Dot-blot Technique Research Articles

Design of dot-blot hybridization assay for simultaneous detection of Campylobacter jejuni and Campylobacter coli: a preliminary study.

Campylobacters are a major cause of gastroenteritis worldwide. These are fastidious in culture and false negative results are seen in many clinical laboratories. Among molecular methods, the dot-blot technique is widely used for a variety of purposes, especially diagnostics. So, the authors aimed to detect C. jejuni and C. coli simultaneously using a dot-blot assay. After evaluating the bioinformatics studies, a cadF-conserved fragment was selected for the design of primers and probe. DNAs from standard strains and a recombinant plasmid, prepared in this study, were used to assess the technique. The specificity of the method was also surveyed using DNAs from other enteric bacteria. The limit of detection was evaluated by recombinant plasmid and different concentrations of the designed probe. A 95-bp fragment of cadF was selected, and in silico analysis studies showed that it is conserved between both species. Also, the non-specific annealing of the primers and probe with other bacteria was not seen theoretically. The technique with recombinant plasmid as well as DNAs of standard strains created black spots on the membrane, confirming that the probe was correctly synthesized. No non-specific reactions with other bacterial species were observed (specificity=100%). The limit of detection of the test was determined to be 50µg/ml. This is the first study to simultaneously detect two important pathogens in the Campylobacter genus and was able to detect C. jejuni and C. coli with acceptable sensitivity and specificity.

Bio-Membrane SELEX as a New Approach for Selecting ss-DNA Aptamers that Bind to the Hydatid Cyst Laminated Layer.

Hydatid cyst (HC) is the larval stage of the canine intestinal tapeworm (cestode), Echinococcus granulosus. In addition to the high global economic cost of livestock farming, the infection can lead to dangerous problems for human health. Therefore, research into new diagnosis and treatment approaches is valuable. This study is set out to explore aptamers that bind to HC antigens. The similarity between HC genotype in sheep and humans was that sheep HCs were collected and used as a biological membrane for aptamer selection. Four Bio- Membrane SELEX rounds were conducted, and ssDNA aptamers were selected. Selected aptamers' affinity and specificity to the laminated layer antigens were evaluated using membrane staining by fluorescein primer as a probe. Biotinylated primer was used as a probe for aptahistochemistry and dot blot techniques. Subsequently, cloning and plasmid extraction was conducted. The affinity and specificity of sequenced aptamers were examined with the dot blot method. Selected aptamers reacted with HC wall in aptahistochemistry, aptahistofluorescent, and dot blot experiments. Following cloning and sequencing, 20 sequences were achieved. A strong reaction between HC total antigens and sequenced aptamers has emerged in the dot blot method. We propose a novel method to determine specific aptamers in this investigation. Bio-Membrane SELEX could be assumed as a practical and sensitive method for aptamer selection. Selected aptamers in this study possibly may be used for specific HC antigens detection.

Anti Perkinsus olseni Monoclonal Antibody Generation Using Hypnospores as Antigens

Antibodies are valuable for the detection, identification, and diagnosis of pathogens in many fields, including aquaculture. In this study, we aimed to produce monoclonal antibodies (mAbs) against the parasite Perkinsus olseni, isolated from the clam Paphia undulata in Thailand. The mAbs were characterized using dot blotting and immunohistochemistry techniques in order to identify their class and their ability to detect the hypnospores and zoospores of P. olseni. Immunoreactions between the obtained mAbs and P. olseni produced black or grey spots according to the concentration of parasites detected by the mAbs. The quantitative detection thresholds of hypnospores for the two most strongly reactive mAbs were 105 cells/mL (producing grey spots) to 107 cells/mL (producing black spots), and more than 107 cells/mL (grey spots) for zoospores. Both mAbs showed immunoreactivity at the surface of trophozoite cells of P. olseni in infected clam tissues as assessed by immunohistochemistry analysis, indicating that they could be used for the detection and identification of P. olseni infection in commercial Pa. undulata production.

