Monoclonal Antibody-based Sandwich ELISA Research Articles

A study on toxin genes and cytotoxicity levels of Bacillus cereus in various ready-to-eat foods and pastry products in Turkey

Bacillus cereus is a spore-forming and toxin-producing gram-positive bacteria widely isolated from soils, meat, milk, and vegetables. It is recognized as one of the pathogenic bacteria that can lead to food poisoning and food spoilage in food service systems due to its ease of contamination of foods and lack of guarantee of elimination by pasteurization and sanitation practices. B. cereus causes two types of diseases mainly characterized by diarrhea and vomiting type syndrome with the toxins it produces. Toxins produced by B. cereus are mainly heat-stable emetic toxin and three different heat-labile enterotoxins. Foodborne illnesses of the diarrheal type are caused by the single protein toxin; cytotoxin K (CytK), and both tripartite toxins; hemolysin BL (Hbl), and the non-hemolytic enterotoxin (Nhe), whereas the emetic type, is caused by an emetic toxin cereulide. In this study, 225 ready-to-eat foods and pastry products were analyzed for B. cereus, its toxin profiles, and cytotoxicity effects. Multiplex PCR is used to identify the presence of the Hbl, CytK, and emetic toxin encoding genes. Component-specific antibody-based ELISA tests were utilized to determine the Hbl-L2 and NheB components. Cytotoxic activity of the B. cereus isolates on Vero cells was also identified. In total, B. cereus was detected in 37 out of 225 (16.4%) food samples. From the positive 37 B. cereus isolates, the ces gene was not identified, whereas 91.9% (34) Nhe, 56.8% (21) Hbl, and 8.1% (3) CytK encoding genes revealed positive results on PCR analysis. PCR results were also compatible with ELISA and Cytotoxicity tests. In a nutshell, 16.4% prevalence of B. cereus in foods is insufficient, and the presence or absence of toxin genes may not yield reliable results. It is critical to detect pathogenic B. cereus toxin gene profiles as well as toxin production ability at the same time. This study presents for the first time, data from a cell culture cytotoxicity test using specific monoclonal antibody-based sandwich ELISA and multiplex PCR for ready-to-eat foods and pastry products in Turkey

The Relationship between Plasma Alpha-1-Antitrypsin Polymers and Lung or Liver Function in ZZ Alpha-1-Antitrypsin-Deficient Patients.

Alpha-1-Antitrypsin (AAT) is a protein of the SERPINA1 gene. A single amino acid mutation (Lys342Glu) results in an expression of misfolded Z-AAT protein, which has a high propensity to intra- and extra-cellular polymerization. Here, we asked whether levels of circulating Z-AAT polymers are associated with the severity of lung disease, liver disease, or both. We obtained cross sectional data from the Dutch part of the Alpha1 International Registry of 52 ZZ-AAT patients who performed a pulmonary function test and donated a blood sample on the same day. From the Alpha-1 Liver Aachen Registry, we obtained a cohort of 40 ZZ-AAT patients with available data on their liver function. The levels of plasma Z-AAT polymers were determined using a LG96 monoclonal antibody-based sandwich ELISA. In a Dutch cohort, the median plasma level of Z-AAT polymers of patients diagnosed for pulmonary disease was 947.5 µg/mL (733.6–1218 µg/mL (95% CI)), which did not correlate with airflow obstruction or gas transfer value. In the Alpha-1 liver patient cohort, the median polymer level was 1245.9 µg/mL (753–2034 µg/mL (95% CI)), which correlated with plasma gamma-glutamyl transferase (GGT, rs = 0.57, p = 0.001), glutamate dehydrogenase (GLDH, rs = 0.48, p = 0.002) and triglycerides (TG, rs = 0.48, p = 0.0046). A Wilcoxon rank test showed higher Z-AAT polymer values for the liver over the lung group (p < 0.0001). These correlations support a possible link between plasma Z-AAT polymers and the liver function.

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.

