Enzyme-linked Immunosorbent Assay System Research Articles

Influence of Abutments Surface (machined vs. laser-microgrooved) in Soft-tissue Response During One Year of Function: Clinical and Biochemical Outcomes of a RCT with Split-Mouth Design

The purpose of this study was to evaluate peri-implant soft tissue response by assessing IL-6, IL-1b and MMP-8 levels in peri-implant crevicular fluid (PICF) around machined vs. laser-microgrooved implants/healing/prosthetic abutments during 1 year of function. Twenty-four patients each received 2 one-stage implants in a split mouth design on the same jaw. In each patient, one implant, one immediate healing, and one prosthetic abutment with a machined surface (M group), and one implant, one immediate healing abutment and one prosthetic abutment with a laser-microgrooved surface (LMS group) were used. PICF sampling, pocket probing depths (PPD) and bleeding on probing (BOP) were assessed at 1, 3, and 12 months. IL-6, IL-1b and MMP-8 levels were determined by specific enzyme-linked immunosorbent assay systems (ELISA). Repeated measure ANOVA was used to run comparisons with groups and between groups months at 1, 3, and 12 months. At 3 and 12 months, the LMS group showed significantly lower PD, BOP and IL-6, IL-1β and MMP-8 levels than the M group (P<.05). This study suggests the presence of more remodeling and/or inflammatory phenomena around implants/abutments with a machined surface than around implants/abutments with a laser-microgrooved surface.

A novel enzyme-linked immunosorbent assay tool to evaluate plasma soluble CD226 in primary Sjögren's syndrome

CD226 is an important receptor constitutively expressed on most immune cells, performing vital functions in immune responses. However, the levels of soluble CD226 (sCD226) and its roles in primary Sjögren syndrome (pSS) remain unclear. In this study, we developed two novel mouse anti-human CD226 monoclonal antibodies (mAbs) and established a novel sandwich enzyme-linked immunosorbent assay (ELISA) system, which proved to be highly effective in detecting human sCD226. We then analyzed the expression of sCD226 in the plasma of pSS patients. Our results showed that the levels of sCD226 were significantly lower in patients with pSS compared to healthy controls. The significant decline was also observed in active group and the patients with high levels of IgG or positive anti-SSB. Additionally, reduced sCD226 was found to be negatively correlated with the disease activity of pSS and several clinical manifestations, including arthralgia, fatigue, decayed tooth and interstitial lung disease (ILD). Furthermore, receiver operator characteristics (ROC) curve analysis showed that sCD226 displayed outstanding capacity in discriminating pSS and predicting the disease activity. Altogether, plasma sCD226 emerges as a promising candidate for diagnostic markers in the context of pSS.

The Potential Mechanism of the Anti-Liver Fibrotic Effect of Curcumin in the Gut-Liver Axis.

This study aimed to explore the curative effect of curcumin on liver fibrosis and its correlation with the gut-liver axis in animal models. Histological staining was utilized to conduct histological analysis of the liver and intestine. An automatic biochemical analyzer or enzyme-linked immunosorbent assay system was utilized for analyzing the biochemical indexes in mice. Western blotting was employed to examine the level of relevant proteins. Furthermore, 16S rRNA high-throughput sequencing was performed to explore the impact of curcumin on intestinal microorganisms in rats with liver fibrosis. Ultrahigh-performance liquid chromatography with quadrupole-orbitrap mass spectrometry was utilized to analyze the effect of curcumin on rat feces metabolites. Our results showed that curcumin reduced the formation of collagen fibers caused by carbon tetrachloride in a dose-dependent manner. In addition, curcumin was able to restore intestinal permeability in rats with liver fibrosis. By adopting α diversity analysis (Chao 1 index, Shannon index, and Simpson index), we observed that both the diversity and the abundance of intestinal flora in rats with liver fibrosis were increased. The principal component analysis diagram demonstrated that curcumin could enhance the abundance and diversity of intestinal flora, and also restore the composition of model rat flora, which was similar to that in normal rats, thereby correcting the imbalance of flora in rats with liver fibrosis. In addition, curcumin regulated feces metabolites and their signaling pathways, including glycerophospholipid metabolism, pantothenate and CoA biosynthesis. Our findings suggest that curcumin exhibits antiliver fibrosis effects, and its antiliver fibrosis effects might correlate with gut-liver axis.