Design of a polytopic construct of LACK, TSA and GP63 proteins for the diagnosis of cutaneous leishmaniasis: An in silico strategy

• The synthesized multi-epitope was successfully expressed into Escherichia coli. • rGLT increased specificity of antigen detection tests by identifying B-cell epitopes. • Our novel multi-epitope antigens represent a potential target for CL diagnostic. Cutaneous leishmaniasis (CL), which is caused by different species of the genus Leishmania, including L. major , is a significant parasitic disease worldwide. Commercial leishmaniasis diagnostic kits frequently target the entire parasite antigen, although this approach can reduce the specificity of the tests. The present study sought to increase the specificity of antigen detection tests by identifying the B-cell epitopes on the surface of the protozoa and developing an antigenic multiepitope construct to react with the system of B-cell epitopes unique to L. major . More specifically, the linear and conformational B-cell epitopes for three L. major proteins, namely, GP63, LACK, and TSA, were predicted using the ABCprd, IEDB, LBtope, CBTOPE, and DiscoTope servers. The three-dimensional structures of these proteins, as well as of the complete multiepitope, were predicted using the I-TASSER server. The multiepitope structure models were validated by means of Ramachandran plots and the Prosa and Verify3D servers. The multiepitope construct was evaluated using the ProtParam and SOLpro servers. The synthesized multiepitope was then successfully expressed into a pET28a plasmid. The expression was confirmed using SDS-PAGE and dot blot techniques. Moreover, the expressed protein was evaluated by means of Western blotting. A band of approximately 39 kDa was observed by SDS-PAGE. The rGLT-pET-28a showed a high response with the 1:1000 dilution of the anti-His-tag monoclonal antibody by Western blot. The results of this study indicated that the integrated GP63, LACK, and TSA multiepitope antigens of L. major represent a potential target for a novel diagnostic kit for CL.

Targeted genetic analysis unveils novel associations between ACE I/D and APO T158C polymorphisms with D-dimer levels in severe COVID-19 patients with pulmonary embolism

Only a percentage of COVID-19 patients develop thrombotic complications. We hypothesized that genetic profiles may explain part of the inter-individual differences. Our goal was to evaluate the genotypic distribution of targeted DNA polymorphisms in COVID-19 patients complicated (PE+) or not (PE−) by pulmonary embolism. We designed a retrospective observational study enrolling N = 94 consecutive patients suffering severe COVID-19 with pulmonary embolism (PE+, N = 47) or not (PE−, N = 47) during hospitalization. A panel of N = 13 prothrombotic DNA polymorphisms (FV R506Q and H1299R, FII G20210A, MTHFR C677T and A1298C, CBS 844ins68, PAI-1 4G/5G, GPIIIa HPA-1 a/b, ACE I/D, AGT T9543C, ATR-1 A1166C, FGB − 455G > A, FXIII103G > T) and N = 2 lipid metabolism-related DNA polymorphisms (APOE T 112C and T158C) were investigated using Reverse Dot Blot technique. Then, we investigated possible associations between genotypic subclasses and demographic, clinical, and laboratory parameters including age, obesity, smoking, pro-inflammatory cytokines, drug therapy, and biomarkers of thrombotic risk such as D-dimer (DD). We found that 58.7% of PE+ had homozygous mutant D/D genotype at ACE I/D locus vs. PE− (40.4%) and 87% of PE+ had homozygous mutant C/C genotype at APOE T158C locus vs. PE− (68.1%). In PE+ group, DD levels were significantly higher in D/D and I/D genotypes at ACE I/D locus (P = 0.00066 and P = 0.00023, respectively) and in C/C and T/C genotypes at APOE T158C locus (P = 1.6e−06 and P = 0.0012, respectively) than PE− group. For the first time, we showed significant associations between higher DD levels and ACE I/D and APOE T158C polymorphisms in PE+ vs. PE− patients suggesting potential useful biomarkers of poor clinical outcome.

