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Systemic Lupus Erythematosus (SLE) is a medical condition characterized by an abnormal immune response that affects multiple organs and tissues, resulting in a range of symptoms and potential problems. To confirm or exclude a diagnosis of systemic lupus erythematosus (SLE), it is necessary to conduct tests for anti-nuclear antibody (ANA) and anti-DFS70 antibody. The most widely accepted and reliable approach for detecting antinuclear antibodies (ANA) is the indirect immunofluorescent test (IIFA), which is known for its complexity and high cost. Therefore, it is imperative to create a more cost-effective and simplified test for the detection of ANA and anti)DFS70 antibodies. Our preliminary study aimed to isolate sensitive and specific antigens for the detection of ANA and anti-DFS70 antibodies using the dot blot method. Nuclear antigens were isolated from leukocytes of SLE patients who underwent leukapheresis. DSF70 antigens were obtained from R&D Systems (RH-LEDGF cat.no 3468-LE, Lot: ODX0819071). Nuclear antigens and DFS70 antigen were attached to the nitrocellulose membrane dot blot with a dilution of 0.01 mcg/mL and 0.1 mcg/mL, respectively. Furthermore, the antigens were tested for sensitivity and specificity in 56 SLE patients and 34 healthy controls. As a comparison for ANA and anti)DFS70 antibody tests, the ELISA methods were used. The sensitivity and specificity of dot blot methods using the nuclear antigen were 61.5% and 84.6% and the DSF70 antigen were 45.5% and 82.3%, respectively. There were moderate sensitivity and good specificity dot blot methods using both nuclear and DFS-70 antigens for ANA and anti-DFS70 antibody detection. The method of nuclear antigen preparation still needs to be modified to improve its sensitivity. [ABSTRACT FROM AUTHOR]

Introduction: The environmental bacterium Burkholderia pseudomallei causes the often fatal and massively underreported infectious disease melioidosis. Antigens inducing protective immunity in experimental models have recently been identified and serodiagnostic tools have been improved. However, further elucidation of the antigenic repertoire of B. pseudomallei during human infection for diagnostic and vaccine purposes is required. The adaptation of B. pseudomallei to very different habitats is reflected by a huge genome and a selective transcriptional response to a variety of conditions. We, therefore, hypothesized that exposure of B. pseudomallei to culture conditions mimicking habitats encountered in the human host might unravel novel antigens that are recognized by melioidosis patients. Methods and results: In this study, B. pseudomallei was exposed to various stress and growth conditions, including anaerobiosis, acid stress, oxidative stress, iron starvation and osmotic stress. Immunogenic proteins were identified by probing two-dimensional Western blots of B. pseudomallei intracellular and extracellular protein extracts with sera from melioidosis patients and controls and subsequent MALDI-TOF MS. Among B. pseudomallei specific immunogenic signals, 90 % (55/61) of extracellular immunogenic proteins were identified by acid, osmotic or oxidative stress. A total of 84 % (44/52) of intracellular antigens originated from the stationary growth phase, acidic, oxidative and anaerobic conditions. The majority of the extracellular and intracellular protein antigens were identified in only one of the various stress conditions. Sixty-three immunoreactive proteins and an additional 38 candidates from a literature screening were heterologously expressed and subjected to dot blot analysis using melioidosis sera and controls. Our experiments confirmed melioidosis-specific signals in 58 of our immunoproteome candidates. These include 15 antigens with average signal ratios (melioidosis:controls) greater than 10 and another 26 with average ratios greater than 5, including new promising serodiagnostic candidates with a very high signal-to-noise ratio. Conclusion: Our study shows that a comprehensive B. pseudomallei immunoproteomics approach, using conditions which are likely to be encountered during infection, can identify novel antibody targets previously unrecognized in human melioidosis. [ABSTRACT FROM AUTHOR]

One of the emerging trends in modern analytical and bioanalytical chemistry involves the substitution of enzyme labels (such as horseradish peroxidase) with nanozymes (nanoparticles possessing enzyme-like catalytic activity). Since enzymes and nanozymes typically operate through different catalytic mechanisms, it is expected that optimal reaction conditions will also differ. The optimization of substrates for nanozymes usually focuses on determining the ideal pH and temperature. However, in some cases, even this step is overlooked, and commercial substrate formulations designed for enzymes are utilized. This paper demonstrates that not only the pH but also the composition of the substrate buffer, including the buffer species and additives, significantly impact the analytical signal generated by nanozymes. The presence of enhancers such as imidazole in commercial substrates diminishes the catalytic activity of nanozymes, which is demonstrated herein through the use of 3,3′-diaminobenzidine (DAB) and Prussian Blue as a model chromogenic substrate and nanozyme. Conversely, a simple modification to the substrate buffer greatly enhances the performance of nanozymes. Specifically, in this paper, it is demonstrated that buffers such as citrate, MES, HEPES, and TRIS, containing 1.5–2 M NaCl or NH4Cl, substantially increase DAB oxidation by Prussian Blue and yield a higher signal compared to commercial DAB formulations. The central message of this paper is that the optimization of substrate composition should be an integral step in the development of nanozyme-based assays. Herein, a step-by-step optimization of the DAB substrate composition for Prussian Blue nanozymes is presented. The optimized substrate outperforms commercial formulations in terms of efficiency. The effectiveness of the optimized DAB substrate is affirmed through its application in several commonly used immunostaining techniques, including tissue staining, Western blotting assays of immunoglobulins, and dot blot assays of antibodies against SARS-CoV-2. [ABSTRACT FROM AUTHOR]

Begomoviruses (Geminiviridae family) are characterized by their high recombination rate and a wide range of hosts, making their control difficult. In Costa Rica, various species of bipartite begomoviruses have been reported, which are Pepper golden mosaic virus (PepGMV), Tomato yellow mottle virus (ToYMoV), Tomato leaf curl Sinaloa virus (ToLCSiV) and the monopartite begomovirus Tomato yellow leaf curl virus (TYLCV). Since the TYLCV first report in Costa Rica, neither additional knowledge has been produced on how this begomovirus has spread in the country's territory nor on the distribution of the other bipartite species. A total of 429 tomato samples collected during the years 2015–2016 were used to study these aspects. Each sample was georeferenced and analysed with various techniques such as nucleic acid hybridization, polymerase chain reaction (PCR) and sequencing for the begomoviruses previously reported in Costa Rica. It was found that the presence/absence of the different species can vary, depending on the province. TYLCV is present in the six provinces analysed in this work, with a proportion from 3.7 to 86.6 per cent. Alajuela, Cartago, and Heredia are the provinces most affected by tomato‐infecting begomoviruses. Fourteen different haplotypes of TYLCV were detected, but all were identified as TYLCV‐IL. The distribution of TYLCV was related to the presence of the whitefly Bemisia tabaci MED, especially in the country's main tomato production areas. This information allows the phytosanitary surveillance services to develop strategies for the integrated management of the disease and to contribute data to the genetic improvement programmes of the crop. [ABSTRACT FROM AUTHOR]

