Monoclonal Antibody-based ELISA Research Articles

The hydro-ethanolic extract of Scoparia dulcis Linn inhibits allergic airway inflammatory responses in murine asthma models

BackgroundInflammatory responses, including airway inflammation, hyper-responsiveness, mucus hyper-secretion, prostaglandins and Th2 cytokines infiltration in the airway submucosa, frequently occur in allergic respiratory disorders. This study sought to investigate the effects of the hydro-ethanolic extract of Scoparia dulcis (SDE) on allergic airway inflammation, airway hyper-responsiveness, goblet cell hyperplasia, mucus hypersecretion, Th2 cytokines and prostaglandin (PG)-D2 using murine asthma models. MethodsAllergic asthma was induced in healthy guinea-pigs, and mice, by exposing them to ovalbumin (OVA) sensitization and challenge, and treatment with either 2 ml/kg normal saline, 10 mg/kg salbutamol, 10 mg/kg prednisolone, 50, 100, or 250 mg/kg of SDE per os. The percentage protection against pre-convulsive dyspnoea after methacholine challenge in OVA-induced asthmatic guinea-pigs was determined for each treatment regime, and histopathological examinations thereafter conducted on the excised lungs. Periodic acid-Schiff staining was also performed to identify goblet cell hyperplasia and mucus hypersecretion in these lung tissues. Lung tissue homogenates and serum from OVA-induced asthmatic mice were also assayed for PGD2, interleukins (IL)-5 and -13 levels using monoclonal antibody-based mouse ELISA kits. ResultsSDE treatments prolonged the time taken for pre-convulsive dyspnoea to occur after methacholine challenge in OVA-induced asthmatic guinea-pigs; indicative of a positive activity against airway hyper-responsiveness. Histopathological studies also showed that SDE significantly inhibited (p ≤ 0.05) pulmonary infiltration of leukocytes, inflammatory cell accumulation, airway smooth muscle thickening and goblet cell hyperplasia. Serum and lung tissue concentrations of PGD2, IL-5 and IL-13, significantly elevated (p ≤ 0.001) following allergic asthma induction in mice, were significantly reduced (p ≤ 0.01) back to within normal levels after SDE treatments. ConclusionThe plant extract exhibited anti-asthmatic and anti-inflammatory properties, as it reduced the severity of allergic inflammation and hyper-reactivity, as well as PGD2 and Th2 cytokines infiltration in the airway; and these effects could be exploited for future management of asthma.

Development of a novel monoclonal antibody-based competitive ELISA for antibody detection against bovine leukemia virus

Infection with bovine leukemia virus (BLV) leads to enzootic bovine leukosis, the most prevalent neoplastic disease in cattle. Due to the lack of commercially available vaccines, reliable eradication of the disease can be achieved through the testing and elimination of BLV antibody-positive animals. In this study, we developed a novel competitive ELISA (cELISA) to detect antibodies against BLV capsid protein p24. Recombinant p24 protein expressed by Escherichia coli, in combination with the monoclonal antibody 2G11 exhibiting exceptional performance, was used for the establishment of the cELISA. Receiver-operating characteristic curve analysis showed that the sensitivity and specificity of the assay were 98.85 % and 98.13 %, respectively. Furthermore, the established cELISA was specific for detecting BLV-specific antibodies, without cross-reactivity to antisera for six other bovine viruses. Significantly, experimental infection of cattle and sheep with BLV revealed that the cELISA accurately monitors seroconversion. In a performance evaluation, the established cELISA displayed a high agreement with Western blotting and the commercial BLV gp51 cELISA kit in the detection of 242 clinical samples, respectively. In conclusion, the novel p24 cELISA exhibited the potential to be a reliable and efficient diagnostic tool for BLV serological detection with a broad application prospect.

Post-Vaccination Sero-Monitoring of Peste des Petits Ruminants in Sheep and Goats in Karnataka: Progress towards PPR Eradication in India.

