Direct Enzyme-linked Immunosorbent Assay Research Articles

Improvement of RBD-FC Immunogenicity by Using Alum-Sodium Alginate Adjuvant Against SARS-COV-2.

Adjuvants use several mechanisms to boost immunogenicity and to modulate immune response. The strength of adsorption of antigen by adjuvants can be a determinant factor for significant improvement of immunopotentiation. We expressed recombinant RBD-FC in PichiaPink Strain 4 and examined the vaccination of mice by vaccine formulation with different adjuvants (sodium alginate and aluminum hydroxide, alone and together). Sodium alginate significantly increased the immunogenicity and stability of RBD-FC antigen, so RBD-FC formulated with combined alginate and alum (AlSa) and sodium alginate alone showed higher antibody titer and stability. Immunogenicity of RBD-FC:AlSa was determined by serological assays including direct enzyme-linked immunosorbent assay (ELISA) and surrogate virus neutralization test (sVNT). High levels of IgGs and neutralizing antibodies were measured in serum of mice immunized with the RBD-FC:AlSa formulation. On the other hand, cytokines IL-10 and INF-γ were severely accumulated in response to RBD-FC:AlSa, and after 10 days, their accumulation was significantly declined, whereas IL-4 showed the highest and the lowest accumulation in response to alum and alginate, respectively. Our data may suggest that combination of alum and sodium alginate has a better compatibility with RBD-FC in vaccine formulation.

Quantitative Intracellular Delivery of Anticancer Nanodrugs Via an Immunoassay Employing Pt-SiO2 Janus-Peroxidase Nanozyme.

The accurate and efficient quantification of nanodrug dosage is crucial for early anticancer therapy. The enzyme-linked immunosorbent assay (ELISA) has emerged as a robust tool for detecting anticancer nanodrug dosage; however, the development of sensing elements to quantify anticancer nanodrugs still poses a challenge. To overcome this problem, we utilize polysuccinimide-loaded curcumin (CUR @PSIOAm) as a model to employ an ELISA based on peroxidase nanozyme Pt-SiO2 Janus nanoparticles (Pt-SiO2 JNPs) for the indirect quantitative analysis of intracellular anticancer nanodrug dosage. This novel approach employs an immunoassay to indirectly quantify the dosage of anticancer nanodrugs while preserving its structural integrity. The silica components of Pt-SiO2 JNPs adsorb intermediates, while the Pt NP components exhibit high catalytic activity. Pt-SiO2 JNPs are functionalized with anti-PSIOAm antibody (Pt-SiO2 JNPs-Ab) to serve as an immunosensor capable of specific recognition of CUR @PSIOAm. Additionally, we employed cytotoxicity assays and confocal imaging techniques to demonstrate the excellent biocompatibility of CUR @PSIOAm, as well as its specific uptake by cancer cells. According to the experimental results, the limit of detection (LOD) for the immunoassay of Pt-SiO2 JNPs as a marker for detecting CUR @PSIOAm is approximately 4.5-fold lower than that of horseradish peroxidase. Therefore, by optimizing the conditions, we established a direct competitive ELISA using Pt-SiO2 JNPs as colorimetric indicators for the quantitative detection of intracellular CUR @PSIOAm. The LOD for this ELISA was determined to be 0.01 ng/mL, while the loaded CUR amount calculated from the drug loading capacity was found to be 0.22 pg/mL. Furthermore, the recoveries obtained from this established ELISA ranged between 94.0 and 108%, demonstrating excellent accuracy. Consequently, the peroxidase mimic Pt-SiO2 JNPs-based ELISA exhibits significant potential for precise quantification of intracellular anticancer nanodrug dosages.

