Enzyme-linked Immunosorbent Assay Plates Research Articles

Reduced infectivity of nervous necrosis virus due to reversible adsorption of serum albumin onto surface protrusions of viral particles

Nervous necrosis virus (NNV) belonging to the genus Betanodavirus is highly lethal to a wide range of fish species. We investigated the reactivity of purified NNV particles with serum albumin and immunoglobulin G (IgG), which are major components in serum. Infectivity of NNV suspended in high-concentration fetal bovine serum (FBS) reduced to approximately 1/60 to 1/200 but that of NNV in ≤10% FBS did not decline. On washing out the FBS, NNV regained its infectivity, suggesting that NNV infectivity was reduced by reversible adsorption of some components in the serum onto the NNV particles. On replacing FBS with bovine serum albumin, NNV infectivity gradually reduced with increasing concentration of serum albumin. Furthermore, albumin adsorbed onto the NNV particles immobilized on enzyme-linked immunosorbent assay (ELISA) plate wells. However, the adsorbed serum albumin was easily washed out by the normal ELISA washing process using phosphate-buffered saline (PBS). Thus, serum albumin reversibly adsorbed onto NNV particles. However, purified rabbit IgG non-specifically adsorbed onto NNV particles regardless of whether conformational structures of NNV surface protrusions were partially or completely denatured, suggesting that IgG is adsorbed onto areas other than the surface protrusions on NNV particles. FBS did not interfere with the adsorption of IgG onto NNV particles. Considering that pocket structures at the apex of the NNV surface protrusions function as binding sites for cellular receptors, serum albumin may get adsorbed onto or near the pocket structures. This could be one of reasons for reduction in infectivity of NNV suspended in high-concentration FBS.

Causal agent for disease symptoms on passionflower in Adim, Cross River State, Nigeria

The stinking passionflower (Passiflora foetida L.) is generally regarded as a weed, although its tender fruits are consumed by a few; while the plant is mostly regarded for its medicinal properties. Cucumber mosaic virus is known not just to cause significant losses among crops, but also present in many weeds species. The objective of this study was to identify the causal agent of the disease symptoms observed from P. foetida in Adim, Cross River State, Nigeria. Leaves of P. foetida with virus symptoms were collected from various locations in Adim. Antigen-coated plate enzyme-linked immunosorbent assay (ACP-ELISA) and reverse transcription polymerase chain reactions (RT-PCR) were used for virus detection and characterisation. The symptomatic leaves reacted positively with the CMV antisera in ACP-ELISA and sequences from RT-PCR products showed the highest identity (98.5%) with a CMV isolate from Xanthosoma sp. (MG021460) in Uganda. Phylogenetic reconstruction aggregated the isolates with other Nigerian CMV sequences in a separate uncharacterised subgroup, closest to MH798809 (Nigeria), MW655577 (Nigeria) and HQ874434 (Austria). The tree also revealed the isolates as members of subgroup IB. This study provides the first molecular evidence of CMV infection on P. foetida in Nigeria and adds to the list of possible natural host for the virus in the country.

Evaluation of cytotoxicity and adaptability of a novel bioceramic root canal sealer: An in vitro and scanning electron microscope study.

Cytotoxicity and adaptability are among the highly imperative tests that should be performed on a novel endodontic material to ensure its successful implementation in endodontic treatment. Assess a recently introduced bioceramic root canal sealer CeramoSeal with TotalFill BC and AH plus sealers regarding the cytotoxicity and adaptability. Five sealer discs were prepared for each sealer and their extracts were cultured in 96-well plates containing human fibroblasts for 24 h. After their incubation, MTT solution was added to each well plate using an enzyme-linked immunosorbent assay plate reader was implemented to calculate the percentage of viable cells. Thirty mandibular single-rooted premolars were prepared using the Edge Endo rotary system, teeth were divided into three groups (n = 10) based on the sealer type: Group 1 CeramoSeal, Group 2 Totalfill, and Group 3 AH plus sealer. Teeth were sectioned longitudinally and viewed under a scanning electron microscope where the region with the gaps was identified and quantified as a percentage of the root canal's overall area. One-way ANOVA test was used for cytotoxicity, while Kruskal-Wallis and Friedman's tests were used for adaptability. Ceramoseal statistically significantly showed the lowest viability, at high concentrations AH plus showed the highest cell viability, while at lower concentration Totalfill BC sealer showed the highest cell viability percentage. The gap percentages were statistically significantly higher in Ceramoseal group, there was no statistically significant difference between AH Plus and Totalfill groups. Ceramoseal sealer exhibited the lowest viability and highest gap percentage compared to the other sealers.

