Maximum Dilution Research Articles

A-097 Body Fluid Validation for 11 Analytes on a Chemistry Analyzer

Abstract Background Most clinical chemistry assays are intended for use in serum, plasma, urine, and/or CSF with no performance characteristics on body fluids. However, testing of body fluids can be medically necessary for certain diagnoses. Such as determining the cause of fluid buildup in peritoneal spaces, pleural effusions, whether effusions are transudates or exudates, and even diagnosing some joint disorders. To provide these medically necessary tests, the burden is on the laboratory to perform regulatory-required validations. Herein is the validation of 11 clinical chemistry analytes in several common body fluids. Methods Mixing and stability studies were performed for the following analytes on the Roche cobas c702 (Roche Diagnostics), using the methods intended for use with serum/plasma samples: Albumin, Amylase, Bilirubin, Urea Nitrogen, Cholesterol, Creatinine, Glucose, Lactate Dehydrogenase, Lipase, Protein, and Triglycerides. Six individual fluid samples were mixed with the intended use matrix in 3 ratios: 1:3, 1:1, and 3:1. The following matrices were evaluated: pleural, peritoneal, JP drainage, synovial, and pancreatic cyst fluids. Stabilities were tested for 7 days at room temperature (RT) and 2-8°C. A maximum dilution study was also performed for Amylase. Results Mixing Study: The results are shown in the table below: Stability: For most body fluids, analytes were stable for 7 days at RT and 2-8 °C. The following analytes displayed shorter stability at RT: Bilirubin (peritoneal: 2 days, pleural: 1 day, JP drainage: 1 day), Urea Nitrogen (JP drain fluid: 2 days), Lipase (peritoneal fluid 5 days), and Triglycerides (peritoneal fluid: 5 days). Lactate dehydrogenase in pleural fluid showed a stability of 5 days at RT and 2 days at 2-8 °C. Maximum Dilution A 1:505 compounded manual and automated dilution displayed a <7% bias. Conclusions No sample matrix interferences were observed for any of the body fluids-analyte pairs tested allowing for this medically necessary testing.

B-316 Evaluation of an FDA-cleared Chromogranin A Assay on an Automated Analyzer

Abstract Background Chromogranin A (CgA) is a hydrophilic and acidic protein, present in the chromaffin granules of neuroendocrine cells. It is the preferred tumor marker for the monitoring of neuroendocrine tumors (NETS), which are often found in the lungs, appendix, pancreas, and gastrointestinal tract. Previous CgA assays were performed by manual ELISA testing, and were not FDA-approved, leading to a longer, more rigorous validation process and longer run times. The introduction of an automated, FDA-cleared CgA assay allows the laboratory to process patient samples more quickly and reliably, shortening a multiple hour process to minutes. The objective of this study was to evaluate the performance of an automated CgA assay recently cleared by the FDA. Methods We validated the the B·R·A·H·M·S™ Chromogranin A II (CGAII) assay on the automated B·R·A·H·M·S KRYPTOR Compact PLUS (Thermo Fisher Scientific Inc.). The CGAII assay is an immunofluorescent assay, which uses Time-Resolved Amplified Cryptate Emission (TRACE) to quantitate CGA in human serum. Assay performance characteristics were evaluated including measuring range (MR) and maximum dilution, precision, method comparison, stability, reference interval, stability, and carryover. Results The MR for CGAII was established with triplicate analysis of serum at 8 levels, ranging from 25.8 - 2,700 ng/mL. Linearity was demonstrated with a slope of 0.907, intercept of 1.948 and observed error of 2.54 ng/mL. Recovery at each point ranged from 83.3-110.3%. Carryover (<5.0 ng/mL) was not observed at a concentration approximately 2 times the upper limit of the MR. A 1:25 or 1:500 automatic dilution was determined to be acceptable (% bias <16%). Intraday precision coefficient of variation (CV) ranged from 4.1-1.7% (SDs from 2.32-6.64 ng/mL) at concentrations of 56.7 and 393.0 ng/mL. The interday CVs ranged from 6.0-7.1% (SDs from 3.43 - 28.84 ng/mL) at concentrations of 57.5 and 403.8 ng/mL. Twenty samples with concentrations ranging from 56.4-2,907 ng/mL were compared to a reference laboratory utilizing a similar methodology. The correlation coefficient was 0.9934, with a slope of 1.014, and intercept of -32.95. An additional twenty samples with concentrations ranging from 67.0-2,446 ng/mL were compared to a CgA ELISA kit (CGA-ELISA-NG, CISBIO) to determine the bias between the two methodologies. The correlation coefficient was 0.9902, with a slope of 0.845, and intercept of -15.31, and a bias of -19.9%. The serum samples were stable for 3 days when stored at 2-8°C. Recovered CgA concentrations fell outside of the 95% confidence interval after day 3 at 2-8°C. The manufacturer provided reference interval of <187 ng/mL was verified by testing 20 healthy adult samples, all of which fell within this range. Conclusions This assay has been validated and shown to be an accurate and precise method for monitoring CgA. Further, the method was validated on an automated analyzer to allow expeditious turnaround time in a core laboratory.

