Antibody Dilution Research Articles

Effect of pretreatment approach on the ELISA-based detection of cyanotoxins in water: Analysis and application

Common cyanotoxins, such as microcystins and nodularins, are produced by frequently occurring harmful cyanobacterial algal blooms in freshwater systems. The required routine monitoring of microcystins and nodularins in drinking water and ambient water demands cost-efficient and reliable enzyme-linked immunosorbent assay kits. We validated the performance of a self-produced broad-spectrum enzyme-linked immunosorbent assay kit and investigated two different methods of mitigating the matrix effects to elucidate the effect of the respective pretreatment approaches recommended by China and the United States on the quantitative detection of cyanotoxins in surface water. We found that the enzyme-linked immunosorbent assay kit achieved a detection limit of 0.15 μg/L with a linear detection range from 0.27 μg/L to 1.87 μg/L for microcystin-LR (the most studied and widely distributed cyanotoxin). The matrix effects could be mitigated both by dilution of water samples with an optimal dilution ratio and dilution of antibody with the buffer containing phosphate buffer solution (10×), bovine serum albumin (1 %) and ethylene diamine tetraacetic acid (0.5 %). In terms of the surface water samples being tested, the concentrations of microcystins and nodularins measured based on pretreatment approach recommended by the United States were 1– 5 times that measured based on pretreatment approach recommended by China, indicating that the pretreatment approach of China overlooks cyanotoxins. In addition, all the measured total microcystins and nodularins of the surface water samples were below the health advisory limit (1.6 μg/L) for microcystins in drinking water proposed by the United States Environmental Protection Agency for school-age children and adults. Our research could provide significant information for outbreak warnings and risk management of harmful cyanobacterial algal blooms.

Determination of Nicotinic Acid in Food and Pharmaceuticals by a Simple and Rapid Fluorescence Polarization Immunoassay (FPIA)

A simple and rapid fluorescence polarization immunoassay (FPIA) based on polyclonal antibodies is reported for the first time for the determination of nicotinic acid. First, six nicotinic acid analogues were coupled with fluorescein isothiocyanate isomer I (FITC) to obtain tracers. The influences of tracer structure on the performance of FPIA as well as the structure-activity relationships were explored. Subsequently, the tracer concentration and antibody dilution ratio on the performance of FPIA were investigated. For the optimal conditions, the detection range of FPIA was from 1.1 to 145.6 μg mL−1 and its IC20 value as the limit of detection (LOD) was 1.1 μg mL−1. The cross-reactivity of antibodies with common vitamins was evaluated. The intra-assay recovery was from 92.0% to 101.2% in food and tablets, with inter-assay recoveries between 92.0% and 103.8%. The developed FPIA provided good sensitivity and reproducibility and therefore provided efficient and economic screening for nicotinic acid in food and tablets.

Establishment of indirect ELISA method for Salmonella antibody detection from ducks based on PagN protein

BackgroundSalmonella as an important food-borne zoonotic bacterial pathogen, infection in ducks is a recessive infection, however, it can also cause high mortality and threat to food safety. Preventing and controlling the infection and transmission of Salmonella in ducks critically require rapid and sensitive detection method. Full-length Salmonella-specific protein PagN was induced and expressed in E.coil BL21 and was purified as an antigen to establish an indirect enzyme-linked immunosorbent assays (iELSA) detection kit.ResultsThe recombinant PagN protein has a molecular weight of 43 kDa containing a His-tag, was recognized by an anti-Salmonella positive serum by Western blot assay. The optimal concentration of PagN as a coating antigen in the iELISA was 1 μg/mL, and the optimal dilution of enzyme-labeled secondary antibody was 1:4000 (0.025 μg/mL). The cutoff OD450 value was established at 0.268. The iELISA kit showed high selectivity since no cross-reaction with E. coli, Staphylococcus aureus and Streptococcus was observed. iELISA method and Dot-blot test were performed on 100 clinical sera samples collected from duck farms, and the actual coincidence rate was 89% (89/100). 613 duck serum samples from 3 different farms were tested using established method and commercial ELISA kit. The concordance between the two methods was 94.1%.ConclusionAnti-PagN based iELISA can serve as a useful tool for diagnosis of Salmonella infection.