Evaluating cross-reactivity of new psychoactive substances (NPS) in human whole blood by enzyme-linked immunosorbent assay (ELISA).

Due to the increase in the use of novel psychoactive substances (NPS) and their overall prevalence, it is important to have effective and reliable screening technologies to detect NPS in biological matrices. Enzyme-linked immunosorbent assays (ELISA) are among the most popular screening methods. To evaluate the effectiveness of ELISA for NPS detection, five subclasses of NPS (novel synthetic opioids, fentanyl analogs, stimulants, benzodiazepines and hallucinogens) were evaluated in whole blood for their cross-reactivity on commercially available ELISA kits. A variety of novel synthetic opioids were tested at concentrations of 1-80 ng/mL and 50-2000 ng/mL and demonstrated no cross-reactivity to a morphine ELISA plate at either concentration range. Fentanyl analogs were tested at concentrations ranging from 0.01 to 1 ng/mL and had cross-reactivities ranging from 8% to 178% on the fentanyl ELISA kit used. Both para-chloro fentanyl (178%) and acryl fentanyl (164%) showed cross-reactivities well above that of fentanyl. Novel stimulants were tested at concentrations of 0.5-40 ng/mL and 20-2,000 ng/mL. 4-Fluoroamphetamine was the only novel stimulant with cross-reactivity (3,354%) to the amphetamine ELISA plate. Novel benzodiazepines were tested at concentrations of 1-40 ng/mL on a benzodiazepine plate. Cross-reactivities ranged from 36.1% to 263%, with desalkylflurazepam having the highest cross-reactivity. Finally, novel hallucinogens were tested at concentrations of 0.5-10 ng/mL on a phencyclidine (PCP) ELISA plate, which produced no cross-reactivity and then with 10-1,000 ng/mL, which gave results from 56.6% to 151%. Both hydroxy-PCP (151%) and chloro-PCP (137%) showed cross-reactivities above that of PCP. This research has demonstrated the utility of using ELISA-based screening for novel benzodiazepines, hallucinogens and for fentanyl analogs; however, there is limited application and risk of false-negative results for the other drug classes due to low or non-existent cross-reactivities.

ELISA for leptospiral 3-hydroxyacyl-CoA dehydrogenase in urine is a promising screening tool for acute leptospirosis.

Introduction. Leptospirosis is a zoonotic disease that is prevalent worldwide. Leptospiral 3-hydroxyacyl-CoA dehydrogenase (3-HADH) is excreted in the urine of infected individuals. However, the potential use of 3-HADH as a biomarker for the diagnosis of leptospirosis using enzyme-linked immunosorbent assay (ELISA) has not been investigated. A technique that identifies Leptospira in a patient in urine sample will be valuable in regular diagnostics and epidemic scenarios, as opposed to existing serological approaches. This study aimed to develop and evaluate an ELISA that can detect 3-HADH in the urine of patients with confirmed acute leptospirosis and to assess its potential as a screening test for leptospirosis. Methods. Laboratory confirmation of acute leptospirosis was done by flaB-nested polymerase chain reaction (PCR) of plasma samples from suspected patients. ELISA-based determination of the presence of 3-HADH in the urine of PCR-positive patients versus PCR-negative patients matched for fever date was performed by coating ELISA plates with urine supernatants and using rabbit anti-3-HADH as the primary antibody. Receiver operating characteristic curve analysis was used to determine the cutoff values for the ELISA. The diagnostic measures between the PCR-positive and PCR-negative patients were compared using the Mann-Whitney U test. Results. In total, 158 febrile patients were assessed, of whom 121 (76.6 %) were male. Of the 15 flaB-nested PCR-positive patients, 12 were in the acute phase of the febrile illness. The best cutoff was an average optical density (ODav) value of 0.2200 for febrile patients. Sensitivity and specificity were 83.33% [95 % confidence interval (CI), 51.59-97.91 %) and 83.33 % (95 % CI, 76.05-89.13 %), respectively. The ODav values for PCR-positive patients in the acute phase of the disease (≤7 days of fever) were significantly higher than those for PCR-negative patients (P<0.001, U=114.0, z=-4.946). Conclusion. Detection of 3-HADH in urine by ELISA appears to be promising for the screening of acute leptospirosis in suspected patients.

