Indirect Enzyme Immunoassay Research Articles

Mycotoxins in vegetating corn plants from experimental mono-sowing

The aim of present study was to reveal the mycotoxin contamination of vegetative maize (Zea mays L.) plants during the periods of leaf formation and panicle emergence. Early maturing hybrids of the varieties Krasnodarsky 194 MV, Ladozhsky 175 MV and Competence®, resistant to fungal diseases, were grown in the spring-summer period of 2023 on the experimental field of the Russian State Agrarian University – Moscow Agricultural Academy (Moscow) with sod-podzolic soil and the application of NPK fertilizers 16:16:16. For mycotoxicological analysis, aerial parts of plants were collected weekly from the phase of formation of the 3rd leaf (18 days after sowing. Samples of seedlings, leaves and stems (total number – 172), after drying, were ground in a laboratory mill and extracted with a mixture of acetonitrile and water in a volumetric ratio of 84:16 with a consumption of 10 ml per 1 g of sample. Mycotoxin content was determined in extracts after 10-fold dilution with phosphate-salt buffer solution pH 7.5 by indirect competitive enzyme immunoassay. T-2 toxin, deoxynivalenol, diacetoxiscirpenol, roridin A, sterigmatocystin and PR toxin were absent in the samples. Corn seedlings and leaves contained cyclopiazonic acid (CPA), emodin (EMO), mycophenolic acid (MPA), alternariol (AOL), ergot alkaloids (EA), aflatoxin B1 (AB1) and single samples – zearalenone (ZEN), ochratoxin A (OA) and citrinin (CIT) in concentrations from 16 up to 35 μg/kg. Fumonisins of group B are found only in early seedlings. The permanent contaminant of the stems was MPA, whereas EA and CIT were absent. In the seedlings and leaves of all hybrids, the detection of AOL and EA remained stable during the change of development phases, as did the average concentrations of CPA (about 100 μg/kg), AOL (from 17 to 27 μg/kg), EMO (35–58 μg/kg), MPA (28–41 μg/kg), EA (6–18 μg/kg) and AB1 (2 μg/kg). In the stems of plants in phases 7–9 of leaf and sweeping, variation in cases of detection of CPA, EMO, AOL, AB1 and OA was noted by varieties.

Expression of the labeled recombinant glycoprotein E2 of the classical swine fever virus in <i>E. Coli</i>

Currently, live attenuated vaccines are used in the Russian Federation for the specific prevention of classical swine fever (CSF), but this strategy contradicts the rules for importing animal products and carries the risks of recombination with field strains. These factors exacerbate the need for further development of candidate recombinant vaccines with similar efficacy and safety. The aim of the presented work was to construct a prokaryotic expression system of the marked glycoprotein E2 of the CSF virus and to evaluate its immunochemical properties. As a result of the conducted studies, a section of the primary amino acid sequence of glycoprotein E2 of a highly pathogenic Shimen strain (subgenotype 1.1) was identified using bioinformatic analysis and a hybrid polypeptide was designed, including a non-specific (marker) part from the modified V5 epitope and a fragment of the BSA sequence. By cloning a codon-optimized fragment into the pET-28a vector, the E. coli BL21(DE3) pLysS/pET-28a/E2-V5 producing strain was constructed. During the optimization of the cultivation conditions of the producer strain, it was found that the highest yield of the target protein is achieved within 5-6 hours after the induction of expression. The immunochemical properties of chromatographically purified recombinant rE2-V5 were determined: for example, it was shown that its activity in indirect enzyme immunoassay exceeds that of native E2. The data obtained indicate the effectiveness of the developed prokaryotic expression system, which consists in proper folding, good solubility and the ability of the target protein to form specific immunocomplexes. The presence of a marker fragment in the expressed hybrid polypeptide in the future will allow using the latter as the basis of a recombinant vaccine that meets the requirements of the DIVA strategy.

Histoplasmosis in domestic cats: new minimally invasive diagnostic techniques.