Immunohistochemical Detection of Estrogen Receptor-Beta (ERβ) with PPZ0506 Antibody in Murine Tissue: From Pitfalls to Optimization.

The estrogen receptor beta (ERβ) is physiologically essential for reproductive biology and is implicated in various diseases. However, despite more than 20 years of intensive research on ERβ, there are still uncertainties about its distribution in tissues and cellular expression. Several studies show contrasts between mRNA and protein levels, and the use of knockout strategies revealed that many commercially available antibodies gave false-positive expression results. Recently, a specific monoclonal antibody against human ERβ (PPZ0506) showed cross-reactivity with rodents and was optimized for the detection of rat ERβ. Herein, we established an immunohistochemical detection protocol for ERβ protein in mouse tissue. Staining was optimized on murine ovaries, as granulosa cells are known to strongly express ERβ. The staining results were confirmed by western blot analysis and RT-PCR. To obtain accurate and reliable staining results, different staining conditions were tested in paraffin-embedded tissues. Different pitfalls were encountered in immunohistochemical detection. Strong heat-induced epitope retrieval (HIER) and appropriate antibody dilution were required to visualize specific nuclear expression of ERβ. Finally, the specificity of the antibody was confirmed by using ovaries from Esr2-depleted mice. However, in some animals, strong (non-specific) background staining appeared. These signals could not be significantly alleviated with commercially available additional blocking solutions and are most likely due to estrus-dependent expression of endogenous immunoglobulins. In summary, our study showed that the antibody PPZ0506, originally directed against human ERβ, is also suitable for reliable detection of murine ERβ. An established staining protocol mitigated ambiguities regarding the expression and distribution of ERβ in different tissues and will contribute to an improved understanding of its role and functions in murine tissues in the future.

Optimized Mouse-on-mouse Immunohistochemical Detection of Mouse ESR2 Proteins with PPZ0506 Monoclonal Antibody.

Despite the physiological significance of ESR2, a lack of well-validated detection systems for ESR2 proteins has hindered progress in ESR2 research. Thus, recent identification of a specific anti-human ESR2 monoclonal antibody (PPZ0506) and its specific cross-reactivity against mouse and rat ESR2 proteins heightened momenta toward development of appropriate immunohistochemical detection systems for rodent ESR2 proteins. Building upon our previous optimization of ESR2 immunohistochemical detection in rats using PPZ0506, in this study, we further aimed to optimize mouse-on-mouse immunohistochemical detection using PPZ0506. Our assessment of several staining conditions using paraffin-embedded ovary sections revealed that intense heat-induced antigen retrieval, appropriate blocking, and appropriate antibody dilutions were necessary for optimization of mouse-on-mouse immunohistochemistry. Subsequently, we applied the optimized immunostaining method to determine expression profiles of mouse ESR2 proteins in peripheral tissues and brain subregions. Our analyses revealed more localized distribution of mouse ESR2 proteins than previously assumed. Moreover, comparison of these results with those obtained in humans and rats using PPZ0506 revealed interspecies differences in ESR2 expression. We expect that our optimized methodology for immunohistochemical staining of mouse ESR2 proteins will help researchers to solve multiple lines of controversial evidence concerning ESR2 expression.