Development of an indirect ELISA system for diagnosis of porcine edema disease using recombinant modified Stx2e antigen.

This study aimed to develop a sensitive and specific recombinant antigen protein indirect enzyme-linked immunosorbent assay (ELISA) kit to detect the Shiga toxin (Stx)-producing Escherichia coli (STEC) antibodies against porcine edema disease (ED). The recombinant antigen was co-expressed with the STEC-derived Stx2e A2-fragment and Stx2e B protein in E. coli BL21(DE3) pLysS cells and purified using maltose-binding protein open columns. We used a Shiga-like toxin 2 antibody to test the specificity of the recombinant antigen in an indirect ELISA, which was detected in antigen-coated wells but not in uncoated wells. We tested the indirect ELISA system using samples from the STEC-immunized pig group, the commercial swine farm group, and healthy aborted fetal pleural effusion group; five and twenty samples, respectively, were positive for STEC in the former, whereas all three samples were negative for STEC in the latter. This newly developed indirect ELISA may be a specific method for diagnosing STEC infections in pigs.

Automatic microdispenser-integrated multiplex enzyme-linked immunosorbent assay device with autonomously driven centrifugal microfluidic system.

In this study, we established the control and design theory of an autonomously driven dispenser at a steady rotation speed and proposed a dispenser-integrated multiplex enzyme-linked immunosorbent assay (ELISA) device. In establishing the theory of the dispenser, we estimated the flow rate in the dispenser and the applied pressure onto the passive valves, so that the suitable burst pressure of the valves and flow rate could be designed. The dispenser-integrated multiplex ELISA device has the potential to perform flow control for executing an ELISA of 6 samples/standards per chip or 18 samples/standards per compact disk by just steadily rotating a chip. In the immunoassay evaluation of the device using mouse IgG detection, it was confirmed that the device could assay 5 μL of several standards in just 30 min without nonspecific reactions, and although this system has a high limit of detection (LOD, 63.4-164 pg mL-1) it is equal to that of manual assay with a titer plate. The device can be fabricated by transferring the microchannel pattern from a mold without complex assembly or alignment, and it can control the liquid operation by just steadily rotating. Thus, the device system developed will contribute to reducing the cost of fabricating chips and control equipment for ELISA systems. Consequently, a compact, portable, and low-cost ELISA system for point-of-care testing is expected to be realized.

Functionalized Carbon Nanotube - Modified ELISA for Early Detection of Heart Attack

A warning issue of heart attacks in young adults needs immediate attention lately. Enzyme-linked immunosorbent assay (ELISA) is an easy and commonly used method for detecting early stages of heart attack. Cardiac troponin I (cTnI) is a responsible biomarker for acute myocardial infarction. However, the conventional ELISA system was only able to detect at 100 pM of cTnI. To improve the system, enhancements were introduced through the integration of functionalized carbon nanotube (fCNT) to amplify cTnI detection signals. By utilizing the advantage of fCNT, a noticeable improvement in results can be obtained. The detection limit was lowered down to an impressive 10 pM. Furthermore, the change of absorbance increased from 31.90% for conventional ELISA surge to 98.61 for modified ELISA system. This three-fold increase in sensitivity shows remarkable improvement through the introduction of fCNT in modified ELISA technique.