The aim of the present study was to evaluate minimally invasive diagnostic techniques, such as the semi-quantitative indirect IgG antibody enzyme immunoassay (EIA) using blood serum and the urinary lateral flow assay (LFA), for the detection of Histoplasma capsulatum in cats with histoplasmosis. Eight client-owned domestic cats diagnosed with histoplasmosis were selected based on cytological, histopathological, mycological, molecular or antigenic techniques. The blood serum of these animals was tested in a semi-quantitative indirect IgG antibody EIA for the detection of H capsulatum. Urine samples were tested for H capsulatum antigen using LFA. Five cats were seropositive on IgG EIA (5/8, with diagnostic sensitivity equal to 62.5%; 95% confidence interval [CI] 24.5-91.5) and five cats were positive on H capsulatum antigen LFA (5/7, with diagnostic sensitivity equal to 71.4%; 95% CI 29.0-96.3). The combined diagnostic sensitivity when interpreted in parallel was 87.5% (7/8, 95% CI 47.3-99.7). The specificity for the anti-Histoplasma IgG EIA was 100% (95% CI 71.5-100) and for the H capsulatum antigen LFA it was also 100% (95% CI 71.5-100). The semi-quantitative indirect IgG antibody EIA for the detection of H capsulatum in blood serum and the urinary LFA for the detection of the same agent emerge as new minimally invasive diagnostic techniques that can assist in the approach to disseminated and pulmonary feline histoplasmosis, especially when both techniques are considered together.

Purification technology design, biochemical and immunological characteristics of the recombinant chimeric antigen for evaluation of T cell immunity against coronavirus infection

Diagnosis of specific T cell immunity to the antigenic determinants of SARS-CoV-2 in patients seems to be an increasingly important task due to accumulation of data about the role of T cell immune response in course of coronavirus infection and clearance of SARS-CoV-2 in case of secondary infection. Previously, we designed the recombinant CorD_PS antigen for evaluation of T cell antiviral immunity, containing conservative and immunogenic sequences of structural proteins of the SARS-CoV-2 coronavirus. E. coli CorD_PS producing strain with stable expression of the recombinant CorD_PS antigen was obtained. Aim of the present work is to design a laboratory technology for the production of recombinant antigen CorD_PS, to control the quality of the obtained chimeric protein and to study its immunological activity. Development of the cultivation conditions for E. coli CorD_PS cells was carried out in conical flasks in LB-M_Km medium at 37 °C, then scaled in a fermenter with a volume of 30 liters. Expression was induced by the addition of IPTG. Expression was controlled in culture lysates in 12% SDS-PAGE. The resulting biomass was lysed using an ultrasonic disintegrator with followed by centrifugation. The compositions of lysing and solubilizing buffers were selected, as well as conditions for refolding of the recombinant protein. Cation exchange (SP-sepharose), hydrophobic (Butylsepharose) and exclusive (Sephacryl S-200 HR) chromatography were used sequentially to purify the dissolved protein. Protein impurities in the preparation were determined by reverse-phase HPLC and electrophoresis in 12% SDS-PAGE, residual lipopolysaccharides were determined using a gel-thrombin variant of the LAL test, residual proteins of the producer strain were determined by enzyme immunoassay, residual DNA of the producer strain was determined by binding with PicoGreen dye. Specificity was controlled by indirect enzyme immunoassay. The evaluation of cytokine production by CD4+T lymphocytes in response to their stimulation by recombinant antigen ex vivo was performed on a flow cytofluorimeter. The biomass yield during cultivation of E. coli CorD_PS in a 30L fermenter was up to 20 g/L for 4 hours of 0.1 mM IPTG induction. Sequential washing of inclusion bodies from bacterial cellular components and their subsequent solubilization in a buffer containing 8 M urea allowed to obtain a solution of denatured antigen with a concentration of 10 mg/mL. The efficiency of refolding by dilution was 75%. After three stages of chromatographic purification, protein samples with a concentration of 1.2-1.4 mg/mL, HPLC purity of 98.43%, corresponding to key quality parameters according to the OFS.1.7.1.0007.15, were obtained. The recombinant antigen showed specific binding to a sample of the First WHO International Standard and sample Company Reference Standard No. 3 of anti- SARS-CoV-2 human immunoglobulins in a specified concentration range. CD4+T lymphocytes effectively responded to recombinant antigen treatment by increasing IFNγ production. The optimal concentration of recombinant coronavirus antigen was 5 μg/mL. The developed technological process makes it possible to obtain 5-7 grams of coronavirus recombinant CorD_PS antigen in one cultivation cycle in 30 liters of fermentation medium with key quality parameters according to the OFS.1.7.1.0007.15. As a result of specific immunological activity studies of the recombinant coronavirus antigen CorD_PS, the concept of its possible use as a diagnostic tool for determining the formation of a T cell immune response was confirmed.