Comparison of different ANCA detection methods in a predominantly MPO-ANCA-associated vasculitis cohort

We compared different antineutrophil cytoplasmic antibody (ANCA) detection methods using a predominantly myeloperoxidase (MPO)-ANCA-associated vasculitis cohort. Stored sera from 147 patients with untreated ANCA-associated vasculitis (AAV), including microscopic polyangiitis and granulomatosis with polyangiitis (n = 115 and 32, respectively), and 124 disease controls were tested for P-ANCA and C-ANCA with immunofluorescence (IIF), and for MPO-ANCA and proteinase 3 (PR3)-ANCA with different antigen-specific immunoassays: direct enzyme-linked immunosorbent assay (ELISA), chemiluminescent enzyme immunoassay (CLEIA), third-generation fluorescent enzyme immunoassay (FEIA), and latex turbidimetrical immunoassay (LTIA). In addition, MPO-ANCA and PR3-ANCA titers were calibrated using certified reference materials (CRMs). The sensitivities and specificities for AAV diagnoses were 95% and 94% (IIF), 86% and 98% (ELISA), 93% and 94% (CLEIA), 92% and 96% (FEIA), and 68% and 88% (LTIA). Dual IIF/antigen-specific immunoassay testing reduced diagnostic accuracies from 94% to 93%. The quantitative agreement between ANCA levels measured using CLEIA and FEIA and calibrated using CRMs was not good. In conclusion, this study demonstrated the high performance of antigen-specific immunoassays for AAV diagnosis in a predominantly MPO-ANCA-associated vasculitis cohort and suggested that the benefit of dual IIF/antigen-specific immunoassay testing is limited. Standardizing ANCA measurements using different immunoassays was difficult, even when using CRMs.

B-292 Detection of natural toxins derived from potatoes by biotin-streptavidin direct ELISA

Abstract Background α-solanine (SO) and α-chaconine (CHA) are natural toxins of potato and often cause food poisoning worldwide. We have previously developed a polyclonal antibody (anti-Sold antibody) that bind to SO and CHA and used it in an enzyme-linked immunosorbent assay (ELISA). Our previously constructed ELISA (conventional ELISA) has a weak point of low sensitivity in detecting SO and CHA, we have tried to improve it in this study. Methods Solanidine, a chemical compound found in both SO and CHA, was conjugated with bovine albumin, and this complex was repeatedly injected into two rabbits for two months. Anti-Sold antibody was then purified from rabbit serum using affinity column chromatography. In our ELISA method, named “B-S direct ELISA”, biotin-labeled anti-Sold antibody is bound to SO and CHA in the sample, followed by the binding of peroxidase-labeled streptavidin. To simulate biological samples from patients with potato food poisoning, SO and CHA powders were mixed with commercially available human serum and urine, and SO and CHA levels were then measured using ELISA. Lastly, SO and CHA levels were detected in extracts from rotten potato (Irish Cobbler) sprouts, peels, and tubers. Results The absorbance of the samples containing SO and CHA prepared in 10 mM phosphate buffer saline (PBS) was about 5-fold higher than that of the conventional ELISA. Furthermore, the absorbance of the samples containing SO and CHA prepared in human serum and urine was enhanced about 2.5- and 1.6-fold, respectively, compared to that of the conventional ELISA. The limit of detection in B-S direct ELISA was 0.32 ng/mL in 10 mM PBS, 2.08 ng/mL in serum, and 4.88 ng/mL in urine, respectively. Using this ELISA, it was also possible to measure the content of SO and CHA in extracts of potato tubers, peels, and sprouts. Both SO and CHA in the extracts from rotten potato tubers and peels were detected as approximately 1.4- and 2.2-fold higher, respectively, in each part than those in the extracts from fresh potatoes. Furthermore, the potato sprout extract contained approximately 50-fold more SO and CHA than the tuber and 9-fold more than the peel. On the other hand, B-S direct ELISA cross-reacted with solanidine and solasodine, which have similar chemical structures to SO and CHA, and also with cholesterol and vitamin D3 in serum. Conclusions Based on our results, B-S direct ELISA presented greater performance in detecting SO and CHA than our conventional ELISA. The non-specific reactions in the serum samples and influence of urea in the urine samples may underlie the reduced detection performance of SO and CHA in ELISA, respectively. Although this ELISA has a detection performance applicable to human biological samples, cross-reactivity should be fully considered when judging the results.