Evaluation of Monoclonal Antibody (IgG2bMAb) for Detection of Coproantigen from Experimentally Infected Rats with Strongyloides ratti

Background and Aim: Highly sensitive and specific diagnostic assay for the detection of Strongyloides is needed due to the intermittent and low concentration of eggs, larvae and adult worms that can be found in a faecal specimen. In some cases, repeated sampling of the faecal specimen is required to obtain satisfactory and reliable results. The aim of the study is to develop and evaluates monoclonal antibody-based Sandwich ELISA for the detection of coproantigen associated with Strongyloides infection using S. ratti as a model.&#x0D; Place and Duration of Study: Department of Medical Microbiology and Parasitology, Faculty of Medicine and Health Sciences, University Malaysia, Between September 2018 and March 2019.&#x0D; Methodology: The monoclonal antibody was raised against a soluble antigen of the infective filariform larvae (iL3) of S. ratti. The monoclonal antibody produced (IgG2bMAb) was evaluated for cross-reactivity against homologous and heterologous helminth antigens such as excretory-secretory (ES), infective larvae (iL3) and coproantigen of S. ratti, adult worms of A. caninum, A. suum, T. canis and T. cati.&#x0D; Results: An IgG2bMAb was observed to react with 30 kDa proteins associated with all homologous antigen from iL3, ES and coproantigen of S. ratti and cross-reacted with one heterologous antigen from adult worm of A. caninum at the same molecular weight. There was no cross-reaction observed with other heterologous antigens from adult worms of T. canis, T. cati and A. suum. The sensitivity of IgG2bMAb for the detection of S. ratti was 85% in Sandwich ELISA. Cross- reaction was observed with hookworm antigen that caused by A. caninum in Western immunoblotting.&#x0D; Conclusion: The results indicated that IgG2b have an immunodiagnostic property as IgG2bMAb and was able to detect antigens from coproantigen related to S. ratti with 85% sensitivity based on Sandwich ELISA) even though cross-reaction was observed with A. caninum. These findings will be very useful to tackle many cases of multiple worms’ infections such as both strongyloidiasis and hookworm. Therefore, we recommend that further evaluation and study in the human area where multiple infections can be common should be carried out.

Development of a robust and standardized immunoserological assay for detection of anti-measles IgG antibodies in human sera

Because of measles outbreaks there is a need for continuous monitoring of immunological protection against infection at population level. For such monitoring to be feasible, a cost-effective, reliable and high-throughput assay is necessary. Herein we describe an ELISA protocol for assessment of anti-measles antibody levels in human serum samples that fulfills the above criteria and is easily adaptable by various laboratories. A serum bank of anonymous patient sera was established (N > 3000 samples). Sera were grouped based on measles immunization schedules and/or changes in vaccine components since the introduction of the first measles vaccine in Hungary in 1969. Newly designed ELISA was performed by using Siemens BEP 2000 Advance System and data were confirmed using commercially available kits. Our indirect ELISA was compared to indirect immunfluoresence and to anti-measles nucleocapsid (N) monoclonal antibody-based sandwich ELISA. The results obtained are in high agreement with the confirmatory methods, and reflect measles vaccination history in Hungary ranging from pre-vaccination era, through the initial period of measles vaccination, to present. Based on measurement of 1985 sera, the highest ratio of low/questionable antibody level samples was detected in cluster ‘1978–1987’ (~25.4%), followed by cluster ‘1969–1977’ (~15.4%).Our assay is suitable for assessment of anti-measles immunity in a large cohort of subjects. The assay is cost-effective, allows high-throughput screening and has superior signal-to-noise ratio. This assay can serve as a first step in assessment of the effectiveness of all three components of the MMR vaccine.

Real-time stability of a hepatitis E vaccine (Hecolin®) demonstrated with potency assays and multifaceted physicochemical methods

The first prophylactic vaccine against hepatitis E virus (HEV), Hecolin®, was licensed in China. Recombinant p239 virus-like particle (VLP) is its active component with dimeric protein as the basic building block harboring the immuno dominant and neutralizing epitopes. The real time and real condition stability of the prefilled syringes for the vaccine was demonstrated using both in vivo mouse potency and in vitro antigenicity assays. A total of 12 lots of Hecolin® were assessed with a set of assays after storage at 2–8°C for 24months. The particle characteristics of p239 VLP recovered from the aluminum-containing adjuvant was assessed with different methods including analytical ultracentrifugation, high performance size exclusion chromatography and transmission electron microscopy. The thermal and conformational stability of the adsorbed antigen was assessed using differential scanning calorimetry. The protein integrity of the recovered p239 antigen was demonstrated using SDS-PAGE with silvering staining, LC-MS and MALDI-TOF MS. Most importantly, the binding activity to the neutralizing antibody or vaccine antigenicity was measured using an epitope-specific and real-time SPR assay and a monoclonal antibody-based sandwich ELISA. Taken together, the overall good stability of the Hecolin® prefilled syringes was demonstrated with unaltered molecular and functional attributes after storage at 2–8°C for 24months.