B-287 Comparing a Recently FDA-Cleared Free Phenytoin Assay to a Laboratory Developed Test

Abstract Background Phenytoin is an anti-convulsant drug that is commonly prescribed to epileptic patients to control seizures. The drug circulates in two forms: protein-bound (90%), and the free, biologically active form (10%). In certain circumstances, such as protein-binding disruption, hypoalbuminemia, and drug-drug interactions, the total phenytoin serum concentration may not accurately reflect active phenytoin levels. Therefore, a free phenytoin level (i.e. non-protein bound) may be indicated. Until recent FDA-clearance, we utilized an in-house, laboratory-development test (LDT) to deliver these needed results. The purpose of this study was to validate the FDA-cleared assay and compare its performance characteristics to a free phenytoin LDT. Methods Validation of the FDA-cleared assay was performed on an automated chemistry analyzer (Roche Diagnostics, cobas c502), utilizing a kinetic interaction of microparticles in a solution (KIMS) method. Both the FDA and LDT assay used the same free phenytoin reagent, however the calibrators and pipetting parameters for each assay were different. FDA assay used a six-point calibration at concentrations of: 0, 2.5, 5, 10, 20, 40 µg/mL (Preciset TDM I Calibrators), whereas the LDT assay used a six-point calibration at concentrations of: 0.0, 0.5, 1.0, 2.0, 3.0, 4.0 µg/mL (cobas FP Free Phenytoin Calibrators). The following performance characteristics were determined for the FDA-cleared assay and compared to the current LDT: analytical measurement range, precision, accuracy, and maximum dilution. Free forms of the drug were obtained as a filtrate through centrifugation using a molecular weight cut-off filter (30 kDa, Millipore), spun at 3600 rpm for twenty minutes. Results Conclusions The FDA-cleared free phenytoin assay is suitable for use in the clinical laboratory producing similar results to our LDT. Switching to the FDA-cleared assay reduces the regulatory burden and eliminates the need for transferring reagent into open channel cassettes and manual dilutions.

Investigating the immunological activity of the Hsp70-P113 fusion protein for Mycoplasma ovipneumoniae detection: a groundbreaking study

BackgroundMycoplasmal pneumonia of sheep and goats (MPSG) is an important infectious disease that threatens sheep and goat production worldwide, and Mycoplasma ovipneumoniae (Movi) is one of the major aetiological agents causing MPSG. The aim of this study was to investigate the immunological activity of the Hsp70‒P113 fusion protein derived from Movi and to develop a serological assay for the detection of Movi.MethodsThis study involved codon optimization of the dominant antigenic regions of Movi heat shock protein 70 (Hsp70) and adhesin P113. Afterwards, the optimized sequences were inserted into the prokaryotic expression vector pET-30a( +) through tandem linking with the aid of a linker. Once a positive recombinant plasmid (pET-30a-rHsp70-P113) was successfully generated, the expression conditions were further refined. The resulting double gene fusion target protein (rHsp70‒P113) was subsequently purified using ProteinIso® Ni–NTA resin, and the reactivity of the protein was confirmed via SDS‒PAGE and Western blot analysis. An indirect enzyme-linked immunosorbent assay (i-ELISA) technique was developed to detect Movi utilizing the fusion protein as the coating antigen. The specificity, sensitivity, and reproducibility of all methods were assessed after each reaction parameter was optimized.ResultsThe resulting rHsp70-P113 protein had a molecular weight of approximately 51 kDa and was predominantly expressed in the supernatant. Western blot analysis demonstrated its favourable reactivity. The optimal parameters for the i-ELISA technique were as follows: the rHsp70-P113 protein was encapsulated at a concentration of 5 μg/mL; the serum was diluted at a ratio of 1:50; the HRP-labelled donkey anti-goat IgG was diluted at a ratio of 1:6,000. The results of the cross-reactivity assays revealed that the i-ELISA was not cross-reactive with other goat-positive sera against Mycoplasma mycodies subsp. capri (Mmc), Mycoplasma capricolum subsp. capripneumoniae (Mccp), Mycoplasma arginini (Marg), orf virus (ORFV) or enzootic nasal tumour virus of goats (ENTV-2). The sensitivity of this method is high, with a maximum dilution of up to 1:640. The results of the intra- and inter-batch replication tests revealed that the coefficients of variation were both less than 10%, indicating excellent reproducibility. The analysis of 108 clinical serum samples via i-ELISA and indirect haemagglutination techniques yielded significant findings. Among these samples, 43 displayed positive results, whereas 65 presented negative results, resulting in a positivity rate of 39.8% for the i-ELISA method. In contrast, the indirect haemagglutination technique identified 20 positive samples and 88 negative samples, resulting in a positivity rate of 18.5%. Moreover, a comparison between the two methods revealed a conformity rate of 78.7%.ConclusionThe results obtained in this study lay the groundwork for advancements in the use of an Movi antibody detection kit, epidemiological inquiry, and subunit vaccines.