IntroductionThe environmental bacterium Burkholderia pseudomallei causes the often fatal and massively underreported infectious disease melioidosis. Antigens inducing protective immunity in experimental models have recently been identified and serodiagnostic tools have been improved. However, further elucidation of the antigenic repertoire of B. pseudomallei during human infection for diagnostic and vaccine purposes is required. The adaptation of B. pseudomallei to very different habitats is reflected by a huge genome and a selective transcriptional response to a variety of conditions. We, therefore, hypothesized that exposure of B. pseudomallei to culture conditions mimicking habitats encountered in the human host might unravel novel antigens that are recognized by melioidosis patients.Methods and resultsIn this study, B. pseudomallei was exposed to various stress and growth conditions, including anaerobiosis, acid stress, oxidative stress, iron starvation and osmotic stress. Immunogenic proteins were identified by probing two-dimensional Western blots of B. pseudomallei intracellular and extracellular protein extracts with sera from melioidosis patients and controls and subsequent MALDI-TOF MS. Among B. pseudomallei specific immunogenic signals, 90 % (55/61) of extracellular immunogenic proteins were identified by acid, osmotic or oxidative stress. A total of 84 % (44/52) of intracellular antigens originated from the stationary growth phase, acidic, oxidative and anaerobic conditions. The majority of the extracellular and intracellular protein antigens were identified in only one of the various stress conditions. Sixty-three immunoreactive proteins and an additional 38 candidates from a literature screening were heterologously expressed and subjected to dot blot analysis using melioidosis sera and controls. Our experiments confirmed melioidosis-specific signals in 58 of our immunoproteome candidates. These include 15 antigens with average signal ratios (melioidosis:controls) greater than 10 and another 26 with average ratios greater than 5, including new promising serodiagnostic candidates with a very high signal-to-noise ratio.ConclusionOur study shows that a comprehensive B. pseudomallei immunoproteomics approach, using conditions which are likely to be encountered during infection, can identify novel antibody targets previously unrecognized in human melioidosis.

Compared with our closest living evolutionary cousins, humans appear unusually prone to develop carcinomas (cancers arising from epithelia). The SIGLEC12 gene, which encodes the Siglec-XII protein expressed on epithelial cells, has several uniquely human features: a fixed homozygous missense mutation inactivating its natural ligand recognition property; a polymorphic frameshift mutation eliminating full-length protein expression in ~60%-70% of worldwide human populations; and, genomic features suggesting a negative selective sweep favoring the pseudogene state. Despite the loss of canonical sialic acid binding, Siglec-XII still recruits Shp2 and accelerates tumor growth in a mouse model. We hypothesized that dysfunctional Siglec-XII facilitates human carcinoma progression, correlating with known tumorigenic signatures of Shp2-dependent cancers. Immunohistochemistry was used to detect Siglec-XII expression on tissue microarrays. PC-3 prostate cancer cells were transfected with Siglec-XII and transcription of genes enriched with Siglec-XII was determined. Genomic SIGLEC12 status was determined for four different cancer cohorts. Finally, a dot blot analysis of human urinary epithelial cells was established to determine the Siglec-XII expressors versus non-expressors. Forced expression in a SIGLEC12 null carcinoma cell line enriched transcription of genes associated with cancer progression. While Siglec-XII was detected as expected in ~30%-40% of normal epithelia, ~80% of advanced carcinomas showed strong expression. Notably, >80% of late-stage colorectal cancers had a functional SIGLEC12 allele, correlating with overall increased mortality. Thus, advanced carcinomas are much more likely to occur in individuals whose genomes have an intact SIGLEC12 gene, likely because the encoded Siglec-XII protein recruits Shp2-related oncogenic pathways. The finding has prognostic, diagnostic, and therapeutic implications.

Background: Diagnosing progressive disseminated histoplasmosis (PDH) is still challenging in many countries where this disease is highly endemic. Definitive diagnosis is established by culture and/or by cytology/histopathology but both procedures have limited sensitivity and cultures are time‐consuming. Antibodies detection by immunodiffusion has a low sensitivity in immunocompromised individuals. Commercially available antigen detection assays have high sensitivity in PDH cases; however, they are expensive and only performed in few laboratories. Aims: To describe the potential use of a novel ELISA for antibodies testing and a dot blot assay for antigen testing for diagnosing PDH using the recombinant 100 kDa protein of Histoplasma capsulatum (Hcp100) and their polyclonal antibodies as novel reagents, respectively. Methods: Serum and urine samples from a cohort of patients with HIV/AIDS and proven PDH were studied for the detection of anti‐Hcp100 antibodies by ELISA and Hcp100 antigen by dot blot, respectively. Sensitivity, specificity and cross‐reactions with other diseases were estimated for each assay and compared with those obtained using histoplasmin (HMN) as a reagent for antibodies detection by ELISA and immunodiffusion, and using a commercial antigenuria test. Results: Antibodies detection by the Hcp100 ELISA demonstrated 78.6% sensitivity and 88.4% specificity, versus 85.7% sensitivity and 81.0% specificity for the HMN ELISA and 26.1% sensitivity and 100% specificity for the immunodiffusion assay. Antigen detection by the Hcp100 dot blot demonstrated 89.3% sensitivity and 97.0% specificity versus 82.1% sensitivity and 90.9% specificity for the commercial test. Conclusion: The immunoassays described herein based on Hcp100 would be a valuable screening tool for diagnosing PDH. [ABSTRACT FROM AUTHOR]

Decapod iridescent virus 1 (DIV1) has emerged as a novel threat to shrimp farming. The development of a rapid, convenient, and accurate method for diagnosing DIV1 is urgently needed, serving as a key factor in the reduction of the risk of viral outbreaks. This study aimed to develop a new variable antigen receptor (vNAR), derived from shark heavy-chain antibodies, directed against DIV1 for use in detection assays. White-spotted bamboo sharks were immunized with DIV1 virions, and an immune phage-display library was constructed. After three rounds of panning, the lead D02 vNAR was isolated from the library. D02 vNAR was identified to target the viral antigen ORF064L through western blotting, LM-SC/SC, and pull-down analyses. Furthermore, indirect enzyme linked immunosorbent assay (indirect ELISA) and dot-blot assays were developed based on D02 vNAR. The detection limits of the indirect ELISA and dot blot methods were 5 ng/mL and 6.25 ng/spot, respectively. Furthermore, in the indirect ELISA method, a standard curve describing the log concentration of DIV1 versus OD value was established, which was linear in the concentration range of 80–10,240 ng/mL, and the linear regression equation was derived as y = 0.3565x − 0.51601. Furthermore, 300 clinical samples were analyzed by the two methods using quantitative real-time polymerase chain reaction (qRT-PCR) as a reference. Compared to qRT-PCR, the diagnostic sensitivity and specificity of the indirect ELISA and dot blot methods were 96.7 % and 100 % and 93.5 % and 100 %, respectively, demonstrating the practicability and reliability of the vNAR-based indirect ELISA and dot blot methods for the robust detection of DIV1. Overall, this is the first study in which a vNAR has been developed against DIV1. The developed vNAR offers a robust tool with great potential for the diagnosis of DIV1 during shrimp cultivation.