ОСОБЕННОСТИ БЕЛОК-ИНДУЦИРОВАННЫХ АССОЦИАТОВ ПРИ СВЕРХВЫСОКОМ РАЗВЕДЕНИИ АНТИТЕЛ К ИНТЕРФЕРОНУ-γ

The possibilities of using the channeling of low-energy (9-13 keV) electrons to determine the nature of changes in the positional order in the arrangement of molecular complexes of water and protein-induced associates at ultrahigh dilution of antibodies to interferon-γ without and in the presence of excipients were studied. We used the method of gas-discharge visualization, which makes it possible to record the parameters of the avalanche-streamer discharge caused by the field emission of electrons passing through the bulk and surface of the drop of the studied solutions. Studies of the structural organization of drug solutions were carried out on solutions of polyclonal affinity-purified human antibodies to interferon-γ. Solutions were obtained by repeated dilution of antibodies in purified water. Used solutions corresponding to four concentration points of antibodies. An analysis of the patterns of the gas-discharge image of solutions made it possible to determine the stereographic projections of the channels of electron motion formed by molecular complexes of solutions, as well as the energy and average concentration of electrons, their energy distribution associated with the features of the short-range order, the specifics of self-organization processes. The results obtained indicate significant changes in the water-dispersed medium during its dilution with human antibodies to interferon-γ.

Humanization and expression of IgG and IgM antibodies in plants as potential diagnostic reagents for Valley Fever.

Monoclonal antibodies (mAbs) are important proteins used in many life science applications, from diagnostics to therapeutics. High demand for mAbs for different applications urges the development of rapid and reliable recombinant production platforms. Plants provide a quick and inexpensive system for producing recombinant mAbs. Moreover, when paired with an established platform for mAb discovery, plants can easily be tailored to produce mAbs of different isotypes against the same target. Here, we demonstrate that a hybridoma-generated mouse mAb against chitinase 1 (CTS1), an antigen from Coccidioides spp., can be biologically engineered for use with serologic diagnostic test kits for coccidioidomycosis (Valley Fever) using plant expression. The original mouse IgG was modified and recombinantly produced in glycoengineered Nicotiana benthamiana plants via transient expression as IgG and IgM isotypes with human kappa, gamma, and mu constant regions. The two mAb isotypes produced in plants were shown to maintain target antigen recognition to CTS1 using similar reagents as the Food and Drug Administration (FDA)-approved Valley Fever diagnostic kits. As none of the currently approved kits provide antibody dilution controls, humanization of antibodies that bind to CTS1, a major component of the diagnostic antigen preparation, may provide a solution to the lack of consistently reactive antibody controls for Valley Fever diagnosis. Furthermore, our work provides a foundation for reproducible and consistent production of recombinant mAbs engineered to have a specific isotype for use in diagnostic assays.

A Novel Methodology for Detecting Variations in Cell Surface Antigens Using Cell-Tearing by Optical Tweezers.

The quantitative analysis of cell surface antigens has attracted increasing attention due to the antigenic variation recognition that can facilitate early diagnoses. This paper presents a novel methodology based on the optical “cell-tearing” and the especially proposed “dilution regulations” to detect variations in cell surface antigens. The cell attaches to the corresponding antibody-coated slide surface. Then, the cell-binding firmness between a single cell and the functionalized surface is assayed by optically tearing using gradually reduced laser powers incorporated with serial antibody dilutions. Groups B and B3 of red blood cells (RBCs) were selected as the experiment subject. The results indicate that a higher dilution called for lower power to tear off the cell binding. According to the proposed relative-quantitative analysis theory, antigenic variation can be intuitively estimated by comparing the maximum allowable dilution folds. The estimation result shows good consistency with the finding in the literature. This study suggests a novel methodology for examining the variation in cell surface antigens, expected to be widely capable with potential sensor applications not only in biochemistry and biophysics, but also in the micro-/nano- engineering field.

Establishment of a time-resolved immunoassay for acute kidney injury based on the detection of Kim-1.