Development of an immunoassay based on a specific antibody for the detection of diphenyl ether herbicide fomesafen

Fomesafen belongs to the diphenyl ether herbicide, and is widely used in the control of broadleaf weeds in crop fields due to its high efficiency and good selectivity. The residual of fomesafen in soil has a toxic effect on subsequent sensitive crops and the microbial community structure because of its long residual period. Therefore, an efficient method for detecting fomesafen is critical to guide the correct and reasonable use of this herbicide. Rapid and sensitive immunoassay methods for fomesafen is unavailable due to the lack of specific antibody. In this study, a specific antibody for fomesafen was generated based on rational design of haptens and a sensitive immunoassay method was established. The half maximal inhibitory concentration (IC50) of the immunoassay was 39 ng/mL with a linear range (IC10–90) of 1.92–779.8 ng/mL. In addition, the developed assay had a good correlation with the standard UPLC-MS/MS both in the spike-recovery studies and in the detection of real soil samples. Overall, the developed indirect competitive enzyme immunoassay reported here is important for detecting and quantifying fomesafen contamination in soil and other environmental samples with good sensitivity and high reproducibility.

Quantitative determination of prothioconazole in wheat grain, soybean, and pond water based on a polyclonal antibody.

Prothioconazole and its metabolite are considered a potential threat to human health and environmental safety. Thus, the development of a sensitive and rapid detection method for prothioconazole is crucial to ensure the safety of agricultural products. In this study, a new hapten of prothioconazole was designed and synthesized, and a selective polyclonal antibody with high affinity against prothioconazole was produced, which was obtained from immunized New Zealand white rabbits. Based on the polyclonal antibody, an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) and indirect competitive chemiluminescence enzyme immunoassay (ic-CLEIA) were developed for detecting prothioconazole pesticides. Under optimized experimental conditions, the limit of quantification (LOQ) values for ic-CLEIA and ic-ELISA were 1.8 and 10.7 ng mL-1, respectively. The results demonstrated that the sensitivity (LOQ) achieved by ic-CLEIA was more than five times higher compared to that obtained with ic-ELISA. In addition, the recoveries obtained by adding standard prothioconazole to wheat grain, soybean, and pond water samples were in the range of 81.9 to 104.7% for ic-ELISA and 89.0 to 118.0% for ic-CLEIA.

Improvement of lifetime and post-mortem diagnostics of trichinellosis in animals of the Suidae family (comparative study)

Trichinosis is one of the most dangerous helminthozoonoses. Methods of compressor trichinelloscopy and peptolysis of animal muscle tissue in artificial gastric juice used for postmortem diagnosis of trichinosis are reliable enough to identify sources of infection and prevent the development of helminthiasis in humans. In addition to these methods, indirect solid-phase enzyme immunoassay (ELISA) and crystalloscopy were used. Meat juice samples obtained from domestic pigs from various regions of the Russian Federation and wild boars were used as the tested biological material. The formulation of ELISA and crystalloscopy, accounting and interpretation of the results obtained were carried out according to the classical method: the result was considered positive, exceeding the negative control by 0.08 units or more in optical density. The specificity of ELISA in all animals with heterologous invasion was 98%, hydrosol-hydrogel reactions - 80% and crystalloscopy - 75%. These methods can be used for testing for trichinosis, but the examination should be comprehensive: immunological and crystallographic methods should be confirmed by compressor trichinelloscopy and peptolysis using artificial gastric juice. Currently, in the system of veterinary and sanitary measures for trichinosis, there is a change in the structure of the sources of invasion. The probability of human infection with hunting products is higher than when eating pork obtained from meat processing enterprises. Therefore, field trichinelloscopic control in the field (CT, HH etc.) occupies a leading place in diagnostics. Thus, the development of portable equipment for veterinary examination and the training of hunters in basic trichinelloscopy skills, as well as the improvement of the formulation and accounting of crystalloscopy and НН are becoming extremely relevant.