One of the emerging trends in modern analytical and bioanalytical chemistry involves the substitution of enzyme labels (such as horseradish peroxidase) with nanozymes (nanoparticles possessing enzyme-like catalytic activity). Since enzymes and nanozymes typically operate through different catalytic mechanisms, it is expected that optimal reaction conditions will also differ. The optimization of substrates for nanozymes usually focuses on determining the ideal pH and temperature. However, in some cases, even this step is overlooked, and commercial substrate formulations designed for enzymes are utilized. This paper demonstrates that not only the pH but also the composition of the substrate buffer, including the buffer species and additives, significantly impact the analytical signal generated by nanozymes. The presence of enhancers such as imidazole in commercial substrates diminishes the catalytic activity of nanozymes, which is demonstrated herein through the use of 3,3′-diaminobenzidine (DAB) and Prussian Blue as a model chromogenic substrate and nanozyme. Conversely, a simple modification to the substrate buffer greatly enhances the performance of nanozymes. Specifically, in this paper, it is demonstrated that buffers such as citrate, MES, HEPES, and TRIS, containing 1.5–2 M NaCl or NH4Cl, substantially increase DAB oxidation by Prussian Blue and yield a higher signal compared to commercial DAB formulations. The central message of this paper is that the optimization of substrate composition should be an integral step in the development of nanozyme-based assays. Herein, a step-by-step optimization of the DAB substrate composition for Prussian Blue nanozymes is presented. The optimized substrate outperforms commercial formulations in terms of efficiency. The effectiveness of the optimized DAB substrate is affirmed through its application in several commonly used immunostaining techniques, including tissue staining, Western blotting assays of immunoglobulins, and dot blot assays of antibodies against SARS-CoV-2.

Linear IgE epitopes play essential roles in persistent allergies, including peanut and tree nut allergies. Using chemically synthesized peptides attached to membranes and microarray experiments is one approach for determining predominant epitopes that has seen success. However, the overall expense of this approach and the inherent challenges in scaling up the production and purification of synthetic peptides precludes the general application of this approach. To overcome this problem, we have constructed a plasmid vector for expressing peptides sandwiched between an N-terminal His-tag and a trimeric protein. The vector was used to make overlapping peptides derived from peanut allergens Ara h 2. All the peptides were successfully expressed and purified. The resulting peptides were applied to identify IgE binding epitopes of Ara h 2 using four sera samples from individuals with known peanut allergies. New and previously defined dominant IgE binding epitopes of Ara h 2 were identified. This system may be readily applied to produce agents for component- and epitope-resolved food allergy diagnosis. [Display omitted] • Overlapping peptides derived from peanut allergen Ara h 2 flanked by human TL1A and a His-tag were successfully expressed in E. coli. • This approach provides an alternative to the chemical synthesis of peptides for epitope mapping. • New and previously defined dominant IgE binding epitopes of Ara h 2 were identified. [ABSTRACT FROM AUTHOR]

Detection of E. coli in water and food constitutes a major challenge. However, the outer membrane proteins of this Gram-negative bacterium represent an excellent choice for their detection and diagnosis. Two outer membrane proteins: A and C, play important roles as drug modulators and in cellular permeability for this bacterium. Here we employed the antibodies raised against these two outer membrane proteins for the development of dot-blot immunoassays to detect this bacterium in water and meat samples. This immune-based assay was not only sensitive and reliable but rapid and cheap to perform. Sensitivities and specificities were demonstrated at the lowest detection limits of just two to three cells per milliliter, while the upper limit reached 107 cells per milliliter. In conclusion, the simplicity and extremely high resolution of this colored-based immunoassay test would be of great importance for untrained food and/or water inspectors or laboratory personnel to use with immediate visual results. [ABSTRACT FROM AUTHOR]

The CRISPR/Cas9 genome engineering system has revolutionized biology by allowing for precise genome editing with little effort. Guided by a single guide RNA (sgRNA) that confers specificity, the Cas9 protein cleaves both DNA strands at the targeted locus. The DNA break can trigger either non-homologous end joining (NHEJ) or homology directed repair (HDR). NHEJ can introduce small deletions or insertions which lead to frame-shift mutations, while HDR allows for larger and more precise perturbations. Here, we present protocols for generating knockout cell lines by coupling established CRISPR/Cas9 methods with two options for downstream selection/screening. The NHEJ approach uses a single sgRNA cut site and selection-independent screening, where protein production is assessed by dot immunoblot in a high-throughput manner. The HDR approach uses two sgRNA cut sites that span the gene of interest. Together with a provided HDR template, this method can achieve deletion of tens of kb, aided by the inserted selectable resistance marker. The appropriate applications and advantages of each method are discussed.

Abstract Compared with our closest living evolutionary cousins, humans appear unusually prone to develop carcinomas (cancers arising from epithelia). The SIGLEC12 gene, which encodes the Siglec‐XII protein expressed on epithelial cells, has several uniquely human features: a fixed homozygous missense mutation inactivating its natural ligand recognition property; a polymorphic frameshift mutation eliminating full‐length protein expression in ~60%–70% of worldwide human populations; and, genomic features suggesting a negative selective sweep favoring the pseudogene state. Despite the loss of canonical sialic acid binding, Siglec‐XII still recruits Shp2 and accelerates tumor growth in a mouse model. We hypothesized that dysfunctional Siglec‐XII facilitates human carcinoma progression, correlating with known tumorigenic signatures of Shp2‐dependent cancers. Immunohistochemistry was used to detect Siglec‐XII expression on tissue microarrays. PC‐3 prostate cancer cells were transfected with Siglec‐XII and transcription of genes enriched with Siglec‐XII was determined. Genomic SIGLEC12 status was determined for four different cancer cohorts. Finally, a dot blot analysis of human urinary epithelial cells was established to determine the Siglec‐XII expressors versus non‐expressors. Forced expression in a SIGLEC12 null carcinoma cell line enriched transcription of genes associated with cancer progression. While Siglec‐XII was detected as expected in ~30%–40% of normal epithelia, ~80% of advanced carcinomas showed strong expression. Notably, >80% of late‐stage colorectal cancers had a functional SIGLEC12 allele, correlating with overall increased mortality. Thus, advanced carcinomas are much more likely to occur in individuals whose genomes have an intact SIGLEC12 gene, likely because the encoded Siglec‐XII protein recruits Shp2‐related oncogenic pathways. The finding has prognostic, diagnostic, and therapeutic implications.