AimTo establish a highly sensitive time‐resolved fluorescence immunoassay (TRFIA) of kidney injury molecule‐1 (Kim‐1) and evaluate its clinical value in acute kidney injury (AKI).MethodsThe Kim‐1‐TRFIA was established by the double‐antibody sandwich method, and the method was evaluated. The established Kim‐1‐TRFIA was used to detect the concentration of Kim‐1 in the serum of healthy controls and patients with AKI.ResultsThe optimal coating antibody concentration and optimal Eu3+‐labeled antibody dilution ratio for Kim‐1‐TRFIA are 1 μg/ml and 1:140, respectively. The linear range is 42.71–4666.69 pg/ml. The intra‐ and inter‐assay coefficients of variation are <10%. The specificity of our Kim‐1‐TRFIA is acceptable. The recovery is between 95.14% and 102.84%. The concentration of Kim‐1 in the serum of patients with AKI is 126.50 ± 67.99 pg/ml, which is significantly higher than that in the serum of healthy controls (49.72 ± 16.40 pg/ml, p < 0.001). Staging patients with AKI by glomerular filtration rate shows that the serum concentration of Kim‐1 increases significantly with increasing disease severity (p < 0.05).ConclusionA highly sensitive Kim‐1‐TRFIA was established. With this immunoassay, a good differential diagnosis can be made, and healthy people and AKI patients can be differentiated by detecting the concentration of Kim‐1 in the serum. Moreover, the severity of AKI patients can be determined.

Detection of SARS-CoV-2 proteins by immunohistochemistry in human tissues Pathology collaborative analysis

Introduction: The COVID-19 (coronavirus disease 2019) caused by SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) is associated with high mortality rates worldwide. Polymerase chain reaction (PCR) is extensively used for virus detection in both infected patients and deceased persons. PCR, however, gives no information about the localization of the virus in cells and tissues. Detection of spike and nucleocapsid proteins and viral ribonucleic acid (RNA) of the SARS-CoV-2 in situ might provide more information and aid in the discovery of the pathomechanism of cellular damage. There are several commercially available anti-spike and anti-nucleocapsid antibodies used to detect immunohistochemical reactions, though each gives different results. Objective: The goal of the present study was to compare the intensity and specificity of several anti-spike and antinucleocapsid antibodies in different dilutions in four Hungarian university departments. Method: Immunohistochemical reactions were performed on coded slides taken from infected lungs of 3 deceased and placenta samples with appropriate negative controls of formalin-fixed paraffin-embedded tissues, scanned, evaluated unanimously and analysed statistically by the assessors. Results: By comparing the intensity, dilution, background and reproducibility of the different primary antibodies, it was possible to select the antibodies with the best results. Conclusion: The antibodies selected with established dilutions can be used in further studies to detect SARS-CoV-2 proteins in surgical materials and in samples obtained during autopsy.

Development of an Indirect ELISA for the Detection of SARS-CoV-2 Antibodies in Cats.

Companion animals are susceptible to a variety of coronaviruses, and recent studies show that felines are highly susceptible to SARS-CoV-2 infection. RT-PCR diagnostic is currently the method of choice to detect the presence of SARS-CoV-2-specific viral nucleic acids in animal samples during an active infection; however, serological assays are critical to determine whether animals were exposed to the virus and to determine the seroprevalence of SARS-CoV-2-specific antibodies in a defined population. In this study, we utilized recombinant nucleocapsid (N) protein and the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2 expressed in E. coli (N) and mammalian cells (N, RBD) to develop indirect ELISA (iELISA) tests using well-characterized SARS-CoV-2-positive and -negative cat serum panels from previous experimental cat challenge studies. The optimal conditions for the iELISA tests were established based on checkerboard dilutions of antigens and antibodies. The diagnostic sensitivity for the detection of feline antibodies specific for the N or RBD proteins of the iELISA tests was between 93.3 and 97.8%, respectively, and the diagnostic specificity 95.5%. The iELISAs developed here can be used for high-throughput screening of cat sera for both antigens. The presence of SARS-CoV-2-specific antibodies in a BSL-2 biocontainment environment, unlike virus neutralization tests with live virus which have to be performed in BSL-3 laboratories.