Establishment of ELISA method for canine adenovirus type 1.

Canine adenovirus (CAdV) had a high prevalence in fox populations and induced fox encephalitis. No ELISA kits specifically for CAdV-1 antigen had been commercialized for foxes in China. It is crucial to develop a rapid and accurate ELISA method for detecting of CAdV-1. The monoclonal antibodies (mAbs, IgG1A) and HRP-labeled polyclonal antibodies (pAbs) were used to establish the ELISA method in this experiment. The results showed that the optimal concentration and coating time for the mAbs (IgG1A) were 2.15 μg/mL and overnight at 4°C, respectively. The dilution ratio of the HRP-labeled pAbs was 1:2000. Five percent skimmed milk was selected as the blocking agent. The optimal incubation times for blocking, CAdV-1, and HRP-labeled pAbs were all 1 h. The cut-off value for negative rectal swab was determined to be 0.366 ± 0.032. The maximum dilution ratio was 100 TCID50/mL. The ELISA method was positive to CAdV-1, and that was negative to CAdV-2, Canine Parvovirus (CPV) and Canine Distempervirus (CDV). The ELISA method showed good repeatability, sensitivity, and specificity. Compared with RT-PCR, the sensitivity, specificity, and coincidence rates of the ELISA method were 93.75, 90.9, and 92.86%, respectively. These results indicate that the established ELISA method can be used for the large-scale screening and epidemiology surveillance of CAdV-1 in foxes.

Development of an ELISA method for detection of antibodies against Mycoplasma hyopneumoniae based on Mhp336 protein

This study aims to establish an ELISA method with high specificity for the detection of antibodies against Mycoplasma hyopneumoniae. Firstly, we constructed a recombinant strain Escherichia coli BL21(DE3)-pET-32a(+)-mhp336 to express the recombinant protein Mhp336 and used the purified Mhp336 as the coating antigen. Then, we optimized the ELISA parameters, including antigen concentration, blocking buffer, blocking time, dilution of serum, incubation time with serum, secondary antibody dilution, secondary antibody incubation time, colorimetric reaction time, and cut-off value. Afterwards, reproducibility experiments were conducted, and the cross reactivity of Mhp366 with other antisera of porcine major pathogens and the maximum dilution ratios of the sera were determined. Finally, 226 porcine serum samples were detected using the method established in this study, a commercial ELISA kit for M. hyopneumoniae antibody detection, and a convalescent serum ELISA kit for M. hyopneumoniae antibody detection. The detection results of the three methods were compared to evaluate the sensitivity and specificity of the ELISA method established in this study. For this method, the optimal antigen concentration, blocking buffer, blocking time, dilution of serum, incubation time with serum, secondary antibody dilution, secondary antibody incubation time, and colorimetric reaction time were 0.05 μg/mL, PBS containing 2.5% skim milk, 1 h, 1:500, 0.5 h, 1:10 000, 1 h, and 5 min, respectively. Validation of the ELISA method based on Mhp336 showed a cut-off value of 0.332. The coefficients of variation of both intra-batch and inter-batch kits were below 7%. The detection results of porcine serum samples indicated that the method established in this study outperformed the commercial ELISA kit and the convalescent serum ELISA kit for M. hyopneumoniae antibody detection in terms of sensitivity and specificity. We successfully established an ELISA method for detecting the antibodies against M. hyopneumoniae based on Mhp336 protein. This method demonstrated high sensitivity and specificity, serving as a tool for the prevention of mycoplasmal pneumonia of swine in pig farms.