Lanthanide doped nanocrystals capable to emit higher energy photons under excitation with lower energy radiation are promising for a broad range of applications including biodetection, biosensing, and bioimaging. However, the adaptation of these nanoparticles to the biological environment that requires good water-solubility, stability and ease of further functionalization still remains a challenge. The application of nanoparticles for biodetection or in various assays encountered many difficulties arising mainly from the strong tendency of nanoparticles for aggregation or nonspecific binding. Here we present a new method to obtain soluble and stable in water-based buffers NaYF4:Yb3+Er3+ nanocrystals with modified surface ready for further conjugation with biomolecules. In the presented approach polyvinylpyrrolidone/vinyl alcohol copolymer (PVP/VA) with photo-activatable linker (N-5-Azido-2-nitrobenzoyl group—ANB-NOS) was used for initial coating due to its high non-covalent affinity to nanoparticles surface. Subsequent coating with aminated dextran by ultraviolet light activation of ANB-NOS was carried out. This step has a significant impact on nanocrystals stability in the physiological buffer as well as on protein conjugation. Conjugation of biomolecules was possible by employing another photo-linker—sulfosuccinimidyl 4,4′-azipentanoate (sulfo-SDA). Bacterial Protein G has been selected to create a universal immune-imaging agent due to its ability to bind Fc fragment of most mammalian immunoglobulins. Moreover, the direct attachment of antibodies to nanoparticles was also examined. The activity of biofunctionalized nanocrystals was tested in immunoblot test, that confirmed preserved activity of attached molecules and lack of undesired nonspecific binding or precipitation on the assay membrane. [ABSTRACT FROM AUTHOR]

Heat shock proteins (HSP) are synthesized in living tissues exposed to transient increase in temperature and play a central role in the protective response against heat and other stresses. In fruits, this response to heat treatment provides resistance to a physiological alteration known as chilling injury. Despite the physiological importance of this group of proteins, publications comparing different methodological alternatives for their analysis are rather scarce. In the present paper, we conducted a comparative study using different electrophoretic and immunological techniques to evaluate the HSP response in fruits. Proteins were extracted from tomato fruit exposed to an HSP-inducing temperature (38 °C) for different times (0, 3, 20, and 27 h). Different alternatives of analysis (SDS-PAGE, SDS-PAGE followed by IEF, Western blot, and dot blot) were performed, and their potential application discussed. The study was complemented with a practical application, in which tomatoes were subjected to heat and anaerobic treatments and then stored in a chill-inducing temperature. This application evidences the relevance of knowing the level of proteins attained by stress treatments which correlates with the acquired tolerance.

Rapid diagnostics provide actionable information for patient care at the time and site of an encounter with the health care system. The mainstay of infectious diseases care is early detection (case finding) and treatment completion, but for many, it is hard to identify positive individuals, as is the case of infection with low burden in schistosomiasis, a parasitic disease common in the tropics and subtropics. We developed a new, accurate, and fast Dot blot methodology (iDot) to indirectly detect Schistosoma mansoni in individuals with very low parasite burden using urine samples. Accuracy of 0.74 was obtained with a significant difference between negative and positive patients and a substantial agreement was found when iDot was compared with five available methods. Our analysis also revealed the superiority of iDot in detecting negative individuals from non-endemic sites, thus, presenting the lowest rate of false positives. This new method called iDot is convenient and suitable for qualitative and quantitative detection of schistosomiasis in individuals with low parasite burden. [ABSTRACT FROM AUTHOR]

ABSTRACT: Group-I Fowl adenovirus (FAdV) is still widespread in China's chicken farms, leading to huge economic losses. The traditional PCR method, which can detect all serotypes at the same time, is not sensitive enough to obtain accurate results, especially in some samples containing only a low titer of virus, such as contaminated live vaccine. In order to solve this problem, this study developed a dot blot assay based on the above PCR method. A total of 6 probes targeting the conserved region of FAdV were designed and systematically optimized through sensitivity, accuracy, and stability analyses. Results showed that it is not only suitable for 12 serotypes, but also effectively improve the sensitivity, which increased more than 100 times in comparison with PCR assay. Moreover, this sensitivity was increased 100 times when detecting contaminated live vaccine samples, showing the great prospect of this method in daily monitoring.

OBJECTIVE: Programmed death ligand-1 (PD-L1) is expressed on tumor cells and macrophages. The detection of PD-L1 expression in cancer and the treatment by targeting the PD-L1/programmed death-1 (PD-1) are of great clinical significance. This work aims to screen the aptamers with high affinity and specificity for recombinant human PD-1 (rhPD-1)/recombinant human PD-L1 extracellular domain (rhPD-L1). MATERIALS AND METHODS: In this study, we have expressed, purified, prepared, and identified rhPD-1 and rhPD-L1. The rhPD-L1/rhPD-1 aptamers with high afïnity and speciïcity were obtained by systematic evolution of ligands by exponential enrichment technique. Ten aptamers sequences to rhPD-L1 and 10 aptamers sequences to rhPD-1 were obtained by cloning and sequencing. The afïnity and speciïcity of candidate aptamers were analyzed by gold nanoparticles-based colorimetric assay, dot blot assay, and electrophoretic mobility shift assay. RESULTS: The aptamers named A6 were picked out as the optimal aptamers that recognize PD-1, speciïcally with the Kd value of 47.84 Â 24.78 nM. The aptamers named B10 were picked out as the optimal aptamers that recognize PD-L1, speciï-cally with the Kd value of 59.72 Â 15.87 nM. CONCLUSIONS: The study lays a foundation for the development of detection methods and therapeutic drugs targeting PD-L1/PD-1. [ABSTRACT FROM AUTHOR]

Commercially available dot blots provide a set of specific antibodies spotted on membranes in a given pattern. If the target analyte is present in the solution that the membrane is incubated with, the detection reaction will result in a chemiluminescence signal which is recorded by film or scanner. In order to know which analytes were detected, the analysis consists of measuring the intensity of the recorded signal on each spot. Manually measuring the entire array (typically ~200 spots) is unreliable and tedious. Fully automatic registration of the blot membrane to the template pattern often fails since there might be only very few positive spots on the membrane. This article presents an ImageJ/Fiji macro that requires minimal user input to perform a robust iterative registration of an adjustable template mask representing the spot pattern to the recorded blot. Once the template mask is matched to the dot blot, the spot intensity of each dot is measured and reported in a results table.