Establishment of a High Content Image Platform to Measure NF-κB Nuclear Translocation in LPS-induced RAW264.7 Macrophages for Screening Anti-inflammatory Drug Candidates

High Content Image (HCI), an automatic imaging and analysis system, provides a fast drug screening method by detecting the subcellular distribution of protein in intact cells. This study established the first standardized HCI platform for lipopolysaccharide (LPS)-induced RAW264.7 macrophages to screen anti-inflammatory compounds by measuring nuclear factor-κB (NF-κB) nuclear translocation. The influence of the cell passages, cell density, LPS induction time and concentration, antibody dilution, serum, dimethyl sulfoxide, and analysis parameters on NF-κB nuclear translocation and HCI data quality was optimized. The BAY-11-7085, the positive control for inhibiting NF-κB, and the Western blot assay were separately employed to verify the stability and reliability of the platform. Lastly, the effect of BHA on NO release, iNOS expression, IL-1β, IL-6, and TNF-α mRNA in LPS-induced RAW264.7 cells was detected. The optimal conditions for measuring NF-κB translocation in LPS-induced RAW264.7 cells by HCI were established. Cells that do not exceed 22 passages were seeded at a density of 10 k cells/well and pretreated with compounds following 200 ng/mL LPS for 40 min. Parameters including the nuclear area of 65 μm2, cell area of 80 μm2, collar of 0.9 μm, and sensitivity of 25% were recommended for image segmentation algorithms in the analysis workstation. Benzoylhypaconine from aconite was screened for the first time as an anti-inflammatory candidate by the established HCI platform. The inhibitory effect of benzoylhypaconine on NF-κB translocation was verified by Western blot. Furthermore, benzoylhypaconine reduced the release of NO, inhibited the expression of iNOS, and decreased the mRNA levels of IL-1β, IL-6, and TNF-α. The established HCI platform could be applied to screen anti-inflammatory compounds by measuring the NF-κB nuclear translocation in LPS-induced RAW264.7 cells.

Development of flow cytometry-based assays to assess the ability of antibodies to bind to SARS-CoV-2-infected and spike-transfected cells and mediate NK cell degranulation.

Since the beginning of the SARS‐CoV‐2 pandemic, antibody responses and antibody effector functions targeting SARS‐CoV‐2‐infected cells have been understudied. Consequently, the role of these types of antibodies in SARS‐CoV‐2 disease (COVID‐19) and immunity is still undetermined. To provide tools to study these responses, we used plasma from SARS‐CoV‐2‐infected individuals (n = 50) and SARS‐CoV‐2 naive healthy controls (n = 20) to develop four specific and reproducible flow cytometry‐based assays: (i) two assessing antibody binding to, and antibody‐mediated NK cell degranulation against, SARS‐CoV‐2‐infected cells and (ii) two assessing antibody binding to, and antibody‐mediated NK cell degranulation against, SARS‐CoV‐2 Spike‐transfected cells. All four assays demonstrated the ability to detect the presence of these functional antibody responses in a specific and reproducible manner. Interestingly, we found weak to moderate correlations between the four assays (Spearman rho ranged from 0.50 to 0.74), suggesting limited overlap in the responses captured by the individual assays. Lastly, while we initially developed each assay with multiple dilutions in an effort to capture the full relationship between antibody titers and assay outcome, we explored the relationship between fewer antibody dilutions and the full dilution series for each assay to reduce assay costs and improve assay efficiency. We found high correlations between the full dilution series and fewer or single dilutions of plasma. Use of single or fewer sample dilutions to accurately determine the response rates and magnitudes of the responses allows for high‐throughput use of these assays platforms to facilitate assessment of antibody responses elicited by SARS‐CoV‐2 infection and vaccination in large clinical studies.

Immunocytochemistry of effusion fluids: Introduction to SCIP approach.