Prediction, Synthesis and Evaluation of a Synthetic Peptide as an Enzyme-Linked Immunosorbent Assay (ELISA) Candidate for Screening of Bovine Antibodies against Theileria annulata.

Tick-borne diseases (TBDs) of livestock are endemic across various parts of tropical countries. Theileriosis is one such economically important TBD, caused by the Theileriidae family of organisms, which is transmitted by ticks. Theileria annulata, the causative agent of tropical theileriosis, contributes a significant loss to the dairy sector by causing anorexia, high fever, anemia, inflammatory changes in vital organs and icterus, thus, a loss in milk yield. Though vaccines are available, their protective efficacy is not absolute, and treatment is limited to early diagnosis of the causative agent. Routinely, microscopic identification of piroplasms in the erythrocytes (Giemsa-stained) of infected animals or schizonts in lymph node biopsies are practiced for diagnosis. PCR-based techniques (multiplex, uniplex, nested and real-time) have been reported to perform well in diagnosing active infection. Several attempts have been made using serological assays like Dot blot, ELISA and ICT, but the results were of variable sensitivity and specificity. Recombinant proteins like the Theileria annulata merozoite surface antigen (Tams1) and Theileria annulata surface protein (TaSP) have been explored as antigenic candidates for these assays. In the present study, we predicted an immunogenic peptide, i.e., TaSP-34, from the TaSP using various computational tools. The predicted peptide was custom synthesized. The diagnostic potential of the peptide was assessed by indirect plate ELISA to detect the bovine-IgM against Theileria annulata. Alongside, a recombinant truncated TaSP (rTaSP(tr)) was expressed and purified, which was used to compare the performance of the peptide as a diagnostic candidate. The IgM-based peptide ELISA was 100% sensitive and 92.77% specific as compared to PCR (Tams1 targeting), while 98.04% sensitivity and 97.44% specificity were observed in comparison with rTaSP(tr) ELISA. Almost perfect agreement between peptide ELISA and Tams1 PCR was observed with a Cohen's kappa coefficient (κ-value) of 0.901 and agreement of 95.31%. Further, the κ-value between the peptide ELISA and rTaSP(tr) ELISA was found to be 0.95, and the agreement was 97.65%, which shows a good correlation between the two tests. The findings suggest that the TaSP-34 peptide can be an efficient and new-generation diagnostic candidate for the diagnosis of T. annulata. Furthermore, the peptide can be synthesized commercially at a larger scale and can be a cost-effective alternative for the protein-based diagnostic candidates for T. annulata.

Development and Comparative Evaluation of Two Highly Sensitive Immunosensor Platforms for Trace Determination of Copper Ions in Drinking Water Using a Monoclonal Antibody Specific to Copper-EDTA Complex.

This study describes the development of two highly sensitive immunosensor platforms for the trace determination of copper ions, Cu(II), in drinking water. These platforms were a microwell-based enzyme-linked immunosorbent assay (ELISA) and a kinetic exclusion assay (KinExA) with a KinExATM 3200 immunosensor. Both ELISA and KinExA were developed utilizing the same antibody and coating reagent. The antibody was a mouse monoclonal antibody, designated as 8D66, that specifically recognized Cu(II)-ethylenediamine tetraacetic acid complex (Cu(II)-EDTA) but did not recognize Cu(II)-free EDTA. The 8D66 monoclonal antibody was generated by the fusion of spleen cells of an immunized BALB/c mouse with SP2/0-Ag14 myeloma cells. The immunogen was a protein conjugate of Cu(II)-EDTA with keyhole limpet hemocyanin protein. The coating reagent was Cu(II)-EDTA covalently linked to bovine serum albumin protein (Cu(II)-EDTA-BSA). Both assays involved the competitive binding reaction between Cu(II)-EDTA complexes, formed in the sample solution, and Cu(II)-EDTA-BSA conjugate which has been immobilized onto ELISA plates (in ELISA) or polymethylmethacrylate beads (in KinExA) for a limited quantity of binding sites of the 8D66 antibody. In ELISA, color signals were generated by a peroxidase-labeled secondary antibody and 3,3',5,5'-tetramethylbenzidine substrate. In KinExA, a fluorescein isothiocyanate-labeled secondary antibody was used to generate KinExAgram (trend-line fluorescence responses vs. time). The conditions of both ELISA and KinExA were investigated, and the optimum procedures were established. Both ELISA and KinExA were validated, and all validation parameters were acceptable. Many different metal ions that are commonly encountered in drinking water did not interfere with the Cu(II) analysis by both ELISA and KinExA. Both assays were applied to the determination of Cu(II) in drinking water with satisfactory accuracy and precision. Both assays were compared favorably with inductively coupled plasma atomic emission spectroscopy in terms of their abilities to accurately and precisely determine Cu(II) in drinking water samples. A comparative evaluation of ELISA and KinExA revealed that KinExA had a higher sensitivity and better precision than ELISA, whereas both assays had comparable accuracy. Both ELISA and KinExA were superior to the existing atomic spectrometric methods for Cu(II) in terms of sensitivity, convenience, and analysis throughputs. The proposed ELISA and KinExA are anticipated to effectively contribute to assessing Cu(II) concentrations and control the exposure of humans to its potential toxicities.

