Antigen Concentration Research Articles

Investigation on the immune effect of a chitosan-based particle-in-oil-in-water emulsion

Particle-in-oil-in-water (P/O/W) multiple emulsion adjuvants introduce particles into the internal water phase of a water-in-oil-in-water emulsion, combining the advantages of both particle and emulsion adjuvants to enhance humoral and cellular immune responses. In this study, we optimized P/O/W multiple emulsion adjuvants. Chitosan, poly (lactic-co-glycolic acid), and aluminum gel were used to prepare the particles, which were introduced into a water-in-oil-in-water emulsion to obtain three P/O/W multiple emulsion adjuvants. The immune enhancement effects and safety of the three adjuvants were compared, and it was proven that the adjuvant with chitosan nanoparticles in the internal water phase had good cellular and humoral immune effects. Simultaneously, the proportion of the internal water phase increased from 13% to 20%, reducing the antigen concentration required for embedding to one-third of the original concentration and expanding the application range of the composite adjuvant.

An innovative transportable immune device for the recognition of α-synuclein using KCC-1-nPr-CS2 modified silver nano-ink: integration of pen-on-paper technology with biosensing toward early-stage diagnosis of Parkinson's disease.

Parkinson's disease (PD), the second most frequent neurodegenerative illness, is a neurological ailment that produces unintentional or uncontrolled body movements, which should be diagnosed in its early stages to hinder the progression. Monitoring the concentration of α-synuclein (α-Syn) in body fluids can be one of the most efficient ways for PD early detection. In this work, a paper-based electrochemical immunosensor was designed for α-Syn bio-assay in human plasma samples based on encapsulation of the biotinylated antibody on novel dendritic fibrous nanosilica ((KCC-1-nPr-CS2)-Ab). For this purpose, a three-electrode system was prepared using stabilization of silver nano-ink on photographic paper. Then, the (KCC-1-NH-CS2)-Ab was immobilized on its surface and used to detect the target antigen (α-Syn). After characterization of the prepared substrate by FE-SEM and EDS, the redox behavior of the biosensor was evaluated using chronoamperometry techniques. Under optimal experimental conditions and using a label-free strategy, the engineered immunosensor showed a linear relationship between peak current and antigen concentration in the linear range from 0.002 to 128 ng mL-1 with the lower limit of quantification of 0.002 ng mL-1. Moreover, this work involves unprecedented use of conductive nano-inks for the manufacture of α-Syn immunosensor, which is aided by the use of a mesoporous silicate dendrimer in encapsulating the α-Syn antibody, thus offering a robust and simple point-of-care device for early PD diagnosis. The ability of the proposed platform to detect small amounts of α-Syn offers a promising approach to developing low-cost, sensitive, and transportable biosensors for Parkinson's disease screening in its early stages.

A Microsphere Immunoassay for the Quantitative Detection of Antigens in Cell Culture Supernatant.

Quantitative immunoassays, such as the traditional enzyme-linked immunosorbent assay (ELISA), are used to determine concentrations of an antigen in a matrix of unknown antigen concentration. Magnetic immunoassays, such as the Luminex xMAP technology, allow for the simultaneous detection of multiple analytes and offer heightened sensitivity, specificity, low sample volume requirements, and high-throughput capabilities. Here, we describe a quantitative immunoassay using the Luminex MAGPIX® System to determine the antigen concentration from liquid samples with unknown concentrations. In detail, we describe a newly developed assay for determining production yields of Drosophila S2-produced Marburg virus (MARV) glycoprotein in insect-cell-culture-derived supernatant. The potential applications of this assay could extend to the quantification of viral antigens in fluids derived from both in vitro and in vivo models infected with live MARV, thereby providing additional applications for virological research.