Quantum dot fluorescent microsphere-based immunochromatographic strip for detecting PRRSV antibodies

Porcine reproductive and respiratory syndrome (PRRS) is an immunosuppressive disease caused by the porcine reproductive and respiratory syndrome virus (PRRSV). Current vaccine prevention and treatment approaches for PRRS are not adequate, and commercial vaccines do not provide sufficient cross-immune protection. Therefore, establishing a precise, sensitive, simple, and rapid serological diagnostic approach for detecting PRRSV antibodies is crucial. The present study used quantum dot fluorescent microspheres (QDFM) as tracers, covalently linked to the PRRSV N protein, to develop an immunochromatography strip (ICS) for detecting PRRSV antibodies. Monoclonal antibodies against PRRSV nucleocapsid (N) and membrane (M) proteins were both coated on nitrocellulose membranes as control (C) and test (T) lines, respectively. QDFM ICS identified PRRSV antibodies under 10 min with high sensitivity and specificity. The specificity assay revealed no cross-reactivity with the other tested viruses. The sensitivity assay revealed that the minimum detection limit was 1.2 ng/mL when the maximum dilution was 1:2,048, comparable to the sensitivity of enzyme-linked immunosorbent assay (ELISA) kits. Moreover, compared to PRRSV ELISA antibody detection kits, the sensitivity, specificity, and accuracy of QDFM ICS after analyzing 189 clinical samples were 96.7%, 97.9%, and 97.4%, respectively. Notably, the test strips can be stored for up to 6 months at 4 °C and up to 4 months at room temperature (18–25 °C). In conclusion, QDFM ICS offers the advantages of rapid detection time, high specificity and sensitivity, and affordability, indicating its potential for on-site PRRS screening.Key points• QDFM ICS is a novel method for on-site and in-lab detection of PRRSV antibodies• Its sensitivity, specificity, and accuracy are on par with commercial ELISA kits• QDFM ICS rapidly identifies PRRSV, aiding the swine industry address the evolving virus

Adaptive laboratory evolution of Clostridium autoethanogenum to metabolize CO2 and H2 enhances growth rates in chemostat and unravels proteome and metabolome alterations.

Gas fermentation of CO2 and H2 is an attractive means to sustainably produce fuels and chemicals. Clostridium autoethanogenum is a model organism for industrial CO to ethanol and presents an opportunity for CO2-to-ethanol processes. As we have previously characterized its CO2/H2 chemostat growth, here we use adaptive laboratory evolution (ALE) with the aim of improving growth with CO2/H2. Seven ALE lineages were generated, all with improved specific growth rates. ALE conducted in the presence of 2% CO along with CO2/H2 generated Evolved lineage D, which showed the highest ethanol titres amongst all the ALE lineages during the fermentation of CO2/H2. Chemostat comparison against the parental strain shows no change in acetate or ethanol production, while Evolved D could achieve a higher maximum dilution rate. Multi-omics analyses at steady state revealed that Evolved D has widespread proteome and intracellular metabolome changes. However, the uptake and production rates and titres remain unaltered until investigating their maximum dilution rate. Yet, we provide numerous insights into CO2/H2 metabolism via these multi-omics data and link these results to mutations, suggesting novel targets for metabolic engineering in this bacterium.

Development and characterization of a novel nanobody with SRMV neutralizing activity

Peste des petits ruminants (PPR) is an acute, contact infectious disease caused by the small ruminant morbillivirus (SRMV), and its morbidity in goats and sheep can be up to 100% with significant mortality. Nanobody generated from camelid animals such as alpaca has attracted wide attention because of its unique advantages compared with conventional antibodies. The main objective of this study was to produce specific nanobodies against SRMV and identify its characteristics. To obtain the coding gene of SRMV-specific nanobodies, we first constructed an immune phage-displayed library from the VHH repertoire of alpaca that was immunized with SRMV-F and -H proteins. By using phage display technology, the target antigen-specific VHHs can be obtained after four consecutive rounds of biopanning. Results showed that the size of this VHH library was 2.26 × 1010 CFU/mL and the SRMV-F and -H specific phage particles were greatly enriched after four rounds of biopanning. The positive phage clones were selected and sequenced, and total of five independent different sequences of SRMV-specific nanobodies were identified. Subsequently, the DNA fragments of the five nanobodies were cloned into E. coli BL21(DE3), respectively, and three of them were successfully expressed and purified. Specificity and affinity towards inactivated SRMV of these purified nanobodies were then evaluated using the ELISA method. Results demonstrated that NbSRMV-1-1, NbSRMV-2-10, and NbSRMV-1-21 showed no cross-reactivity with other antigens, such as inactivated BTV, inactivated FMDV, His-tag labeled protein, and BSA. The ELISA titer of these three nanobodies against inactivated SRMV was up to 1:1000. However, only NbSRMV-1-21 displayed SRMV neutralizing activity at a maximum dilution of 1:4. The results indicate that the nanobodies against SRMV generated in this study could be useful in future applications. This study provided a novel antibody tool and laid a foundation for the treatment and detection of SRMV.