DNA Probe as Biosensor Candidate for Clavibacter michiganensis subsp. michiganensis on Tomato Plants

The study was conducted to evaluate gene detection technique using a specific DNA probe to detect tomA gene in Clavibacter michiganensis subsp. michiganensis, a bacteria causing cancer in tomatoes. The probe was designed using Primer3Plus program, labeled with the non-radioactive molecule digoxigenin (DIG) and used in the hybridization method with the dot blot technique. The test samples consisted of two types, i.e. genomic DNA samples from pure bacterial cultures and from artificially infected tomato seeds with C. michiganensis subsp. michiganensis. Samples derived from pure bacterial cultures showed positive hybridization results at all levels of DNA concentration; while samples from tomato seeds only showed positive reactions at concentrations of 10, 8, 6, and 4 g L-1. This study concludes that the designed probe has the potential to be used in the development of biosensor-based detection methods for C. michiganensis subsp. michiganensis in tomato seeds and is quite specific because there is no cross-reaction with non-target bacterial groups.

Identification of G-quadruplex anti-Interleukin-2 aptamer with high specificity through SELEX stringency

Aptamers are short single-stranded oligonucleotides capable of binding to various targets with high specificity and affinity. This study aimed to identify an aptamer against mouse interleukin-2 (mIL-2) as one of the most important cytokines in autoimmune diseases for diagnostic and therapeutic purposes. For this purpose, 14 SELEX rounds were performed on recombinant mIL-2 with high stringency. The dot blot and flow cytometry techniques were conducted to determine affinity, dissociation constant (Kd), specificity, and SELEX rounds screening. The stringency of rounds was considered based on aptamer/target incubation time, washing steps, and target proteins. Finally, the aptamer's structure was mapped and predicted by M-fold and QGRS Mapper web-based software. After 14 rounds, the flow cytometry analysis revealed that the 11th round was a proper round. The high-affinity aptamers M20 and M15 were chosen for their ability to bind mIL-2. According to DNA folding software, M20 and M15 aptamers had G-quadruplex and stem-loop structures, respectively. The M20 aptamer affinity was greater than M15, and its predicted Kd was 91 nM. A simple SELEX protocol with round stringency was explained to identify DNA aptamers against protein targets. The reported G-quadruplex aptamer might have potential diagnostic or therapeutic application in IL-2–related disorders.

Extracellular Vesicle (EV) Dot Blotting for Multiplexed EV Protein Detection in Complex Biofluids.

Extracellular vesicles (EVs) are nanoscale vesicles secreted from cells, carrying biomolecular cargos similar to their cells of origin. Measuring the protein content of EVs in biofluids can offer a crucial insight into human health and disease. For example, detecting tumor-derived EVs' protein markers can aid in early diagnosis of cancer, which is life-saving. In order to use these EV proteins for diagnosis, sensitive and multiplexed methods are required. The current methods for EV protein detection typically require large sample consumption due to challenges with sensitivity and often need an EV isolation step for complex biofluid samples such as blood plasma. In this work, we have developed a simple and sensitive method for multiplexed detection of protein markers on EV membrane surfaces, which we call "EV dot blotting", inspired by conventional dot blotting techniques. After optimization of multiple factors such as antibody concentration, blocking reagent, type of 3D membranes, and use of gold nanoparticles for signal enhancement, cancer-cell-derived EVs were spiked in pooled normal human plasma for conducting a multiplexed assay in a microarray format. Without the need of isolating EVs from blood plasma, a limit of detection of 3.1 × 105 EVs/mL or 1863 EVs/sample was achieved for CD9 protein, 4.7 × 104 EVs/mL or 281 EVs/sample for CD24, and 9.0 × 104 EVs/mL or 538 EVs/sample for EpCAM, up to 4 orders of magnitude lower than those of conventional ELISA. This platform offers sensitive, multiplexed, simple, and low-cost EV protein detection directly from complex biofluids with minimal sample consumption, providing a useful tool for multiplexed EV protein quantification for a variety of applications.