Peste des petits ruminants (PPR) presents economic challenges in enzootic countries impacting small ruminant productivity. The state of Karnataka, India, implemented a mass vaccination campaign in alignment with the PPR-Global Eradication Programme (GEP) and the National Strategic Plan for PPR eradication. This study was conducted from January to March 2023 to assess seroconversion in post-vaccinated goats and sheep at the epidemiological unit (epi-unit) level, aligning with the World Organisation for Animal Health (WOAH) and the Food and Agriculture Organization (FAO) guidelines in the PPR Global Control and Eradication Strategy (GCES). Before vaccination, 3466 random serum samples were collected from small ruminants of three age groups (6-12 months, 1-2 years, and >2 years) across 116 epi-units, spanning 82 taluks in 28 districts. Post-vaccination sero-monitoring included 1102 serum samples collected from small ruminants of the 6-12-month age group only, across 111 epi-units covering 64 taluks in 23 districts. The PPRV antibody status was determined using an indigenous hemagglutinin (H) protein monoclonal antibody-based competitive ELISA kit. Pre-vaccination, the PPR seropositivity rates were 55%, 62%, and 66% in the age groups of 6-12 months, 1-2 years, and >2 years, respectively, with a 61% PPRV antibody prevalence across all the age groups. Notably, 41% of the epi-units exhibited antibody prevalence rates of ≥70%, indicating a substantial population immunity, possibly attributed to the previous vaccination program in the state since 2011. In contrast, only 17% of the epi-units had below 30% seroprevalence rates, emphasizing the need for intensified vaccination. Statistical analysis of the data revealed significant correlations (p < 0.05) between the presence of PPRV antibodies and host factors such as species, breed, and sex. Post-vaccination seroprevalence in the 6-12 months age group was found to be 73.4%, indicating the use of an efficacious vaccine. On the evaluation of vaccination immunity in the 6-12 months age group, it was revealed that over 69% of the epi-units achieved a response surpassing ≥70%, indicating a significant improvement from 42% of the epi-units in pre-vaccination. For active PPR eradication, a mass vaccination campaign (>95% coverage) targeting small ruminant populations aged >4 months is advocated, aiming to achieve the desired herd immunity of >80%. This study offers crucial insights into PPR baseline seroprevalence/immunity status and vaccine efficacy, guiding national strategies towards a PPR-free India and further supporting the global eradication initiative.

Development and evaluation of a monoclonal antibody-based blocking ELISA to detect antibodies against the E2 protein of bovine viral diarrhea virus-1

With the rapid development of cattle industry, bovine viral diarrhea virus (BVDV) is becoming widespread in China, which causes serious economic losses to the industry. Effective vaccination and viral surveillance are critical for the prevent and control of BVDV infection. In the present study, the immunogenic domain of E2 protein of BVDV-1 was expressed by prokaryotic pET-28a vector. Monoclonal antibodies (mAbs) against E2 protein were prepared and systemically examined by western blot, immunofluorescence assay, blocking ELISA (bELISA) and virus neutralization test (VNT). The mAb 1E2B3, which showed good reactivity and neutralizing activity to BVDV-1 strains, was selected for ELISA establishment. After a series of screening and optimization, a novel bELISA for highly sensitive and specific detection of BVDV-1 antibodies was established, using HRP-labeled 1E2B3 and recombinant E2 protein. ROC analysis of 91 positive and 84 negative reference bovine serum samples yielded the area under the curve (AUC) of 0.9903. A diagnostic specificity of 96.43 % and a sensitivity of 95.6 % were achieved when the cutoff value was set at 24.31 %. There was no cross reaction to the positive sera of classical swine fever virus (CSFV), BVDV-2, border disease virus (BDV), bovine parainfluenza virus type 3 (BPIV3), infectious bovine rhinotracheitis virus (IBRV), foot-and-mouth disease virus (FMDV), Mycoplasma bovis (M.bovis) and Brucella. The total agreement rate of bELISA with VNT was 93.96 % (249/265). In addition, the result of bELISA was positively correlated with neutralizing antibody titer, and the bELISA could well distinguish the serum samples before and after BVDV vaccination. These results indicate that the established bELISA in this study is specific, sensitive, simple and convenient, which provides technical support for the vaccine efficacy evaluation, prevention and control of BVD in the future.

Development of monoclonal antibody-based blocking ELISA for detecting SARS-CoV-2 exposure in animals.