Tiêu chảy sau cai sữa (post-weaning diarrhea, PWD) là bệnh thường gặp ở heo con, gây tổn thất kinh tế nặng nề. Enterotoxigenic Escherichia coli (ETEC) là một trong những nguyên nhân chủ yếu gây PWD. Chủng vi khuẩn này đặc trưng bởi hai yếu tố gây bệnh là nhân tố bám dính thành ruột, và các độc tố gây mất nước. Các nghiên cứu gần đây đã chỉ ra rằng tiên mao F18 và F4 là hai kháng nguyên bám dính phổ biến nhất ở các chủng ETEC trong nước và trên thế giới. Vaccine là biện pháp ngăn ngừa PWD hữu hiệu và kinh tế, đặc biệt là vaccine uống với khả năng kích thích hệ miễn dịch đường ruột. Hiện nay vacccine uống phòng ETEC/F4 đã được thương mại hóa, tuy nhiên nhân tố bám dính F18 vẫn gặp nhiều khó khăn trong việc kích thích miễn dịch niêm mạc ruột. Ở nghiên cứu này, hệ thống nấm men Pichia pastoris biểu hiện nhân tố bám dính F18 trên bề mặt được phát triển và đánh giá khả năng biểu hiện trên màng. Kết quả cho thấy F18 đã được biểu hiện trong phân đoạn màng của P. pastoris. Hệ thống biểu hiện trên bề mặt nấm men P. pastoris này có thể được ứng dụng nhằm tạo ra vaccine uống giá rẻ và hiệu quả.

Despite the availability of a wide range of commercial kits, protein quantification is often unreliable, especially for tissue-derived samples, leading to uneven loading in subsequent experiments. Here we show that the widely used Bicinchoninic Acid (BCA) assay tends to underestimate protein concentrations of tissue samples. We present a Ponceau S staining-based dot-blot assay as an alternative for protein quantification. This method is simple, rapid, more reliable than the BCA assay, compatible with biological samples lysed in RIPA or 2x SDS gel-loading buffer, and also inexpensive.

Abstract: Candidiasis or infection that is caused by Candida has become a new list of the therapeutical problems recently. The difficulties in diagnosing are the main cause of the unsatisfactory results from common therapies and diagnosis methods. This has urged researchers to find alternative ways in candidiasis diagnosis such as serology-based detection using antigen or antibody development. The aim of this study was to evaluate the potential of protein derived from Candida albicans biofilm as bioreceptor on candidiasis immunoassay through Dot Blot method. The research method used descriptive method with the following stages: (1) preparation of Candida albicans biofilm (2) extraction of Candida albicans protein through enzymatic and mechanical methods, (3) determination of protein molecular weight with SDS-PAGE (4) production of polyclonal anti- candida and (5) analysis of protein extract as bioreeceptor on dot blot. Profile of biofilm proteins on SDS-PAGE analysis were shown on molecular weight 27,42; 29,89; 38,10; 44,90; 48,75; 52,92; 55,14; 59,86; 70,56; 87,36; 102,54;115,05; 130,14;143,14;181,53 kD. There were differences in the intensity of dots in the control group (44070) and treatment groups (63170.5). It is noticeable that biofilm protein extract of C. albicans can be used for induction of anti-Candida polyclonal antibody production as the potential candidate of bioreceptor in candidiasis immunoassay. Keywords: SDS-PAGE, polyclonal antibody, immunoassay, dot blot, biofilm

Objective: To access the performance of the Tellgenplex human papillomavirus (HPV) DNA test compared to the polymerase chain reaction-reverse dot blot (PCR-RDB) assay for the HPV genotyping. Methods: Sixty cervical swab samples were genotyped by the Tellgenplex HPV DNA test and the PCR-RDB assay. The Tellgenplex HPV DNA test and the PCR-RDB assay can detect 26 and 23 HPV genotypes, respectively. Each sample showed discrepancy was genotyped using sequencing. Results: The percent agreement between the two tests ranged from 83.3% to 100.0% according to different genotype. This showed perfect agreement (>0.81) for high-risk HPV genotypes (35, 39, 45, 53, 56, 59, 66, 68, and 82), substantial agreement (>0.65) for high-risk HPV genotypes (16, 18, 33, 52, and 58) and low-risk HPV genotype 43 between the two assays by the kappa analysis. The positive rates of the two assays for frequent HPV genotypes (16, 35, 39, 45, 52, 53, 58, 59, 66, and 82) were not statistically different, but the PCR-RDB assay showed higher positive rates than the Tellgenplex HPV DNA test for HPV genotypes 81 (P

• PWN-SA571 is a biomarker for PWN. • Specific Mabs for PWN-SA571 were generated. • PWN-SA571 Mabs could detect PWN and PWNIPT by ELISA and dot blots. • PWN-SA571 Mabs could detect antigens by LFA. The biochemical characteristics of pine wood nematode (PWN)-secretory antigen 571 (PWN-SA571) identified from the PWN secretome and PWN-infected pine tree extracts (PWNIPT) were investigated. PWN-SA571 has homologs in various nematodes in diverse families and a signal peptide at the amino-terminal end. Since it was not possible to express the full-length PWN-SA571 protein, two peptides showing high antigenicity were synthesized and used as antigens for generating monoclonal antibodies (Mabs). Mab-secreting fusion hybridoma cell lines were selected based on immunoreactivity to free peptide and BSA-conjugated peptide (BSA pep) antigens by enzyme-linked immunosorbent assay for establishing Mab-secreting hybridoma lines. Ascites containing PWN-SA571-specific Mabs showed stronger immunoreactivity to PWNIPT than to healthy pine tree extracts. In addition, PWN-SA571-specific Mabs could recognize less than 20 ng of BSA pep in lateral flow assays. Furthermore, purified biotin-conjugated PWN-SA571-specific Mab IgG or IgM were able to detect BSA pep (<15 ng) and PWN extracts (<600 ng). Taken together, these results demonstrate that PWN-SA571-specific Mabs could detect PWN or PWNIPT extracts by ELISA, LFA, or dot blot analysis. [ABSTRACT FROM AUTHOR]

