Direct Enzyme-linked Immunosorbent Assay Research Articles

Cross-Reactivity of 24 Cannabinoids and Metabolites in blood using the immunalysis cannabinoids direct enzyme-linked immunosorbent assay kit.

With a 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.

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.

Aptamers for nanobodies: A nontoxic alternative to toxic ochratoxin A in immunoassays

To measure toxins using immunoassays, hazardous toxin standards need to be added for quantification. To solve this problem, we propose to use aptamers as competitors to replace toxin standards. In this work, aptamers specific for ochratoxin A (OTA) nanobodies were selected using a DNA library containing a 36 nucleotide random region. The obtained sequences were highly aligned and the best competitor was identified to be a sequence named apt2-OT based on an indirect competitive enzyme-linked immunosorbent assay (ELISA). The Kd of apt2-OT was measured to be 2.86 μM using local surface plasmon resonance spectroscopy. The optimal apt2-OT was identified to substitute the OTA standard with a concentration needed for 50% inhibition of binding (IC50) of 3.26 μM based on a nontoxic direct competitive ELISA. The equivalence relationship between the aptamer and OTA was established in a flour sample, and a recovery experiment was performed. The detection limit for this method was 0.23 ng/mL, with a linear range from 0.25 to 10.50 ng/mL. The recovery rate was 97.5%–115.5%. This study provides a low-cost, rapid and environmentally friendly alternative to the development of immunoassays for toxins.

Novel hapten design, highly sensitive monoclonal antibody production, and immunoassay development for rapid screening of illegally added chloramphenicol in cosmetics

Hapten design and synthesis have been regarded as the key factor to generate high-quality antibodies. In the present study, a novel hapten of chloramphenicol was synthesized, characterized and compared with two conventional haptens. The new hapten generated mAb 4B5 showed higher sensitivity and titer than the other two haptens-based mAbs. The haptens synthesized with the structure of chloramphenicol base generated more sensitive antibodies than the hapten with chloramphenicol succinate, and the spacer arm linked to the phenyl group hapten elicited the strongest antibody response. After optimization, a direct competitive enzyme-linked immunosorbent assay (dcELISA) and a lateral flow immunoassay (LFIA), both based on the mAb 4B5, were developed. The dcELISA had a half maximum inhibition concentration of 0.23 ng/mL and the LFIA showed a cutoff value of 5–10 ng/mL. The LFIA was applied to detect illegally-added chloramphenicol samples in anti-acne cosmetics, five out of 19 samples were tested chloramphenicol containing within 10 min, which result was confirmed with the dcELISA and HPLC. The LFIA has an adequate sensitivity and can be used as a point of care diagnostic device for rapidly screening chloramphenicol in cosmetics.

Circulation of Crimean-Congo Haemorrhagic Fever Virus in Ticks in Upper Guinea-Republic of Guinea

The aim of this study was to map the distribution of agents carrying the Crimean-Congo haemorrhagic fever virus (arbovirus-tica) in the natural region of Upper Guinea. The prefectures of Kankan, Dabola and Faranah were used as collection areas. Random sampling of different types of animals was used to collect the biomaterial. Two types of analysis methods (RT-PCR and ELISA) were used. Out of a total of 578 ticks collected and divided into 254 pools, the genus Amblyomma was the most frequently encountered with 83 tick pools. Molecular analysis (RT-PCR) for the detection of virus RNA revealed 2 positive cases (0.8%). Direct enzyme-linked immunosorbent assays (ELISA) for the detection of Ag yielded only one positive case (0.4%). We found that the species Rhipicephalus geigyi was the main vector and reservoir of the pathogen in Upper Guinea.

Antibody-free and selective detection of okadaic acid using an affinity peptide-based indirect assay.