A comparative study of tumor markers and dynamic contrast-enhanced CT and their combined evaluation in the diagnosis of renal cancer patients

The objectives of this study were to investigate the diagnostic efficacy of dynamic contrast-enhanced Computed Tomography (CT) and tumor markers and their combined evaluation in renal cancer patients. A total of eighty male patients with solid renal lesions were selected from our hospital and categorized into two groups based on pathological examination findings: renal cancer group and benign lesion group. A comparison was made between the findings of dynamic contrast-enhanced CT and tumor markers and the diagnostic efficacy of either mono or combined evaluation were compared. Receiver operating characteristic curves were plotted to better analyze and compare different diagnostic techniques. The serum concentrations of carcinoembryonic antigen (CEA), Cytokeratin 19 fragment (CYFRA21-1), midkine (MK) and matrix metalloproteinase 9 (MMP-9) in renal cancer patients were significantly higher than those with benign lesions (p < 0.05). The Receiver Operating Characteristic (ROC) curve analysis demonstrated that the combination of dynamic contrast-enhanced CT and tumor markers had the highest area under the curve. This difference was statistically significant (p < 0.05). The analysis of the overall model quality results revealed that the model values for both single and combination indicators exceeded 0.5, with the combined indicators having the highest value of (0.84). The diagnostic efficacy of dynamic contrast-enhanced CT combined with tumor markers for renal cancer is significantly higher than either the dynamic contrast-enhanced CT or tumor markers alone. The combination of dynamic contrast-enhanced CT and tumor markers has a significant therapeutic benefit in diagnosing renal cancer. We envisage that, this approach can offer valuable guidance for the clinical diagnosis and treatment of renal cancer.

Recurrent herpetic erosion of the cornea: diagnosis, treatment and prevention of recurrences

A group of patients was found to have a special form of recurrent corneal erosion caused by types I and II herpes virus. This form represents an independent form of ophthalmic herpes - herpetic recurrent erosion (HRE) of the cornea. The herpetic etiology of recurrent corneal erosion was confirmed by the immunofluorescence study of scraping from the conjunctiva, which revealed a high concentration of the herpes simplex virus antigen. Treatment of patients (171 patients, 182 eyes) with HRE included 2 consecutive stages: stage I - relief of acute symptoms of the disease with the help of conservative treatment (instillations of interferon inducers, autologous serum, corneal protectors, tear substitutes, use of therapeutic soft contact lenses); in some cases, phototherapeutic keratectomy was used in the absence of the effect of conservative therapy, as well as in the localization of the focus in the optical zone. Stage II involved anti-relapse therapy based on the use of a Russian-produced herpes vaccine in the intercurrent period. After vaccination, observation for 2 years or more showed that 81.3% of patients achieved clinical recovery (complete cessation of HRE recurrences), 15.8% had a decrease in the frequency and severity of relapses, while 2.9% of patients did not respond to the treatment.

Follicular fluid concentration of soluble Human-G Leukocytic Antigen (sHLA-G) in in vitro fertilization cycles of women with and without peritoneal endometriosis.

The objective of this research is to investigate the association between the concentrations of soluble human leukocyte G antigen (sHLA-G) in the follicular fluid (FF) in infertile patients with peritoneal endometriosis submitted to in vitro fertilization. We performed a cross-sectional study, including ninety-six women undergoing in vitro fertilization (IVF) ageing ≤ 40 years. Infertile patients were classified into two groups: with endometriosis diagnosed by laparoscopy and without endometriosis due to tubal factor. ELISA measured soluble HLA-G in the FF of a pool of punctured (more than 17mm) follicles from women with endometriosis and without endometriosis who were subjected to ovulation induction for IVF. Embryos obtained after fertilization were classified according to the graduated embryo score (GES). Groups were comparables in terms of age, the number of follicles, AMH, FSH and all included reproductive outcomes. There was no association between sHLA-G concentrations and the average score of the generated embryos (p>0.05). Measurement of sHLA-G in the follicle fluid in women with endometriosis and without endometriosis (tubal factor) showed no significant difference (p>0.05). We also compared sHLA-G per follicle and per embryo, which were not different between both groups (p>0.05). Patients with peritoneal endometriosis submitted to IVF did not demonstrate an altered sHLA-G in the follicular fluid compared to the follicular fluid sHLA-G concentration in tubal factor patients. Also, this molecule was not linked to any other reproductive outcome.