A solid phase NMR-based sandwich immunoassay for the prostate-specific antigen (PSA) is presented. Carbon-encapsulated iron nanoparticles were functionalized with bovine serum albumin, coupled to monoclonal antibodies, and then used as magnetic labels. A nitrocellulose membrane with 8-μm pores was coated with capture antibodies and subsequently incubated with a serum sample and a suspension of the nanoconjugate. Test strips were placed in a portable homemade NMR relaxometer. Magnetic nanoparticles attached to nitrocellulose decrease the T2 relaxation time of the water protons located inside the pores of the membrane. Thus, T2 is inversely proportional to the concentration of the antigen (PSA) in the sample. The assay can be performed within 4 h. The detection limit is 0.44 ng mL−1. Kallikrein 2, human chorionic gonadotropin, and α-fetoprotein do not interfere. [ABSTRACT FROM AUTHOR]

Knowledge of dispersal and spatial dynamics of pest populations is fundamental for implementation of integrated pest management and integrated resistance management. This study evaluated 1) the effectiveness of egg white albumin protein to mark larvae and adults of two polyphagous and highly mobile pests, Spodoptera frugiperda (J.E. Smith) (fall armyworm) and Helicoverpa zea (Boddie) (corn earworm) (Lepidoptera: Noctuidae), and 2) the sensitivity of polyvinylidene difluoride membrane (dot blot) in detecting albumin on marked insects. Laboratory and field experiments tested egg albumin as a protein marker, which was detected using two enzyme-linked immunosorbent assay (ELISA), microplate, and dot blot. In the laboratory, 100% of the moths sprayed with 20% egg white solution acquired the albumin marker, which was detected through the last time point tested (5 d) after application. Egg albumin was not effective at long-term marking of larvae, detected only prior the molting to the next instar. Albumin application in field cages resulted in a high percentage of moths detected as marked at 24 h and 5 d for both species. Egg albumin applied in the open field resulted in 15% of the recaptured corn earworm moths marked with most of them collected 150 m from the application area, although some were captured as far as 1,600 m within approximately 6 d after adult emergence. The results indicated egg albumin is a suitable marker to study the dispersion of fall armyworm and corn earworm in the agroecosystem and dot blot was as effective to detect egg albumin as was indirect ELISA. [ABSTRACT FROM AUTHOR]

The mortality rate of cholangiocarcinoma (CCA) is high since there is a lack of a non-invasive technique to accurately detect tumors at the early stage. CCA biomarkers are consistently needed for various purposes including screening, early diagnosis, prognosis and follow-up. Herein, using bioinformatic analysis of our mitochondrial proteome database of CCA tissues, we identified cytokine-induced apoptosis inhibitor 1 (CIAPIN1) as a potential prognostic biomarker for CCA. CIAPIN1 levels in the sera of 159 CCA patients and 93 healthy controls (HC) were measured using a dot blot assay. The median level ± quartile deviation of CIAPIN1 level in the sera of CCA patient group was 0.5144 ± 0.34 µg/µL, which was significantly higher than 0.2427 ± 0.09 µg/µL of the HC group (p < 0.0001). In CCA patients, higher serum CIAPIN1 level was significantly associated with lymph node metastasis (p = 0.024) and shorter overall survival time (p = 0.001, Kaplan–Meier test). Cox regression analysis showed that the serum CIAPIN1 level can be an independent prognostic indicator for the survival of CCA patients. Moreover, for the prediction of CCA prognosis, CIAPIN1 is superior to CEA, CA19-9 and ALP. In conclusion, CIAPIN1 can be a serum biomarker candidate for the poor prognosis of CCA.

Aim: The present study was undertaken to clone, express and study the immunogenicity of P67 protein of Mycoplasma leachii. Materials and Methods: P67 gene was amplified from genomic DNA of M. leachii. The polymerase chain reaction (PCR) product was inserted in pRham N-His SUMO Kan vector and was used to transform competent Escherichia cloni 10G cells. Recombinant protein expression was done by inducing cells with 0.2% Rhamnose. Purification was done using nickel nitrilotriacetic acid affinity chromatography. Western blot and dot blot analysis were performed to assess the immunoreactivity of P67 protein. Results: PCR amplicon size of P67 gene was found to be 1500 base pair. The size of the fusion protein with SUMO tag was 79 kDa in sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis. The recombinant P67 fusion protein expressed in pRham N-His SUMO Kan vector was found to be immunogenic in both western blot and dot blot analysis. Conclusion: Western blot and dot blot analysis of P67 protein of M. leachii revealed that the protein is immunogenic. Further work is needed to evaluate the role of P67 antigen of M. leachii as an immunodiagnostic agent.

El polisacárido Vi (PsVi) de Salmonella Typhi es un antígeno T-independiente y ha demostrado ser protector en adultos jóvenes. Sin embargo, para aumentar la respuesta de anticuerpos y conferir propiedades T-dependientes al polisacárido, se ha conjugado a proteínas. Dentro de los controles exigidos por los organismos regulatorios para estas vacunas está la identidad antigénica de sus componentes y para eso se recomiendan el uso de técnicas de Resonancia Magnética Nuclear o técnicas serológicas. El objetivo del presente trabajo, fue establecer las condiciones óptimas de trabajo de un Dot Blot que permitiera determinar, rápidamente, la identidad de los antígenos en vacunas conjugadas contra S. Typhi. Para ello, se estudiaron los tiempos de incubación, las concentraciones óptimas de anticuerpo monoclonal (AcM) y del ingrediente farmacéutico activo (IFA), así como los volúmenes de aplicación óptimos para las IFAs y formulaciones vacunales, tanto para el PsVi como para el toxoide diftérico (TD). Los resultados mostraron que para la determinación de la identidad antigénica fueron suficientes 5 µL de muestras de los conjugados monovalentes en una dilución de 1/10 (vol/vol) e igual volumen para las formulaciones vacunales. Quedó demostrado que la concentración de 2,5 µg/mL para el AcM contra el PsVi y a 2 µg/mL para el AcM contra TD fueron suficientes para la determinación; mientras que los tiempos de incubación fueron ajustados a 15 min con incubación a 37 ºC. Como conclusión del trabajo se puede decir que quedaron establecidas las condiciones óptimas de trabajo para la determinación rápida de la identidad antigénica del PsVi y del TD presentes en IFA y formulaciones vacunales conjugadas.