Thermal analysis of AISI 1020 low carbon steel plate agglutinated by gas tungsten arc welding technique: a computational study of weld dilution using finite element method

An important goal in a number of optimization studies is a high-quality weld joint. Thermal analysis of AISI 1020 low carbon steel plate agglutinated by gas tungsten arc welding technique was carried out using S 2021 version. With SOLIDWORKS Premium, the simulation was run. The simulation was performed using the Thermal Simulation programme with 20 weld runs. With the findings of the initial study serving as a sensor, a design study was conducted. A total of 15 runs were completed, and the weld dilution and thermal conductivity responses were available. A range of welding temperatures including 3397 to 3688 °C were experimentally applied in the joining process of AISI 1020 low carbon steel plate of 10 mm thickness, and a strain gauge indicator was used to measure the thermal stresses induced in the steel plate. However, minimum and maximum weld dilution values of 73.1 and 46.8% were obtained with FEM at an input of arc heat of 66.4 and 37.2 J/mm, while the minimum and maximum weld dilution values of 71.55 and 45.5% were computed using experimental approach at the same heat input. On the other hand, maximum and minimum weld dilution of 71.55 and 44.5% were computed from experimental process at minimum and maximum welding current of 199.77 and 250.23 A, while 73.1 and 46.8% were obtained for the maximum and minimum weld dilution through FEM procedure at the same welding input variables. Hence, gas tungsten arc welding input parameters should be properly selected and controlled during welding operation, in order to minimize thermal effects and welding flaws such as high dilution rate.

Mycoplasma synoviae LP78 is a fibronectin/plasminogen binding protein, putative adhesion, and potential diagnostic antigen.

Mycoplasma synoviae (M. synoviae) is one of the major poultry pathogens causing infectious synovitis, airsacculitis, a high incidence of shell breakage, and egg production loss. However, the pathogenesis of M. synoviae remains unclear. Adhesion of mycoplasmas to host cells is a crucial step in infection and colonization. The purpose of this study was to determine the adhesive function of a putative P80 family lipoprotein (LP78) and evaluate its application in the detection of antibodies against M. synoviae. Recombinant LP78 (rLP78) was expressed in the supernatant component of Escherichia coli and mouse anti-rLP78 serum was prepared. Bioinformatic analysis and western blotting results revealed that LP78 was conservative among M. synoviae strains. It was distributed not only in the cytoplasm but also on the membrane of M. synoviae through western blotting and indirect immunofluorescence (IFA). The adherence of M. synoviae to DF-1 cells was significantly inhibited by mouse anti-rLP78 serum (p < 0.01). IFA revealed that rLP78 adhered to DF-1 cells, and this adherence was prevented by mouse anti-rLP78 serum. Furthermore, rLP78 was found to bind to the DF-1 cells membrane proteins in a dose-dependent manner by enzyme-linked immunosorbent assay (ELISA). Screening of DF-1 cells membrane proteins by western blotting showed that proteins with molecular weight of 35-40 kDa and 55-70 kDa bound to rLP78. Moreover, rLP78 was identified to be a fibronectin/plasminogen binding protein. The sensitivity and specificity of rLP78-based iELISA were 85.7 and 94.1%, respectively. The maximum dilution of positive serum (HI titer, 1:128) detected via rLP78-based iELISA was 1:6,400, whereas that detected using a commercial ELISA kit was 1:12,800-1:25,600. Both rLP78-based iELISA and the commercial ELISA kit detected seroconversion after 7 days of challenge and immunization. No cross-reactivity with positive sera against other avian pathogens was observed in rLP78-based iELISA. Collectively, these results indicate that LP78 is a fibronectin/plasminogen-binding adhesion protein of M. synoviae and a potential diagnostic antigen. The present study will facilitate a better understanding of the pathogenesis of M. synoviae and the development of new diagnostic.