Immunodiagnostic monoclonal antibody-based sandwich ELISA of fasciolosis by detection of Fasciola gigantica circulating fatty acid binding protein.

Up to now, parasitological diagnosis of fasciolosis is often unreliable and possesses low sensitivity. Hence, the detection of circulating parasite antigens is thought to be a better alternative for diagnosis of fasciolosis, as it reflects the real parasite burden. In the present study, a monoclonal antibody (MoAb) against recombinant Fasciola gigantica fatty acid binding protein (rFgFABP) has been produced. As well, a reliable sandwich enzyme-linked immunosorbent assay (sandwich ELISA) has been developed for the detection of circulating FABP in the sera of mice experimentally and cattle naturally infected with F. gigantica. MoAb 3A3 and biotinylated rabbit anti-recombinant FABP antibody were selected due to their high reactivities and specificities. The lower detection limit of sandwich ELISA was 5 pg mL-1, and no cross-reaction with other parasite antigens was observed. This assay could detect F. gigantica infection from day 1 post infection. In experimental mice, the sensitivity, specificity and accuracy of this assay were 93·3, 100 and 98·2%, while in natural cattle they were 96·7, 100 and 99·1%. Hence, this sandwich ELISA method showed high efficiencies and precisions for diagnosis of fasciolosis by F. gigantica.

Immunodiagnosis of Fasciola gigantica Infection Using Monoclonal Antibody-Based Sandwich ELISA and Immunochromatographic Assay for Detection of Circulating Cathepsin L1 Protease.

BackgroundTropical fasciolosis caused by Fasciola gigantica infection is one of the major diseases infecting ruminants in the tropical regions of Africa and Asia including Thailand. Parasitological diagnosis of fasciolosis is often unreliable and possesses low sensitivity. Therefore, the detection of circulating parasite antigens is thought to be a better alternative for diagnosis of fasciolosis, as it reflects the real parasite burden.MethodsIn this study, we have produced a monoclonal antibody (MoAb) against recombinant F. gigantica cathepsin L1 (rFgCatL1), and developed both sandwich enzyme-linked immunosorbent assay (sandwich ELISA) and immunochromatographic (IC) test for rapid detection of circulating cathepsin L1 protease (CatL1) in the sera from mice experimentally and cattle naturally infected with Fasciola gigantica. MoAb 4E3 and biotinylated rabbit anti-recombinant CatL1 antibody were selected due to their high reactivities and specificities.ResultsThe lower detection limits of sandwich ELISA and IC test were 3 pg/ml and 0.256 ng/ml, respectively. Sandwich ELISA and IC test could detect F. gigantica infection from day 1 to 35 post infection. In experimental mice, the sensitivity, specificity and accuracy were 95%, 100% and 98.6% (for sandwich ELISA), and 93%, 100% and 98.2% (for IC test), while in natural cattle they were 98.3%, 100% and 99.5% (for sandwich ELISA), and 96.7%, 100% and 99.1% (for IC test).ConclusionsThese two assay methods showed high efficiencies and precisions for diagnosis of fasciolosis by F. gigantica.

Comparative Diagnosis of Serum IgG1 and Coproantigen ELISA for Fasciolosis Detection of Goats in Mexico.

The objective of present study was to determine the prevalence of natural caprine fasciolosis in the Mixteca region of Mexico using coproantigen and serum IgG1 ELISA tests for comparative purposes. A total of 1070 serum and faecal samples were analyzed for IgG1 antibodies and coproantigens, using ELISA with E/S products as antigen and a monoclonal antibody-based sandwich ELISA. Prevalence of 73.46% was found using the serological ELISA and a percentage of 77.20 was found for coproantigen ELISA. The diagnostic sensitivity and specificity for serum ELISA were 86.7% and 96.4%, and for the coproantigen ELISA they were 93.1% and 97.8%, respectively. The seropositive samples were further categorized as low, medium, or high positivity. Results show a great proportion of low and medium positive goats when the serum ELISA test was used. Correlation coefficients between coproantigens and seropositivity were statistically significant (P < 0.01) for low seropositivity (r = 0.93) and medium seropositivity (r = 0.84). The accuracy of faecal antigen ELISA was higher compared to indirect ELISA serological test. Two ELISAs were shown to be useful for demonstrating the current status of F. hepatica infection in the endemic areas and can be employed in studies on epidemiology as well as anthelmintics treatment for preventing economic loss and the risk of transmission to humans.