Human placental mesenchymal stromal cell therapy restores the cytokine efflux and insulin signaling in the skeletal muscle of obesity-induced type 2 diabetes rat model.

Obesity poses a significant risk factor for the onset of metabolic syndrome with allied complications, wherein mesenchymal stem cell therapy is seen as a promising treatment for obesity-induced metabolic syndrome. In the present study, we aim to explore the beneficial effects of the human placental mesenchymal stromal cells (P-MSCs) on obesity-associated insulin resistance (IR) including inflammation. To understand this, we have analyzed the peripheral blood glucose, serum insulin levels by ELISA, and the glucose uptake capacity of skeletal muscle by a 2-NBDG assay using flow cytometry in WNIN/GR-Ob rats treated with and without P-MSCs. Also, we have studied insulin signaling and cytokine profile in the skeletal muscle by western blotting, dot blotting, and Multiplex-ELISA techniques. The skeletal muscle of WNIN/GR-Ob rats demonstrates dysregulation of cytokines, altered glucose uptake vis-a-vis insulin signaling. However, P-MSCs' treatment was effective in WNIN/GR-Ob rats as compared to its control, to restore HOMA-IR, re-establishes dysregulated cytokines and PI3K-Akt pathway in addition to enhanced Glut4 expression and glucose uptake studied in skeletal muscle. Overall, our data advocate the beneficial effects of P-MSCs to ameliorate inflammatory milieu, improve insulin sensitivity, and normalize glucose homeostasis underlining the Ob-T2D conditions, and we attribute for immunomodulatory, paracrine, autocrine, and multipotent functions of P-MSCs.

Prevalence and molecular characterization of alpha and beta-Thalassemia mutations among Hakka people in southern China.

Our aim was to investigate molecular features of thalassemia for proper clinical consultation and prevention in Heyuan. In our research, a total of 25,437 positive screening subjects were further subjected to a genetic analysis of α-thalassemia (α-thal) and β-thalassemia (β-thal). The deletion of α-thal mutation was tested by Gap-PCR, while the non-deletion of α-thal and β-thal mutation were identified by the PCR-reverse dot blot (PCR-RDB) technique. Nested PCR detected Hkαα/-- SEA and Hkαα/αα. Among the 25,437 positive screening subjects, 44.09% (11216/25437) subjects were bearers of thalassemia variations, and 30.85% (7847/25437) subjects showed α-thal changes alone. Among the 23 genotypes with α-thal mutation alone, the three common genotypes were --SEA/αα(68.34%), -α3.7/αα(16.44%), and -α4.2/αα(6.38%). Of the 11.50% (2924/25437) subjects and 29 genotypes with β-thal mutation alone, the three common genotypes were βCD41-42/βN(36.22%), βIVS-II-654/βN(30.88%), and β-28/βN(13.47%). Additionally, of the 1.75% (445/25437) subjects and 55 genotypes showed both α- and β-thal mutations. We also identified 269 cases of Hb H and six patients of Hkαα. Furthermore, the common genotypes of α-thal and β-thal mutations were consistent with allele frequencies of mutations. Our study establishes molecular features of thalassemia among Hakka people in Heyuan. It will be useful for developing strategies to prevent thalassemia.