Due to the remarkably wide morphologic spectrum of reactive mesothelial cells, some of the effusion fluids may be difficult to interpret with objective certainty by cytomorphology alone. Cytomorphology of well to moderately differentiated adenocarcinomas (responsible for the bulk of malignant effusions) may overlap with floridly reactive mesothelial cells. Even mesotheliomas including diffuse malignant epithelioid mesothelioma, are usually cytomorphologically bland without unequivocal features of malignancy.The intensity of challenge depends on the interpreter’s training or experience level, institutional demographics of patients (such as type of prevalent diseases, predominant sex and age group), technical support, and quality of cytopreparatory processing. In general immunocytochemistry is valuable adjunct to facilitate objective interpretation with or without other ancillary techniques as indicated. An increasing number of immunomarkers is further refining the contribution of immunohistochemistry to this field.However, application of immunohistochemistry to effusion fluids is relatively challenging because of many variables. Multiple factors such as delay after specimen collection, specimen processing related factors including fixation and storage; ambient conditions under which paraffin blocks are archived (for retrospective testing); antigen retrieval method; duration of antigen retrieval step; antibody clone and dilution; and antibody application time are identical to application of immunohistochemistry in other areas. The significant challenge related to the potential compromization of the immunoreactivity pattern due to exposure to non-formalin fixatives / reagents is also applicable to effusion fluid specimens.The immunoreactivity results would be compared and corelated with cumulative metadata based on the reported studies performed and validated on formalin-fixed paraffin-embedded tissue sections. Deviating from such protocols may lead to suboptimal results, which is not uncommon in clinical practice with potential compromization of patient care and related liability. Because of this, it is critical to perform immunocytochemistry on formalin-fixed cell-block sections only.In addition, unless the interpretation criteria for immunohistochemical evaluation of effusion fluids are not modified specifically, it may not be productive in resolving some challenging cases. However, this aspect is not well elaborated in the literature. A basic and critical challenge is finding and locating the cells of interest in cell-block sections of effusion fluids. A unique approach is to choose a fundamental immunopanel which highlight the mesothelial and inflammatory cells in reactive effusion fluids to create the basic map. This allows detection of a ‘second-foreign’ population which can be immunocharacterized further with the help of subtractive coordinate immunoreactivity pattern (SCIP) approach elaborated here.

Immunocytochemistry of effusions: Processing and commonly used immunomarkers.

Definitive cytopathological interpretation of some of the effusion fluids may not be possible based on cytomorphological evaluation alone. As discussed in other reviews, this is due to various reasons specifically applicable to effusion fluids including remarkably wide morphologic spectrum of reactive mesothelial cells overlapping with some well to moderately differentiated metastatic carcinoma. The challenge is subject to various factors including level of interpreter training or experience, institutional demographics (such as type of prevalent diseases, predominant sex and age group), technical advances in ancillary support, and expertise in cytopreparatory processing. In such cases immunohistochemistry performed on cell-block sections is simple objective adjunct with or without other ancillary techniques. Ongoing increase in number of immunomarkers along with rabbit monoclonal antibodies with relatively higher affinity is further refining this field. SCIP (subtractive coordinate immunoreactivity pattern) approach, discussed as separate dedicated review article, facilitates refined interpretation of immunoreactivity pattern in coordinate manner on various serial sections of cell-blocks. However, many variables such as delay after specimen collection, specimen processing related factors including fixation and storage; ambient conditions under which paraffin blocks are archived (for retrospective testing); antigen retrieval method; duration of antigen retrieval step; antibody clone and dilution; and antibody application time are common with application of immunohistochemistry in other areas. This review is dedicated to highlight technical aspects including processing of effusion specimens for optimum immunocytochemical evaluation along with commonly used immunomarkers in effusion cytopathology. This review focuses on the technical and general information about various immunomarkers.

Fast and real-time electrical transistor assay for quantifying SARS-CoV-2 neutralizing antibodies

Due to the SARS-CoV-2 pandemic renewed attention has been directed towards viral neutralization assays and neutralizing antibodies quantification, for vaccine pre-clinical trials and determining vaccine efficacy over time. The gold standard to assess antibody titer is the plaque reduction neutralization test, an end-point assay which evaluates the highest serum antibody dilution that neutralizes viral replication, by inspecting the cytopathic effect induced on cell cultures. Here, we use planar, PEDOT:PSS-based organic electrochemical transistors for real-time, remote-controlled, reliable and fast electrical monitoring of the cytopathic effect induced by SARS29 CoV-2 on Vero E6 cell lines, allowing the quantification of serum neutralizing titer. Our low-cost and scalable device has the potential to speed-up large-scale viral neutralization screening without the need for cancerous staining or highly specialized operators. Finally, the technology could be easily transferred to assess neutralizing antibody response towards different viruses in their permissive cell substrates.