Okadaic acid (OA) is a type of marine biotoxin produced by some species of dinoflagellates in marine environments. Consumption of shellfish contaminated with OA can cause diarrhetic shellfish poisoning (DSP) in humans with symptoms that typically include abdominal pain, diarrhea and vomiting. In this study, we developed an affinity peptide-based direct competition enzyme-linked immunosorbent assay (dc-ELISA) for the detection of OA in real samples. The OA-specific peptide was successfully identified via M13 biopanning and a series of peptides were chemically synthesized and characterized their recognition activities. The dc-ELISA system showed good sensitivity and selectivity with a half-maximal inhibitory concentration (IC50) of 148.7ng/mL and a limit of detection (LOD) of 5.41ng/mL (equivalent, 21.52ng/g). Moreover, the effectiveness of the developed dc-ELISA was validated using OA-spiked shellfish samples, and the developed dc-ELISA showed a high recovery rate. These results suggest that the affinity peptide-based dc-ELISA can be a promising tool for detecting OA in shellfish samples.

B-052 Establishment of New Antibodies and ELISA System to Detect the Potato Alkaloids α-Solanine and α-Chaconine

Abstract Background Food poisoning by potato alkaloids, α-solanine (SO) and α-chaconine (CHA), currently occurs worldwide, however laboratory methods have not yet been established to detect these toxins in biological samples. In this study, we established new antibodies to detect SO and CHA and evaluated the practicality of enzyme-linked immunosorbent assays (ELISAs) using these antibodies in laboratory medicine. 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. Two types of polyclonal solanidine antibodies were then purified from rabbit serum using affinity column chromatography. Using these antibodies, two forms of direct ELISAs (Sold ELISA-1, 2) were constructed. The basic detection performance of the ELISAs was confirmed by measuring the levels of SO and CHA diluted in phosphate-buffered saline (PBS). 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 In samples of SO diluted in PBS, the limit of quantification (LoQ) was determined as 2.56 ng/mL and 9.55 ng/mL for Sold ELISA-1 and -2, respectively. In CHA diluted in PBS, the LoQs of Sold ELISA-1 and -2 were 1.36 ng/mL and 4.8 ng/mL, respectively. The LoQs of SO in the serum were calculated as 14.25 ng/mL and 19.41 ng/mL using Sold ELISA-1 and -2, respectively. In contrast, the LoQs of CHA in the serum were 12.48 ng/mL and 16.92 ng/mL using Sold ELISA-1 and -2, respectively. The LoQs of SO in the urine were determined as 30.28 ng/mL and 45.16 ng/mL using Sold ELISA-1 and -2, respectively. Conversely, the LoQs of CHA in the urine were 22.92 ng/mL and 38.15 ng/mL using Sold ELISA-1 and -2, respectively. Using Sold ELISA-1, both SO and CHA in the extracts from rotten potato tubers and peels were detected as approximately 1.7- and 2.3-fold higher, respectively, in each part than those in the extracts from fresh potatoes. When both SO and CHA were measured in the same samples using Sold ELISA-2, the levels were approximately 1.6- and 2.4-fold higher, respectively, in the tubers and peels than those in fresh potato samples. Furthermore, the potato sprout extract contained approximately 80-fold more SO and CHA than the tuber and 8-fold more than the peel. Discussion and Conclusion Based on our results, Sold ELISA-1 presented greater performance in detecting SO and CHA than Sold ELISA-2. 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 the sensitivity depends on the sample type, these ELISAs may be effective as future clinical and food testing methods.