The global pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses a significant threat to public health. Besides humans, SARS-CoV-2 can infect several animal species. Highly sensitive and specific diagnostic reagents and assays are urgently needed for rapid detection and implementation of strategies for prevention and control of the infection in animals. In this study, we initially developed a panel of monoclonal antibodies (mAbs) against SARS-CoV-2 nucleocapsid protein. To detect SARS-CoV-2 antibodies in a broad spectrum of animal species, an mAb-based blocking enzyme-linked immunosorbent assay (bELISA) was developed. Test validation using a set of animal serum samples with known infection status obtained an optimal percentage of inhibition cut-off value of 17.6% with diagnostic sensitivity of 97.8% and diagnostic specificity of 98.9%. The assay demonstrates high repeatability as determined by a low coefficient of variation (7.23%, 4.89%, and 3.16%) between-runs, within-run, and within-plate, respectively. Testing of samples collected over time from experimentally infected cats showed that the bELISA was able to detect seroconversion as early as 7 days post-infection. Subsequently, the bELISA was applied for testing pet animals with coronavirus disease 2019 (COVID-19)-like symptoms and specific antibody responses were detected in two dogs. The panel of mAbs generated in this study provides a valuable tool for SARS-CoV-2 diagnostics and research. The mAb-based bELISA provides a serological test in aid of COVID-19 surveillance in animals. IMPORTANCE Antibody tests are commonly used as a diagnostic tool for detecting host immune response following infection. Serology (antibody) tests complement nucleic acid assays by providing a history of virus exposure, no matter symptoms developed from infection or the infection was asymptomatic. Serology tests for COVID-19 are in high demand, especially when the vaccines become available. They are important to determine the prevalence of the viral infection in a population and identify individuals who have been infected or vaccinated. ELISA is a simple and practically reliable serological test, which allows high-throughput implementation in surveillance studies. Several COVID-19 ELISA kits are available. However, they are mostly designed for human samples and species-specific secondary antibody is required for indirect ELISA format. This paper describes the development of an all species applicable monoclonal antibody (mAb)-based blocking ELISA to facilitate the detection and surveillance of COVID-19 in animals.

Evaluation of protection and immunity induced by infectious bronchitis vaccines administered by oculonasal, spray or gel routes in commercial broiler chicks

Broiler chicks’ responses following combined IBV live attenuated Massachusetts and 793B strains through gel, spray or oculonasal (ON) vaccination routes were cross-compared. Subsequently, the responses following IBV M41 challenge of the unvaccinated and vaccinated groups were also assessed. Post-vaccination humoral and mucosal immune responses, alongside viral load kinetics in swabs and tissues, were determined using commercial ELISA assays, monoclonal antibody-based IgG and IgA ELISA assays and qRT-PCR respectively. After challenged with IBV-M41 strain, humoral and mucosal immune responses, ciliary protection, viral load kinetics, and immune gene mRNA transcriptions between the three vaccination methods were examined and compared. Findings showed that post-vaccinal humoral and mucosal immune responses were similar in all three vaccination methods. Post vaccinal viral load kinetics is influenced by method of administration. The viral load peaked in the ON group within the tissues and the OP/CL swabs in the first and third weeks respectively. Following M41 challenge, ciliary protection and mucosal immune responses were not influenced by vaccination methods as all three methods offered equal ciliary protection. Immune gene mRNA transcriptions varied by vaccination methods. Significant up-regulation of MDA5, TLR3, IL-6, IFN-α and IFN-β genes were recorded for ON method. For both spray and gel methods, significant up-regulation of only MDA5 and IL-6 genes were noted. The spray and gel-based vaccination methods gave equivalent levels of ciliary protection and mucosal immunity to M41 virulent challenge comparable to those provided by the ON vaccination. Analysis of viral load and patterns of immune gene transcription of the vaccinated-challenged groups revealed high similarity between turbinate and choanal cleft tissues compared to HG and trachea. With regards to immune gene mRNA transcription, for all the vaccinated-challenged groups, similar results were found except for IFN-α, IFN-β and TLR3, which were up-regulated only in ON compared to gel and spray vaccination methods.