B-027 Adaptation of Neonatal Dried Blood Spot Assays for Use on Automated Microplate Instruments

Abstract Background Newborn screening (NBS) is routinely conducted to identify various inherited metabolic disorders which can affect a child’s long-term health or survival. The assay methods used include enzyme-linked immunosorbent assays (ELISA) as well as enzymatic biochemical assays. Dried blood spots (DBS) prepared by applying whole blood to filter paper cards are typically used as the sample for these screening assays. In this study we have adapted an openly programmable automated ELISA processing instrument to perform these types of assays with a high level of accuracy and precision. Method The Awareness Technology (Palm City, FL) ChemWell-2 instrument was chosen for this study. This instrument is a two plate ELISA processing system which has the capability of performing the DBS extraction in an uncoated round bottom 96-well microplate and transferring a small volume of extracted sample to be assayed to a second flat bottom 96-well microplate. The following enzymatic assay kits manufactured by ZenTech (Liège, Belgium) were used in this study: total galactose, phenylalanine, glucose-6-phosphate dehydrogenase (G-6-PD) and biotinidase. Results In order to evaluate the assay performance controls and additional standard DBS were run as samples. Three to four different sample concentration levels were run four to five times in each assay. The runs were repeated 4 times each to determine inter-assay (between run) precision. The results are summarized in the table below: Conclusion Enzymatic NBS methods for total galactose, phenylalanine, G-6-PD and biotinidase including the DBS sample extraction step can be processed with clinically useful accuracy and precision using the Awareness Technology ChemWell-2 automated analyzer.

Recent Advances in Electrochemical Biosensors for the Detection of Foodborne Pathogens: Current Perspective and Challenges.

Foodborne pathogens cause many diseases and significantly impact human health and the economy. Foodborne pathogens mainly include Salmonella spp., Escherichia coli, Staphylococcus aureus, Shigella spp., Campylobacter spp. and Listeria monocytogenes, which are present in agricultural products, dairy products, animal-derived foods and the environment. Various pathogens in many different types of food and water can cause potentially life-threatening diseases and develop resistance to various types of antibiotics. The harm of foodborne pathogens is increasing, necessitating effective and efficient methods for early monitoring and detection. Traditional methods, such as real-time polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA) and culture plate, are time-consuming, labour-intensive and expensive and cannot satisfy the demands of rapid food testing. Therefore, new fast detection methods are urgently needed. Electrochemical biosensors provide consumer-friendly methods to quickly detect foodborne pathogens in food and the environment and achieve extensive accuracy and reproducible results. In this paper, by focusing on various mechanisms of electrochemical transducers, we present a comprehensive overview of electrochemical biosensors for the detection of foodborne pathogens. Furthermore, the review introduces the hazards of foodborne pathogens, risk analysis methods and measures of control. Finally, the review also emphasizes the recent research progress and solutions regarding the use of electrochemical biosensors to detect foodborne pathogens in food and the environment, evaluates limitations and challenges experienced during the development of biosensors to detect foodborne pathogens and discusses future possibilities.