Preparation of monoclonal antibody and establishment of indirect competitive chemiluminescence enzyme immunoassay for sodium pentachlorophenolate

In this study, three haptens of sodium pentachlorophenolate (PCP-Na) were designed and synthesized, and the monoclonal antibody against PCP-Na with high specificity and affinity was successfully prepared. Based on this monoclonal antibody, a highly sensitive indirect competitive chemiluminescence enzyme immunoassay (ic-CLEIA) was developed for the determination of PCP-Na and its metabolite pentachlorophenol (PCP). The ic-CLEIA method based on optimal conditions showed that the half maximal inhibitory concentration (IC50), linear range (IC20-IC80) and the detection limit (LOD, IC10) for PCP-Na were 0.083 ng/mL, 0.0076–1.5111 ng/mL, and 0.0032 ng/mL, respectively, and 0.29 ng/mL, 0.0983–0.7008 ng/mL, and 0.0192 ng/mL for PCP, respectively. The results of cross reaction showed that the method had no obvious cross reaction with other six chlorophenols compounds (phenol, 2-chlorophenol, 4-chlorophenol, 2, 4-dichlorophenol, 2, 4, 5-trichlorophenol, 2, 4, 6-trichlorophenol) except the metabolite PCP. The average recoveries in chicken, pork and fish samples were 81.3–105.8 %, with coefficient of variation (CV) lower than 7.6 %. In this study, a new highly rapid, sensitive and accurate ic-CLEIA method was established, which can be used for the sensitive detection of PCP-Na and its metabolite PCP in animal tissues.

Accuracy of serological tests for bovine brucellosis: A systematic review and meta-analysis

The direct methods for diagnosis of bovine brucellosis have several limitations, therefore serological tests are the basis for the diagnosis of the disease. However, a meta-analysis estimating the diagnostic sensitivity (DSe) and diagnostic specificity (DSp) on the main tests used in bovine brucellosis control programs worldwide has not been performed. This systematic review and meta-analysis aimed to estimate the DSe, DSp and thereby accuracy of serological tests individually used in the diagnosis of bovine brucellosis. The databases CABI, Cochrane Library, PubMed/MEDLINE, SciELO, Scopus and Web of Science were used to select articles. The search resulted in 5308 studies, of which 71 were selected for systematic review using quality assessment tools and 65 studies were included in the meta-analysis. For the meta-analysis, 178 assays and 11 different serological tests were considered. To estimate DSe and DSp of the tests, studies were divided according to animal selection for the studies: (1) studies that carried out a random or consecutive selection of participants (noncasecontrol studies) and (2) all studies, including casecontrol studies. Considering only the non-case-control studies to estimate the DSe, the tests that exhibited the best and worst performance were the iELISA test (indirect enzyme immunoassay - bacterial suspension as antigen - BS) (96.5%, 95% CI: 94.1–97.9%) and 2ME (2- mercaptoethanol test) (85.0%, 95% CI: 79.6–89.1%), respectively; while for DSp, the FPA (fluorescence polarization assay) (99, 7%, 95% CI: 99.5–99.8%) and PCFIA tests (protein concentration fluorescence immunoassay) (78.5%, 95% CI: 70.0–85.1%) showed better and worse performance, respectively. Overall, our results showed an overestimation in the DSe and DSp of the eleven serological tests assessed when casecontrol studies were included in the meta-analysis, which is a concern considering its impacts on the time and costs associated with populational diagnosis of the diseases, since several of these tests are routinely used in the control and eradication programs of bovine brucellosis worldwide. Furthermore, the tests that exhibited the best DSe and DSp, iELISA (BS) and FPA, respectively, are relatively easy to perform and interpret and the test which showed the best overall accuracy was FPA.