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

Monoclonal antibody-based indirect competitive ELISA for quantitative detection of Enterobacteriaceae siderophore enterobactin

Enterobactin (Ent) is a promising indicator to monitor intestinal level of Enterobacteriaceae for assessment of gut inflammation. In this study, we developed a monoclonal antibody (mAb)-based ELISA for Ent quantification. We immunized mice with an Ent conjugate vaccine. An mAb named 2E4, with the highest anti-Ent antibody titer, was selected for developing indirect competitive ELISA (ic-ELISA). The purified mAb 2E4 showed high affinity (3.1 × 10−10 M) and specificity to Ent. The limit of detection of ic-ELISA was 0.39 μg/mL. The intra- and inter-assay recovery rates of standard curve were up to 94.6% with the coefficients of variation between 4.0% and 12.3%, indicating high accuracy, repeatability, and reproducibility of the ic-ELISA. In addition, the ic-ELISA was able to quantitatively detect Ent produced in different bacterial cultures. Collectively, this study developed an ic-ELISA with excellent performance in Ent quantification, laying a solid foundation for Ent-based diagnostics of gut health.

Evaluation of the diagnostic performance and the utility of Helicobacter pylori stool antigen lateral immunochromatography assay

BackgroundHelicobacter pylori causes the most common human gastric infection. H. pylori Stool Antigen Lateral Flow Immunochromatography assay (HpSA-LFIA) is considered one of the most cost-effective and rapid non-invasive assays (active tests). Evaluating HpSA-LFIA is of crucial for ensuring accuracy and utility assurance. This study aimed to evaluate the polyclonal antibody-based HpSA-LFIA in comparison to a monoclonal antibody-based ELISA kit. MethodologyStool samples were collected from 200 gastric patients for HpSA-LFIA and semiquantitative HpSA-ELISA tests. A statistical analysis of the diagnostic performance was performed using MedCalc software. Chi-square tests were performed to determine the effects of gender and age. Results and conclusionThe results showed that HpSA-LFIA achieved remarkable sensitivity (93.75%) and NPV (98.00%). However, it had poor specificity, PPV, and accuracy of 59.76%, 31.25%, and 65.31%, respectively. LR+ and LR-were 2.33% & 0.1%, respectively. Gender didn't affect the diagnostic performance of HpSA-LFIA. Age groups had irrelevant sensitivity; however, specificity was significantly higher in patients aged >45 years. We can conclude that HpSA-LFIA was not accurate enough to be the sole test for diagnosis and suggest developing other confirmatory tests in case of positive conditions.

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.

Development and Evaluation of Two Double-Antibody Sandwich ELISAs for Detecting Severe Fever with Thrombocytopenia Syndrome Virus Infection.

Severe fever with thrombocytopenia syndrome virus (SFTSV) is a newly emerging tick-borne virus with a fatality rate between 12% and 50%. Currently, effective vaccines or antiviral drugs are not available to treat SFTSV infection, and a diagnostic method for its detecting is urgently needed. Monoclonal (MAb) and polyclonal antibodies (PAbs) against SFTSV were prepared by immunizing animals with the SFTSV nucleocapsid protein (NP). We developed 2 double-antibody sandwich enzyme-linked immunosorbent assays (DAS-ELISAs) to detect the SFTSV NP, which was captured using the MAbs and PAbs generated. Both methods were applicable for the diagnosis of SFTSV-infected patients, as confirmed by a quantitative polymerase chain reaction. Furthermore, the sensitivity and specificity of the 2 assays for diagnosing severe fever with thrombocytopenia syndrome were 100% and the antibodies did not react with recombinant Dabieshan NP or recombinant dengue virus NS1 subtype 1 and 2 proteins. In addition, 2 standard curves were established for quantitative detection of the NP: the monoclonal antibody-based ELISA (MAb-based ELISA) had a lower limit of detection than the polyclonal-based ELISA. Therefore, the MAb-based ELISA can be employed for convenient and effective detection of SFTSV.

Estimating Vaccine Potency Using Antibody-Based Competition Assays.

The issues of vaccine potency and stability constitute formidable challenges associated with the development and readiness of vaccines for biodefense. In most instances, the vaccines will be stockpiled (at considerable cost) for years and used only in the rare event of a public health emergency. It is therefore imperative that there be means to readily monitor overall stability of the stockpiled vaccines, preferably using reliable in vitro assays, without the need for expensive and labor-intensive animal studies. In this chapter, we describe an in vitro monoclonalantibody-based competition ELISA known as RiCoE for assessing the potency of a ricin toxin subunit vaccine. RiCoE can be applied to drug substance and drug products adsorbed to aluminum salts adjuvant. While RiCoE is specific for ricin toxin, the general methodologies and protocols described herein are amenable to virtually any subunit or even virus-like particle-based vaccine. Ultimately, RiCoE-like assays may replace or at least reduce the need for animal studies in vaccine potency determinations.