A single synthetic lipid antigen for improved serological diagnosis of Buruli ulcer

SETTING: The diagnosis of Buruli ulcer (BU) is frequently made by experienced health workers in rural regions. This leads to long turnaround times to confirm the diagnosis as it requires specialised laboratory infrastructure to perform confirmatory testing.BACKGROUND: Given the lack of success with protein antigens to detect BU in human sera, the aim of this study was to evaluate a range of single synthetic lipid antigens using an enzyme-linked immunosorbent assay (ELISA). The ELISA system used was initially developed to detect TB using single synthetic lipid antigens.METHODS: Thirty polymerase chain reaction (PCR) positive BU samples and 30 PCR-negative healthy contact samples collected from Asante Akim North and Ahafo Ano North Districts, Ghana, that are endemic for BU between 2013 and 2016 were used to evaluate the synthetic lipid antigen ELISA. A Quantikine ELISA was also conducted on a randomly blinded sub-set of 30 samples.RESULTS: The synthetic lipid ELISA evaluated here outperforms all other ELISA tests using protein antigens to detect BU to date and has shown potential as a fast (2 h) test for BU which may be adapted for use at the point of care. A sensitivity of 63% and specificity of 80% was observed for 30 BU-positive and 30 BU-negative samples, with significantly reduced interleukin-8 (IL-8) levels in a subset of patients with BU.CONCLUSION: A single lipid was shown for the first time to have the ability to distinguish between PCR-positive BU and negative sera using ELISA. The low lipid antibody load detected may be a result of immune suppression caused by the presence of mycolactone in patients with BU, given that levels of IL-8 were significantly reduced in patients with BU compared to the control serum samples.

B-052 Establishment of New Antibodies and ELISA System to Detect the Potato Alkaloids α-Solanine and α-Chaconine

Abstract Background Food poisoning by potato alkaloids, α-solanine (SO) and α-chaconine (CHA), currently occurs worldwide, however laboratory methods have not yet been established to detect these toxins in biological samples. In this study, we established new antibodies to detect SO and CHA and evaluated the practicality of enzyme-linked immunosorbent assays (ELISAs) using these antibodies in laboratory medicine. Methods Solanidine, a chemical compound found in both SO and CHA, was conjugated with bovine albumin, and this complex was repeatedly injected into two rabbits for two months. Two types of polyclonal solanidine antibodies were then purified from rabbit serum using affinity column chromatography. Using these antibodies, two forms of direct ELISAs (Sold ELISA-1, 2) were constructed. The basic detection performance of the ELISAs was confirmed by measuring the levels of SO and CHA diluted in phosphate-buffered saline (PBS). To simulate biological samples from patients with potato food poisoning, SO and CHA powders were mixed with commercially available human serum and urine, and SO and CHA levels were then measured using ELISA. Lastly, SO and CHA levels were detected in extracts from rotten potato (Irish Cobbler) sprouts, peels, and tubers. Results In samples of SO diluted in PBS, the limit of quantification (LoQ) was determined as 2.56 ng/mL and 9.55 ng/mL for Sold ELISA-1 and -2, respectively. In CHA diluted in PBS, the LoQs of Sold ELISA-1 and -2 were 1.36 ng/mL and 4.8 ng/mL, respectively. The LoQs of SO in the serum were calculated as 14.25 ng/mL and 19.41 ng/mL using Sold ELISA-1 and -2, respectively. In contrast, the LoQs of CHA in the serum were 12.48 ng/mL and 16.92 ng/mL using Sold ELISA-1 and -2, respectively. The LoQs of SO in the urine were determined as 30.28 ng/mL and 45.16 ng/mL using Sold ELISA-1 and -2, respectively. Conversely, the LoQs of CHA in the urine were 22.92 ng/mL and 38.15 ng/mL using Sold ELISA-1 and -2, respectively. Using Sold ELISA-1, both SO and CHA in the extracts from rotten potato tubers and peels were detected as approximately 1.7- and 2.3-fold higher, respectively, in each part than those in the extracts from fresh potatoes. When both SO and CHA were measured in the same samples using Sold ELISA-2, the levels were approximately 1.6- and 2.4-fold higher, respectively, in the tubers and peels than those in fresh potato samples. Furthermore, the potato sprout extract contained approximately 80-fold more SO and CHA than the tuber and 8-fold more than the peel. Discussion and Conclusion Based on our results, Sold ELISA-1 presented greater performance in detecting SO and CHA than Sold ELISA-2. The non-specific reactions in the serum samples and influence of urea in the urine samples may underlie the reduced detection performance of SO and CHA in ELISA, respectively. Although the sensitivity depends on the sample type, these ELISAs may be effective as future clinical and food testing methods.