Mycotoxins in Macroalgae from the Kandalaksha Gulf of the White Sea after Natural and Experimental Alienation from the Substrate

Samples of macrophyte algae were collected from an ecotope of Kandalaksha Gulf, the White Sea. These algae were selected from substrates, storm wracks and after long-term storage in the soil, and they were assessed by indirect competitive enzyme immunoassay for the presence of low-molecular metabolites of micromycetes (mycotoxins). In Fucus distichus, F. serratus, F. vesiculosus, and Ascophyllum nodosum, the concentrations of all analyzed toxic substances from storm wracks were uniformly reduced compared to those in living thalli. For Laminaria digitata, Saccharina latissima and Ahnfeltia plicata, in living individuals of which mycotoxins were not found or were extremely rare, cases of contamination with citrinin, mycophenolic acid, emodin and sterigmatocystin were found in storm wracks. After 12 months of burial in soil F. distichus samples had only 9 components out of 16 analyzed. In F. serratus, F. vesiculosus, A. nodosum, and Pelvetia canaliculata, only emodin, mycophenolic acid, alternariol, and ergot alkaloids were determined.

A Chemiluminescence Enzyme Immunoassay Based on Biotinylated Nanobody and Streptavidin Amplification for Diazinon Sensitive Quantification

The advantages of genetic modification and preferable physicochemical qualities make nanobody (Nb) easy to develop a sensitive and stable immunosensor platform. Herein, an indirect competitive chemiluminescence enzyme immunoassay (ic-CLEIA) based on biotinylated Nb was established for the quantification of diazinon (DAZ). The anti-DAZ Nb, named Nb-EQ1, with good sensitivity and specificity, was obtained from an immunized library via a phage display technique, where the molecular docking results indicated that the hydrogen bond and hydrophobic interactions between DAZ and complementarity-determining region 3 and framework region 2 in Nb-EQ1 played a critical role in the Nb-DAZ affinity processes. Subsequently, the Nb-EQ1 was further biotinylated to generate a bi-functional Nb-biotin, and then an ic-CLEIA was developed for DAZ determination via signal amplification of the biotin–streptavidin platform. The results showed that the proposed method based on Nb-biotin had a high specificity and sensitivity to DAZ, with a relative broader linear range of 0.12–25.96 ng/mL. After being 2-folds dilution of the vegetable samples matrix, the average recoveries were 85.7–113.9% with a coefficient of variation of 4.2–19.2%. Moreover, the results for the analysis of real samples by the developed ic-CLEIA correlated well with that obtained by reference method GC-MS (R2 ≥ 0.97). In summary, the ic-CLEIA based on biotinylated Nb-EQ1 and streptavidin recognition demonstrated itself to be a convenient tool for the quantification of DAZ in vegetables.

Effect of commercial tannins on parasitic infection and immunity of lambs naturally infected with Haemonchus contortus.

This study aimed to evaluate the effects of different doses of quebracho extract tannins (Schinopsis lorenzii) (EQ) included in the basal diet (DB) on the count of eggs per gram of feces (EPG), count of parasites in the abomasum, hematological, and immunological parameters in lambs naturally infected with Haemonchus contortus. Forty-eight lambs were evaluated for 42days and divided into four groups (n=12): CONT- lambs that received DB without EQ supplementation; G1%- DB with EQ at 1% of dry matter intake (DMI) level; G3%- DB with EQ at the 3% of DMI level; G6%- DB with EQ at the 6% DMI level. For EPG counts, feces were collected weekly. For counting abomasal parasites, at the end of the experiment (D42) half of the lambs in each group were slaughtered. For hematological and immunological analysis, venous blood samples were collected at D0, D21, and D42 to perform the erythrocyte count, mean cell volume (MCV), hematocrit, differential leukocyte count, total plasma protein dosage, and serum IgA class anti-H. contortus using an indirect enzyme immunoassay (ELISA). In the EPG count, there was no significant difference (p>0.05) between the groups compared with the control. Analysis of the abomasal parasite count showed Haemonchus contortus in its entirety; however, there was no significant difference (p>0.05) in the number of males, females, and total parasites. No differences (p>0.05) were observed in erythrocyte count, MCV, hematocrit, and total plasma protein. The average total leukocyte, monocyte, eosinophil, and basophil counts showed no difference (p>0.05). However, the averages of segmented neutrophils in the G1% and G3% groups presented a reduction in the percentage of cells (p<0.05) between D21 and D42. Regarding the lymphocyte count, a difference was observed in the CONT and G3% groups at D21 and D42 (p<0.05), when compared to D0. The analyses of the average absorbance of anti-Haemonchus contortus serum IgA showed no difference (p>0.05), when the groups were analyzed over time or when compared between them. It can be concluded that the inclusion of different doses of tannins from EQ did not alter the parasitological, hematological, and immunological parameters of lambs infected with H. contortus.