Antigen-Capture Enzyme-Linked Immunosorbent Assay for Specific Detection of &lt;em&gt;Mycoplasma pneumoniae&lt;/em&gt;

Mycoplasma pneumoniae is a cell wall-deficient prokaryote, mainly known to colonize the human respiratory tract and to be endemic, with epidemic peaks every 6 years, in older children and young adults. Diagnosis of M. pneumoniae is challenging because of the fastidious nature of the pathogen and the possibility of asymptomatic carriage. Laboratory diagnosis of M. pneumoniae infection based on antibody titration in the serum samples of patients remains the most practiced method. Because of the potential problem of immunological cross-reactivity with the use of polyclonal serum for M. pneumoniae, an antigen-capture enzyme-linked immunosorbent assay (ELISA) has been developed to improve the specificity of serological diagnosis. ELISA plates are coated with M. pneumoniae polyclonal antibodies, raised in rabbits and rendered specific after adsorption against a panel of heterologous bacteria that share antigens with M. pneumoniae species and/or are known to colonize the respiratory tract. The reacted M. pneumoniae homologous antigens are then specifically recognized by their corresponding antibodies in the serum samples. Further optimization of the physicochemical parameters to which the antigen-capture ELISA is subjected led to a highly specific, sensitive, and reproducible ELISA.

Antigen-Capture Enzyme-Linked Immunosorbent Assay for Specific Detection of &lt;em&gt;Mycoplasma pneumoniae&lt;/em&gt;

Mycoplasma pneumoniae is a cell wall-deficient prokaryote, mainly known to colonize the human respiratory tract and to be endemic, with epidemic peaks every 6 years, in older children and young adults. Diagnosis of M. pneumoniae is challenging because of the fastidious nature of the pathogen and the possibility of asymptomatic carriage. Laboratory diagnosis of M. pneumoniae infection based on antibody titration in the serum samples of patients remains the most practiced method. Because of the potential problem of immunological cross-reactivity with the use of polyclonal serum for M. pneumoniae, an antigen-capture enzyme-linked immunosorbent assay (ELISA) has been developed to improve the specificity of serological diagnosis. ELISA plates are coated with M. pneumoniae polyclonal antibodies, raised in rabbits and rendered specific after adsorption against a panel of heterologous bacteria that share antigens with M. pneumoniae species and/or are known to colonize the respiratory tract. The reacted M. pneumoniae homologous antigens are then specifically recognized by their corresponding antibodies in the serum samples. Further optimization of the physicochemical parameters to which the antigen-capture ELISA is subjected led to a highly specific, sensitive, and reproducible ELISA.

Highly Sensitive Hepatitis B Virus Identification by Antibody-Aptamer Sandwich Enzyme-Linked Immunosorbent Assay

Hepatitis B virus (HBV) infection is considered a major global health problem, causing various health issues, including cirrhosis, hepatitis, and liver cancer. The risk of developing such complications increases when the viral load is above 105 copies/mL. Early identification of HBV infection is imperative to preventing the spread of infection to other parts of the body. Although various sensing methods have been developed to identify HBV, researchers are still working toward developing cheap, easy, and sensitive detection methods. We developed a highly sensitive and rapid HBV detection method using nanomaterials on enzyme-linked immunosorbent assay (ELISA). Aptamer-antibody was utilized as the detection probe and immobilized on a zeolite-modified ELISA plate to detect the HBV biomarker hepatitis B surface antigen (HBsAg). To enhance the detection of HBsAg, aptamer and antibody were attached to gold nanoparticle through electrostatic interaction and immobilized on the zeolite-modified ELISA plate through amine linker. This probe-modified ELISA plate detected low levels of HBsAg, with a detection limit of 0.1 ng/mL. Furthermore, serum spiked experiments showed increment of absorbance with increasing HBsAg concentration, but control trials with other biomolecules showed no increment of absorbance, showing the specific and selective detection of HBsAg. This nanomaterial-modified ELISA plate can detect low levels of HBsAg and help in the diagnosis of HBV infection in its early stages.

Cell-Free Dot Blot: an Ultra-Low-Cost and Practical Immunoassay Platform for Detection of Anti-SARS-CoV-2 Antibodies in Human and Animal Sera.