Quantification of Peach Fruit Allergen Lipid Transfer Protein by a Double Monoclonal Antibody-based Sandwich ELISA

Fruit is one of the most commonly reported food allergy sources in China, and peach lipid transfer protein (Pru p 3) has been identified as the major allergen inducing systematic symptoms. Crude allergen extracts and single component allergens have been used in food allergy diagnosis and immunotherapy. Reliable and sensitive analytical methods to quantify Pru p 3 content in fruit will help to identify low-allergenic cultivars among the abundant peach genetic resources in China. In this study, we developed a sensitive sandwich ELISA method to measure Pru p 3 in peach fruit based on two monoclonal antibodies with high sensitivity and linearity. Significant variability was observed in peach cultivars, with a much higher Pru p 3 level in peach peel than in pulp. This method will be very useful to select peach varieties with low Pru p 3 content and as a diagnostic product to detect hidden allergens in processed food.

Taenia solium Human Cysticercosis: A Systematic Review of Sero-epidemiological Data from Endemic Zones around the World.

Background Taenia solium cysticercosis is a zoonotic neglected disease responsible for severe health disorders such as seizures and death. Understanding the epidemiology of human cysticercosis (HCC) in endemic regions will help to expose critical information about the transmission of the disease, which could be used to design efficient control programs. This review gathered serological data on apparent prevalence of T. solium circulating antigens and/or seroprevalence of T. solium antibodies, apparent prevalence of human taeniasis and risk factors for HCC from endemic communities in order to understand the differences in exposure to the parasite and active infections with T. solium metacestodes in endemic areas around the world.MethodsThree databases were used to search sero-epidemiological data from community-based studies conducted between 1989 and 2014 in cysticercosis endemic communities worldwide. The search focused on data obtained from T. solium circulating antigen detection by monoclonal antibody-based sandwich ELISA and/or T. solium antibody seroprevalence determined by Enzyme-linked Immunoelectrotransfer Blot (EITB). A meta-analysis was performed per continent.Principal FindingsA total of 39,271 participants from 19 countries, described in 37 articles were studied. The estimates for the prevalence of circulating T. solium antigens for Africa, Latin America and Asia were: 7.30% (95% CI [4.23–12.31]), 4.08% (95% CI [2.77–5.95]) and 3.98% (95% CI [2.81–5.61]), respectively. Seroprevalence estimates of T. solium antibodies were 17.37% (95% CI [3.33–56.20]), 13.03% (95% CI [9.95–16.88]) and 15.68% (95% CI [10.25–23.24]) respectively. Taeniasis reported prevalences ranged from 0 (95% CI [0.00–1.62]) to 17.25% (95% CI [14.55–20.23]).SignificanceA significant variation in the sero-epidemiological data was observed within each continent, with African countries reporting the highest apparent prevalences of active infections. Intrinsic factors in the human host such as age and immunity were main determinants for the occurrence of infections, while exposure was mostly related to environmental factors which varied from community to community.

Development of ELISA Assays for Measurement of Can f 1 and Can f 3 in Dog Allergen Extracts