Determination of 3-Phenoxybenzoic Acid by an Improved Enzyme-Linked Immunosorbent Assay (ELISA) by the Immobilization of Nanobodies with Streptavidin and Biotin

Nanobodies (Nbs) are single domain antibodies that are frequently utilized in enzyme-linked immunosorbent assays (ELISAs). However, a major limitation of nanobodies is their low sensitivity in capture assays. To address this issue, the combination of streptavidin and biotin can be employed to immobilize the nanobodies on plates in an oriented manner, thereby exposing their binding sites. In this study, nanobodies specific to 3-phenoxybenzoic acid (3-PBA) were biotinylated at their C-termini and immobilized onto streptavidin-coated microplates. A direct competitive ELISA was developed for the determination of 3-phenoxybenzoic acid in ovine and bovine urine. Compared to directly coating the nanobodies, the streptavidin-bridged nanobody capture-based ELISA demonstrated improved sensitivity, with a 16.6% enhancement, and a half-maximum inhibition concentration (IC50) of 2.05 ng/mL. The developed assay provided a linear range from 0.27 to 20.2 ng/mL (IC20-IC80) and a limit of detection (LOD) of 0.1 ng/mL. The average recoveries of 3-phenoxybenzoic acid spiked in the urine ranged from 82.57% to 102.75% and were consistent with values obtained by liquid chromatography – mass spectrometry (LC-MS). These findings demonstrate that the streptavidin-bridged capture nanobody-based ELISA significantly enhanced the sensitivity for the determination of 3-phenoxybenzoic acid residues in animal urine.

Heartworm (Dirofilaria immitis) Prevalence in Dogs Determined by In-House ELISA Based on Filaria-Specific Antibodies in Tropical and Temperate Regions of Mexico

Dirofilaria immitis is a mosquito-borne nematode of dogs, other carnivores and, occasionally, humans. Globally, D. immitis infection (which causes heartworm) is typically more prevalent in tropical than temperate regions. In this study, the seroprevalence of D. immitis was determined from a sample of 335 non-stray dogs from four municipalities, two each from the states of Puebla and Guerrero in Mexico, using polyclonal antibodies to detect serum antigens using an enzyme-linked immunosorbent assay (ELISA). The accuracy of the assay was compared with the modified Knott’s test. The polyclonal antibody used in the direct ELISA had a high sensitivity (100%) with variable specificity (98.2–98.8%) in the municipalities of Puebla and Guerrero. The area under the curve for the four municipalities was 1.0, indicating a high accuracy test, with a cut-off value ranging from 0.45 to 0.50. The overall prevalence of D. immitis infection was 17.56% (59 out of 335). The highest prevalence was in Acapulco (24.78%), followed by Chilpancingo (20.93%), Tecamachalco (10.81%) and Quecholac (8.06%). The highest percentage of positive samples was detected in tropical regions (23.12%) and the lowest in temperate regions (9.56%). This study demonstrates that polyclonal anti-D. immitis antibodies can successfully diagnose heartworm-infected dogs and be used to monitor prevalence effectively and develop prevention strategies against Dirofilaria infection.

Revolutionizing ELISA: A one-step blocking approach using lipid bilayer coatings

Enzyme-linked immunosorbent assay (ELISA) is an essential technique for biomolecule detection in diagnostics and research, but traditional protocols are time-consuming and variable. Conventional blocking agents like bovine serum albumin (BSA) pose ethical issues and potential assay interference. This study presents a one-step lipid-blocking approach to simplify the ELISA procedure. Compared to conventional blockers, the Lipid Universal Coating Assembly (LUCA) antifouling blocker showed comparable sensitivity while significantly reducing processing time and steps. Furthermore, it showed improved co-blocking efficacy and signal intensity in conventional protocols when combined with minimal BSA concentrations. Stability tests under accelerated aging confirmed the robustness of the LUCA antifouling blocker. Additionally, it effectively facilitated antibody detection for COVID-19 antigens in direct ELISA and human IL-6 antigen in Sandwich ELISA, suggesting its versatile applicability. Taken together, our findings reveal that one-step lipid blocking not only streamlines the ELISA process but also maintains high sensitivity and specificity, providing an efficient, user-friendly, and environmentally friendly alternative to traditional ELISA blockers and a robust platform for rapid diagnostics.