Breast Cancers Secreting Sialyl-fibronectin Are Less Likely to Cause Epithelial-mesenchymal Transition and Have Good Prognoses.

Elevated blood fibronectin (FN) levels have been observed in various cancers; however, their significance is controversial. We measured sialyl-fibronectin (S-FN), a type of FN secreted by tumor cells in the blood, and investigated whether blood S-FN secretion is associated with cancer malignancy and recurrent metastases. We constructed an enzyme-linked immunosorbent assay (ELISA) system that recognizes S-FN as an antigen and measured the amount of S-FN secreted into the blood of 89 patients with breast tumors. The relationship between S-FN secretion and prognostic predictors was analyzed. Immunostaining was performed to identify the site of S-FN secretion in the breast tissue. Among the 82 patients, 21 (25.6%, 21/82) and 61 (74.4%, 61/82) were blood S-FN-positive and S-FN-negative, respectively. Regarding prognostic predictors, blood S-FN-positive and S-FN-negative patients showed significant difference in locoregional recurrence (p=0.026), remote metastases (p=0.049), and histological margins (p=0.001). Locoregional recurrence was associated with positive histological margins in S-FN-positive patients. However, remote metastases were associated with N-factor and histological classification (HC) in S-FN-negative patients. Furthermore, S-FN particles were detected in the cytoplasm of breast cancer cells through immunostaining. After the onset of recurrent metastases, two S-FN-positive and six S-FN-negative patients received anticancer drug treatment; however, further progression was observed in five S-FN-negative patients. S-FN-positive patients are less likely to develop distant metastases, have a better prognosis, and may be less resistant to therapeutic agents than S-FN-negative patients, which contain many epithelial-mesenchymal transition cells.

Urinary titin is not an early biomarker of skeletal muscle atrophy induced by muscle denervation in mice.

Early detection of skeletal muscle atrophy is important to prevent further muscle weakness. However, there are few non-invasive biomarkers for skeletal muscle atrophy. Recent studies have reported that the N-terminal fragment (N-titin) of titin, a giant sarcomeric protein, is detected in the urine of patients with muscle damage. In this study, we hypothesized that urinary N-titin would be a potential early biomarker of skeletal muscle atrophy in mice caused by sciatic nerve denervation. Male mice were randomly divided into control and denervation groups, and urinary N-titin levels were assessed daily for 9 days using an enzyme-linked immunosorbent assay system. Despite reduced titin protein levels in atrophic muscles 10 days after denervation, cleaved N-titin fragments were not increased in the urine of mice with denervation-induced muscle atrophy. Furthermore, we found no uptake of Evans blue dye from the extracellular space into the cytoplasm in atrophic muscles, suggesting that the sarcomeric membrane is intact in those muscles. The present results suggest that cleaved N-titin in the urine is not suitable as an early biomarker of skeletal muscle atrophy.

Development of a Novel Enzyme-Linked Immunosorbent Assay and Lateral Flow Test System for Improved Serodiagnosis of Visceral Leishmaniasis in Different Areas of Endemicity.