Phosphate-triggered ratiometric fluoroimmunoassay based on nanobody-alkaline phosphatase fusion for sensitive detection of 1-naphthol for the exposure assessment of pesticide carbaryl

The excessive use of carbaryl has resulted in the risk of its exposure. In this study, we isolated six nanobodies (Nbs) from a camelid phage display library against the biomarker of carbaryl, 1-naphthol (1-NAP). Owing to its characteristics of easy genetic modifications, we produced a nanobody-alkaline phosphatase (Nb-CC4-ALP) fusion protein with good stability. A dual-emission system based ratiometric fluoroimmunoassay (RFIA) for quick and highly sensitive determination of 1-NAP was developed. Silicon nanoparticles (SiNPs) was used as an internal reference and for aggregation-induced emission enhancement (AIEE) of gold nanoclusters (AuNCs), while AuNCs could be quenched by MnO2 via oxidation. In the presence of ALP, ascorbic acid phosphate (AAP) can be transformed into ascorbic acid (AA), the later can etch MnO2 to recover the fluorescence of the AuNCs. Based on optimal conditions, the proposed assay showed 220-fold sensitivity improvement in comparison with conventional monoclonal antibody-based ELISA. The recovery test of urine samples and the validation by standard HPLC-FLD demonstrated the proposed assay was an ideal tool for screening 1-NAP and provided technical support for the monitoring of carbaryl exposure.

Proteome Modulation in Peripheral Blood Mononuclear Cells of Peste des Petits Ruminants Vaccinated Goats and Sheep.

In the present study, healthy goats and sheep (n = 5) that were confirmed negative for peste des petits ruminants virus (PPRV) antibodies by monoclonal antibody-based competitive ELISA and by serum neutralization test and for PPRV antigen by s-ELISA were vaccinated with Sungri/96. A quantitative study was carried out to compare the proteome of peripheral blood mononuclear cells (PBMCs) of vaccinated goat and sheep [5 days post-vaccination (dpv) and 14 dpv] vs. unvaccinated (0 day) to divulge the alteration in protein expression following vaccination. A total of 232 and 915 proteins were differentially expressed at 5 and 14 dpv, respectively, in goats. Similarly, 167 and 207 proteins were differentially expressed at 5 and 14 dpv, respectively, in sheep. Network generated by Ingenuity Pathway Analysis was “infectious diseases, antimicrobial response, and inflammatory response,” which includes the highest number of focus molecules. The bio functions, cell-mediated immune response, and humoral immune response were highly enriched in goats at 5 dpv and at 14 dpv. At the molecular level, the immune response produced by the PPRV vaccine virus in goats is effectively coordinated and stronger than that in sheep, though the vaccine provides protection from virulent virus challenge in both. The altered expression of certain PBMC proteins especially ISG15 and IRF7 induces marked changes in cellular signaling pathways to coordinate host immune responses.

Development and evaluation of a monoclonal antibody-based competitive ELISA for the detection of antibodies against H7 avian influenza virus

BackgroundH7 subtype avian influenza has caused great concern in the global poultry industry and public health. The conventional serological subtype-specific diagnostics is implemented by hemagglutination inhibition (HI) assay despite lengthy operation time. In this study, an efficient, rapid and high-throughput competitive enzyme-linked immunosorbent assay (cELISA) was developed for detection of antibodies against H7 avian influenza virus (AIV) based on a novel monoclonal antibody specific to the hemagglutinin (HA) protein of H7 AIV.ResultsThe reaction parameters including antigen coating concentration, monoclonal antibody concentration and serum dilution ratio were optimized for H7 antibody detection. The specificity of the cELISA was tested using antisera against H1 ~ H9, H11 ~ H14 AIVs and other avian viruses. The selected cut-off values of inhibition rates for chicken, duck and peacock sera were 30.11, 26.85 and 45.66% by receiver-operating characteristic (ROC) curve analysis, respectively. With HI test as the reference method, the minimum detection limits for chicken, duck and peacock positive serum reached 20, 21 and 2− 1 HI titer, respectively. Compared to HI test, the diagnostic accuracy reached 100, 98.6, and 99.3% for chicken, duck and peacock by testing a total of 400 clinical serum samples, respectively.ConclusionsIn summary, the cELISA assay developed in this study provided a reliable, specific, sensitive and species-independent serological technique for rapid detection of H7 antibody, which was applicable for large-scale serological surveillance and vaccination efficacy evaluation programs.