SARS-CoV-2 nucleocapsid antigen in plasma of children hospitalized for COVID-19 or with incidental detection of SARS-CoV-2 infection.

In hospitalized children, SARS-CoV-2 infection can present as either a primary reason for admission (patients admitted for COVID-19) or an incidental finding during follow-up (patients admitted with COVID-19). We conducted a nested case-control study within a cohort of pediatric patients with confirmed SARS-CoV-2 infection, to investigate the concentration of plasma nucleocapsid antigen (N-Ag) in children admitted for COVID-19 or with COVID-19. While reverse transcriptase polymerase chain reaction Ct values in nasopharyngeal swab were similar between the two groups, children admitted for COVID-19 had a higher rate of detectable N-Ag (12/18 (60.7%) versus 6/18 (33.3%), p = 0.0455) and a higher concentration of N-Ag (medians: 19.51 g/mL vs. 1.08 pg/mL, p = 0.0105). In children hospitalized for COVID-19, the youngest had higher concentration of N-Ag (r = -0.74, p = 0.0004). We also observed a lower prevalence of detectable spike antibodies in children hospitalized for COVID-19 compared to those hospitalized for other medical reasons (3/15 [20%] vs. 13/16 [81.25%], respectively, p = < 0.0011), but similar rates of IgG nucleocapsid antibodies (5/14 [35.7%] vs. 6/17 [35.3%], respectively, p = 0.99). Our findings indicate that N-Ag is associated with COVID-19-related hospitalizations in pediatric patients, and less frequently detected in children tested positive for SARS-CoV-2 but hospitalized for another medical reason. Further studies are needed to confirm the value of N-Ag in identifying COVID-19 disease infections in which SARS-CoV-2 is the main pathogen responsible for symptoms.

Fluorescence anisotropic probe for sensing cardiac troponin-I antigen through target-specific antibody-conjugated gold nanoclusters.

Fluorescence anisotropy (FA) is a versatile and efficient platform for developing biosensors that rely on the rate of rotations of fluorescence molecular entities in biochemical systems. However, by virtue of its intricate complexity, FA is a neglected and less explored area for developing biosensors. Herein, we experimented with the possibility of developing a fluorescence anisotropic probe to detect cardiac troponin I (cTnI), the gold standard biomarker for acute myocardial infarction, via target-specific monoclonal antibody-conjugated gold nanoclusters. The successful detection of cTnI antigen in clinically relevant concentration with a low detection limit of 0.91 ng mL-1 was achieved. The specific molecular interaction between the cTnI antigen and its monoclonal antibody tagged at the surface of gold nanoclusters has restricted the free rotation of gold nanoclusters and increased the FA value. This incremental increase in FA can be correlated to the concentration of cTnI antigen in the sample, thereby achieving the quantitative linear detection of cTnI.

Histological and immunohistochemical characteristic of the gingival stroma in the portion of the third molars in children of various age.