Optical Properties of Crystalline Lactose Fluidized with Dilutions of Various Substances in the Terahertz Frequency Range

Lactose is a commonly used component of pharmaceutical medications in tablet form. It was previously shown that lactose changes conformationally after saturation in fluidized beds with active pharmaceutical ingredients obtained by repeated dilution of antibodies to interferon-gamma in combination with an external intensive vibration treatment. Moreover, it was revealed that these solutions are self-organized dispersed systems in which nano-objects are formed. Their biological activity and mechanism of action were previously established as well. The current work was dedicated to investigating the optical properties of fluidized lactose powders in the terahertz frequency range. Spectral analyses of powders of crystalline lactose saturated in fluidized beds with a diluted solution of either glycine buffer, antibodies to interferon-gamma, or water were carried out, intact lactose served as a control. All powders were tableted before testing. In the course of the study, the macroscopic parameters of the tablets were established, at which they had a stable shape and their THz optical properties had no parasitic diffraction losses. These tablets were analyzed using terahertz time-domain spectroscopy in the frequency range of 0.2–2.6 THz. The differentiation between the spectra was conducted using a principal component analysis. The differences between intact lactose and the lactose saturated with any of studied solutions were demonstrated. Additionally, lactose saturated with solutions of multiple dilutions of a substance (antibodies or glycine buffer) differed not only from intact lactose, but also from lactose saturated with a diluted solution of water. Moreover, discrimination of lactose formulations saturated with different substances (antibodies or glycine buffer) was also possible. Additionally, intact lactose differed from lactose saturated with diluted water. The methods reported could be useful for the quality control of the medications based on the technology of repeated dilution of an original substance.

Development of an indirect ELISA method based on the VP4 protein for detection antibody against duck hepatitis A virus type 1

In Picornavirus, the VP4 gene is located inside the viral capsid, but the antibodies it produces have neutralizing activity. At the same time, we know that the VP4 gene is relatively conserved among the four structural proteins, which makes the usage VP4 protein-based antigen potentially meaningful. Therefore, we used purified duck hepatitis A virus type 1 (DHVA-1) recombinant VP4 protein as the coating antigen to establish an indirect method. The optimal antigen, serum and enzyme-labeled antibody dilutions were 1:400 (3.375 μg/mL), 1:80 and 1:1600, respectively. The optimal blocking buffer was 1% skimmed milk powder. The cutoff value was determined to be 0.203, and the analytical sensitivity was 1:1600. The established ELISA method has good specificity, repeatability and sensitivity. It has a high coincidence rate of 70.8 % with the DHVA-1 as the coating antigen. So, it can be used for the detection of DHAV-1 serum antibodies.

Current epidemiological situation on leptospirosis in the territories adjacent to Lake Baikal