Development of an LC-MS/MS Proposed Candidate Reference Method for the Standardization of Analytical Methods to Measure Lipoprotein(a).

Use of lipoprotein(a) concentrations for identification of individuals at high risk of cardiovascular diseases is hampered by the size polymorphism of apolipoprotein(a), which strongly impacts immunochemical methods, resulting in discordant values. The availability of a reference method with accurate values expressed in SI units is essential for implementing a strategy for assay standardization. A targeted LC-MS/MS method for the quantification of apolipoprotein(a) was developed based on selected proteotypic peptides quantified by isotope dilution. To achieve accurate measurements, a reference material constituted of a human recombinant apolipoprotein(a) was used for calibration. Its concentration was assigned using an amino acid analysis reference method directly traceable to SI units through an unbroken traceability chain. Digestion time-course, repeatability, intermediate precision, parallelism, and comparability to the designated gold standard method for lipoprotein(a) quantification, a monoclonal antibody-based ELISA, were assessed. A digestion protocol providing comparable kinetics of digestion was established, robust quantification peptides were selected, and their stability was ascertained. Method intermediate imprecision was below 10% and linearity was validated in the 20-400 nmol/L range. Parallelism of responses and equivalency between the recombinant and endogenous apo(a) were established. Deming regression analysis comparing the results obtained by the LC-MS/MS method and those obtained by the gold standard ELISA yielded y = 0.98*ELISA +3.18 (n = 64). Our method for the absolute quantification of lipoprotein(a) in plasma has the required attributes to be proposed as a candidate reference method with the potential to be used for the standardization of lipoprotein(a) assays.

Development of a Monoclonal Antibody-Based ELISA for the Detection of Alternaria Mycotoxin Tenuazonic Acid in Food Samples

In this study, a specific monoclonal antibody (mAb) against Alternaria mycotoxin tenuazonic acid (TeA) was prepared and a sensitive indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) for the detection of TeA was developed. Tenuazonic acid coupled with carboxymethoxylamine hemihydrochloride (TeA-CMO) was conjugated to keyhole limpet hemocyanin (KLH) as an immunogen for Balb/c mice injection. The hybridoma cell line 3F10 secreting the mAb specific to TeA was obtained, and then by intraperitoneal injection and the caprylic acid-ammonium sulfate precipitation, the purified mAb was prepared and identified as the immunoglobulin G1 isotypes. The affinity constant (kD) of the mAb was 3.045 E−3 M by SPR analysis. Based on this mAb, an ic-ELISA was established after optimization of assay condition, namely the concentration of coating antigen and antibody were 250 ng/mL and 125 ng/mL respectively, competition time of antigen-antibody was 40 min, and incubation time of secondary antibody was 40 min in 0.01 M PBS buffer (pH 7.4). Under the optimized condition, the IC50 value and the detection limit (LOD) were 18.50 ng/mL and 1.00 ng/mL respectively. The average recovery rate from spiked beer, apple juice, and grape juice was from 85.0 to 120.0%. A squared coefficient of correlation (R2) between ic-ELISA and HPLC method was 0.9732. The established ic-ELISA provides an acceptable technique for the detection of TeA residue in food samples.