Since its emergence in late 2019, the coronavirus disease 2019 (COVID-19) pandemic has caused severe disruption to key aspects of human life globally and highlighted the need for timely, adaptive, and accessible pandemic response strategies. Here, we introduce the cell-free dot blot (CFDB) method, a practical and ultra-low-cost immune diagnostic platform capable of rapid response and mass immunity screening for the current and future pandemics. Similar in mechanism to the widely used enzyme-linked immunosorbent assays (ELISAs), our method is novel and advantageous in that (i) it uses linear DNA to produce the target viral antigen fused to a SpyTag peptide in a cell-free expression system without the need for traditional cloning and antigen purification, (ii) it uses SpyCatcher2-Apex2, an Escherichia coli-produced peroxidase conjugate as a universal secondary detection reagent, obviating the need for commercial or sophisticated enzyme conjugates, and (iii) sera are spotted directly on a nitrocellulose membrane, enabling a simple "dipping" mechanism for downstream incubation and washing steps, as opposed to individual processing of wells in a multiwell plate. To demonstrate the utility of our method, we performed CFDB to detect anti-severe acute respiratory syndrome coronavirus 2 nucleocapsid protein antibodies in precharacterized human sera (23 negative and 36 positive for COVID-19) and hamster sera (16 negative and 36 positive for COVID-19), including independent testing at a collaborating laboratory, and we show assay performance comparable to that of conventional ELISAs. At a similar capacity to 96-well plate ELISA kits, one CFDB assay costs only ~$3 USD. We believe that CFDB can become a valuable pandemic response tool for adaptive and accessible sero-surveillance in human and animal populations. IMPORTANCE The recent COVID-19 pandemic has highlighted the need for diagnostic platforms that are rapidly adaptable, affordable, and accessible globally, especially for low-resource settings. To address this need, we describe the development and functional validation of a novel immunoassay technique termed the cell-free dot blot (CFDB) method. Based on the principles of cell-free synthetic biology and alternative dot blotting procedures, our CFDB immunoassay is designed to provide for timely, practical, and low-cost responses to existing and emerging public health threats, such as the COVID-19 pandemic, at a similar throughput and comparable performance as conventional ELISAs. Notably, the molecular detection reagents used in CFDB can be produced rapidly in-house, using established protocols and basic laboratory infrastructure, minimizing reliance on strained commercial reagents. In addition, the materials and imaging instruments required for CFDB are the same as those used for common Western blotting experiments, further expanding the reach of CFDB in decentralized facilities.

Monkeypox infection elicits strong antibody and B cell response against A35R and H3L antigens

Monkeypox virus (MPXV) resides in two forms; mature and enveloped, and depending on it, distinct proteins are displayed on the viral surface. Here, we expressed two MPXV antigens from the mature, and one from the enveloped form, and tested their reactivity to sera of 11 MPXV recoverees while comparing to sera from recently and past vaccinated individuals. 8 out of 11 recoverees exhibited detectable neutralization levels against Vaccinia Lister. Sera from all recoverees bound strongly to A35R and H3L antigens. Moreover, the responses to A35R were significantly higher within the recoverees compared to both recently and past vaccinated donors. Lastly, A35R- and H3L-specific IgG+ B cells ranging from 0.03-0.46% and 0.11-0.36%, respectively, were detected in all recoverees (A35R), and in 9 out of 11 recoverees (H3L). Therefore, A35R and H3L represent MPXV immune targets and could be used in a heat-inactivated serological ELISA for the identification of recent MPXV infection.

B-Cell Epitope Mapping of the Vibrio cholera Toxins A, B, and P and an ELISA Assay.

Oral immunization with the choleric toxin (CT) elicits a high level of protection against its enterotoxin activities and can control cholera in endemic settings. However, the complete B-cell epitope map of the CT that is responsible for protection remains to be clarified. A library of one-hundred, twenty-two 15-mer peptides covering the entire sequence of the three chains of the CT protein (CTP) was prepared by SPOT synthesis. The immunoreactivity of membrane-bound peptides with sera from mice vaccinated with an oral inactivated vaccine (Schankol™) allowed the mapping of continuous B-cell epitopes, topological studies, multi-antigen peptide (MAP) synthesis, and Enzyme-Linked Immunosorbent Assay (ELISA) development. Eighteen IgG epitopes were identified; eight in the CTA, three in the CTB, and seven in the protein P. Three V. cholera specific epitopes, Vc/TxA-3, Vc/TxB-11, and Vc/TxP-16, were synthesized as MAP4 and used to coat ELISA plates in order to screen immunized mouse sera. Sensitivities and specificities of 100% were obtained with the MAP4s of Vc/TxA-3 and Vc/TxB-11. The results revealed a set of peptides whose immunoreactivity reflects the immune response to vaccination. The array of peptide data can be applied to develop improved serological tests in order to detect cholera toxin exposure, as well as next generation vaccines to induce more specific antibodies against the cholera toxin.

The use of filamentous hemagglutinin adhesin to detect immune responses to Campylobacter hepaticus infections in layer hens.