Visceral leishmaniasis (VL) is caused by protozoan parasites of the Leishmania donovani complex and is one of the most prominent vector-borne infectious diseases with epidemic and mortality potential if not correctly diagnosed and treated. East African countries suffer from a very high incidence of VL, and although several diagnostic tests are available for VL, diagnosis continues to represent a big challenge in these countries due to the lack of sensitivity and specificity of current serological tools. Based on bioinformatic analysis, a new recombinant kinesin antigen from Leishmania infantum (rKLi8.3) was developed. The diagnostic performance of rKLi8.3 was evaluated by enzyme-linked immunosorbent assay (ELISA) and lateral flow test (LFT) on a panel of sera from Sudanese, Indian, and South American patients diagnosed with VL or other diseases, including tuberculosis, malaria, and trypanosomiasis. The diagnostic accuracy of rKLi8.3 was compared with rK39 and rKLO8 antigens. The VL-specific sensitivity of rK39, rKLO8, and rKLi8.3 ranged from 91.2% over 92.4% to 97.1% and specificity ranged from 93.6% over 97.6% to 99.2%, respectively. In India, all tests showed a comparable specificity of 90.9%, while the sensitivity ranged from 94.7% to 100% (rKLi8.3). In contrast to commercial serodiagnostic tests, rKLi8.3-based ELISA and LFT showed improved sensitivity and no cross-reactivity with other parasitic diseases. Thus, rKLi8.3-based ELISA and LFT offer improved VL serodiagnostic efficiency in East Africa and other areas of endemicity. IMPORTANCE Reliable and field suitable serodiagnosis of visceral leishmaniasis (VL) in East Africa has until now been a big challenge due to low sensitivity and cross-reactivity with other pathogens. To improve VL serodiagnosis, a new recombinant kinesin antigen from Leishmania infantum (rKLi8.3) was developed and tested with a panel of sera from Sudanese, Indian, and South American patients diagnosed with VL or other infectious diseases. Both prototype rKLi8.3-based enzyme-linked immunosorbent assay (ELISA) and lateral flow test (LFT) showed improved sensitivity and no cross-reactivity with other parasitic diseases. Thus, rKLi8.3-based ELISA and LFT offer substantially increased diagnostic efficiency for VL in East Africa and other areas of endemicity, compared to currently commercially available serodiagnostic tests.

Development of New Antibodies and an ELISA System to Detect the Potato Alkaloids α-Solanine and α-Chaconine.

Food poisoning can be caused by the potato alkaloids α-solanine (SO) and α-chaconine (CHA). Therefore, this study aimed to establish new enzyme-linked immunosorbent assays (ELISAs) for detecting these two toxins in biological samples and potato extracts. Two antibodies that bind to solanidine, a chemical compound found in both SO and CHA, were newly developed, and two types of ELISAs (Sold1 ELISA and Sold2 ELISA) were constructed. We measured SO and CHA diluted in phosphate-buffered saline (PBS), serum, and urine. The detection performance of the two ELISAs for SO and CHA in PBS was higher than in serum and urine, and the sensitivity of Sold2 ELISA was lower than that of Sold1 ELISA. Thus, we used these ELISAs to measure SO and CHA in potato part extracts and found that potato sprouts contained approximately 80-fold more SO and CHA than tubers and 8-fold more SO and CHA than peels. Although the detection sensitivity of SO and CHA depends on the sample types, these ELISAs may be effective as future clinical and food testing methods after further improvements.

Antibiotic-enzyme-inorganic nanoflowers based immunoassay for the ultrasensitive detection of Staphylococcus aureus

In this work, we constructed a moderate and convenient approach for the determination of staphylococcus aureus (S. aureus) by using organic-inorganic flower-like hybrid nanoflowers and Pig IgG together in an enzyme-linked immunosorbent assay (ELISA) system. To ensure efficient capture, the hybrid nanoflowers were prepared by encapsulating horseradish peroxidase (HRP) and vancomycin (VAN) in the inorganic nanocrystal composites (calcium ion solution), just like the mimic biomineralization process. Owing to the self-assembly technique, the synthesized VAN-HRP-CaHPO4 nanoflowers (NFs) can not only retain the ability to particularly capture the gram-positive bacteria but also enhance the stability and enzymatic activity to achieve the signal output amplification. Then, taking advantage of the integration of signal amplification elements (HRP) and biorecognition unit (VAN), the VAN-HRP-CaHPO4 NFs were utilized as a new kind of capture & signal regent in the procedure of S. aureus detection. Based on this ELISA system, S. aureus could be clearly detected within the concentration ranging from 1.0 × 102 to 1.0 × 107 CFU mL−1. The detection limit was defined as 4.3 CFU mL−1, which performance is superior to some commercial ELISA kits. Additionally, this system detected the S. aureus in food samples and showed an acceptable recovery. As a cost-effective and sensitive platform, this proposed assay was enable to fulfill the requirement of a quick and effective detection of S. aureus.