Generation of Synthetic Peptide-Specific Antibody for the Development of A Southern Rice Black-Streaked Dwarf Virus Diagnostic Test

Southern rice black-streaked dwarf virus (SRBSDV) causes severe epidemical disease on rice with the infected area up to millions of hectares in South China and North and Central of Vietnam. So far, there are no effective, cheap, quick, and practicable methods for diagnosing SRBSDV. The conventional RT-PCR technique is the most popular method for detecting SRBSDV with high accuracy. However, it is hard to apply this method for large-scale SDBSDV diagnosis because of the requirements of expensive reagents and instruments, as well as complex procedures. Meanwhile, SRBSDV diagnostic techniques based on antigen detection have outstanding advantages due to their low cost, easy manipulation, and wide application possibility. Today, there are still no commercially available specific antibodies to SRBSDV. In a previous study, to develop the SRBSDV diagnostic technique by the ELISA technique, a SRBSDV specific antibody was generated by a recombinant P10 envelope protein (66kDa), which has a titer of 1:5,000. In this study, we continued to study the production of SRBSDV specific polyclonal antibodies from small antigen–rich peptides from the SRBSDV P10 envelope protein. The resulting purified antibody can specifically bind to the P10 protein and at the diluted concentration of 1:100,000 it can detect SRBSDV in infected rice samples via the dot-blot technique. Our research results open up new opportunities for proactive antibodies to develop a SRBSDV membrane rapid diagnostic kit.

Development and standardization of the Dot Blot test for serological diagnosis of bovine leptospirosis

Leptospirosis a public health problem and an endemic zoonosis in Brazil, is diagnosed by serological methods. Therefore, low-cost and easy to execute methodologies with good/high sensitivity, such as Dot Blot, are an important diagnostic tool. The aim of this study was to standardize and validation the dot-blot technique for the serological diagnosis of bovine leptospirosis. Several concentrations of antigens applied to nitrocellulose membranes, and different dilutions of conjugated bovine serum were evaluated to develop and standardize the test. The best distinction/contrast between positive and negative samples was observed for 1μL antigen (0.11μg/μL outer membrane protein of the Hardjo serovar (OMPH) and 0.08 μg/μL outer membrane protein of the Wolffi serovar (OMPW)), 1:500 and 1:10000 bovine serum dilution and conjugate, respectively. The Dot Blot test efficiency was 71.87% and kappa index, 0.46 (p<0.0001). The other parameters measured were: sensitivity 91.89%; specificity 59.32%; positive predictive value 58.62%; and, negative predictive value 59.32%. In addition to high sensitivity, other advantages of the Dot Blot technique have been identified, such as practicality, low cost since it does not require sophisticated devices and the fact that the Hardjo and Wolffi OMP also reacted with serovars from other pathogenic serogroups. The results provided positive expectations for the use of Dot Blot as support in the diagnosis of bovine leptospirosis, especially if used as a screening test, stimulating further research for the future development of kits for diagnostic purposes.

TP53 drives abscopal effect by secretion of senescence-associated molecular signals in non-small cell lung cancer

BackgroundRecent developments in abscopal effect strongly support the use of radiotherapy for the treatment of metastatic disease. However, deeper understanding of the molecular mechanisms underlying the abscopal effect are required to best benefit a larger proportion of patients with metastasis. Several groups including ours, reported the involvement of wild-type (wt) p53 in radiation-induced abscopal effects, however very little is known on the role of wtp53 dependent molecular mechanisms.MethodsWe investigated through in vivo and in vitro approaches how wtp53 orchestrates radiation-induced abscopal effects. Wtp53 bearing (A549) and p53-null (H1299) NSCLC lines were xenotransplanted in nude mice, and cultured in 2D monolayers and 3D tumor spheroids. Extracellular vesicles (EVs) were isolated from medium cell culture by ultracentrifugation protocol followed by Nanoparticle Tracking Analysis. Gene expression was evaluated by RT-Real Time, digital qRT-PCR, and dot blot technique. Protein levels were determined by immunohistochemistry, confocal anlysis, western blot techniques, and immunoassay.ResultsWe demonstrated that single high-dose irradiation (20 Gy) induces significant tumor growth inhibition in contralateral non-irradiated (NIR) A549 xenograft tumors but not in NIR p53-null H1299 or p53-silenced A549 (A549sh/p53) xenografts. We further demonstrates that irradiation of A549 cells in vitro induces a senescence-associated secretory phenotype (SASP) producing extracellular vesicles (EVs) expressing CD63 and carrying DNA:RNA hybrids and LINE-1 retrotransposon. IR-A549 EVs also hamper the colony-forming capability of recipient NIR A549 cells, induce senescent phenotype, nuclear expression of DNA:RNA hybrids, and M1 macrophage polarization.ConclusionsIn our models, we demonstrate that high radiation dose in wtp53 tumors induce the onset of SASP and secretion of CD63+ EVs loaded with DNA:RNA hybrids and LINE-1 retrotransposons that convey senescence messages out of the irradiation field triggering abscopal effect in NIR tumors.