Pembrolizumab-induced erosive lichenoid reaction in a patient with metastatic lung adenocarcinoma: A case report

Introduction: Immune-related adverse events induced by immune checkpoint inhibitors are quite common. Cutaneous lichenoid immune-related adverse events are among the most frequent. However, oral lichenoid adverse reactions are extremely rare. We herein describe a patient who was treated with pembrolizumab for metastatic lung cancer and developed an erosive oral lichenoid reaction induced by immunotherapy. Case presentation: An 87-year-old man treated with pembrolizumab for metastatic lung adenocarcinoma (stage IVa based on AJCC 2018) developed multifocal erosions of the oral mucosa mainly located on the buccal mucosa, palate, and inner portion of the lips with multiple small, irregular, hyperkeratotic areas. Histopathological examination showed epithelial necrosis and a dense band-like layer of an inflammatory infiltrate of lymphocytes and histiocytes within the upper dermis. Direct immunofluorescence was negative for both IgG and C3. A diagnosis of erosive oral lichenoid reaction of the mucosa induced by immunotherapy was established. Given the severity of the condition, pembrolizumab treatment was withheld and concomitant topical and systemic steroids were started. After 1 month, the drug-related toxicity was ameliorated and immunotherapy was re-introduced. Discussion: Only one other case of pembrolizumab-induced erosive lichen planus of the oral mucosa has been described to date. Previously reported drug-induced lichenoid rashes were mainly localized on the skin. Clinically, the main differential diagnoses of lichenoid erosive lesions are bullous immune-related disorders and should be excluded. In our patient, histological examination combined with negative results of both direct immunofluorescence and enzyme-linked immunosorbent assays confirmed the diagnosis of erosive lichenoid drug reaction. Conclusion: Clinicians should be aware of lichenoid involvement of the oral mucosa because related pain and food intake difficulties may seriously compromise treatment compliance. Prompt treatment of oral drug-related reactions may prevent interruption of immunotherapy and improve patients’ quality of life.

Generation of a Single-Chain Variable Fragment Antibody against Feline Immunoglobulin G for Biosensor Applications.

For many decades, feline infectious disease has been among the most common health problems and a leading cause of death in cats. These diseases include toxoplasmosis, feline leukemia virus (FeLV), and particularly feline immunodeficiency virus (FIV) disease. Early diagnosis is essential to increase the chance of successful treatment. Generally, measurement of the IgG level is considered to be indicative of an individual's immune status for a particular pathogen. The antibodies specific to feline IgG are crucial components for the development of a detection kit. In this study, feline IgG-bound scFv was selected using phage display technology. Three rounds of biopanning were conducted against purified feline IgG. Through an indirect enzyme-linked immunosorbent assay (ELISA), two scFv clones demonstrating the best binding ability to feline IgG were chosen for biochemical characterization. In addition, the selected scFv (N14) was expressed and purified in a bacterial system. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the size of the purified N14 was 29 kDa. A sandwich ELISA was used to evaluate the binding capacity of the purified scFv to feline IgG. As expected, the purified N14 had the capacity to bind feline IgG. Furthermore, N14 was modified to create a scFv-alkaline phosphatase (scFv-AP) fusion platform. The surface plasmon resonance (SPR) results revealed that N14-AP bound to feline IgG with an affinity binding value of 0.3 ± 0.496 μM. Additionally, the direct ELISA demonstrated the binding capacity of N14-AP to feline IgG in both cell lysate and purified protein. Moreover, N14-AP could be applied to detect feline IgG based on electrosensing with a detection limit of 10.42 nM. Overall, this study successfully selected a feline IgG-bound scFv and developed a scFv-AP platform that could be further engineered and applied in a feline infectious disease detection kit.