Aim: To evaluate the state of the gingival stromal elements in the portion of the third molars requiring extraction of these teeth due to orthodontic indications considering the stage of tooth germ formation. Materials and Methods: The surgery to extract third molars due to orthodontic indications was performed on 95 children aged 11 to 18 years. The three groups of observation were isolated according to clinical-radiological signs: І (n=30) - children aged 11-13 years; ІІ (n=35) - children aged 13-16 years, and ІІІ (n=30) - children aged 16-18 years. During surgery, the samples of gums were taken from the adjacent areas for examination. The samples were fixed, dehydrated, paraffinized for further histological processing. Immunohistochemical methods were used according to the protocols supplied by a producer. In particular, by means of immunohistochemical method, Ki-67, CD-34 antigens and vimentin with primary antibodies against them were determined. The primary antibodies were visualized by the polymeric visualization system with diaminobenzidine giving a brown color to the places of location of the antigens examined. The data obtained were statistically processed. Results: The results of the study showed that specific gravity of the vascular bed in the gingival papillary layer of children was the most variable. It ranges from (12,7±0,09) % at the stage of "D" root formation to (54,8±0,17) % at the "H" stage. Lower concentrations of CD-34 antigens and vimentin are found in the endotheliocytes of children aged 13-16 and 16-18 years, compared to the children aged 11-13 years (p<0,05). No changes were found in the specific volume of the blood vessels, CD-34 antigens and vimentin in the reticular gingival layer of children from the groups of observation. Conclusions: Therefore, the conducted histological and immunohistochemical study of the connective gingival tissues in the portion of the third molars in children enables to draw a conclusion that in the process of formation of the root of this tooth a number of changes occur in the gingival stroma. They include an increase of the blood flow volume in the papillary gingival layer on the background of a decreased concentration of CD-34 genes and vimentin, a longer stage of development of the third molar root. The specific volume of the islets of neoangiogenesis of the papillary gingival layer is the largest in children aged 13-16 years.

Kelvin probe force microscopy on patterned large-area biofunctionalized surfaces: a reliable ultrasensitive platform for biomarker detection.

Kelvin probe force microscopy (KPFM) allows the detection of single binding events between immunoglobulins (IgM, IgG) and their cognate antibodies (anti-IgM, anti-IgG). Here an insight into the reliability and robustness of the methodology is provided. Our method is based on imaging the surface potential shift occurring on a dense layer of ∼5 × 107 antibodies physisorbed on a 50 μm × 90 μm area when assayed with increasing concentrations of antigens in phosphate buffer saline (PBS) standard solutions, in air and at a fixed scanning location. A comprehensive investigation of the influence of the main experimental parameters that may interfere with the outcomes of KPFM immune-assay is provided, showing the robustness and reliability of our approach. The data are supported also by a thorough polarization modulation infrared reflection-absorption spectroscopy (PM-IRRAS) analysis of the physisorbed biolayer, in the spectral region of the amide I, amide II and amide A bands. Our findings demonstrate that a 10 min incubation in 500 μL PBS encompassing ≈ 30 antigens (100 zM) triggers an extended surface potential shift that involves the whole investigated area. Such a shift quickly saturates at increasing ligand concentration, showing that the developed sensing platform works as an OFF/ON detector, capable of assessing the presence of a few specific biomarkers in a given assay volume. The reliability of the developed methodology KPFM is an important asset in single molecule detections at a wide electrode interface.

A new immunosensing platform based on conjugated Poly(ThidEp-co-EDOT) copolymer for resistin detection, a new obesity biomarker.

Thedesign of a novel electrochemical impedimetric biosensor for label-free analysis of resistin, a biomarker for obesity, is reported. For the fabrication of the immunosensor, a novel approach composed of electrochemical copolymerization of double epoxy groups-substituted thiophene (ThidEp) and 3,4-Ethylenedioxythiophene (EDOT) monomers was utilized. Anti-resistin antibodies were covalently attached to the copolymer-coated electrode. The capture of resistin antigens by anti-resistin antibodies caused significant variations in charge transfer resistance (Rct) because of the immunoreactions between these proteins. Under optimum experimental variables, the changes in impedance signals were employed for the determination of resistin antigen concentration, and the prepared immunosensor based on conjugated copolymer illustrated a wide linear range between 0.0125 and 22.5 pg/mL, a low detection limit (LOD) of 3.71 fg/mL, and a good sensitivity of 1.22 kΩ pg-1mL cm2. The excellent analytical performance of the resistin immunosensor in terms of selectivity, sensitivity, repeatability, reproducibility, storage stability, and low detection limit might be attributed to the conductive copolymer film layer generation on the disposable indium tin oxide (ITO) platform. The capability of this system for the determination of resistin in human serum and saliva samples was also tested. The immunosensor results were in accordance with the enzyme-linked immunosorbent assay (ELISA) results. The matrix effects of human serum and saliva were also investigated, and the proposed immunosensor displayed good recovery ranging from 95.91 to 106.25%. Theengineered immunosensor could open new avenues for obesity monitoring.