Identification of Allergic Epitopes of Soybean β-Conglycinin in Different Animal Species.

Soybean can cause allergy in both humans and animals. The herein study aims to identify the antigenic determinants (epitopes) of β-conglycinin that lead to allergy in different animal species (swine, bovine, and rats). The epitopes of β-conglycinin were identified through co-immunoprecipitation and mass spectrometry. The binding abilities of seven identified epitope peptides to allergic sera of three animal species were compared by ELISA and dot-blot techniques. Some epitope peptides could be recognized by the three animal allergic sera, while most epitopes showed some differences in binding abilities to the different animal sera. The strongest reaction using swine sera was detected with peptides α2, β2, and β3, but the biggest sensitive regions for bovine and rats were peptides α2, β1, and β4. Most epitopes of β-conglycinin exhibited different binding abilities to the three animal sera, in which the biggest sensitive regions were peptides α2, β2, and β3 for swine, but peptides α2, β1, and β4 were detected for bovine and rats.

Overexpression of bacterial katE gene improves the resistance of modified tomato plant against Fusarium oxysporum f. sp. lycopersici

ABSTRACT Tomato (Solanum lycopersicum L.) yield is severely affected by Fusarium fungal disease. To improve the resistance of tomato against Fusarium oxysporum f. sp. lycopersici (FOL), Escherichia coli katE gene was transformed into two tomato cultivars, namely Castle Rock and Super strain B, via Agrobacterium tumefaciens; the transformation efficiency was 5.6% and 3.5%, respectively. The integration of the katE gene into T0, T1, and T2 transgenic tomato lines was confirmed using PCR. In addition, DNA dot blot technique confirmed the integration of the katE gene into T2 transgenic tomato lines. The RT-PCR analysis confirmed that the katE gene could be expressed normally in the T2 modified lines. Under artificial infection with FOL, the non-modified plants exhibited more severe fungal disease symptoms than those observed in katE overexpression (OE) lines. Our analysis showed that the levels of three defense enzymes, namely superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD), were increased during transgenic T2 generation pre-treated with FOL. The bioassay of modified lines revealed that an average of 52.56% of the modified Castle Rock cultivar and 50.28% of the modified Super Strain B cultivar showed resistance under Fusarium infection. These results clearly indicate that the modified tomato plants, in which the katE gene was overexpressed, became more resistant to the infection by FOL than the wild-type plants. Our study has proven that the overexpression of the E. coli katE gene in the OE lines could be utilized to develop and improve the resistance against fungal diseases in the modified crops.

Acetylcholinesterase (AChE) monoclonal antibody generation and validation for use as a biomarker of glyphosate-based herbicide exposure in commercial freshwater fish

