in a dose-dependent manner, with a detection limit of 1 ng/mL. Collectively, our results indicated that the mAbs and pAb against chTNF-α are specific and could be used for the study of the biological functions of chTNF-ɑ and the detection of chTNF-ɑ.

Development of an in-house capture ELISA: An attempt to detect CagA antigen in sera of Helicobacter pylori infected patients.

The CagA protein one of the key virulence factors of Helicobacter pylori plays an important role in the pathogenesis of peptic ulcer diseases. Unfortunately the cagA gene status can only be determined by PCR while serology is an alternative approach to detect antigens or antibodies. Our aim is to detect the CagA antigen in sera of infected subjects by the development of an in-house capture ELISA test. Gastric antral biopsies and serum samples were collected from 63 patients. PCR was used to determine the cagA status. Our previously developed recombinant CagA protein and monoclonal antibody were used for setting up the capture ELISA test. H. pylori positive [(38 gastritis, 14 duodenal ulcers (DU), 11 gastric ulcer (GU)] patients were determined by PCR. The cagA gene was detected in 21 (55%) of gastritis, 11 (78%) of DU and 7 (60%) of GU patients. The reagents used in setting up the capture ELISA test following optimization displayed high performance. This study showed that our developed in-house capture ELISA has the potential to detect the CagA antigen at very low concentrations even though not detected in our H. pylori infected patients sera but we are also intended to use it in saliva and stool samples.

Immunological studies on chicken interferon-kappa using an antigen-capture ELISA developed using new mouse monoclonal antibodies.

Interferon (IFN)-κ is a type I IFN that plays a central role in anti-viral defense and host immune response. The functions of type I IFNs have not been clearly defined in chickens compared to those of their mammalian counterparts. In this study, we developed an antigen-capture ELISA using newly developed mouse monoclonal antibodies (mAbs) which are specific for chicken IFN-κ (chIFN-κ) and showed that this ELISA can measure native chIFN-κ production during the activation of macrophages by polyinosinic:polycytidylic acid (poly I:C). The recombinant chicken IFN-κ expressed in Escherichia coli was used to immunize mice. Five mAbs that specifically recognized chIFN-κ were selected and characterized based on their specificity and binding activity toward chIFN-κ via indirect ELISA and western blotting. To develop a capture ELISA for chicken IFN-κ, two sets of the best capture and detection mAbs combinations were identified via pairing assays. To validate the antigen-capture assay, the production of native IFN-κ was induced in chicken HD11 macrophages using poly I:C. Furthermore, qRT-PCR was used to confirm the transcript-level expression of IFN-κ in HD11 cells at 24 and 48 h. The neutralizing effects of anti-chIFN-κ mAbs were evaluated based on their ability to block the induction of IFN-stimulated genes (ISGs) in DF-1 fibroblast cells stimulated with recombinant chIFN-κ proteins. All five mAbs blocked the mRNA expression of ISGs in a dose-dependent manner. Our results validate the specificity and utility of these newly developed mAbs for the detection of native chicken IFN-κ. This novel antigen-capture ELISA will be a valuable tool for fundamental and applied research involving IFN-κ in the normal and diseased states.

Zika Virus Non-Structural Protein 1 Antigen-Capture Immunoassay.

Infection with Zika virus (ZIKV), a member of the Flavivirus genus of the Flaviviridae family, typically results in mild self-limited illness, but severe neurological disease occurs in a limited subset of patients. In contrast, serious outcomes commonly occur in pregnancy that affect the developing fetus, including microcephaly and other major birth defects. The genetic similarity of ZIKV to other widespread flaviviruses, such as dengue virus (DENV), presents a challenge to the development of specific ZIKV diagnostic assays. Nonstructural protein 1 (NS1) is established for use in immunodiagnostic assays for flaviviruses. To address the cross-reactivity of ZIKV NS1 with proteins from other flaviviruses we used site-directed mutagenesis to modify putative epitopes. Goat polyclonal antibodies to variant ZIKV NS1 were affinity-purified to remove antibodies binding to the closely related NS1 protein of DENV. An antigen-capture ELISA configured with the affinity-purified polyclonal antibody showed a linear dynamic range between approximately 500 and 30 ng/mL, with a limit of detection of between 1.95 and 7.8 ng/mL. NS1 proteins from DENV, yellow fever virus, St. Louis encephalitis virus and West Nile virus showed significantly reduced reactivity in the ZIKV antigen-capture ELISA. Refinement of approaches similar to those employed here could lead to development of ZIKV-specific immunoassays suitable for use in areas where infections with related flaviviruses are common.