Изменения плазменной концентрации фактора Виллебранда и активности фактора VIII у больных гемобластозами с инфекционными осложнениями

Infectious complications (IC) and sepsis are among the main causes of mortality in patients with haemoblastosis. The mechanisms of von Willebrand factor (vWF) involvement in inflammatory processes revealed in recent decades determine the possibility of its use as a marker of poor prognosis, along with factor VIII (FVIII), which closely interacts with vWF in the implementation of hemostasis and refers to the acute phase proteins. Objective. To determine the relationship of changes in vWF concentration and FVIII activity with mortality in IC of hemoblastoses. Material and methods. The study included 90 patients with haemoblastosis and IC in the intensive care unit. We assessed parameters necessary to calculate the SOFA score, C-reactive protein and procalcitonin content, coagulogram parameters, as well as vWF antigen concentration (vWF:Ag) and FVIII activity (FVIII:C). Results. Mortality in the study group was 35.5%, sepsis was diagnosed in 77 patients out of 90 (85.5%) included in the study. There was an increase in vWF:Ag to 361% (262–439) and FVIII:C to 373% (240–600). vWF:Ag was found to be significantly higher in the cohort of deceased patients than in survivors. vWF:Ag was found to correlate with C-reactive protein concentration, procalcitonin and the severity of organ disorders on the SOFA scale. Univariate and multivariate analysis revealed a predictive role of elevated vWF:Ag in the mortality prognosis in patients with IC along with D-dimer and antithrombin III concentrations. Conclusion. Increased vWF:Ag and FVIII:C are noted in patients with haemoblastosis and IC. Increased vWF:Ag, as well as increased D-dimer and decreased antithrombin III concentrations are independent mortality predictors in oncohematological patients with IC

Label-free detection of Aβ-42: a liquid crystal droplet approach for Alzheimer's disease diagnosis.

This study introduces a biosensor based on liquid crystals (LC) designed to detect the Aβ-42 biomarker, commonly associated with Alzheimer's disease. The sensor utilizes LC droplets created using a PEI/Tween-20 surfactant mixture, arranged radially in an aqueous solution. These droplets are coated with the Aβ1-16 antibody, enabling the detection of the Aβ1-42 biomarker. The key advantage of this biosensor lies in its ability to directly translate the antigen-antibody interaction into a change in the molecular orientation of the LC droplets, simplifying the detection process by removing additional procedural steps. Specifically, this immunoassay induces a transformation in the nematic droplets orientation from radial to bipolar upon successful antigen binding. When only the Aβ1-16 antibody coated the LC droplets, no change in orientation was detected, confirming the reaction's specificity. The orientation shift in the LC droplets indicates the formation of an immunocomplex between the Aβ1-16 antibody and the Aβ1-42 antigen. The LC droplet immunoassay effectively detected Aβ1-42 antigen concentrations ranging from 45 to 112.5 μM, with the Aβ1-16 antibody immobilized on the droplets at a concentration of 1 μg mL-1. These findings suggest that the LC microdroplets' orientational behavior can be harnessed to develop a biosensor for the in vivo detection of various proteins or pathogens in a PBS aqueous medium. Owing to its label-free nature and distinct optical signaling, this LC droplet-based immunoassay holds promise for further development into a cost-effective, portable diagnostic tool.