Quercetin Attenuates the Production of Pro-Inflammatory Cytokines in H292 Human Lung Epithelial Cells Infected with Pseudomonas aeruginosa by Modulating ExoS Production.

The type three secretion system (T3SS) is a major virulence system of Pseudomonas aeruginosa (P. aeruginosa). The effector protein Exotoxin S (ExoS) produced by P. aeruginosa is secreted into the host cells via the T3SS. For the purpose of an experiment on inhibitors with regard to ExoS secretion, we developed a sandwich-type enzyme-linked immunosorbent assay (ELISA) system. Quercetin was selected because it has a prominent ExoS inhibition effect and also is known to have anti-inflammatory and antioxidant effects on mammalian cells. In this study, we investigated the effects of quercetin on the expression and secretion of ExoS using ELISA and Western blot analysis methods. The results showed that the secretion of ExoS was significantly decreased by 10 and 20 μM of quercetin. Also, popB, popD, pscF, and pcrV which are composed of the T3SS needle, are reduced by quercetin at the mRNA level. We also confirmed the inhibitory effect of quercetin on cytokines (IL-6, IL-1β, and IL-18) in P. aeruginosa-infected H292 cells by real-time polymerase chain reaction (PCR) and ELISA. Collectively, quercetin inhibits the secretion of ExoS by reducing both ExoS production and the expression of the needle protein of T3SS. Furthermore, these results suggest that quercetin has the potential to be used as an anti-toxic treatment for the inflammatory disease caused by P. aeruginosa infection.

Production and characterisation of a SARS-CoV-2 S-protein RBD homodimer with increased avidity for specific antibodies

Monitoring of the proportion of immune individuals and the effectiveness of vaccination in a population involves evaluation of several important parameters, including the level of virus-neutralising antibodies. In order to combat the COVID-19 pandemic, it is essential to develop approaches to detecting SARS-CoV-2 neutralising antibodies by safe, simple and rapid methods that do not require live viruses. To develop a test system for enzyme-linked immunosorbent assay (ELISA) that detects potential neutralising antibodies, it is necessary to obtain a highly purified recombinant receptor-binding domain (RBD) of the spike (S) protein with high avidity for specific antibodies.The aim of the study was to obtain and characterise a SARS-CoV-2 S-protein RBD homodimer and a recombinant RBD-expressing cell line, as well as to create an ELISA system for detecting potential neutralising antibodies.Materials and methods: the genetic construct was designed in silico. To generate a stable producer cell line, the authors transfected CHO-S cells, subjected them to antibiotic pressure, and selected the optimal clone. To isolate monomeric and homodimeric RBD forms, the authors purified the recombinant RBD by chromatographic methods. Further, they analysed the activity of the RBD forms by Western blotting, bio-layer interferometry, and indirect ELISA. The analysis involved mono clonal antibodies GamXRH19, GamP2C5, and h6g3, as well as serum samples from volunteers vaccinated with Gam-COVID-Vac (Sputnik V) and unvaccinated ones.Results: the authors produced the CHO-S cell line for stable expression of the recombinant SARS-CoV-2 S-protein RBD. The study demonstrated the recombinant RBD’s ability to homodimerise after fed-batch cultivation of the cell line for more than 7 days due to the presence of unpaired cysteines. The purified recombinant RBD yield from culture broth was 30–50 mg/L. Monomeric and homodimeric RBD forms were separated using gel-filtration chromatography and characterised by their ability to interact with specific monoclonal antibodies, as well as with serum samples from vaccinated volunteers. The homodimeric recombinant RBD showed increased avidity for both monoclonal and immune sera antibodies.Conclusions: the homodimeric recombinant RBD may be more preferable for the analysis of levels of antibodies to the receptor-binding domain of the SARS-CoV-2 S protein.

Clinical Significance of Soluble LAG-3 (sLAG-3) in Patients With Cervical Cancer Determined via Enzyme-Linked Immunosorbent Assay With Monoclonal Antibodies.