RationaleDog allergen extract is a non-standardized extract with no potency measures. Understanding dog allergen extract potency will enhance the safety and efficacy of this product. Our goal is to develop monoclonal antibody-based sandwich ELISA assays for the measurement of Can f 1 and Can f 3.MethodsMouse monoclonal antibodies were generated against natural Can f 1 and dog serum albumin (Can f 3). Anti-Can f 1 and anti-Can f 3 hybridoma supernatants were screened against dog hair and dog epithelium extracts. Purified IgG antibodies were biotinylated for further pairwise screening and optimization. The defined concentrations of capture (non-biotinylated) and primary (biotinylated) antibodies were used for initial testing of various commercially available dog allergen extracts.ResultsScreening of Can f 1 antibodies revealed consistent strong responses with natural protein and dog extracts during indirect ELISA. Three pairs were selected for Can f 1 measurement: 6G1 as a capture antibody (2.5 μg/mL) and 4C3, 7D12, or 9A9 as biotinylated primary antibody (each at 1.0 μg/mL). Anti Can f 3 antibodies exhibit greater variability and less affinity: 10-20 μg/mL of each capture and primary antibody are required for consistent measurement of Can f 3. Further studies are needed to optimize both the assays.ConclusionssELISA assays have been developed for determining Can f 1 and Can f 3 contents of non-standardized dog allergen extracts. RationaleDog allergen extract is a non-standardized extract with no potency measures. Understanding dog allergen extract potency will enhance the safety and efficacy of this product. Our goal is to develop monoclonal antibody-based sandwich ELISA assays for the measurement of Can f 1 and Can f 3. Dog allergen extract is a non-standardized extract with no potency measures. Understanding dog allergen extract potency will enhance the safety and efficacy of this product. Our goal is to develop monoclonal antibody-based sandwich ELISA assays for the measurement of Can f 1 and Can f 3. MethodsMouse monoclonal antibodies were generated against natural Can f 1 and dog serum albumin (Can f 3). Anti-Can f 1 and anti-Can f 3 hybridoma supernatants were screened against dog hair and dog epithelium extracts. Purified IgG antibodies were biotinylated for further pairwise screening and optimization. The defined concentrations of capture (non-biotinylated) and primary (biotinylated) antibodies were used for initial testing of various commercially available dog allergen extracts. Mouse monoclonal antibodies were generated against natural Can f 1 and dog serum albumin (Can f 3). Anti-Can f 1 and anti-Can f 3 hybridoma supernatants were screened against dog hair and dog epithelium extracts. Purified IgG antibodies were biotinylated for further pairwise screening and optimization. The defined concentrations of capture (non-biotinylated) and primary (biotinylated) antibodies were used for initial testing of various commercially available dog allergen extracts. ResultsScreening of Can f 1 antibodies revealed consistent strong responses with natural protein and dog extracts during indirect ELISA. Three pairs were selected for Can f 1 measurement: 6G1 as a capture antibody (2.5 μg/mL) and 4C3, 7D12, or 9A9 as biotinylated primary antibody (each at 1.0 μg/mL). Anti Can f 3 antibodies exhibit greater variability and less affinity: 10-20 μg/mL of each capture and primary antibody are required for consistent measurement of Can f 3. Further studies are needed to optimize both the assays. Screening of Can f 1 antibodies revealed consistent strong responses with natural protein and dog extracts during indirect ELISA. Three pairs were selected for Can f 1 measurement: 6G1 as a capture antibody (2.5 μg/mL) and 4C3, 7D12, or 9A9 as biotinylated primary antibody (each at 1.0 μg/mL). Anti Can f 3 antibodies exhibit greater variability and less affinity: 10-20 μg/mL of each capture and primary antibody are required for consistent measurement of Can f 3. Further studies are needed to optimize both the assays. ConclusionssELISA assays have been developed for determining Can f 1 and Can f 3 contents of non-standardized dog allergen extracts. sELISA assays have been developed for determining Can f 1 and Can f 3 contents of non-standardized dog allergen extracts.

Monoclonal antibody-based cross-reactive sandwich ELISA for the detection of Salmonella spp. in milk samples

An immunogen consisting ofSalmonellalipopolysaccharide and bovine serum albumin was prepared by periodate oxidation.

Development and evaluation of NS1 specific monoclonal antibody based antigen capture ELISA and its implications in clinical diagnosis of West Nile virus infection