ИММУНОФЕРМЕНТНАЯ ТЕСТ-СИСТЕМА ДЛЯ ОЦЕНКИ НАПРЯЖЕННОСТИ ПОСТВАКЦИНАЛЬНОГО ИММУНИТЕТА К ВИРУСУ КЛАССИЧЕСКОЙ ЧУМЫ СВИНЕЙ

Classical swine fever (CSF), being a particularly dangerous transboundary disease of pigs of all breeds and ages, remains one of the most important threats to the global pig industry. An urgent task is the development of new recombinant vaccines that meet the requirements of the DIVA strategy. No less important is the issue of equipping preventive means with accurate and rapid diagnostic test systems that allow assessing the intensity of post-vaccination immunity in target animals. The purpose of this study is to construct an enzyme-linked immunosorbent test system for indicating antibodies to the CSF virus based on a recombinant antigen. Specific components of the test system were obtained using genetic engineering and biochemical methods. Optimization of the ELISA protocol was carried out on a sample of immune and normal pig sera with variations in parameters (time and temperature of sensitization, working concentrations of specific components, reaction time). Determination of the analytical characteristics of the test system included tests for specificity, sensitivity limit, positive-negative threshold, and intra-laboratory reproducibility. It was found that the following parameters are optimal for performing ELISA: sensitizing antigen concentration – 50 ng per well, sensitization time – 3 hours at 37 °C or 12 hours at 4 °C; dilution of the antispecies conjugate is 1:50000, binding time to the enzymatic label is 45 min. The analytical sensitivity limit of the test system was 190 ng/ml for protein, the intra-laboratory reproducibility of results between wells of one plate was no less than 95 %, between individual plates was no less than 93 %. The developed ELISA test system, which has high sensitivity and specificity, can be recommended for assessing the effectiveness of vaccination against CSF.

Confounding mitigation for the exposure-response relationship of bevacizumab in colorectal cancer patients.

The exposure-response relationship of bevacizumab may be confounded by various factors, including baseline characteristics, time-dependent target engagement and recursive relationships between exposure and response, requiring effective mitigation. This study aimed to investigate the exposure-response relationships of bevacizumab in metastatic colorectal cancer (mCRC) patients while mitigating potential biases. Bevacizumab pharmacokinetics was described using target-mediated drug disposition modelling. Relationships between target kinetics, progression-free (PFS) and overall (OS) survivals were assessed using joint pharmacokinetic and parametric hazard function models. Both prognostic-driven and response-driven potential biases were mitigated. These models evaluated the impact of increased antigen target levels, clearance and intensified dosing regimen on survival. Estimated target-mediated pharmacokinetic parameters in 130 assessed patients were baseline target levels (R0 = 8.4nM), steady-state dissociation constant (KSS = 10 nM) and antibody-target complexes elimination constant (kint = 0.52 day-1). The distribution of R0 was significantly associated with increased baseline concentrations of carcinoembryonic antigen, circulating vascular endothelial growth factor and the presence of extrahepatic metastases. Unbound target levels (R) significantly influenced both progression and death hazard functions. Increasing baseline target levels and/or clearance values led to decreased bevacizumab unbound concentrations, increased R levels and shortened PFS and OS, while increasing bevacizumab dose led to decreased R and longer survival. This study is the first to demonstrate the relationship between bevacizumab concentrations, target involvement and clinical efficacy by effectively mitigating potential sources of bias. Most of the target amount may be tumoural in mCRC. Future studies should provide a more in-depth description of this relationship.

Pharmacokinetics and coagulation biomarkers in children and adults with hemophilia A receiving emicizumab prophylaxis every 1, 2, or 4 weeks