Las autoridades regulatorias recomiendan el uso de técnicas de Resonancia Magnética Nuclear o técnicas serológicas para la determinación de la identidad de los antígenos presentes en las vacunas conjugadas. Con la aparición de las vacunas conjugadas multivalentes, se ha hecho necesario recurrir a técnicas inmunoquímicas con la utilización de anticuerpos monoclonales para aumentar la sensibilidad en la determinación de la identidad de los antígenos en dichas vacunas conjugadas. El objetivo del presente trabajo fue establecer las condiciones óptimas de trabajo que permitieran utilizar la técnica del Dot Blot para determinar la identidad de los antígenos en vacunas conjugadas de Streptococcus pneumoniae serotipo 19F. Para ello se estudiaron los tiempos de incubación, la influencia del reactivo en la solución de bloqueo; también las concentraciones óptimas del anticuerpo monoclonal y de los ingredientes farmacéuticos activos, así como los volúmenes de aplicación óptimos para estos y vacunas. Se utilizó un anticuerpo monoclonal contra el polisacárido capsular del serotipo 19F de neumococo. Las muestras empleadas en este trabajo fueron lotes de ingredientes farmacéuticos activos de conjugados de polisacárido capsular 19F y lotes de un candidato vacunal cubano conjugado heptavalente contra neumococos. Los resultados mostraron que para la determinación de la identidad antigénica fueron suficientes 10 µL de muestras de los principios activos a una concentración de 125 µg/mL e igual volumen para las vacunas heptavalentes. Quedó demostrado que una concentración de 1 µg/mL para el anticuerpo monoclonal y tiempos de incubación de 30 min a 37 °C fueron suficientes para la determinación. Estos resultados permiten concluir que quedaron establecidas las condiciones óptimas de trabajo para determinar la identidad antigénica por Dot Blot del polisacárido capsular de S. pneumoniae serotipo 19F presente en las vacunas conjugadas.

The development of DNA probes started from 1950's for diagnostic purposes and it is still growing. DNA probes are applied in several fields such as food, medical, veterinary, environment and security, with the aim of prevention, diagnosis and treatment. The use of DNA probes permits microorganism identification, including pathogen detection, and their quantification when used in specific systems. Various techniques obtained success by the utilization of specific DNA probes, that allowed the obtainment of rapid and specific results. From PCR, qPCR and blotting techniques that were first used in well equipped laboratories to biosensors such as fiber optic, surface plasmon resonance (SPR), electrochemical, and quartz crystal microbalance (QCM) biosensors that use different transduction systems. This review describes i) the design and production of primers and probes, and their utilization from the traditional techniques to the new emerging techniques like biosensors used in current applications; ii) the possibility to use labelled-free probes and probes labelled with an enzyme/fluorophore, etc.; iii) the different sensitivity obtained by using specific systems; and iv) the advantage obtained by using biosensors.

The appearance of uracil in the deoxyuridine moiety of DNA is among the most frequently occurring genomic modifications. Three different routes can result in genomic uracil, two of which do not require specific enzymes: spontaneous cytosine deamination due to the inherent chemical reactivity of living cells, and thymine-replacing incorporation upon nucleotide pool imbalances. There is also an enzymatic pathway of cytosine deamination with multiple DNA (cytosine) deaminases involved in this process. In order to describe potential roles of genomic uracil, it is of key importance to utilize efficient uracil-DNA detection methods. In this review, we provide a comprehensive and critical assessment of currently available uracil detection methods with special focus on genome-wide mapping solutions. Recent developments in PCR-based and in situ detection as well as the quantitation of genomic uracil are also discussed.

Objective To assess the practicality of dot-blot testing for rapid and sensitive detection of the antiacetylcholine receptor (anti-AChR) antibodies in myasthenia gravis (MG). Methods In this case-control study, we tested serum specimens of 85 patients with MG, 85 healthy control individuals, and 85 patients without MG who have other autoimmune and neurological illnesses. All the serum specimens were tested for anti-AChR antibodies using 3 assays: in-house enzyme-linked immunosorbent assay (ELISA), the dot-blot assay, and commercial ELISA. Results In-house ELISA, commercial ELISA, and dot-blot test results were positive for anti-AChR antibodies in 65 (76.5%) patients with MG. The results of all 3 tests were negative for anti-AChR antibodies in healthy controls and patients without MG. We observed perfect concordance (K = 1, P <.001) between all 3 tests. In-house ELISA correlated significantly (r = 0.873, P <.001) with commercial ELISA. In-house ELISA and the dot-blot test demonstrated similar diagnostic performance in detecting anti-AChR antibodies. Conclusions The dot-blot assay is a simple, nonradioactive immune assay for rapid detection of anti-AChR antibodies in MG. [ABSTRACT FROM AUTHOR]

In this paper, we describe the labelling of antibodies by gold nanoparticles (AuNPs) with diameters of 10 and 60 nm with detection by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). Additionally, the AuNPs labelling strategy is compared with commercially available labelling reagents based on MeCAT (metal coded affinity tagging). Proof of principle experiments based on dot blot experiments were performed. The two labelling methods investigated were compared by sensitivity and limit of detection (LOD). The absolute LODs achieved were in the range of tens of picograms for AuNP labelling compared to a few hundred picograms by the MeCAT labelling. [ABSTRACT FROM AUTHOR]

OBJECTIVE: Bauhinia purpurea (BP) Linn. (Caesalpiniaceae) is a plant of great medicinal importance and has been used since ancient times for treating many inflammatory conditions including arthritis. This study investigates the anti-arthritic potential of the hydroalcoholic extract from the stem bark of BP. MATERIALS AND METHODS: The anti-inflammatory and anti-arthritic activity of BP at various doses was used to evaluate its anti-inflammatory activity and anti-arthritic activity. Serum of arthritic rats was collected at day 21 for detecting serum cytokines level and to evaluate the effect of BP on its serum level. Furthermore, the safety of BP was evaluated in acute (5 days) and subacute (28 days) toxicity study in rats. RESULTS: There was a significant inhibition (P < 0.01) in paw edema at a different time scale with different doses of BP (50, 100, and 200 mg/kg). BP also demonstrated dose-dependent anti-arthritic activity on all observation days (3, 7, 14, and 21). In addition, there was also a significant decrease (P < 0.01) in oxidative stress markers, circulating pro-inflammatory cytokine (tumor necrosis factor alpha from 45.91 to 37.44, interleukin-1 (IL-1) β from 18.24 to 16.06, and IL-6 from 69.77 to 58.44) and an increase in anti-inflammatory cytokine (IL-10 from 8.07 to 12.07) levels. BP was found to be safe with an oral LD50 value of >2 g/kg in acute toxicity study and also no toxicological effect was observed in the oral subacute toxicity study. CONCLUSION: This study demonstrates that BP bark possesses anti-arthritic activity potential and confirm its folklore use in the treatment of inflammatory conditions. [ABSTRACT FROM AUTHOR]