Development and validation of an enzyme-linked immunosorbent assay for the quantification of a recombinant humanized anti-IL-4Rα monoclonal antibody CM310 in serum and its application to pharmacokinetic study in Sprague-Dawley Rats

CM310 is a recombinant humanized monoclonal antibody targeting Interleukin (IL)-4 receptor alpha (IL-4Rα). IL-4Rα blockade prevents IL-4 and IL-13 from binding to their receptor, thereby inhibiting downstream signaling pathways that drive Type 2 helper T-cell (Th2) inflammation. CM310 holds potential for treating Th2-related inflammatory diseases, such as asthma, atopic dermatitis and chronic sinusitis with nasal polyposis. In this study, a direct enzyme-linked immunosorbent assay (ELISA) was developed to measure the concentrations of CM310 in rat serum. Seven calibration standards (ranging from 25 to 1600 ng/mL) and three quality controls (70, 500 and 1250 ng/mL) were defined. The limit of detection (LOD), lower limit of quantification (LLOQ) and upper limit of quantification (ULOQ) were 13, 25 and 1600 ng/mL, respectively. The method exhibited excellent precision and accuracy and successfully applied to in vitro serum stability and pharmacokinetic (PK) studies. In conclusion, we have developed and validated a highly sensitive and selective method for measuring CM310 in Sprague-Dawley rats. The development and validation ELISA method met the acceptable criteria, which suggested that these can be applied to quantify CM310, as well as in PK studies.

Development of a broad-spectrum therapeutic Fc-nanobody for human noroviruses.

Human norovirus was discovered more than five decades ago and is a widespread cause of outbreaks of acute gastroenteritis. There are no approved vaccines or antivirals currently available. However, norovirus inhibitors, including capsid-specific monoclonal antibodies (Mabs) and nanobodies, have recently shown promising results. Several Mabs and nanobodies were found to inhibit norovirus replication using a human intestinal enteroid (HIE) culture system and/or could block norovirus attachment to histo-blood group antigen (HBGA) co-factors. In our pursuit to develop a single broad-spectrum norovirus therapeutic, we continued our analysis and development of a cross-reactive and HBGA interfering nanobody (NB26). To improve NB26 binding capacity and therapeutic potential, we conjugated NB26 onto a human IgG Fc domain (Fc-NB26). We confirmed that Fc-NB26 cross-reacts with genetically diverse GII genotype capsid protruding (P) domains (GII.8, GII.14, GII.17, GII.24, GII.26, and GII.NA1) using a direct enzyme-linked immunosorbent assay. Furthermore, X-ray crystallography structures of these P domains and structures of other GII genotypes reveal that the NB26 binding site is largely conserved, validating its broad reactivity. We showed that Fc-NB26 has ~100-fold higher affinity toward the norovirus P domain compared to native NB26. We also found that both NB26 and Fc-NB26 neutralize human norovirus replication in the HIE culture system. Furthermore, the mode of inhibition confirmed that like NB26, Fc-NB26 caused norovirus particle disassembly and aggregation. Overall, these new findings demonstrate that structural modifications to nanobodies can improve their therapeutic potential.IMPORTANCEDeveloping vaccines and antivirals against norovirus remains a challenge, mainly due to the constant genetic and antigenic evolution. Moreover, re-infection with genetically related and/or antigenic variants is not uncommon. We further developed our leading norovirus nanobody (NB26) that indirectly interfered with norovirus binding to HBGAs, by converting NB26 into a dimeric Fc-linked Nanobody (Fc-NB26). We found that Fc-NB26 had improved binding affinity and neutralization capacity compared with native NB26. Using X-ray crystallography, we showed this nanobody engaged highly conserved capsid residues among genetically diverse noroviruses. Development of such broadly reactive potent therapeutic nanobodies delivered as a slow-releasing prophylactic could be of exceptional value for norovirus outbreaks, especially for the prevention or treatment of severe acute gastroenteritis in high-risk groups such as the young, elderly, and immunocompromised.

Influence of climatic factors on spatio-temporal variabilities in the occurrence of aflatoxins in major food grains available at retail shops in Bangladesh