Monoclonal antibody specific to acetylcholinesterase (AChE) was extracted from the brain of hybrid catfish after exposure to glyphosate-based herbicide for 24 h. AChE was partially purified using hydroxyapatite and chromatography columns. The specific characteristics of AChE were studied by western blot using commercial polyclonal antibody (Rabbit anti-Fish AChE). It was found that the protein band had a molecular weight of 71 kDa. After mice were injected with AChE 4 times, the spleen showed a response to the induction. Polyclonal B cells from the mouse's spleen were taken and fused with myeloma cells to produce hybrid cells. After two fusions were performed, the clones specific to AChE were selected by dot blot, ELISA, immunohistochemistry and western blot techniques. Two clones, ACHE 33 and ACHE 99, which had the isotype of IgM were found. These two produced monoclonal antibodies specific to AChE in both denatured and native forms. The ACHE 33 monoclonal antibody clone from hybrid catfish could be cross-react with two commercial freshwater fishes, Nile tilapia and climbing perch, based on dot blot, immunohistochemistry, and western blot techniques. Moreover, AChE in Nile tilapia and climbing perch with glyphosate- based herbicide exposure gave a positive result with ACHE 33 as protein with molecular weight of 66 kDa. Based on our results, the produced monoclonal antibody showed specificity and could be applied to test AChE expression to assess glyphosate-based herbicide contamination in hybrid catfish, Nile tilapia and climbing perch. It could be also be a useful tool in indicating the quality of water resources.

Study of the genotypic and hematological feature of hemoglobin H disease in West Guangxi area

To analyze the incidence, genotype and hematological feature of hemoglobin H (HbH) disease in West Guangxi region. A total of 1246 patients diagnosed with HbH disease from January 2013 to December 2018 in our hospital were enrolled. Red blood cell parameters, hemoglobin electrophoresis, Gap-polymerase china reaction (Gap-PCR) and polymerase chain reaction-reverse dot blot (PCR-RDB) techniques were used to detect the 6 common α-thalassemia mutations and 17 common β-thalassemia mutations. The results were compared with those of other regions. The detection rate for HbH disease was 5.66%. Among the 1246 patients, 614 (49.28%) had deletion-type HbH disease, including -α 3.7/--SEA (35.32%),-α 4.2/--SEA(13.72%) and -a 3.7/--THAI(0.24%), 632(50.72%) had non-deleted HbH disease, mainly α CS α /--SEA (44.86%), followed by α WS α/--SEA (4.33%), α QS α /--SEA (1.45%) and α CS α/--THAI(0.08%). Co-committent HbH disease and β-thalassemia were detected in 54 cases (4.33%). Most patients with HbH disease showed mild to moderate anemia. Very few had severe anemia. Among these, patients with HbH-CS had the most severe anemia, and HbH-WS were the mildest. Hb levels in patients with HbH disease alone were lower than those with co-committent HbH and β-thalassemia. Compared with other regions, the incidence and genotype of HbH disease of West Guangxi are different. The prevalence of HbH disease is high in West Guangxi region, and the main genotypeis non-deletion. α CS α /--SEA is the most common, and most of them had moderate anemia. Compared with the deletion-type HbH disease, non-deleted HbH patients were more severe. When HbH disease co-committed with β-thalassemia, the severity of anemia is reduced. The difference between West Guangxi and other regions may account for the variance of clinical manifestations and incidence of HbH disease in this region.

Designing Diagnostic Kit for Toxoplasma gondii Based on GRA7, SAG1, and ROP1 Antigens: An In Silico Strategy