BackgroundWest Nile virus (WNV) is a neurotropic flavivirus that causes viral encephalitis. Recent epidemics of WNV around the world have been associated with significant rates of mortality and morbidity in humans. The early confirmatory diagnosis of WNV infection is important for timely clinical management and epidemiological control in areas where multiple flaviviruses are endemic. ObjectiveThe aim of this study is to develop an monoclonal antibody based antigen capture ELISA for early confirmatory diagnosis of WNV infection with high degree of specificity and sensitivity having no cross reactivity with any of the closely related members of other circulating viruses. Study designThe gene coding for the NS1 protein of WNV was cloned and expressed in pET-28a expression vector. Purified recombinant protein was then utilized for generation of mice monoclonal antibody (Mab) and hyper immune sera (HIS) in rabbit. The sandwich ELISA was developed using the rabbit HIS and mice Mab as capture and detector antibody respectively and the results were compared with real time RT-PCR by evaluating 105 suspected clinical samples. ResultsThe comparative evaluation of the sandwich ELISA with real time RT-PCR revealed 97% concordance with sensitivity and specificity of 90% and 98% respectively. ConclusionThe WN NS1 antigen was detectable in the blood from the first day up to day 9 after the onset of symptoms. The higher sensitivity and specificity of this monoclonal Antibody based sandwich ELISA makes it useful for early diagnosis of WN infection in endemic areas during outbreaks.

Diagnosis of Fasciola gigantica infection using a monoclonal antibody-based sandwich ELISA for detection of circulating cathepsin B3 protease

A reliable monoclonal antibody (MoAb)-based sandwich enzyme-linked immunosorbent assay (sandwich ELISA) was developed for the detection of circulating cathepsin B3 protease (CatB3) in the sera from mice experimentally infected with Fasciola gigantica and cattle naturally infected with the same parasite. The MoAb 2F9 and biotinylated rabbit polyclonal anti-recombinant CatB3 antibody were selected due to their high reactivities and specificities to F. gigantica CatB3 antigen based on indirect ELISA and immunoblotting. The lower detection limit of the sandwich ELISA assay was 10, 100 and 400pg/ml, when applied for the detection of rCatB3 antigen and CatB3 in whole body (WB) of newly excysted juveniles (NEJ) and metacercariae (Met) of F. gigantica, respectively. This sandwich ELISA assay could detect F. gigantica infection from day 1 to 35 post infection and revealed that circulating level of CatB3 peaked at day 1 post infection. In contrast, the antibody detection by indirect ELISA could only demonstrate the antibody level from 35 days post infection. The reliability of the assay method was evaluated using serum samples from mice infected with F. gigantica or Schistosoma mansoni, and hamsters infected with Opisthorchis viverrini, as well as normal mice and hamsters. In addition, sera from cattle infected with Paramphistomum cervi, Strongylid, Trichuris sp. and Strongyloides sp., as well as sera from normal cattle were also assessed. In experimental mice, the diagnostic sensitivity, specificity, positive predictive value, negative predictive value, false positive rate, false negative rate and accuracy of ELISA were 95%, 100%, 100%, 97.9%, 0%, 5.3% and 98.5%, while in natural cattle they were 96.7%, 100%, 100%, 98.5%, 0%, 3.4% and 98.9%, respectively. Hence, this assay method showed high efficient and precision for early diagnosis of fasciolosis by F. gigantica.

Prevalence ofpeste des petits ruminantsamong sheep and goats in India

This study measured the clinical prevalence of peste des petits ruminants (PPR) among sheep and goats in India between 2003 and 2009 by analyzing clinical samples from suspected cases of PPR that were submitted to the Rinderpest and Allied Disease Laboratory, Division of Virology, IVRI, Mukteswar for PPR diagnosis. PPR outbreaks were confirmed by detecting PPR virus (PPRV)-specific antigen in the clinical samples. Clinical samples (blood, nasal swabs, spleen, lymph node, kidney, liver, intestine, and pooled tissue materials) were taken from a total of 592 sheep and 912 goats in different states of India and screened for the presence of PPRV antigen using a monoclonal antibody-based sandwich ELISA kit. A total of 20, 38, and 11 laboratory-confirmed PPR outbreaks occurred among sheep, goat, and combined sheep and goat populations, respectively. Our findings provide evidence of widespread PPR endemicity in India. The underlying reasons could be variations in husbandry practices in different geographical regions, agro-climatic conditions, and livestock migration. Furthermore, decrease in the number of PPR outbreaks over time might be due to the effectiveness of current live PPR vaccines and timely vaccination of target species. Vaccination against PPR has been practiced in India since 2002 to control this disease.

Diagnosis of botulism types C and D in cattle by a monoclonal antibody-based sandwich ELISA

This study was undertaken to evaluate two monoclonal antibody-based sandwich ELISAs (sELISAs) for the detection of Clostridium botulinum neurotoxins (BoNTs) types C and D from culture-enriched intestinal content samples from...