A neutralizing monoclonal antibody-based competitive ELISA for classical swine fever C-strain post\u2013vaccination monitoring

BackgroundVirus neutralization test (VNT) is widely used for serological survey of classical swine fever (CSF) and efficacy evaluation of CSF vaccines. However, VNT is a time consuming procedure that requires cell culture and live virus manipulation. C-strain CSF vaccine is the most frequently used vaccine for CSF control and prevention. In this study, we presented a neutralizing monoclonal antibody (mAb) based competitive enzyme-linked immunosorbent assay (cELISA) with the emphasis on the replacement of VNT for C-strain post–vaccination monitoring.ResultsOne monoclonal antibody (6B211) which has potent neutralizing activity against C-strain was generated. A novel cELISA was established and optimized based on the strategy that 6B211 can compete with C-strain induced neutralizing antibodies in pig serum to bind capture antigen C-strain E2. By testing C-strain VNT negative pig sera (n = 445) and C-strain VNT positive pig sera (n = 70), the 6B211 based cELISA showed 100% sensitivity (95% confidence interval: 94.87 to 100%) and 100% specificity (95% confidence interval: 100 to 100%). The C-strain antibody can be tested in pigs as early as 7 days post vaccination with the cELISA. By testing pig sera (n = 139) in parallel, the cELISA showed excellent agreement (Kappa = 0.957) with VNT. The inhibition rate of serum samples in the cELISA is highly correlated with their titers in VNT (r2 = 0.903, p < 0.001). In addition, intra- and inter-assays of the cELISA exhibited acceptable repeatability with low coefficient of variations (CVs).ConclusionsThis novel cELISA demonstrated excellent agreement and high level correlation with VNT. It is a reliable tool for sero-monitoring of C-strain vaccination campaign because it is a rapid, simple, safe and cost effective assay that can be used to monitor vaccination-induced immune response at the population level.

Hypoallergen Peanut Lines Identified Through Large-Scale Phenotyping of Global Diversity Panel: Providing Hope Toward Addressing One of the Major Global Food Safety Concerns.

Peanut allergy is one of the serious health concern and affects more than 1% of the world’s population mainly in Americas, Australia, and Europe. Peanut allergy is sometimes life-threatening and adversely affect the life quality of allergic individuals and their families. Consumption of hypoallergen peanuts is the best solution, however, not much effort has been made in this direction for identifying or developing hypoallergen peanut varieties. A highly diverse peanut germplasm panel was phenotyped using a recently developed monoclonal antibody-based ELISA protocol to quantify five major allergens. Results revealed a wide phenotypic variation for all the five allergens studied i.e., Ara h 1 (4–36,833 µg/g), Ara h 2 (41–77,041 µg/g), Ara h 3 (22–106,765 µg/g), Ara h 6 (829–103,892 µg/g), and Ara h 8 (0.01–70.12 µg/g). The hypoallergen peanut genotypes with low levels of allergen proteins for Ara h 1 (4 µg/g), Ara h 2 (41 µg/g), Ara h 3 (22 µg/g), Ara h 6 (829 µg/g), and Ara h 8 (0.01 µg/g) have paved the way for their use in breeding and genomics studies. In addition, these hypoallergen peanut genotypes are available for use in cultivation and industry, thus opened up new vistas for fighting against peanut allergy problem across the world.

Monoclonal antibody-based capture ELISA in the diagnosis of previous dengue infection

BackgroundDengue is an important mosquito-borne disease. There is currently only one licensed vaccine for dengue prevention. The vaccine provides higher efficacy in pre-vaccination dengue-seropositive persons but a higher risk of subsequent more severe dengue in dengue-seronegative persons. It is recommended that the dengue vaccine may be given in dengue-seropositive individuals or as mass vaccination without individual pre-vaccination screening in areas where the dengue seroprevalence is > 80% in children aged 9 years. We evaluated a dengue specific immunoglobulin G monoclonal antibody-based capture enzyme-linked immunosorbent assay (MAb-ELISA) in the diagnosis of previous dengue infection using serum samples from the cohort study in Ratchaburi Province, Thailand.MethodsThe MAb-ELISA was compared to 70% plaque reduction neutralization test (PRNT70) in 453 serum samples from children aged 3–11 years in Ratchaburi Province, Thailand.ResultsThe sensitivity and specificity of MAb-ELISA at the positive to negative (P/N) ratio cut-off level of > 3 were both 0.91 in the diagnosis of previous dengue infection, compared to PRNT70. The false positivity was mainly in Japanese encephalitis (JE) seropositive subjects.ConclusionsThis research provides evidence that MAb-ELISA is useful for dengue seroprevalence study and dengue pre-vaccination screening. JE seropositivity was the major cause of false positive result in the study population.