Background: The tumor microenvironment and tumor immunity have become the focus of research on tumor diagnosis and treatment. Lymphocyte activation gene-3 (LAG-3, CD223) is a newly discovered immunosuppressive receptor that is abnormally expressed in various tumor microenvironments and plays an important role as an immune checkpoint in the tumor immune response. Objective: We developed a novel enzyme-linked immunosorbent assay kit, examined the levels of soluble LAG-3 (sLAG-3) in the serum of patients with cervical cancer, and identified new biomarkers for cervical cancer development. Methods: To investigate the potential biological function of sLAG-3, we generated and characterized 2 novel anti-LAG-3 monoclonal antibodies, namely 4F4 and 4E12. We performed western blotting, immunofluorescence, and immunohistochemistry using hybridoma technology and an enzyme-linked immunosorbent assay kit for detecting human sLAG-3 based on an improved double-antibody sandwich enzyme-linked immunosorbent assay method. The stability and sensitivity of these kits were also assessed. Results: We screened and characterized 2 novel monoclonal antibodies against human LAG-3. The enzyme-linked immunosorbent assay kit also includes a wide range of tests. Using this enzyme-linked immunosorbent assay system, we found that the expression level of sLAG-3 in the peripheral blood of patients with cervical cancer significantly decreased as the disease progressed (P < .0001). Multivariate logistic regression analysis revealed that low sLAG-3 expression was an independent predictor of cervical cancer and related diseases (P < .05). Furthermore, receiver operating characteristic curve analysis showed that sLAG-3 had diagnostic value for cervical cancer metastasis (P < .0001). Conclusion: These data suggest that sLAG-3 is a potential biomarker for cervical cancer development. Therefore, this kit has a certain application value in the diagnosis of cervical cancer.

Establishment of a Monoclonal Antibody-Based Enzyme-Linked Immunosorbent Assay to Measure Soluble B7-H5 in Patients with Cancer.

B7-H5, an immune checkpoint molecule, is markedly upregulated in multiple cancers and plays an important role in tumor progression and immune escape. However, the expression and significance of soluble B7-H5 (sB7-H5) in cancer remain unclear. Herein, we generated two novel mouse anti-human B7-H5 monoclonal antibodies (mAbs) 2E5 and 7B10, which had different epitopes. Based on the two mAbs, a sandwich enzyme-linked immunosorbent assay (ELISA) system was developed. Using this ELISA, we found that compared with healthy controls (HCs), sB7-H5 levels were significantly increased in the serum of patients with gastric cancer (GC), colorectal cancer (CRC), and lung cancer (LC) and were associated with TNM stage and metastasis. Receiver operating characteristic (ROC) curve analysis showed that sB7-H5 has diagnostic value for GC, CRC, and LC. Collectively, our findings delineate that sB7-H5 may be used as a predictor for diagnosis of cancer and a potential therapeutic target for cancer treatment.

Development of monoclonal antibody based IgG and IgM ELISA for diagnosis of severe fever with thrombocytopenia syndrome virus infection

IntroductionSevere fever with thrombocytopenia syndrome virus (SFTSV) is a newly emerged virus that poses a great threat to human health because of high fatality rate. MethodsTo develop sensitive and specific sero-diagnostic systems for SFTSV infections, monoclonal antibodies (MAbs) against recombinant SFTSV nucleocapsid (rSFTSV-N) protein were developed by immunizing BALB/C mice with rSFTSV-N protein and fusing the spleen cells with SP2/0 myeloma cells. Three hybridoma cell lines secreting MAbs against rSFTSV-N were obtained. MAb based IgG sandwich enzyme linked immunosorbent assay (ELISA) and IgM capture ELISA systems were established by using the newly developed MAbs. One hundred fifteen clinical suspected SFTS patients serum samples were used to evaluate the newly established systems by comparing with the total antibody detecting sandwich ELISA system and indirect ELISA systems. ResultsThe MAbs based sandwich IgG ELISA was perfectly matched with that of the total antibody sandwich ELISA and the indirect IgG ELISA. IgM capture ELISA results perfectly matched with that of the total antibody sandwich ELISA while detecting eight additional positive samples missed by the indirect IgM ELISA. ConclusionsThe MAbs against rSFTSV-N protein offer new tools for SFTSV studies and our newly developed MAb-based IgG and IgM capture ELISA systems would offer safe and useful tools for diagnosis of SFTS virus infections and epidemiological investigations.