Cucumber mosaic virus among ornamental crops in the Russian Far East

Aim. To summarise and analyse scientific data on Cucumber mosaic virus strains (CMV – Cucumber mosaic virus) (Martellivirales: Bromoviridae, Cucumovirus) isolated from ornamental plants in the Far East of the Russian Federation. Discussion. The paper describes the genome structure and tripartite organization of CMV virions. Strains of this virus isolated from ornamental cultures in the south of the Russian Far East are described: Garden balsam, Common snapdragon, Dahlias, Hybrid gladiolus, Hybrid delphinium, Cambria, Indian canna, Cattleya, Tiger lily, Garden petunia, Primula obconica, Moth orchids, Fatshedera from Lize Freres, Weeping fig, Common hollyhock, Purple coneflower. The physicochemical properties (i. e. point of thermal inactivation, period of preservation of infectivity at a temperature of 20 °C, maximum dilution of juice causing disease of healthy plants) and the symptoms of these strains on a wide range of species and varieties of indicator plants are systematised according to the Russian Collection of East Asian Viruses, functioning at the Laboratory of Virology of the Federal Scientific Center for East Asia Terrestrial Biodiversity, Far Eastern Branch, Russian Academy of Sciences. Conclusions. The limiting factor in the development of floriculture is infectious diseases, among which viral infections (including CMV) are of the greatest importance, demonstrating a high strain diversity in the south of the Russian Far East. Thus, the study of the biological, physico‐chemical, immunochemical and molecular biological properties of CMV is an urgent task, as it opens up the possibility of studying isolates of this virus and classifying its strains taking into account individual characteristics and kinship relationships.

Continuous fermentation using high cell density cell recycle system for L-lactic acid production.

For complete utilization of high glucose at ∼100 g/L, a high cell density (HCD) continuous fermentation system was established using Lb. delbrueckii NCIM 2025 for the bioproduction of lactic acid (LA). An integrated membrane cell recycling system coupled with the continuous bioreactor, aided to achieve the highest 34.77 g/L h LA productivity and 0.94-0.98 g/g yield. ∼34 times higher productivity was observed (in comparison to batch fermentation conducted in this study), when the continuous operations were carried out at the maximum dilution rate and wet cell weight i.e. 0.36 h-1 and 230 g/L, respectively. These results show the potential of this method for large-scale lactic acid production because it not only produces high titers but also ensures that glucose is used effectively. The method's superior performance in comparison to earlier studies suggests it as an affordable and sustainable alternative for the production of LA.

Изучение токсичности отходов бурения методом биотестирования

The extraction of hydrocarbon raw materials has always been accompanied by a negative impact on environmental components. The object of the research is waste generated during the drilling process. The subject of the research is toxicity of drilling fluids for Daphnia magna. The urgency of the problem lies in solving the problem of their use and disposal. As a rule, these are multicomponent mixtures, which, when released into water bodies and soil cover, can exhibit toxic properties towards hydrobionts and pedofauna. The purpose of the research is to study the toxicity of drilling waste to the cladocera Daphnia magna. The objectives are as follows: to study the effect of drilling fluids on the survival of daphnia; determine the effect of the toxicant on the reproductive function of crustaceans. The article presents the results of a study of the effect of drilling waste on the freshwater crustacean Daphnia magna. During the experiment, the effect on daphnia of the liquid phase of both the original drilling waste and in the dilution range of 1:1 – 1:100 was studied using biotesting. Acute and chronic toxicity (4 and 30 days, respectively) was assessed by changes in the survival and reproductive function of crustaceans. It has been determined that in a short experiment, by the 4th day of observation, 40 and 64 % of crustaceans survived in the variants with the maximum dilution (1:10 and 1:100, respectively) of the drilling fluid. In a chronic experiment, the survival rate of daphnia decreased in proportion to the substance content. The toxicity of drilling waste also manifested itself in disruption of the reproductive function of crustaceans: delayed onset of sexual maturity and a decrease in the total number of juveniles. Based on the conducted research, the following conclusions are obtained. Drilling fluids exhibit acute and chronic toxicity to Daphnia magna, reducing their survival by 40 % when the toxicant is diluted 10 and 100 times, respectively. The effect of drilling solutions on the reproductive function of crustaceans is manifested in the inhibition of the timing of puberty (by 2–3 days compared to the control variant) and the overall fertility of daphnia: the number of juveniles that has appeared by the 30th day of the experiment was lower than the control values by 33–50 % (depending on drilling fluid concentrations).

Experimental evaluation of the possibility of determining bacterial endotoxins in typhoid Vi polysaccharide vaccines using the gel-clot test