The simple immunoprecipitation method was used to isolate tilapia immunoglobulin (Ig) for immunization in order to produce monoclonal antibodies (MAbs) specific to tilapia Ig. First, the tilapia antiserum against bovine serum albumin (BSA) was prepared by peritoneal injection of BSA into tilapia, and the tilapia anti‐BSA antiserum was used to precipitate BSA to form the Ig/BSA immune complex. The Ig/BSA immune complex was then injected into Swiss mice for hybridoma production. After fusion, three hybridoma clones producing MAbs specific to the tilapia antibody were selected by dot blot and Western blot. All MAbs (101A, 59G, and 11A) were bound specifically to the heavy chain of immunoglobulin M (IgM). The MAbs 101A and 59G demonstrated twofold higher affinity than MAb 11A and the commercialized antibody. However, MAbs 11A could also bind to the heavy chain of IgM in Asian seabass, Lates calcarifer, as well. These MAbs can be used to monitor the immune responses of individual fish by indirect ELISA upon exposure to various antigens. [ABSTRACT FROM AUTHOR]

In recent years, Brucella suis has been sporadically reported in Italy in domestic and wild swine. Since standard serological tests can determine false positive results, the development of alternative tests with improved sensitivity and specificity is rather essential. We analyzed 1212 sera collected at slaughterhouse from healthy pigs belonging to 62 farms of North-Central Italy. Sera were tested by Rose Bengal Test, Complement Fixation Test, and subsequently by a Dot Blot (DB) and Western Blot assays (WB). Only one serum resulted positive to all tests, indicating that swine brucellosis has a very limited spread. DB andWBcould represent a support to the available serological tests; however, further studies to validate these tests are needed. In the presence of reemerging diseases, a prompt and continuous monitoring design is necessary to acquire epidemiological information for the subsequent application of specific health emergency plans. [ABSTRACT FROM AUTHOR]

مقدمه: Cryptosporidiosis یکی از علل مهم اسهال در انسان و حیوانات می‌باشد. تشخیص این بیماری، بر پایه‌ی استفاده از روش‌های میکروسکوپی با حساسیت پایین است. هدف از انجام این مطالعه، استفاده از روش Dot blot جهت شناسایی انگل بود. روش‌ها: 131 نمونه‌ی مدفوع از گوساله‌های شیرخوار جمع‌آوری شد و با استفاده از روش فرمالین اتر، روی آن‌ها تغلیظ انجام گرفت. سپس، نمونه‌ها به روش Ziehl-Neelsen اصلاح شده، رنگ‌آمیزی شدند و در نهایت، کل نمونه‌ها به روش Dot blot مورد آزمایش قرار گرفتند. یافته‌ها: اواوسیست گونه‌های Cryptosporidium، در 40 (5/30 درصد) نمونه از 131 نمونه‌ی مدفوع با استفاده از روش رنگ‌آمیزی Ziehl-Neelsen تشخیص داده شدند، اما 49 (4/37 درصد) نمونه از نمونه‌ها با استفاده از روش Dot blot از نظر آنتی‌ژن Cryptosporidium مثبت بود حساسیت روش Dot blot حدود 87 درصد و ویژگی آن حدود 86 درصد برآورد گردید. نتیجه‌گیری: روش Dot blot، عملکردی به نسبت مناسب در تشخیص آلودگی Cryptosporidium دارد.

Background and Purpose: Definitive diagnosis of Creutzfeldt–Jakob disease (CJD) requires demonstration of infective prion protein (PrPSc) in brain tissues by immunohistochemistry or immunoblot, making antemortem diagnosis of CJD difficult. The World Health Organization (WHO) recommends detection of 14-3-3 protein in cerebrospinal fluid (CSF) in cases of dementia, with clinical correlation, as a useful diagnostic marker for CJD, obviating the need for brain biopsy.This facility is currently available in only a few specialized centers in the West and no commercial kit is available for clinical diagnostic use in India. Hence the objective of this study was to develop an in-house sensitive assay for quantitation of 14-3-3 protein and to evaluate its diagnostic potential to detect 14-3-3 proteins in CSF as a biomarker in suspected cases of CJD. Materials and Methods: A minigene expressing the “core” 14-3-3 protein was synthesized by overlapping polymerase chain reaction (PCR) and the recombinant protein was produced by employing a bacterial expression system. Polyclonal antibodies raised in rabbit against the purified recombinant protein were used for developing a dot blot assay with avidin-biotin technology for signal amplification and quantitation of 14-3-3 protein in CSF. Results: The results in the present study suggest the diagnostic potential of the dot blot method with about 10-fold difference (P< 0.001) in the CSF levels of 14-3-3 protein between the CJD cases (N= 50) and disease controls (N= 70). The receiver operating characteristic (ROC) analysis of the results suggested an optimal cutoff value of 2 ng/mL. Conclusions: We have developed an indigenous, economical, and sensitive dot blot method for the quantitation of 14-3-3 protein in CSF.

Neisseria meningitidis serogroups A, B, C, W, Y and X are the main responsible of meningococcal disease. Vaccines are necessary to prevent this disease. The Finlay Institute is developing polysaccharide vaccines against serogroups A, C, W, Y and X. In the development of vaccines, the identity assay is a mandatory requirement for final product release. This test is performed by Dot Blot technique using commercial polyclonal antibodies (PAb). However, the Finlay Institute has monoclonal antibodies (mAbs) against capsular polysaccharides from N. meningitidis (CPs). The objective of this work was to use these mAbs in identity tests to replace commercial PAb. The specificity of the mAbs, the cross-reactivity with other CPs and the identity of the CPs present in four multivalent polysaccharide vaccines, were evaluated. All tested mAbs showed high specificity, and no cross-reactivity was found. Also all mAbs were able to identify the presence of homologous PsC in multivalent vaccine formulations. We conclude that the mAbs obtained in our institution can be used for identity assays of vaccines

Prognosis of cholangiocarcinoma (CCA) patients is absolutely poor. Since improvement of prognosis and/or response to treatment by personalized and precision treatments requires earlier and precise diagnostic markers, discovery of prognostic markers attracts more attention. Apoptosis-inducing factor, mitochondrion-associated 3 (AIFM3) is highly expressed in several cancers including CCA. The present study investigated whether the serum AIFM3 level can be used as a potential marker for CCA prognosis. For this purpose, we first determined secretory protein nature of AIFM3 using bioinformatic tools. The results show that although AIFM3 lacks signal peptide, it can be secreted into plasma/serum via an unconventional pathway. Then, the AIFM3 levels in the sera of 141 CCA patients and 70 healthy controls (HC) were measured using a semi-quantitative dot blot assay. The results show that the AIFM3 level in the sera of CCA group was significantly higher than that of HC. When correlation between serum AIFM3 levels and the clinicopathological parameters of CCA patients were examined, serum AIFM3 levels correlated significantly with lymph node metastasis, age, and the patients’ overall survival (OS). Higher AIFM3 levels were significantly associated with shorter OS, and only AIFM3 was an independent prognostic marker for CCA. In conclusion, AIFM3 can be used as a prognostic marker for CCA.