Toxoplasma gondii is an intracellular protozoan that infects a wide range of mammalians. Commercial diagnostic kits usually use the whole parasite antigen to, which can reduce the specificity of the test. B-cell epitopes are antigenic structural index and react specifically with antibody receptors. Prediction of B-cell epitopes and designing of antigenic multi-epitope may have a potential role for more precise diagnosis. Currently, bioinformatics methods play an important role in designing the antigenic multi-epitope to develop a serological diagnosis kit. In the current study, liner and conformational B-cell epitopes prediction of Dense granule protein 7 (GRA7), Surface Antigen 1 (SAG1), and Rhoptry protein 1 (ROP1) antigens have conducted by the ABCpred, IEDB, LBtope, CBTOPE and DiscoTope servers, respectively. Prediction of the 3D structure of three toxoplasma proteins(SAG1,GRA7,ROP1) and multi-epitope (final structure) was performed using -TASSER server. Model 1 was selected as the best model with C-Score: -3.89. The modeled GRA7, SAG1, and ROP1 structure and multi-epitope structure were validated by the Ramachandran plots Prosa and Verify 3D server. Epitopic regions of 3D protein structure were identified by Chimera UCSF software. Physicochemical properties of the Multi epitope were evaluated using ProtParam and SOLpro server. The synthesized multi-epitope was directly cloned into a pET28b expression plasmid and transformed into Escherichia coli BL21 (DE3) and induced by 1 mM IPTG. The expressed protein was finally confirmed by SDS-PAGE and dot-blot techniques. SDS-PAGE results of recombinant multi-epitope peptide (rMEP)-pET-28b showed a band of about 24.088 kDa. The western blot results indicated that rMEP had a high response with 1:50 with positive serum. The current results indicated that the integrated recombinant (SAG1, GRA7, and ROP1) multi-epitope antigens of T. gondii might be useful to develop practical diagnostic kits for acute and chronic toxoplasmosis.

Conformational Change in Herpes Simplex Virus Entry Glycoproteins Detected by Dot Blot.

Conformational changes in viral membrane proteins drive membrane fusion, a critical step in virus entry and infection. Here we describe a simple and rapid virus blotting immunoassay to define conformational changes with a panel of monoclonal antibodies to distinct sites across a viral glycoprotein. This dot blot technique has been utilized to define low pH-triggered changes in the prefusion form of the herpesviral fusogen gB. At pH of <6.2 there are specific changes in herpes simplex virus 1 gB domains I and V. This corresponds broadly to host cell endosomal pH. Many of the identified changes are at least partially reversible. This method can be adapted to document changes in viral proteins that are not fusion proteins, including those induced by alternate triggers such as receptor-binding or protease cleavage.

IgE Cross-Reactivity of Cashew Nut Allergens

Background: Allergic sensitisation towards cashew nut often happens without a clear history of eating cashew nut. IgE cross-reactivity between cashew and pistachio nut is well described; however, the ability of cashew nut-specific IgE to cross-react to common tree nut species and other Anacardiaceae, like mango, pink peppercorn, or sumac is largely unknown. Objectives: Cashew nut allergic individuals may cross-react to foods that are phylogenetically related to cashew. We aimed to determine IgE cross-sensitisation and cross-reactivity profiles in cashew nut-sensitised subjects, towards botanically related proteins of other Anacardiaceae family members and related tree nut species. Method: Sera from children with a suspected cashew nut allergy (n = 56) were assessed for IgE sensitisation to common tree nuts, mango, pink peppercorn, and sumac using dot blot technique. Allergen cross-reactivity patterns between Anacardiaceae species were subsequently examined by SDS-PAGE and immunoblot inhibition, and IgE-reactive allergens were identified by LC-MS/MS. Results: From the 56 subjects analysed, 36 were positive on dot blot for cashew nut (63%). Of these, 50% were mono-sensitised to cashew nuts, 19% were co-sensitised to Anacardiaceae species, and 31% were co-sensitised to tree nuts. Subjects co-sensitised to Anacardiaceae species displayed a different allergen recognition pattern than subjects sensitised to common tree nuts. In pink peppercorn, putative albumin- and legumin-type seed storage proteins were found to cross-react with serum of cashew nut-sensitised subjects in vitro. In addition, a putative luminal binding protein was identified, which, among others, may be involved in cross-reactivity between several Anacardiaceae species. Conclusions: Results demonstrate the in vitro presence of IgE cross-sensitisation in children towards multiple Anacardiaceae species. In this study, putative novel allergens were identified in cashew, pistachio, and pink peppercorn, which may pose factors that underlie the observed cross-sensitivity to these species. The clinical relevance of this widespread cross-sensitisation is unknown.