In Bangladesh, little is known about the role of climatic factors influencing spatio-temporal occurrence among different food grains of aflatoxins, which have been linked to liver cancer cases and other health hazards. In this study, 252 food grain samples of 10 types were collected from retail markets at 18 districts, pre-monsoon, monsoon and post-monsoon seasons during February-September, 2018. Occurrence and concentration of total aflatoxins in the samples were analysed by a direct competitive enzyme-linked immunosorbent assay, and compared with climatic data (temperature, humidity, rainfall, and sunshine hours). Contamination of aflatoxins was found in approx. 38% (n=96) of the total samples, with highest occurrence in groundnuts (82%), followed by corn grains (70%), chick pea (54.5%), wheat grain (50%) and green gram (42.9%). Aflatoxins occurrence was moderate in split chick pea (30.8%), yellow pea (28.2%), black gram (27.8%), and lentils (27.6%), while the lowest in grass pea (17.6%). Aflatoxins concentration was found to exceed the European Union permissible limit (≤5 µg kg-1) in 33 (13.1%) samples. Overall, approximately 5.6%, 7.5%, and 25.0% samples were contaminated with high (10-19 ppb), medium (5-9 ppb) and low (1-4 ppb) concentrations of aflatoxins, respectively. High level concentration of aflatoxins was more frequent in groundnut (36%) and wheat grain (20%), in comparison to other types. Occurrence of aflatoxins during pre-monsoon was detected among 21% (19/91) samples, which increased to ca. 30% (36/119) during monsoon, and 98% (41/42) during post-monsoon. However, aflatoxins mean concentration during post-monsoon was comparatively low than the other seasons. Variations in aflatoxins monthly prevalence correlated significantly with relative humidity (p ≤ 0.01) and rainfall (p≤ 0.05), when a 1-month time lag was considered. Therefore, predisposing climatic conditions, i.e., rainfall incidences and persistence of higher relative humidity in the previous month(s), have salient influence on aflatoxins occurrence, potentially impacting both pre-harvest and stored food grains. This study underscores the need of a more holistic monitoring of aflatoxins in agricultural products for a longer term, and adoption of proper intervention measures for food grains while being stored and before consumption. Asian Australas. J. Food Saf. Secur. 2024, 8(2), 32-47

Comparative detection of goat's milk casein through various enzyme‐linked immunosorbent assay (ELISA) approaches using caprine alpha‐S1 casein peptides and bovine casein polyclonal antibodies

SummaryCasein causes allergic reactions in cow's milk protein allergy (CMPA) individuals. Casein can cross‐react with cows and goats. Hence, a rapid and highly specific technique is required for its detection. The study aims to evaluate and optimise multiple Enzyme‐Linked Immunosorbent Assay (ELISA) approaches for the detection of casein isolated from goat's milk. The study utilised and optimised various ELISA approaches, including direct, indirect and sandwich with various antibodies. Several parameters of ELISA including pre‐treatments of casein, blocking reagent, concentrations of antibodies and background signal were evaluated. The results showed that sandwich ELISA revealed a good signal‐to‐noise ratio at 1 mg mL−1. Indirect ELISA showed a higher signal‐to‐noise ratio without any heat treatment in comparison to the direct ELISA despite utilising an equivalent concentration (i.e., 10 μg mL−1) of goat's milk casein. The findings provide a theoretical foundation for obtaining a high signal‐to‐noise ratio for antigenicity studies of goat's milk casein to minimise its immunological response.

A computationally supported designer benzodiazepine strategy for public toxicology laboratories.

Public laboratories must balance innovative and existing methods to keep up with designer drug trends. This article presents a strategy for handling designer benzodiazepines (DBZDs) in casework from screening to interpretation. The cross-reactivity of 22 DBZDs and metabolites was tested against the Immunalysis™ Benzodiazepine Direct Enzyme-Linked Immunosorbent Assay kit. The kit had high intra-analyte precision (coefficients of variation < 15%). Inter-analyte performance varied, triggering confirmation testing at concentrations ranging from 35 to 460 μg/L. The CCRFSL implemented a 40-analyte benzodiazepine and Z-drug confirmation method in 2019. Ten additional analytes were later validated for qualitative reporting, and the limits of detection (LODs) for 13 analytes were lowered by 60%. The method of standard addition was also optimized for as-needed quantitation. Equal and 1/x weighting factors correlated well with target concentrations (coefficients of determination (r2) > 0.98), but 1/x weighting provided the most consistently accurate concentrations. Six computational models were developed to predict DBZD binding affinity to the γ-aminobutyric acid-A receptor to assist in case interpretation (r2 > 0.7 for cross-validation and test set prediction). These models were used to predict the binding affinity of analytes in the confirmation method. Other public laboratories can use this same practical strategy to adapt to any designer drug class (e.g., benzodiazepines, opioids, cannabinoids, and stimulants).