Campylobacter hepaticus is the aetiological agent of Spotty Liver Disease (SLD). SLD can cause significant production loss and mortalities among layer hens at and around peak of lay. We previously developed an enzyme linked immunosorbent assay (ELISA), SLD-ELISA1, to detect C. hepaticus specific antibodies from bird sera using C. hepaticus total proteins and sera pre-absorbed with Campylobacter jejuni proteins. The high specificity achieved with SLD-ELISA1 indicated the presence of C. hepaticus specific antibodies in sera of infected birds. However, some of the reagents used in SLD-ELISA1 are time consuming to prepare and difficult to quality control. This understanding led to the search for C. hepaticus specific immunogenic proteins that could be used in recombinant forms as antibody capture antigens in immunoassay design. In this study, an immunoproteomic approach that combined bioinformatics analysis, western blotting, and LC MS/MS protein profiling was used, and a fragment of filamentous hemagglutinin adhesin (FHA), FHA1,628-1,899 with C. hepaticus specific antigenicity was identified. Recombinant FHA1,628-1,899 was used as antigen coating on ELISA plates to capture FHA1,628-1,899 specific antibodies in sera of infected birds. SLD-ELISA2, based on the purified recombinant FHA fragment, is more user-friendly and standardizable than SLD-ELISA1 for screening antibody responses to C. hepaticus exposure in hens. This study is the first report of the use of FHA from a Campylobacter species in immunoassays, and it also opens future research directions to investigate the role of FHA in C. hepaticus pathogenesis and its effectiveness as a vaccine candidate.

Production of polyclonal antibodies against leek yellow stripe virus (LYSV) coat protein expressed in Escherichia coli and its application in serological diagnostics.

Leek yellow stripe virus (LYSV) is one of the most important potyviruses, associated with garlic throughout the world, including India. LYSV causes stunting and yellow streaks in garlic and leek leaves and with other coinfecting viruses leading to severe symptom expression and yield reduction. In this study, we have made the first reported attempt to produce specific polyclonal antibodies to LYSV using expressed recombinant coat protein (CP), which would be useful for screening and routine indexing of the garlic germplasm. The CP gene was cloned, sequenced, and further subcloned in pET-28a(+) expression vector, which yielded ∼35 kDa fusion protein. The fusion protein was obtained in insoluble fraction after purification and its identity was confirmed by SDS-PAGE and western blotting. The purified protein was used as immunogen for production of polyclonal antisera in New Zealand white rabbit. Antisera raised, was able to recognize the corresponding recombinant proteins in western blotting, immunosorbent electron microscopy and dot immunobinding assay (DIBA). Developed antisera to LYSV (titer 1:2000) was used for screening of 21 garlic accessions in antigen coated plate enzyme-linked immunosorbent assay (ACP-ELISA) and 16 accessions were found positive for LYSV, indicating its widespread presence within the collection tested. To the best of our knowledge, this is the first report of a polyclonal antiserum against the in-vitro expressed CP of LYSV and its successful application in diagnosis of LYSV in garlic accessions in India.

Cytokines and neoangiogenesis parameters in young patients with acute discogenic lumbosacral radiculopathy

Introduction. Discogenic lumbosacral pain in young people has a complex and multicomponent pathogenesis. Evidence of the content of cytokines and neoangiogenesis factors in patients with acute discogenic lumbosacral radiculopathy are deficiency and often contradictory. Objective: to evaluate the cytokine and neoangiogenesis parameters in the blood of young patients with acute discogenic lumbosacral radiculopathy.Materials and methods. The study involved 49 patients (27 (55.1%) men and 22 (44.9%) women) with a mean age of 36 [27; 45] years with acute lumbosacral pain caused by degenerative changes in the spine and signs of compression of the spinal nerves, according to MRI. The control group consisted of 17 healthy individuals (10 (58.8%) men and 7 (41.2%) women) with a mean age of 33 [25; 41] years. The level of C-reactive protein was measured by an automatic biochemical analyzer Konelab 30Iprime (ThermoFisher, Finland). The levels of interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-8 (IL-8), tumor necrosis factor alpha (TNF-α), vascular endothelial growth factor A (VEGF-A) in blood were determined by enzyme-linked immunosorbent assay (ELISA) on a plate ELISA analyzer Real-best (Russia) using reagent kits Cloud-Clone Corp. (USA, China).Results. Patients with acute discogenic lumbosacral radiculopathy, compared with the control group, has an increase in the levels of C-reactive protein (11.2 [7.1; 15.3] vs. 4.2 [3.5; 4.9] mg/ml; р = 0.011), TNF-α (23.1 [16.8; 29.5] vs. 9.7 [6.9; 12,5] pg/ml; р = 0.001), IL-1β (4.7 [3.1; 6.3] vs. 3.2 [2.3; 4.1] pg/ml; р = 0.041), IL-6 (11.2 [6.1; 16.3] vs. 4.5 [3.1; 5.9] pg/ml; р = 0.007), IL-8 (30. [21.9; 48.8] vs. 20.5 [8.5; 32.6] pg/ml; р = 0.023) and VEGF-A (318 [260; 570] vs.168 [100; 240] pg/ml; р = 0.002).Conclusion. The obtained results confirm the importance of pro-inflammatory factors and indicators of neoangiogenesis in the development of acute discogenic lumbosacral radiculopathy in young patients.