Rapid detection of zearalenone in Coicis Semen based on ELISA

Zearalenone(ZEN) is a toxic metabolite produced by Fusarium culmorum, F. graminearum, F. tricinctum, and other fungi, with estrogenic characteristics. Exposure to or ingestion of ZEN during pregnancy can cause reproductive dysfunction, miscarriage, stillbirth, and malformation, and seriously endanger human life and health. The detection methods for ZEN in the Chinese Pharmacopoeia(2020 edition) are liquid chromatography(LC) and liquid chromatography-mass spectrometry(LC-MS), and it is stipulated that ZEN should not exceed 500 μg in 1 000 g of Coicis Semen. Although these detection methods by instruments can achieve the qualitative and quantitative analysis of ZEN in Coicis Semen, their high detection cost and long periods hinder the rapid screening of a large number of samples in the field. In this study, the synthesized ZEN hapten was conjugated with bovine serum albumin(BSA) and ovalbumin(OVA) to obtain the complete ZEN antigen. By virtue of antibody preparation techniques, ZEN monoclonal antibody 4F6 was prepared, which showed 177.5%, 137.1%, and 109.7% cross-reactivity with ZEN structural analogs zearalanol, zearalenone, and α-zearalenol, respectively, and no cross-reactivity with other fungal toxins such as aflatoxin. Direct competitive enzyme-linked immunosorbent assay(dcELISA) based on ZEN monoclonal antibody 4F6 was developed for the determination of ZEN in Coicis Semen with an IC_(50) of 1.3 μg·L~(-1) and a detection range of 0.22-21.92 μg·L~(-1). The recoveries were 83.91%-105.3% and the RSD was 4.4%-8.0%. The established dcELISA method was used to determine the ZEN residuals in nine batches of Coicis Semen samples, and the results were validated by LC-MS. The correlation between the two detection methods was found to be 0.993 9, indicating that the established dcELISA could be used for the rapid qualitative and quantitative detection of ZEN residuals in Coicis Semen.

Validation of an ELISA test kit for the quantitative analyses of total Aflatoxins in spices marketed in Cameroon

Aflatoxins (mold-synthetized secondary metabolites) contamination associated with food such as spices continue to be a global problem in the world. The aim of this study was to measure the standard parameters of validation (Linearity, occuracy, repeatability, intermediate precision, reproducibility, limit of detection, limit of quantification and robustness) of the total Aflatoxins (AFt) low-matrix Enzyme-Linked ImmunoSorbent Assay (ELISA) Kit (Lot No. 062218, RENEKABIO, USA) and used this method to evaluate the contamination degree of AFt ( AFB1, AFB2, AFG1 and AFG2) in 4 different spice matrices [white pepper (Piper nigrum), black pepper (Piper nigrum), njansang (Ricinodendron heudelottii) and super épice mbongo ( Mix of Aframomum citratum, Monodora myristica and Afrostyrax lepidophyllus] marketed in Cameroon and commonly used as ingredients in Cameroonian sauces. The test was performed as a solid phase direct competitive ELISA using a horse radish peroxidase conjugate as the competing, measurable entity. Data obtained from validation showed linearity in the range between 0 mg kg-1 and 20 mg kg-1; an accuracy marked by a recovery rate ranging from 95.09 % to 110.29 %; a coefficient of variation (CV) of repeatability of 3.14 %, a coefficient of variation (CV) of intermediate precision of 6.82 % and a coefficient of variation (CV) of reproducibility of 13.96 %. Detection and quantification limits of 0.15 mg kg -1 and 0.50 mg kg -1 were determined, respectively. The robustness test revealed a critical point of the method as an incubation for 30 minutes at room temperature. The application of the method to our spices analyzed revealed 100 % contamination of 2.34 ± 0.25 µg kg -1 (white pepper), 2.37 ± 0.38 µg kg -1 (black pepper), 2.58 ± 0.87 µg kg -1 (Njansang) and 6.60 ± 5.21 µg kg -1 (Super épice mbongo). The RENEKABIO low matrix ELISA kit used for this research enables an accurate, reliable and faithful screening and quantification of AFt in spice matrices. This will be an asset in quality control of spices marketed in Cameroon.

Borrelia miyamotoi a neglected tick-borne relapsing fever spirochete in Thailand.