Cross-reactivity of 24 cannabinoids and metabolites in blood using the Immunalysis Cannabinoids Direct enzyme-linked immunosorbent assay.

With wider availability of synthetic and semi-synthetic cannabinoids in the consumer space, there is a growing impact on public health and safety. Forensic toxicology laboratories should keep these compounds in mind as they attempt to remain effective in screening for potential sources of human performance impairment. Enzyme-linked immunosorbent assay (ELISA) is a commonly utilized tool in forensic toxicology, as its efficiency and sensitivity make it useful for rapid and easy screening for a large number of drugs. This screening technique has lower specificity, which allows for broad cross-reactivity among structurally similar compounds. In this study, the Cannabinoids Direct ELISA kit from Immunalysis was utilized to assess the cross-reactivities of 24 cannabinoids and metabolites in whole blood. The assay was calibrated with 5 ng/mL of 11-nor-9-carboxy-Δ9-tetrahydrocannabinol and the analytes of interest were evaluated at concentrations ranging from 5 to 500 ng/mL. Most parent compounds demonstrated cross-reactivity ≥20 ng/mL, with increasing alkyl side-chain length relative to Δ9-tetrahydrocannabinol resulting in decreased cross-reactivity. Of the 24 analytes, only the carboxylic acid metabolites, 11-nor-9-carboxy-Δ8-tetrahydrocannabinol, 11-nor-9(R)-carboxy-hexahydrocannabinol and 11-nor-9(S)-carboxy-hexahydrocannabinol, were cross-reactive at levels ≤10 ng/mL. Interestingly, 11-nor-9(R)-carboxy-hexahydrocannabinol demonstrated cross-reactivity at 5 ng/mL, where its stereoisomer 11-nor-9(S)-carboxy-hexahydrocannabinol, did not. As more information emerges about the prevalence of these analytes in blood specimens, it is important to understand and characterize their impact on current testing paradigms.

Plasma renin as a novel prognostic biomarker of sepsis-associated acute respiratory distress syndrome

Sepsis-associated acute respiratory distress syndrome (ARDS) is a life-threatening condition in critical care medicine for which there is a substantial need for early prognostic biomarkers of outcome. The present study seeks to link plasma renin levels and 30-day mortality in sepsis-associated ARDS patients treated at our institution. The Registry of Critical Illness (RoCI) prospectively enrolled patients from the intensive care units (ICU) within a single academic medical center, and a convenience sample of patients with sepsis-associated ARDS was analyzed from this cohort. This study was approved by the Mass General Brigham Institutional Review Boards (IRB) as part of the RoCI, and all procedures performed were in accordance with the ethical standards of the institutional board. From April 2012 to February 2019, a cohort of 32 adult sepsis-associated ARDS patients with 500 µL of plasma samples available on Day 0 and Day 3 of their ICU stay were enrolled. Renin levels were measured twice, on Day 0 and Day 3 via the direct renin enzyme-linked immunosorbent assay (ELISA EIA-525) by DRG diagnostics. Day 0 and Day 3 renin were statistically evaluated via logistic regression to predict 30-day mortality. Direct renin levels of 64 samples were assayed from 32 sepsis-associated ARDS patients (50% male; mean ± SD, 55 ± 13.8 years old). The 30-day hospital mortality rate was 59.4%. Patients who died within 30 days of admission were more likely to have an elevated Day 3 Renin (Odds ratio [OR] = 6, 95% CI 1.25–28.84) and have received vasopressors (OR = 13.33, 95% CI 1.43–123.95). Adjusting for vasopressor use as a proxy for septic shock status, patients with an Elevated Day 3 Renin had a 6.85 (95% CI 1.07–43.75) greater odds of death than those with Low-Normal Day 3 Renin. Patients with sustained Elevated Renin levels from Day 0 to Day 3 had the highest risk of death in a 30-day window. In this study, we found that renin may be a novel biomarker that has prognostic value for patients with sepsis-associated ARDS. Future studies evaluating renin levels in patients with sepsis-associated ARDS are needed to validate these findings.

Development of alkaline phosphatase-linked single-chain variable fragment fusion proteins for one-step immunodetection of deoxynivalenol in cereals.