BackgroundEmicizumab is a bispecific antibody that bridges activated factor (F)IX and FX, mimicking the function of missing activated FVIII and thus improving hemostasis in people with hemophilia A. The efficacy and safety of emicizumab were demonstrated in 4 phase III clinical trials (HAVEN 1-4). ObjectivesHere, we describe pharmacokinetics (PKs), pharmacodynamics (PDs), and exploratory safety biomarkers in HAVEN 1 to 4. MethodsParticipants received emicizumab at a loading dose of 3 mg/kg weekly for 4 weeks, followed by maintenance doses of 1.5 mg/kg weekly, 3 mg/kg every 2 weeks, or 6 mg/kg every 4 weeks. PKs, PDs, and safety biomarkers were assessed in samples collected at regular intervals during the trials. ResultsEmicizumab plasma trough concentrations increased during the loading dose period, reaching a mean of 52.9 μg/mL (SD, 13.6 μg/mL) at week 5, and were sustained at 42.1 to 52.3 μg/mL thereafter with maintenance dosing. Activated partial thromboplastin time shortened following the first emicizumab dose. Mean FVIII-like activity and thrombin generation peak height increased to 25.2 IU/dL (SD, 6.9 IU/dL) and 115.2 nM (SD, 42.5 nM) at week 5, with levels sustained at 17 to 23 IU/dL and >116 nM thereafter, respectively. Emicizumab did not notably affect FIX or FX plasma antigen levels, prothrombin time, or concentrations of exploratory safety markers of coagulation activation (D-dimer, prothrombin fragment 1 + 2, and fibrinogen). ConclusionIn HAVEN 1 to 4, emicizumab demonstrated sustained PKs and PDs and improved coagulation parameters without affecting safety biomarkers.

Prognostic evaluation of relapse based on squamous cell carcinoma antigen, CXCR2, and CD44V6 blood levels in patients with Stage I–II squamous cell lung cancer

Lung cancer, the leading cause of cancer-related mortality, predominantly exists as non-small cell lung cancer, accounting for approximately 85% of cases and comprising adenocarcinoma, squamous cell carcinoma (SCC), and large cell carcinoma as the three most prevalent histological subtypes. This study focused on investigating pre- and post-operative concentrations of SCC antigen in blood serum, as well as the percentage of CXCR2-containing lymphocytes and CD44v6-containing monocytes in blood cell populations among patients with Stages I&amp;ndash;II squamous cell lung cancer (SCLC) within 1 year after tumor resection. The primary objective was to assess their potential for predicting relapse. The study cohort comprised 57 patients (32 men and 25 women) with newly diagnosed squamous cell lung cancer (21 at stage I and 36 at stage II). Following tumor resection, categorized as R0 in terms of surgical intervention, all parameters were examined before surgery and at 3 weeks, 3 months, and 6 months postoperatively. Analysis revealed that the probability of relapse could be accurately predicted, ranging from 68.4% to 89.5%, based on differences in SCC antigen concentration, the percentage of lymphocytes with CXCR2, and monocytes with the CD44v6 receptor during various post-operative intervals. Subsequent regression analysis and the formulation of a combined model incorporating the above-mentioned parameters led to an enhanced predictive value for tumor recurrence, reaching 96.5% accuracy (with specificity at 95.6% and sensitivity at 100%). These results indicate the potential utility of the combined model as an additional marker for predicting postoperative relapse in patients with Stage I&amp;ndash;II SCLC.

A species-independent lateral flow microarray immunoassay to detect WNV and USUV NS1-specific antibodies in serum

Arboviruses such as West Nile Virus (WNV) and Usutu Virus (USUV) are emerging pathogens that circulate between mosquitoes and birds, occasionally spilling over into humans and horses. Current serological screening methods require access to a well-equipped laboratory and are not currently available for on-site analysis. As a proof of concept, we propose here a species-independent lateral flow microarray immunoassay (LMIA) able to quickly detect and distinguish between WNV Non-Structural 1 (NS1) and USUV NS1-specific antibodies. A double antigen approach was used to test sera collected from humans, horses, European jackdaws (Corvus monedula), and common blackbirds (Turdus merula). Optimization of the concentration of capture antigen spotted on the LMIA membrane and the amount of detection antigen conjugated to detector particles indicated that maximizing both parameters increased assay sensitivity. Upon screening of a larger serum panel, the optimized LMIA showed significantly higher spot intensity for a homologous binding event. Using a Receiver Operating Characteristics (ROC) curve, WNV NS1 LMIA results in humans, horses, and C. monedula showed good correlation when compared to “gold standard” WNV FRNT90. The most optimal derived sensitivity and specificity of the WNV NS1 LMIA relative to corresponding WNV FRNT90-confirmed sera were determined to be 96% and 86%, respectively. While further optimization is required, this study demonstrates the feasibility of developing a species-independent LMIA for on-site analysis of WNV, USUV, and other arboviruses. Such a tool would be useful for the on-site screening and monitoring of relevant species in more remote or low-income regions.