РАПСОВЫЙ ЖМЫХ И ШРОТ: ПЕРВЫЕ РЕЗУЛЬТАТЫ МИКОТОКСИКОЛОГИЧЕСКОГО ОБСЛЕДОВАНИЯ

The article presents the results of a survey of 26 production batches of rapeseed cake and meal for microscopic fungi and mycotoxin contamination. The indicator of the total number of fungi in most samples corresponded to the regulated norm of 5000 CFU/g, Penicillium spp., Aspergillus spp. prevailed in the composition of the mycobiota, Cladosporium spp., Scopulariopsis spp. were detected less often, and flour fungi and yeasts were found in small amounts. The presence of the anthraquinone toxin emodin in all samples was established by the method of indirect competitive enzyme immunoassay, alternariol, mycophenolic acid and ergoalkaloids were detected somewhat less frequently, in isolated cases – aflatoxin B1, cyclopiazonic acid and T-2 toxin. The prospects for further research and the need to assess the potential of toxin formation by sanitary-significant micromycetes are discussed.

Development of indirect competitive ELISA and CLEIA for quantitative analysis of melatonin in health products.

Melatonin (MT), as a hormone regulating the rhythm of sleep, is widely used in health products. However, illicit and excessive use of MT might cause undesirable effects. Therefore, it is essential to establish highly sensitive and specific rapid methods for MT analysis in health products. In this study, we established indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) and indirect competitive chemiluminescent enzyme immunoassay (ic-CLEIA) for sensitive and selective detection of MT in health products. Under optimal conditions, half-maximum inhibitory concentration (IC50 value) and limit of detection (LOD, IC10 value) for MT by ic-ELISA were 0.25 ng mL-1 and 0.03 ng mL-1 respectively, while the IC50 and LOD of ic-CLEIA were lower at 0.17 ng mL-1 and 0.03 ng mL-1 respectively. Three MT-free Chinese patent medicines were spiked with MT and the recovery rates ranged from 71.89% to 117% (ic-ELISA) and 83.66% to 107.17% (ic-CLEIA). The level of MT in six MT-containing health products was assessed in parallel using the developed methods and HPLC. Both ic-ELISA and ic-CLEIA showed good consistencies (R2 = 0.999 and 0.993, respectively) with HPLC, indicating that the two methods developed were sensitive, fast, and reliable for application in MT analysis.

Lassa fever. Part 2. Laboratory diagnostics, treatment, development of medications

Globalization and high-speed means of transportation contribute to the spread of infections dangerous to humans. Airborne pathogens have pandemic potential as currently shown in case of the novel coronavirus SARS-CoV-2. Natural focal Lassa fever (LF) common in West African countries, in 35 cases was registered in non-endemic geographical areas because any person infected with Lassa virus (LASV) is a long-term source of infection (up to two months). Cases of person-to-person infection in endemic territories are described. In Germany, the facts of secondary virus transmission from patients to doctors have been recorded during the examination and blood collection from an apparently healthy person as well as during the autopsy of a deceased subjects due to severe LF course. Nonspecific malaise symptoms in LF are also characteristic of numerous other diseases common on the African continent, e.g., malaria and typhoid fever or viral infections such as yellow fever, Chikungunya, dengue and Zika, monkey pox and Ebola virus disease. In this regard, there may be similar dermatological manifestations. Timely detection of cases and differential diagnosis are crucial to ensure safe patient care and use of affordable antiviral therapy for LL provided by the drug Ribavirin. Research methods for studying LASV use polymerase chain reaction (PCR) for detecting viral RNA, electron microscopy, isolation of infectious virus cultured sensitive cells, indirect immunofluorescence reaction, enzyme immunoassay (ELISA) and immunochromatographic assays for the detection of antibodies and/or antigen as well as immunoblotting. Currently, test kits based on molecular and genetic methods are mainly used for LF laboratory diagnostics. Since the 1980s, ribavirin has been used to treat patients with LF. The serum accumulation of the drug in large quantities causes hemolysis, development of anemia and impaired renal function. In this regard, treatment options are being considered with decline in its concentration due to combined use with other antiviral drugs. A search for new therapeutic agents capable of inhibiting viral replication at disease early stage has been in progress due to lack of any approved vaccines.