Anticancer effect of Illicium verum (star anise fruit) against human breast cancer MCF-7 cell line.

To investigate the anticancer effect of Illicium verum against human breast cancer MCF-7 cell line. An experimental study was conducted in Multidisciplinary and Tissue Culture Laboratory, Aga Khan University in collaboration with Pharmacology Department of Bahria University Medical and Dental College, Karachi, Pakistan from January 2021 to June 2021. MCF-7 cells of Luminal-A breast cancer were seeded in 96-well plate and treated with I.verum methanol extract. After incubation, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) dye was used for cell viability and cell proliferation assays to determine the number of dead and viable cells, and the absorbance was measured using an enzyme-linked immunosorbent assay (ELISA) plate reader. In cell viability assay, different doses of I. verum methanol extract were used to treat the MCF-7 (0.25, 0.5, 1, 3, 6, 12, 25, and 50μg/ml) cells. For apoptosis analysis, the cells were processed with 4´, 6-diamidino-2-phenylindole fluorescent nuclear dye (DAPI) and were examined for fluorescence intensity and apoptotic cells. For cell proliferation assay and apoptosis the IC50 dose of 5.5μg/ml I. verum methanol extract was used. The MCF-7 cells showed a significant reduction (p-value <0.01) in cell viability in the presence of all tested doses of I. verum methanol extract, except for the dose of 0.25μg/ml. The IC50 dose 5.5μg/ml of same extract also showed a significant reduction (p-value <0.01) in cell proliferation and apoptosis induction in MCF-7 cells. Illicium verum methanol extract possesses very potent anticancer action against MCF-7 cells through cytotoxicity, reduction, and inhibition of cancer cells and by inducing apoptosis.

Screening of sperm antigen epitopes by phage display technique and its preliminary clinical application

BackgroundAt present, there is a lack of standardized preparation methods of sperm antigen for the detection of antisperm antibody (AsAb). To screen sperm antigen mimotopes from a phage display random peptide library and use them to establish an enzyme-linked immunosorbent assay (ELISA) for the detection of AsAb, immunoglobulins were extracted from the sera of rabbits with positive AsAb and negative AsAb, respectively, by the saturated ammonium sulfate method, and a phage display 12-mer peptide library was affinity panned by the extracted immunoglobins coated on the ELISA plate. Then, the obtained positive phage clones were identified by ELISA and sent for sequencing and peptides synthesis. Last, a diagnostic ELISA was established to detect clinical serum and seminal plasma samples.ResultsA total of sixty phage clones were chosen by affinity panning, and sixteen of them reacted positively with AsAb in indirect ELISA and sandwich ELISA. Following DNA sequencing and translation, the peptide sequences of the sixteen positive clones were obtained. By comparison in Blast database, four of sixteen positive clones were found to be closely related to male reproduction. Two (#1 and #25) of four mimotopes were synthesized, and an ELISA method was established using the two mimotopes as sperm specific antigens. One hundred and thirty-four serum samples and seventy-four seminal plasma samples from infertile couples were analyzed by the established ELISA with #1 and #25 mimotopes, respectively. The positive rates of AsAb in serum samples were 20.15% (27/134) for #1 and 11.19% (15/134) for #25, respectively, and the coincidence rate between them was 91.04% (122/134). The positive rates of AsAb in seminal plasma samples were 1.35% (1/74) for both #1 and #25, and the coincidence rate was 100%.ConclusionSperm antigen mimotopes can be obtained successfully by the phage display technique, and can be used as standard sperm specific antigens to establish an ELISA method for the detection of AsAb.