Scientific relevance. Currently, only the rabbit pyrogen test is used to test the Vianvac® typhoid Vi polysaccharide vaccine for pyrogenicity. As part of the product specification file, the gel-clot test for bacterial endotoxins (BE) will improve the reliability of quality control, as well as harmonise the requirements for the vaccine with the requirements outlined for this group of medicinal products by leading world pharmacopoeias.Aim. This study aimed at an experimental assessment of the applicability of the gel-clot test to the quantification of BE in the typhoid Vi polysaccharide vaccine.Materials and methods. This study used samples from 5 batches of the typhoid Vi polysaccharide vaccine (0.5 mL/dose, solution for subcutaneous injection), LAL and TAL reagents. The analysis included the gel-clot test and the pyrogenicity test according to the State Pharmacopoeia of the Russian Federation (OFS.1.2.4.0006.15 and OFS.1.2.4.0005.15, respectively).Results. According to calculations, the BE limit for the tested vaccine was 96 EU/mL, and the maximum valid dilution (MVD) was 3200. The authors determined the regulatory requirements for typhoid Vi polysaccharide vaccine quality in terms of BE (not more than 48 EU/dose). The in vitro BE tests were positive at vaccine dilutions of 1/16 to 1/32 and negative at 1/64 to 1/256. The authors selected and validated a working vaccine dilution of 1/128. The BE content measured in the tested samples ranged from 0.24 to 0.48 EU/dose. The in vivo pyrogen tests were positive at dilutions of 1/16 to 1/128 and negative at 1/256 in all experiments with samples from 5 vaccine batches at dilutions ranging from 1/16 to 1/256.Conclusions. This study has experimentally proven that the gel-clot test can quantify BE in the typhoid Vi polysaccharide vaccine. The authors have recommended introducing the gel-clot BE test in the monograph of the State Pharmacopoeia of the Russian Federation on the typhoid Vi polysaccharide vaccine (FS.3.3.1.0012.15).

Computational Study of Deflagration-to-Detonation Transition in a Semi-Confined Slit Combustor

Systematic three-dimensional numerical simulations of flame acceleration and deflagration-to-detonation transition (DDT) in a semi-confined flat slit combustor are performed. The combustor is assumed to be partly filled with the stoichiometric ethylene–oxygen mixture at normal pressure and temperature conditions. The objective of the study is to reveal the conditions for DDT in terms of the minimum height of the combustible mixture layer in the slit, the maximum dilution of the mixture with nitrogen and the maximum slit width. The results of the calculations are compared with the available experimental data. The calculation results are shown to agree satisfactorily with the experimental data on the slit-filling dynamics, flame structure, the occurrence of the preflame self-ignition center, DDT, and detonation propagation. DDT occurs in the layer at a time instant when the flame accelerates to a velocity close to 750 m/s. DDT occurs near the slit bottom due to the formation of the self-ignition center ahead of the leading edge of the flame as a result of shock wave reflections from the walls of injector holes at the slit bottom and from the corners of the conjugation of the slit bottom and side walls. The decrease in the height of the mixture layer, the dilution of the mixture with nitrogen, and the increase in the slit width are shown to slow down flame acceleration in the slit and increase the DDT run-up distance and time until DDT failure. The obtained results are important for determining the conditions for mild initiation of detonation via DDT in semi-confined annular RDE combustors.

B-055 Comparison of Sensitivity Between Tissue Anti-Transglutaminase and Anti-Endomysium IgA and IgG in Serological Screening for Celiac Disease in a Large Clinical Laboratory in Brazil

Abstract Background Celiac Disease (CD) is a genetic and autoimmune disorder characterized by permanent intolerance to gluten found in foods, resulting in small intestine injury in children and adults. Anti-tissue transglutaminase (anti-tTG) and anti-endomysium (anti-EM) antibodies are important serological markers of this disease. The aim was to analyze the analytical sensitivity of these markers in a large-scale Clinical Laboratory routine. Methods We employed a pure sample and a series of dilutions in the analysis of the functional sensitivity of each marker (anti-tTG IgA/IgG and anti-EM IgA/IgG) and a database survey of a large Clinical Laboratory in Sao Paulo/Brazil, from January to December 2021, analyzing 60 000 samples with concomitant results of anti-tTG IGA and anti-EM IgA and 7075 samples with results of anti-tTG IgG and anti-EM IgG, from patients with no defined clinic condition. The anti-tTG tests were performed by fluorescence enzyme immunoassay (FEIA) and the anti-EM tests by indirect immunofluorescence (IFI), with an initial dilution of 1:10, following the manufacturer's recommendations. Results The determination of functional sensitivity by checking the maximum dilution of antibody detection showed higher sensitivity of the anti-tTG IgA and IgG test (dilution factor 0.3 for both) compared to the anti-EM IgA and IgG (dilution factor 0.7 for both). Of the 60 000 samples analyzed for IgA markers, 59 083 (98.5%) were negative for both tests. Of the remaining 917 samples (1.5%), 609 (66.4%) were positive for both anti-tTG IgA and anti-EM IgA, 308 (33.6%) were positive only for anti-tTG IgA, and we did not observe any sample anti-tTG IgA negative with anti-EM IgA positive. Of the 7075 samples analyzed for IgG markers, 7032 (99.4%) were negative for both tests. Of the remaining 43 samples (0.6%), 18 (41.9%) were positive for anti-tTG IgG and anti-EM IgG, 14 (32.6%) were positive only for anti-tTG IgG, and 1 (2.3%) were positive only for anti-EM IgG. Conclusion The study showed greater sensitivity of anti-tTG compared to anti-EM for both IgA and IgG, in agreement with the literature data. The anti-tTG IgA test, in individuals without immunoglobulin A (IgA) deficiency, is recommended as the initial test in suspicion of celiac disease by current scientific guidelines on celiac disease. Based on our findings and literature data, in individuals with IgA deficiency, we suggest a screening using two automated IgG markers, tTG IgG and deamidated gliadin IgG (DG IgG). In addition to the lower sensitivity of anti-EM, indirect immunofluorescence (IFI), compared to the fully automated FEIA method, is a more subjective, observer-dependent, and labor-intensive methodology to be used as screening in large-scale Clinical Laboratory routines.