Serum CYFRA21–1 and SCC-Ag levels in women during pregnancy and their diagnostic value for cervical cancer

ObjectivesThe incidence of cervical cancer increases every year during pregnancy. Cervical cytology in pregnant women has a unique morphology and liquid-based cytology methods are prone to cause false positives. The aim of this study was to investigate the serum cytokeratin 19 fragment antigen 21–1 (CYFRA21–1) and squamous cell carcinoma associated antigen (SCC-Ag) concentrations in healthy pregnant women during pregnancy and to assess their diagnostic value for cervical cancer in pregnancy. MethodsIn this prospective study, 165 healthy non-pregnant women, 441 healthy pregnant women and 22 patients with cervical cancer in pregnancy were recruited. The healthy pregnant women group included 143 women in the first trimester (T1), 147 in the second (T2) and 151 in the third (T3). ResultsBoth SCC-Ag and CYFRA21–1 levels were significantly different in the healthy pregnant women group compared to the control group. The CYFRA21–1 and SCC-Ag were higher in the T1 and T3 than in the control groups. However, there was no statistically significant difference in serum CYFRA21–1 and SCC-Ag levels in the T2 group compared to the control group. The AUCs of CYFRA21–1, SCC-Ag and CYFRA21–1 combined with SCC-Ag were 0.674, 0.792, and 0.805, respectively. The cut-off values of CYFRA21–1 and SCC-Ag were 6.64 ng/mL and 1.75 ng/mL, respectively. ConclusionsSerum CYFRA21–1 and SCC-Ag levels were higher in pregnant women during early and late pregnancy compared to non-pregnant individuals, while they were not statistically different from non-pregnant women during mid-trimester. CYFRA21–1 and SCC-Ag have diagnostic value for cervical cancer in pregnancy.

Development of recombinant oyster heat shock protein 70 mediated in situ capture RT-qPCR to detect human norovirus and Tulane virus

Human noroviruses (HuNoVs) are the major foodborne pathogens that cause non-bacterial gastroenteritis globally. Conventional RT-qPCR is prone to counting free RNA in the sample, resulting in an inflated infectious virus titer. Porcine gastric mucin (PGM), broadly used as the capture unit of in situ capture RT-qPCR (ISC-RT-qPCR), can precisely capture intact norovirus without adsorbing free RNA. However, PGM is a complex mixture that is difficult to obtain, and the concentration of histo-blood group antigens in it is unclear, hampering its widespread use. This study introduced a newly discovered capture unit, recombinant oyster heat shock protein 70 (roHSP70), to replace PGM. The roHSP70-mediated ISC-RT-qPCR (roHSP-ISC-RT-qPCR) were developed and compared with the PGM-mediated ISC-RT-qPCR (PGM-ISC-RT-qPCR) and conventional column-based RNA extraction RT-qPCR (CBE-RT-qPCR). The results showed that roHSP70 can capture various genotypes of HuNoVs. The smaller concentration of roHSP70 (20.0 μg/mL) captured HuNoVs to a similar extent compared to PGM (1.0 mg/mL). In addition, roHSP-ISC-RT-qPCR can accurately detect the amount of authentic intact virus particles in heat-treated Tulane virus (TV) samples with an accuracy comparable to that of TCID50. Interestingly, roHSP-ISC-RT-qPCR gave lower titers than CBE-RT-qPCR when measuring cell-cultured TV with high titers. However, when measuring lower-titer TV in artificially contaminated oysters, roHSP-ISC-RT-qPCR instead gave significantly higher titers than CBE-RT-qPCR. Overall, roHSP-ISC-RT-qPCR had a similar excellent performance as the PGM-ISC-RT-qPCR in detecting intact norovirus in samples. Moreover, roHSP70 has broader application scenarios than PGM due to its single-identified component and easier accessibility.