Borrelia miyamotoi is a relapsing fever spirochete that shares the same vector as Lyme disease causing Borrelia. This epidemiological study of B. miyamotoi was conducted in rodent reservoirs, tick vectors and human populations simultaneously. A total of 640 rodents and 43 ticks were collected from Phop Phra district, Tak province, Thailand. The prevalence rate for all Borrelia species was 2.3% and for B. miyamotoi was 1.1% in the rodent population, while the prevalence rate was quite high in ticks collected from rodents with an infection rate of 14.5% (95% CI: 6.3-27.6%). Borrelia miyamotoi was detected in Ixodes granulatus collected from Mus caroli and Berylmys bowersi, and was also detected in several rodent species (Bandicota indica, Mus spp., and Leopoldamys sabanus) that live in a cultivated land, increasing the risk of human exposure. Phylogenetic analysis revealed that the B. miyamotoi isolates detected in rodents and I. granulatus ticks in this study were similar to isolates detected in European countries. Further investigation was conducted to determine the serological reactivity to B. miyamotoi in human samples received from Phop Phra hospital, Tak province and in rodents captured from Phop Phra district using an in-house, direct enzyme-linked immunosorbent assay (ELISA) assay with B. miyamotoi recombinant glycerophosphodiester-phosphodiesterase (rGlpQ) protein as coated antigen. The results showed that 17.9% (15/84) of human patients and 9.0% (41/456) of captured rodents had serological reactivity to B. miyamotoi rGlpQ protein in the study area. While a low level of IgG antibody titers (100-200) was observed in the majority of seroreactive samples, higher titers (400-1,600) were also detected in both humans and rodents. This study provides the first evidence of B. miyamotoi exposure in human and rodent populations in Thailand and the possible roles of local rodent species and Ixodes granulatus tick in its enzootic transmission cycle in nature.

Comparison of Serum Levels of Interleukin 17 and Interleukin 9 in Leprosy Patients with and without Lepra Reaction: A Cross-Sectional Analytical Study from Central India.

New pathways of host defence have emerged in leprosy, such as T helper (Th) -17, Th-9, T regulatory cells, and other factors like transforming growth factor-beta, etc. Interleukin (IL) 17 produced by Th17 cells has been found to be elevated in lepra reaction, especially type 2 lepra reaction (T2R). Role of IL-9 has not been studied widely in leprosy reactions so far. The study aimed to compare serum levels of IL-17 and IL-9 in leprosy patients with and without lepra reaction. This was a cross-sectional analytical study including untreated adult leprosy patients with and without lepra reaction. A total of 65 patients were included in the study with 30 leprosy patients without reaction and 35 with lepra reaction. Serum levels of IL-17 and IL-9 were measured in these patients using direct enzyme-linked immunosorbent assay and were compared. Borderline tuberculoid (BT) leprosy with type 1 and Lepromatous (LL) leprosy with T2R patients showed significantly higher levels of IL-17 than BT and LL leprosy patients without lepra reaction, respectively. LL patients with T2R showed significantly lower levels of IL-9 than lepromatous cases without reaction. IL-9 levels were higher in BT patients with T1R as compared to BT patients without reaction but the difference was not significant. We found evidence in support of role of IL-17 in the pathogenesis of T2R, which might serve as useful serum markers for the same. IL-17 might have a role in BT leprosy with T1R. IL-9 seems to have a protective role in T2R as opposed to IL-17, working in synergism with Th1 cytokines.

Comparative evaluation of some techniques used for the detection of rabies virus.