Deoxynivalenol (DON) is a mycotoxin that widely distributes in various foods and seriously threatens food safety. To minimize the consumers' dietary exposure to DON, there is an urgent demand for developing rapid and sensitive detection methods for DON in food. In this study, a bifunctional single-chain variable fragment (scFv) linked alkaline phosphatase (ALP) fusion protein was developed for rapid and sensitive detection of deoxynivalenol (DON). The scFv gene was chemically synthesized and cloned into the expression vector pET25b containing the ALP gene by homologous recombination. The prokaryotic expression, purification, and activity analysis of fusion proteins (scFv-ALP and ALP-scFv) were well characterized and performed. The interactions between scFv and DON were investigated by computer-assisted simulation, which included hydrogen bonds, hydrophobic interactions, and van der Waals forces. The scFv-ALP which showed better bifunctional activity was selected for developing a direct competitive enzyme-linked immunosorbent assay (dc-ELISA) for DON in cereals. The dc-ELISA takes 90 min for one test and exhibits a half inhibitory concentration (IC50) of 11.72 ng/mL, of which the IC50 was 3.08-fold lower than that of the scFv-based dc-ELISA. The developed method showed high selectivity for DON, and good accuracy was obtained from the spike experiments. Furthermore, the detection results of actual cereal samples analyzed by the method correlated well with that determined by high-performance liquid chromatography (R2=0.97165). These results indicated that the scFv-ALP is a promising bifunctional probe for developing the one-step colorimetric immunoassay, providing a new strategy for rapid and sensitive detection of DON in cereals.

Development of Polyclonal Antibodies-Based Serological Method for the Detection of Calanthe Mild Mosaic Virus and Application in Virus Certification Programme.

Calanthe mild mosaic virus (CalMMV) infecting orchids is an important potyvirus which is known to cause mild leaf mosaic and flower colour-breaking symptoms in Calanthe and other orchid plants. The present study reports the production of polyclonal antibodies against CalMMV using bacterially expressed recombinant coat protein as immunogen, which in turn would be useful in routine indexing and screening of orchid germplasm. The coat protein (CP) gene (~ 807bp) of CalMMV isolated from infected orchid sample was cloned in expression vector, pET-28a ( +) that yielded ~ 31kDa fusion protein with Histidine tag (His6BP). The expression of fusion CP was confirmed through SDS-PAGE and Western blotting. The His6BP-CalMMV-CP obtained in soluble state after purification was used to immunize New Zealand white rabbit for the production of polyclonal antibodies (PAb). The PAb produced against the purified fusion protein successfully detected CAlMMV in the orchid samples at a dilution of 1:2000 in direct antigen-coated enzyme-linked immunosorbent assay (DAC-ELISA). This study presents the first report of Histidine tag (His6BP) fusion CalMMV-CP-based antibody production and its successful application in the identification of the virus in orchid plants. Outcome of this study will be helpful in routine certification programmes, screening of orchid germplasm and production of CalMMV-free planting materials of orchids.

In Vivo Evolution of a Klebsiella pneumoniae Capsule Defect With wcaJ Mutation Promotes Complement-Mediated Opsonophagocytosis During Recurrent Infection.

Klebsiella pneumoniae carbapenemase-producing K pneumoniae (KPC-Kp) bloodstream infections are associated with high mortality. We studied clinical bloodstream KPC-Kp isolates to investigate mechanisms of resistance to complement, a key host defense against bloodstream infection. We tested growth of KPC-Kp isolates in human serum. In serial isolates from a single patient, we performed whole genome sequencing and tested for complement resistance and binding by mixing study, direct enzyme-linked immunosorbent assay, flow cytometry, and electron microscopy. We utilized an isogenic deletion mutant in phagocytosis assays and an acute lung infection model. We found serum resistance in 16 of 59 (27%) KPC-Kp clinical bloodstream isolates. In 5 genetically related bloodstream isolates from a single patient, we noted a loss-of-function mutation in the capsule biosynthesis gene, wcaJ. Disruption of wcaJ was associated with decreased polysaccharide capsule, resistance to complement-mediated killing, and surprisingly, increased binding of complement proteins. Furthermore, an isogenic wcaJ deletion mutant exhibited increased opsonophagocytosis in vitro and impaired in vivo control in the lung after airspace macrophage depletion in mice. Loss of function in wcaJ led to increased complement resistance, complement binding, and opsonophagocytosis, which may promote KPC-Kp persistence by enabling coexistence of increased bloodstream fitness and reduced tissue virulence.