Non-Invasive Detection of SARS-CoV-2 Antigen in Saliva versus Nasopharyngeal Swabs Using Nanobodies Conjugated Gold Nanoparticles.

The development of sensitive, non-invasive tests for the detection of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) antigens is imperative, and it is still challenging to manage the extent of infection throughout the population. Here, we designed and optimized a sandwich enzyme-linked immunosorbent assay (ELISA) protocol for SARS-CoV-2 S1 antigen detection in saliva. Both saliva samples and nasopharyngeal swabs were collected from 220 real-time quantitative polymerase chain reaction (RT-qPCR)-confirmed positive and negative cases. S1 protein receptor-binding domain (RBD) nanobodies were efficiently conjugated with 40 nm gold nanoparticles (AuNPs) and employed as antigen detection probes in the developed system, while recombinant S1 monoclonal antibodies (S1mAbs) were employed as antigen capture probes. After checkerboard assays and system optimization, the clinical samples were tested. In saliva, the developed ELISA system showed the highest sensitivity (93.3) for samples with cycle threshold (Ct) values ≤ 30; interestingly, high sensitivity (87.5 and 86%) was also achieved for samples with Ct values ≤ 35 and ≤40, respectively, compared with 90, 80 and 88% sensitivity rates for nasopharyngeal swabs with the same categorized Ct values. However, the specificity was 100%, and no cross-reactions were detected with Middle East respiratory syndrome coronavirus (MERS-CoV) or SARS-CoV antigens. These results reveal that our protocol could be established as an efficient and sensitive, non-invasive diagnostic tool for the early detection of SARS-CoV-2 infection using easily collectable saliva samples.

ИЗМЕНЕНИЕ ПРОФИЛЯ МИКОТОКСИНОВ В БУРЫХ ВОДОРОСЛЯХ ИЗ ШТОРМОВЫХ ВЫБРОСОВ

Brown algae Fucus vesiculosus Linnaeus, Fucus serratus Linnaeus, Ascophyllum nodosum (Linnaeus) Le Jolis and Saccharina latissima (Linnaeus) C.E. Lane, C. Mayes, Druehl &amp; G.W. Saunders collected from natural substrates and from storm emissions in one of the ecotopes of the Kandalaksha Bay of the White Sea (66°31' N 33°11' E) were used for comparative assessment of the content of low-molecular metabolites of micromycetes beloning to the genera Fusarium Link, Alternaria Nees, Penicillium Link, Aspergillus P. Micheli ex Haller, Myrothecium Tode, Cladosporium Link and others. After drying, the samples were crushed in a laboratory mill, a mixture of acetonitrile and water was used for extraction in a volume ratio of 84:16 with a consumption rate 10 mL per 1 g specimen. Extracts after 10-fold dilution with the buffer solution were analyzed using a set of certified enzyme-linked immunosorbent assay systems (Russia). The lower limit of quantitative measurements corresponded to an 85% level of antibody binding. All analyzed compounds – T-2 toxin, diacetoxiscirpenol, deoxynivalenol, zearalenone, fumonisins, alternariol, ochratoxin A, citrinin, PR-toxin, mycophenolic acid, aflatoxin B1, sterigmatocystin, cyclopiazonic acid, emodin, roridin A and ergot alkaloids – were found in the fresh thalli of F. vesiculosus, F. serratus, and A. nodosum. In the samples from the emissions, the profile of mycotoxins has been significantly changed. In F. vesiculosus and F. serratus the content of mycotoxins decreased sharply and uniformly and, as a result, the incidence of detection reduced to 8% and 15%. In A. nodosum, alternariol, aflatoxin B1 and mycophenolic acid were revealed in 17% of samples near the limits of determination of methods, and the other components of the complex could not be found. The mycotoxins were absent in the fresh thalli of S. latissima, and only some of the samples from the emissions had weak contamination with mycophenolic acid and emodin.