Developing an Indirect ELISA for the Detection of African Swine Fever Virus Antibodies Using a Tag-Free p15 Protein Antigen.

African swine fever (ASF) is one of the most severe diseases caused by the ASF virus (ASFV), causing massive economic losses to the global pig industry. Serological tests are important in ASF epidemiological surveillance, and more antigen targets are needed to meet market demand for ASFV antibody detection. In the present study, ASFV p15 protein was fusion-expressed in Escherichia coli (E. coli) with elastin-like polypeptide (ELP), and the ELP-p15 protein was purified using a simple inverse transition cycling (ITC) process. The ELP tag was cleaved off using tobacco etch virus protease (TEVp), resulting in a tag-free p15 protein. Western blot analysis demonstrated that the p15 protein reacted strongly with ASFV-positive serum. The p15 protein was used as a coating antigen in an indirect ELISA (iELISA) for detecting ASFV antibodies. The p15-iELISA method demonstrated high specificity to ASFV-positive sera, with a maximum detection dilution of 1:1600. Moreover, the method exhibited good reproducibility, with less intra-assay and inter-assay CV values than 10%. Therefore, p15-iELISA offers a novel approach for accurately detecting ASFV antibodies with significant clinical application potential.

Establishment of a Dual-Antigen Indirect ELISA Based on p30 and pB602L to Detect Antibodies against African Swine Fever Virus.

African swine fever (ASF) is an acute, virulent, and highly fatal infectious disease caused by the African swine fever virus (ASFV). There is no effective vaccine or diagnostic method to prevent and control this disease currently, which highlights the significance of ASF early detection. In this study, we chose an early antigen and a late-expressed antigen to co-detect the target antibody, which not only helps in early detection but also improves accuracy and sensitivity. CP204L and B602L were successfully expressed as soluble proteins in an Escherichia coli vector system. By optimizing various conditions, a dual-antigen indirect ELISA for ASFV antibodies was established. The assay was non-cross-reactive with antibodies against the porcine reproductive and respiratory syndrome virus, classical swine fever virus, porcine circovirus type 2, and pseudorabies virus. The maximum serum dilution for detection of ASFV-positive sera was 1:1600. The intra-batch reproducibility coefficient of variation was <5% and the inter-batch reproducibility coefficient of variation was <10%. Compared with commercial kits, the dual-antigen indirect ELISA had good detection performance. In conclusion, we established a detection method with low cost, streamlined production process, and fewer instruments. It provides a new method for the serological diagnosis of ASF.

Dynamical Simulation, Sensitivity, and Productivity Analysis of a Light-Photoacclimation Model for Microalgae-Based Carbohydrate Production in Continuous Photobioreactors

The world’s human population is increasing as is the demand for new sustainable sources of energy. Accordingly, microalgae-based carbohydrates for biofuel production are being considered as an alternative source of raw materials for producing biofuels. Microalgae grow in photobioreactors under constantly changing conditions. Models improve our understanding of microalgae growth. In this paper, a photoacclimated model for continuous microalgae cultures in photobioreactors was used to study the time-varying behavior and sensitivity of solutions under optimal productivity conditions. From the perspective of dynamic simulation in this work, light intensity was found to play an influential role in modifying metabolic pathways as a cell stressor. Enhancing carbohydrate productivity by combining nutritional deficiency and light intensity regulation modeling strategies could be helpful to optimize the process for the highest yield in large-scale cultivation systems. Under the proposed simulation conditions, a maximum carbohydrate productivity of 48.11 gCm−3d−1 was achieved using an optimal dilution rate of 0.2625 d−1 and 350 μmolm−2s−1 of light intensity. However, it is important to note that, a particular set of manipulated inputs can generate multiple outputs at a steady state. A numerical solution of the sensitivity functions indicated that the model outputs were especially sensitive to changes in parameters corresponding to a minimum nitrogen quota, maximum nitrogen intake rate, dilution rate, and maximum nitrogen quota compared to to other model parameters.