A rapid monoclonal antibody-based direct immunoassay and a lateral-flow device for on-site sensitive analysis of the mycotoxin alternariol

Alternariol is an emerging mycotoxin that may be present in many foods at relatively high concentrations. Therefore, sensitive analytical methods are needed to reduce human exposure to this secondary fungal metabolite. The aim of the present study was to develop alternative immunochemical methods for the rapid analysis of alternariol using novel homemade monoclonal antibodies. In this study, a direct competitive enzyme-linked immunosorbent assay for alternariol is reported for the first time, with an IC50 value and a detection limit of 0.020 ng/mL and 0.004 ng/mL, respectively. Under the optimized immunoassay conditions, alternariol could be accurately and precisely quantified in fruit juice samples in only 25 min. In addition, a nanoparticle-based lateral-flow immunochromatography assay with an IC50 value and a visual detection limit of 0.24 ng/mL and 1 ng/mL, respectively, was developed. This immunoassay was validated according to European guidelines for portable semiquantitative analytical methods. Thus, juice samples with alternariol levels above 4 ng/mL were detected by this rapid method in only 15 min, with a negligible false suspect rate.

Direct immunoassay on a polyester microwell plate for colorimetric detection of the spike protein in swab and saliva samples.

This study presents the development of a polyester microplate for detecting the S-protein of the SARS-CoV-2 virus in saliva and nasopharyngeal swab samples using direct enzyme-linked immunosorbent assay (ELISA) technology. The polyester microplate was designed to contain 96 zones with a 3 mm diameter each, and a volume of 2-3 μL. The experimental conditions including reagent concentration and reaction time were optimized. The microplate image was digitized and analyzed using graphical software. The linear range obtained between protein S concentrations and pixel intensity was 0-10 μg mL-1, with a correlation coefficient of 0.99 and a limit of detection of 0.44 μg mL-1. The developed methodology showed satisfactory intraplate and interplate repeatability with RSD values lower than 7.8%. The results achieved through immunoassay performed on polyester microplates were consistent with those of the RT-PCR method and showed a sensitivity of 100% and 90% and specificity of 85.71% and 100% for saliva and nasopharyngeal samples, respectively. The proposed direct immunoassay on polyester microplates emerges as an alternative to conventional immunoassays performed on commercial polystyrene plates, given the low cost of the device, low consumption of samples and reagents, lower waste generation, and shorter analysis time. Moreover, the immunoassay has shown great potential for diagnosing COVID-19 with precision and accuracy.

Absolute concentration estimation of COVID-19 convalescent and post-vaccination IgG antibodies.

Soon after commencement of the SARS-CoV-2 disease outbreak of 2019 (COVID-19), it became evident that the receptor-binding domain of the viral spike protein is the target of neutralizing antibodies that comprise a critical element of protective immunity to the virus. This study addresses the relative lack of information regarding actual antibody concentrations and binding affinities in convalescent plasma (CP) samples from COVID-19 patients and extends these analyses to post-vaccination (PV) samples to estimate protective IgG antibody (Ab) levels. A direct enzyme-linked immunosorbent assay (ELISA) was used to measure IgG anti-spike protein (SP) antibodies (Abs) relative to human chimeric spike S1 Ab standards. Microplate wells were coated with recombinant SP. Affinities of Ab binding to SP were determined by previously described methods. Binding affinities were also determined in an RBD-specific sandwich ELISA. Two indices of protective immunity were determined as permutations of Ab molar concentration divided by affinity as dissociation constant (KD). The range and geometric means of Ab concentrations in 21 CP and 21 PV samples were similar and a protective Ab level of 7.5 μg/ml was determined for the latter population, based on 95% of the normal distribution of the PV population. A population (n = 21) of plasma samples from individuals receiving only one vaccination with the BNT162b2 or mRNA-1273 vaccines (PtV) exhibited a geometric mean Ab concentration significantly (p < 0.03) lower than the PV population. The results of this study have implications for future vaccine development, projection of protective efficacy duration, and understanding of the immune response to SARS-CoV-2 infection.