Avidin-Biotin recombinant antigen capture ELISA for the detection of peste des petits ruminants virus in the clinical specimens of sheep and goats.

This study describes the development of Avidin-Biotin recombinant Antigen Capture ELISA (ABrAC ELISA) for the detection of the peste des petits ruminants virus (PPRV) antigens in the clinical specimens of sheep and goats. The assay uses the truncated recombinant PPRV N-terminal immunogenic region of nucleoprotein (rPPRV-NPN) as a reference positive antigen and its polyclonal antibodies as capture/detective antibodies and the rabbit PPRV polyclonal antibodies as coating antibodies. The cut-off value was determined as double times the mean reactivity of blank control based on the reactivity of the PPR confirmed negative and positive control panel samples. On assessing the specificity with the related differential diagnosis of the disease-causing viruses and bacteria, the assay showed specific detective reactivity to PPRV. Further, on evaluation using clinical specimens (n-274) of sheep and goats, the assay showed that the relative diagnostic sensitivity of 86.49 % (95 % confidence interval (CI): 71.23-95.46 %) and diagnostic specificity of 96.20 % (95 % CI: 92.91-98.25 %) against PPRV nucleoprotein-specific monoclonal antibody-based sandwich-ELISA (PPR s-ELISA) kit, with an accuracy of 94.89 % (95 % CI: 91.58-97.18 %) and Cohen's Kappa value of 0.791 + 0.055 SE (95 % CI: 0.68-0.90) with substantial agreements. The ABrAC-ELISA is an alternative method of an immunoassay for the rapid, sensitive, and specific detection of the PPRV antigens m the clinical specimens of sheep and goats for surveillance or diagnosis of PPR. This study also shows that the rPPRV-NPN and its specific polyclonal antibodies could be the sustainable source of safe diagnostic reagents without the need to handle the infectious virus during the eradication and post-eradication phases in endemic countries like India or PPR non-endemic countries.

Adsorption onto aluminum hydroxide adjuvant protects antigens from degradation.

Aluminum based adjuvants are widely used in commercial vaccines, since they are known to be safe and effective with a variety of antigens. The effect of antigen adsorption onto Aluminum Hydroxide is a complex area, since several mechanisms are involved simultaneously, whose impact is both antigen and formulation conditions dependent. Moreover, the mode of action of Aluminum Hydroxide is itself complex, with many mechanisms operating simultaneously. Within the literature there are contrasting theories regarding the effect of adsorption on antigen integrity and stability, with reports of antigen being stabilized by adsorption onto Aluminum Hydroxide, but also with contrary reports of antigen being destabilized. With the aim to understand the impact of adsorption on three recombinant proteins which, following in vivo immunization, are able to induce functional bactericidal antibodies against Neisseria meningitidis type B, we used a range of physico-chemical tools, such as DSC and UPLC, along with in vitro binding of antibodies that recognize structural elements of the proteins, and supported the in vitro data with in vivo evaluation in mice studies. We showed that, following exposure to accelerated degradation conditions involving heat, the recombinant proteins, although robust, were stabilized by adsorption onto Aluminum Hydroxide and retain their structural integrity unlike the not adsorbed proteins. The measure of the Melting Temperature was a useful tool to compare the behavior of proteins adsorbed and not adsorbed on Aluminum Hydroxide and to predict protein stability.