Comparison of Indirect Immunofluorescence and Enzyme Immunoassay for the Detection of Antinuclear Antibodies.

The detection of autoantibodies directed towardnuclear antigens is one of the main criteria for the diagnosis of systemic lupus erythematosus (SLE), for which the most commonly used techniques are the enzyme immunoassay and immunofluorescence assay (IFA). However, the sensitivity and specificity of these tests vary between different techniques. Thus, in this study, we aimed to determine the superior method fordetecting antinuclear antibodies (ANAs)and compare the accuracy of tests ordered by rheumatologists versus non-rheumatologists. We compared the sensitivity and specificity of the two assays in 149 patients from a non-selected population, who were sent to the immunology laboratory of King Abdulaziz Medical City, Jeddah from 2018 to 2019. The sensitivity and specificity of the indirect IFAwere 77.78 % and 58.65%, respectively. The positive and negative predictive values of IFA for SLEwere 44.87% and 85.92%, respectively. The sensitivity and specificity of the enzyme-linked immunosorbent assay (ELISA) were 77.78% and 80.77%, respectively. The negative and positive predictive values of ELISA for SLE were 63.64% and 89.36%, respectively. The highest number of false-positive IFA tests was requested by family physicians and the lowest was requested by rheumatologists. Our data show that IFA has a higher negative predictive value, while ELISA has a higher positive predictive value. The positive predictive value of the test can be improved by pre-selecting patients by specialist rheumatologists.

Production of high affinity monoclonal antibody and development of indirect competitive chemiluminescence enzyme immunoassay for gentamicin residue in animal tissues

To determine gentamicin residues in animal tissues, a monoclonal antibody (Mab) was produced and a sensitive indirect competitive chemiluminescent enzyme immunoassay (icCLEIA) was developed. At first, gentamicin was conjugated with bovine serum albumin as immunogens which were used to immunize BALB/c mice. Then, an anti-gentamicin Mab was prepared by hybridoma technology. Finally, a sensitive icCLEIA was developed with an 50% inhibition concentration (IC50) of 0.067 ng/mL for gentamicin. The limit of detection of the icCLEIA was 0.002 ng/mL. The cross reactivity of the Mab with structural analogues were<0.01%. The recoveries of gentamicin ranged from 80 to 101% and coefficient of variation was <6.4% in pork and fish samples. Samples were detected by UPLC-MS/MS for evaluating reliability of the icCLEIA. The results suggested that the prepared anti-gentamicin Mab can be used for rapid and convenient immunoassays to detect gentamicin residues in animal tissues.

Dynamics of changes in the number of SARS-CoV-2 seropositive patients over two years of the COVID-19 pandemic