Detection of bluetongue virus group-specific antigen using monoclonal antibody based sandwich ELISA

A monoclonal antibody (MAb) specific for the bluetongue virus (BTV) group specific antigen (VP7) was characterized for its reactivity with purified virus and recombinant BTV VP7 (rVP7) protein and its suitability for use in the sandwich ELISA. The MAb, designated as 5B5 was specific to VP7 and belongs to IgG2a subclass and was selected for the development of the sELISA in this study. The MAb had a titer of 1:25 with BTV and 1:2 with the rVP7 protein. The sELISA is based on capturing of BTV antigen with VP7 specific MAb followed by detection using BTV polyclonal antiserum raised in rabbits. The assay was evaluated with six cell culture adapted serotypes of BTV that have been isolated from India, 1, 2, 15, 17, 18 and 23. The assay could detect BTV antigen as early as day 8 in blood. It was also successfully applied for the detection of BTV group specific antigen in clinical samples of blood, washed RBCs, buffy coat and plasma. A total of 102 field samples from animals, suspected of being infected with BTV, were tested and 29.42% were positive. The blood samples were also amplified in cell culture which improved the sensitivity of the assay. Results confirmed that the sELISA is rapid and specific.

Serum soluble CD36, assessed by a novel monoclonal antibody-based sandwich ELISA, predicts cardiovascular mortality in dialysis patients

Aim Accelerated atherosclerosis is a characteristic feature of chronic kidney disease (CKD). CD36 is a scavenger receptor which contributes to foam cell formation, an early crucial step in atherosclerosis development. Recently, a soluble form of CD36 (sCD36) has been discovered. The aim of the study was to develop an ELISA method for quantitative sCD36 evaluation and to measure it in a cohort of CKD stage 5 patients. Method A novel monoclonal antibody-based sandwich ELISA for sCD36 evaluation was developed and verified by repeated optimization procedures. Serum concentration of sCD36 was then analyzed in a cohort of 228 CKD stage 5 patients prior to dialysis initiation. Additionally, samples from a control group of 73 healthy, age and gender-matched subjects were evaluated. Results The novel CD36 ELISA assay had a recovery of at least 90%, and intra- and inter-assay variability of 6 and 11%, respectively. Concentration of serum sCD36 in CKD patients was significantly increased as compared to controls, and associated with the use of HMG-CoA reductase inhibitors (statins) and the presence of diabetes mellitus (DM). Patients above the 75th percentile of sCD36 concentration were at increased risk of 3-year cardiovascular mortality, as compared to the rest of the cohort [HR 2.85 (1.09–7.59) p = 0.03]. Conclusion For the first time, sCD36 was assessed quantitatively in a group of patients and showed associations with DM, CKD, and statin use. Furthermore, the concentration of sCD36 predicted cardiovascular mortality in CKD stage 5 patients.

Culture enrichment assists the diagnosis of cattle botulism by a monoclonal antibody based sandwich ELISA

Monoclonal antibodies (MAbs) obtained from a mouse immunised with Clostridium botulinum type D toxoid were developed into a sandwich ELISA (sELISA) format that was able to detect type D toxin and types C and D toxin complexes. The sELISA was examined for its potential to replace the mouse bioassay as an alternative in vitro assay for the diagnosis of cattle botulism. Its application directly to intestinal samples collected from suspect cattle botulism cases and prepared for testing for the standard mouse bioassay showed poor correlation and sensitivity with the mouse bioassay results. However, anaerobic pre-enrichment of the samples after heat treatment at 80 °C for 10 min to activate any residual C. botulinum spores greatly improved the sELISA detection rate of the toxin by increasing the sample toxin levels. All of the mouse bioassay positive cattle cases tested were detected by the sELISA from the heated and pre-enriched samples tested after 24 h incubation. Toxin was detected by sELISA and subsequently confirmed by mouse bioassay in samples from an additional 3 cases that had been originally mouse bioassay negative. The results indicate that the application of this procedure for screening intestinal samples for C. botulinum strains that produce types C and D toxins from suspect cattle botulism cases would improve the diagnostic rate as well as significantly reduce the number of mice involved in diagnosis.