Neurotropic viruses in the family Rhabdoviridae, genus Lyssavirus, are what cause rabies, an acute, progressive, and highly lethal encephalomyelitis. Evaluation of the used diagnostic techniques to determine the most simple; rapid and accurate test for rabies virus (RABV) recognition in different specimens aiming to reach a rapid diagnosis as a step aid in the disease control and to prevent or even minimize the suspected hazard. The used techniques included an infection trial of Swiss mice with the mice-adapted challenge rabies virus followed by the detection of the virus in the infected mices' brains. Virus detection was carried out through the application BHK21 cell line infection; fluorescent antibody technique; latex agglutination test (LAT); direct enzyme-linked immunosorbent assay (ELISA); rabies antigen detection kit ELISA; conventional polymerase chain reaction (PCR). It was found that virus inoculation in mice and BHK21 cell lines needs 5-7 days with positivity of 90% and 100%, respectively. Rapid antigen kit was able to detect rabies antigen in mice brains suspension and BHK21 infected fluid within 3-5 minutes with percentages of 60% and 55.5%, respectively. In 1-1.5 hours, the direct fluorescent antibody method (DFAT) detected 90% and 100% of the rabies antigen in BHK21 cell line infection and brain impressions, respectively. Latex agglutination showed clear results with 88.8% with BHK21 infected fluid within 3-5 minutes while it did not carry out on brain emulsions to prevent falsely positive results brought on by the presence of tissue fragments. Conventional one-step PCR revealed 100% positivity with either brain or cell culture preparations within 2 days. Direct ELISA showed 88.8% positivity with BHK21 infected fluid with 1 day of work. Mice inoculation test, cell culture infection; DFAT and PCR are the most accurate techniques for the detection of RABV with a positivity of 90%-100% followed by LAT and ELISA with a positivity of 88.8%, and lastly, rabies antigen ELISA kit (RAK) with a positivity of 55.5%-60% taking in consideration the required time for each. In addition, the positivity % of the applied tests revealed their sensitivity and specificity.

Identification and Characterization of Clostridium perfringens Atypical CPB2 Toxin in Cell Cultures and Field Samples Using Monoclonal Antibodies.

A direct sandwich enzyme-linked immunosorbent assay (sELISA) was developed for the detection of the atypical β2-toxin (CPB2) of Clostridium perfringens. Polyclonal (PAbs) and monoclonal (MAbs) antibodies were previously obtained employing recombinant CPB2 produced in the baculovirus system as antigen. In the current study, PAbs were used as capture molecules, while purified MAbs conjugated to horseradish peroxidase (MAbs-HRP) were used for the detection of atypical CPB2 toxin. MAbs 5C11E6 and 2G3G6 showed high reactivity, sensitivity and specificity when tested on 232 C. perfringens cell culture isolates. In addition, a reactivity variation among different strains producing atypical CPB2 toxin was observed using the conformation-dependent MAb 23E6E6, suggesting the hypothesis of high instability and/or the existence of different three-dimensional structures of this toxin. Results obtained by sELISA and Western blotting performed on experimentally CPB2-contaminated feces revealed a time-dependent proteolytic degradation as previously observed with the consensus allelic form of CPB2. Finally, the sELISA and an end-point PCR, specific for the atypical cpb2 gene, were used to test field samples (feces, rectal swabs and intestinal contents) from different dead animal species with suspected or confirmed clostridiosis. The comparison of sELISA data with those obtained with end-point PCR suggests this method as a promising tool for the detection of atypical CPB2 toxin.

Ultrasensitive Immunoassay for the Determination of Imidacloprid in Medicinal Herbs

The safety of medicinal herbs is critical to human health, and the monitoring of pesticide residues in medicinal herbs is necessary. In the present study, a direct competitive enzyme-linked immunosorbent assay (dc-ELISA) was developed for the determination of imidacloprid in medicinal herbs. Using the optimal conditions, the 50% inhibiting concentration and detection range (20–80% inhibiting concentration) of the established dc-ELISA were 0.08 and 0.03–0.29 ng/mL, respectively. Except for clothianidin (4.64%) and acetamiprid (2.52%), the cross-reactivity with analyte analogs was negligible. In purple perilla, cordate houttuynia, water lily, bitter melon, and mung bean, the average imidacloprid recoveries were from 62.97 to 134.09%, with relative standard deviations between 0.21 and 14.58%. Furthermore, the immunoassay results correlated well with spiked samples analyzed by high-performance liquid chromatography – tandem mass spectrometry (HPLC-MS/MS). The established immunoassay is rapid, sensitive, and specific and suitable to monitor imidacloprid residues in medicinal herbs.