Serological assays, being rapid and relatively inexpensive methods for detecting COVID-19, may play an important role in combating the SARS-CoV-2 pandemic. The aim of the present study was to assess dynamics of changes in the number of seropositive patients for SARS-CoV-2 antibodies over 2.5 years of the evolving COVID-19 pandemic. The study included 6051 persons (2840 women and 3211 males). Their mean age was 41.680.17 years (MSEM). At the time of this survey, all participants were residents of the Chelyabinsk region. General information was collected over the period from 06/01/2020 to 01/18/2022. Seropositivity for SARS-C0V-2 was assessed by test kits for IgG, IgM and IgA antibodies (JSC Vector-Best, Novosibirsk, Russia) against SARS-CoV-2 using indirect two-stage enzyme immunoassay (ELISA). Over the entire period, 27 cases were seronegative (20.45%); 99 samples were positive for IgA to SARS-CoV-2 (75%), and 6 samples (4.55%) yielded questionable ELISA results. IgG testing for SARS-Cov-2 antibodies was negative in 2433 cases (42.35%); 3245 samples (56.48%) were positive, and 67 specimens provided (1.17%) doubtful results using ELISA tests. IgM antibodies were not revealed in 2710 (70.41%) cases; 996 were positive (25.88%), and 143 specimens (3.72%) yielded doubtful results by ELISA technique. In general, the highest proportion of positive results was found in class A immunoglobulins. The wave-like distribution of the density among all antibody-positive patients was revealed, which, however, was not associated with peak values of COVID-19 morbidity in Chelyabinsk Region. Most waves of seroprevalence were detected before the waves of SARS-CoV-2 infection. A positive relationship was established between IgG and IgM seropositivity against SARS-CoV-2 with age and female gender. Conclusion. In general, serological testing and regular monitoring of antibodies against SARS-CoV-2 may play an important role in assessing its prevalence during the coronavirus pandemic and immune response to the infection at a population level.

Determination of Acetamiprid Residues in Vegetables by Indirect Competitive Chemiluminescence Enzyme Immunoassay.

Acetamiprid (ACE) is widely used in various vegetables to control pests, resulting in residues and posing a threat to human health. For the rapid detection of ACE residues in vegetables, an indirect competitive chemiluminescence enzyme immunoassay (ic-CLEIA) was established. The optimized experimental parameters were as follows: the concentrations of coating antigen (ACE-BSA) and anti-ACE monoclonal antibody were 0.4 and 0.6 µg/mL, respectively; the pre-incubation time of anti-ACE monoclonal antibody and ACE (sample) solution was 30 min; the dilution ratio of goat anti-mouse-HRP antibody was 1:2500; and the reaction time of chemiluminescence was 20 min. The half-maximum inhibition concentration (IC50), the detection range (IC10–IC90), and the detection limit (LOD, IC10) of the ic-CLEIA were 10.24, 0.70–96.31, and 0.70 ng/mL, respectively. The cross-reactivity rates of four neonicotinoid structural analogues (nitenpyram, thiacloprid, thiamethoxam, and clothianidin) were all less than 10%, showing good specificity. The average recovery rates in Chinese cabbage and cucumber were 82.7–112.2%, with the coefficient of variation (CV) lower than 9.19%, which was highly correlated with the results of high-performance liquid chromatography (HPLC). The established ic-CLEIA has the advantages of simple pretreatment and detection process, good sensitivity and accuracy, and can meet the needs of rapid screening of ACE residues in vegetables.

Herpes Simplex Virus Seroprevalence among Pregnant Finnish Women and Their Spouses-A Six-Year Follow-Up Cohort Study.

The aim was to evaluate the herpes simplex virus (HSV) seroprevalence and seroconversion among 285 pregnant women and their 120 male spouses in Finland during a six-year follow-up (FU) between 1998–2008. We also studied the effect of sexual habits, pregnancy, and other demographic factors on the acquisition of HSV infection. Combined HSV-1 and HSV-2-IgG antibodies were assessed in the first baseline serum samples with an indirect enzyme immunoassay method. The individuals with seronegative or borderline HSV serology at baseline were additionally tested using their latest FU serum sample available. The overall HSV seroprevalence during the FU was 58.9% (168/285) among the women and 53.3% (64/120) among their spouses. The seroconversion rate was 11.4% (15/132) and 12.5% (8/64) among women and their spouses, respectively. Both spouses were HSV seropositive in 39.2% (47/120). To determine the HSV-2 seroprevalence, we also tested all HSV-seropositive participants using HSV-2-specific antigen. HSV-2 seropositivity was detected in 10.9% (44/405) of the participants. The age (p = 0.006) and history of genital warts (p = 0.006) of the women were associated with combined HSV-1 and/or HSV-2 seropositivity, while a younger age was related to HSV seroconversion (p = 0.023). Among the male spouses, HSV seropositivity was associated with the practice of oral sex (p = 0.033). To conclude, women of childbearing age acquire primary HSV infections and the presence of HSV in oral epithelium is common among HSV-seropositive individuals.