Background. In presence the epizootiological situation on leptospirosis in the territories adjacent to Lake Baikal is not studied well. The showing up of natural focus, discovering of host animal’s species composition and etiological structure of leptospirosis causative agents are essential to plan any epidemiological response and to organize the specific prophylaxis.Aim: to analyze contemporary characteristics of leptospirosis natural foci at the territories adjacent to Lake Baikal.Materials and methods. From 2011 till 2020 the territory of nine Irkutsk Region's administrative districts was investigated. Trapping has been done at four kind of sites (wetland, meadow, forest-shrub, steppificated meadow) and boroughs. 1152 small mammals which belonged to 35 species were collected. Collected samples were studied by complex of methods. Serogroup belonging was detected by micro agglutination and decomposition test, and genomic species – by Microflex LT mass analyzer.Results. A leptospirosis natural foci in the Irkutsk city boundary was newly founded and there were isolated (from small mammals) four pathogenic cultures of leptospiras belonging to Javanica serogroup. Main hosts of causative agents at the territories adjacent to Lake Baikal were defined (insect-eating mammals: tundra shrew, largetoothed Siberian shrew, even-toothed shrew, shrew-mouse, and rodents: root vole, ground vole, narrow-skulled vole, sewer rat). By micro agglutination were defined pathogenic leptospirosis serogroups (Icterohaemorrhagiae, Grippotyphosa, Javanica, Pomona, Sejroe, Autumnalis, Australis), all over 2.4 ± 0.45 % seropositive. Antibody dilution at survey animals ranged from 1 : 20 to 1 : 640. On the grounds of PCR results the mean value of infected animals in studied sample was 16.4 ± 1.14 %. Positive PCR findings were at 19 from 35 animal species.Conclusion. At the territories adjacent to Lake Baikal In the Baikal region, the proportion of animals that have had contact with pathogenic leptospira is quite high. Natural focus of this infection are highly pervasive in this region, they are evolved mainly in dewy sites.

Super Resolution Localization Microscopy Enables Quantitative Diagnosis of Multiple Myeloma

Multiple myeloma (MM) is a bone marrow-based malignancy that has a range of consequences resulting from either its direct effects on the bone marrow microenvironment- causing anaemia, more extensive myelosuppression and bone lysis - or its indirect effects on the kidney and other organ systems. Over the past several years, the introduction of autologous stem cell transplantation, immunomodulatory drugs, proteasome inhibitors, histone deacetylase inhibitors, and monoclonal antibodies has substantially improved survival outcomes. However, MM remains biologically heterogeneous with significant variability among patients in terms of clinical features, response to therapy and overall survival (OS). Several prognostic variables can help predict this variability in outcomes ranging from clinical based systems such as the International Staging System (ISS) to more advanced molecular characterizations of the myeloma PCs by cytogenetics and gene expression profiling. However with the advancement in technology utilized for laboratory testing and the emergence of new treatments for MM, there has been an evolution in the significance of these previously defined prognostic markers with time. Assessment of disease activity and depth of response continues to be a moving target in MM. Super-resolution fluorescence imaging is a major breakthrough in the field of optical imaging in this century. Super-resolution fluorescence imaging has been applied to a variety of biological imaging applications, including membrane, cytoskeletal and cytosolic proteins in fixed and living cells. Molecular motions can be quantified.To establish the detection limit and sensitivity threshold of dSTORM and FC, we used serial dilutions of anti-CD38 antibody to detect expression of CD38 on 8226 cells by Super Resolution localization Microscopy and flow cytometry (FC). Design six different antibody concentrations (300ng/ml, 30ng/ml, 10ng/ml, 3ng/ml, 1ng/ml, 0.3ng/ml) to label MM cells with immunofluorescence, and then detect them by flow cytometry. Similarly, design eight different antibody concentrations (1μg/ml, 300ng/ml, 30ng/ml, 10ng/ml, 3ng/ml, 1ng/ml, 0.3ng/ml, 0.1ng/ml), and perform the same treatment, perform immunofluorescence labeling on MM cells, and then perform super-resolution imaging, and calculate the density of CD38 protein on the surface of MM cells of each concentration. Figure 1A detects the ratio of the number of cells to the total number of cells, it can be seen from Figure 1A that when the antibody concentration is not less than 30ng/ml, almost all MM thinners can detect the signal, but when the antibody concentration is less than at 30ng/ml, only a few or no cells can detect the signal. This shows that when the antibody concentration is lower than 30ng/ml, the sensitivity of flow cytometry is no longer sufficient to detect CD38 protein on the surface of MM cells. From Figure 1B, it can be seen that even when the CD38 antibody concentration is 0.3ng/ml, super-resolution imaging can still accurately identify each CD38 antigen molecule on the surface of MM cells, and the statistical CD38 protein density is 8.5864±1.4180/μm2. This shows that the sensitivity of super-resolution is much higher than that of streaming. DisclosuresNo relevant conflicts of interest to declare.