Direct Immunoassay Research Articles

A-048 Accurate Measurement of Free Testosterone in Serum using Equilibrium Dialysis Coupled with ID-UHPLC-MS/MS

Abstract Background Assessment of free testosterone (FT) has been recommended in recent clinical practice guidelines as a biomarker for diagnosis and management of testosterone related disorders such as male testosterone deficiency due to hypogonadism and female androgen excess due to polycystic ovary syndrome (PCOS). FT is currently measured using direct commercial immunoassays, assays using equilibrium dialysis (ED) or other separation techniques. Though the ED approach is recognized as the recommended approach, it is laborious and technically challenging for routine clinical use. The CDC Clinical Standardization Program is developing a high throughput automated method using the ED procedure coupled with isotope dilution ultra-high-performance liquid chromatography tandem mass spectrometry (ID-UHPLC-MS/MS) that is suitable for routine patient care and large-scale epidemiologic studies. Methods Serum samples are dialyzed in a custom-designed multi-well plate against a protein-free HEPES buffer (pH 7.4) at 37 °C until equilibrium. After isolating endogenous FT from protein-bound testosterone by ED, isotope-labeled internal standard (13C3-testosterone) was added to the dialysate for quantification. Certified pure primary reference material (National Measurement Institute-M914) was used to prepare calibrators, enabling traceability and ensuring measurement trueness. FT was further isolated from the dialysate matrix using supported liquid extraction and a chromatographic separation from interfering compounds and quantitation by tandem MS. Results The measurement ranges covered 0.2–1000 ng/dL for testosterone, with the bias within ±5% and precision less than 10% CV. A total of 45 samples with a wide range of total testosterone (TT, 21–912 ng/dL) and SHBG (11–129 nmol/L) were analyzed and showed the suitability of this assay to measure the serum free testosterone levels in normal, hypogonadal males as well as in the majority of normal females and females with androgen excess. The application of 48-well format plate and automated liquid handler system significantly improves the throughput of sample preparation. In addition, comparison of the TT results measured with the presented TT method with calculated TT concentrations, suggest that on average calculation overestimates TT concentration by 34%. Conclusion The described high throughput method for FT allows for sufficiently accurate and precise measurement for routine applications including large epidemiologic studies to help with establishing reference intervals.

A-080 Overcoming Challenges of Equilibrium Dialysis-Based Free Thyroxine Measurements

Abstract Background Thyroid function tests are widely used to detect diseases of the thyroid. Concerns about the accuracy and reliability of free thyroxine (FT4) measurements have been stated by the clinical laboratory community. To address these concerns, CDC established a reference measurement procedure (RMP) for FT4 based on equilibrium dialysis (ED). ED-based methods have advantages over traditional direct immunoassays. As with all analytical methods, factors affecting accuracy and reliability of ED-based methods need identification and understanding. To investigate potential sources of error, the CDC FT4 RMP was used to evaluate T4 stability at different of storage conditions, adsorption of FT4 to common labware surfaces, and the effect of dialysis membrane materials on the measured FT4 in serum. Methods FT4 was measured in serum using the IFCC RMP (1). FT4 was separated from the protein-bound form during the equilibrium dialysis step of the RMP. Dialysates were spiked with internal standard (13C6-T4) and the analyte was further isolated by C18 solid phase extraction (SPE) and liquid-liquid extraction (LLE) in ethyl acetate prior to injection on an UHPLC system coupled with a triple quadrupole mass spectrometer. Bracketed calibration and primary reference materials were used to determine FT4 concentration in serum. This procedure was used to evaluate stability of serum FT4 during storage at −70 to 5 °C. Short- and long-term T4 stability of calibrator solutions at −70 °C to ambient temperature and the extent of adsorption of T4 calibrator solutions to the commonly used labware surfaces were evaluated. Furthermore, the suitability of 4 different dialysis membranes was assessed. In addition, three alternatives to the currently used SPE and LLE extraction steps were tested: LLE using cyclohexane and ethyl acetate, LLE with ethyl acetate, and supported liquid extraction with cyclohexane and ethyl acetate. Results T4 calibrator solution was stable in 1.7% ammonium hydroxide in ethanol for at least 2.9 years at −70 °C and up to 7 days at ambient temperature. Exposure of various calibrator solutions such as T4 in ethanol, ethanol with 1.7% ammonium hydroxide, 10% acetonitrile with 0.1% formic acid to common labware resulted in up to 10.1% loss of the analyte. FT4 in serum and T4 in dialysates and extracts were stable at −70 to 5 °C. Use of alternative dialysis membranes resulted in up to −8.8% mean bias to the reference membranes. Recoveries of the analyte using four extraction methods were 85.4%–95.0%. None of the tested extraction conditions significantly changed the results obtained with the original FT4 RMPs. Conclusion Although FT4 RMP must strictly adhere to the ED conditions described for the RMP, other parts of the sample preparation procedures can deviate from the originally published procedure. Careful selection of procedure for calibrator solvents, dialysis membranes, labware, and storage conditions is required to ensure accurate and reproducible FT4 measurements.

Risk factors associated with blood vitamin D levels in COVID-19 patients

The two main factors associated with infectious diseases are the presence of antigens that enter the body and the immune system. It has been recently reported that vitamin D may be associated with the immune system. This study aimed to analyze the relationship between blood vitamin D levels and the occurrence of COVID-19 infection. This study used a cross-sectional approach for analytical survey research. The population in this study were all lecturers at Malahayati University. The total population participating in this study was 62 lecturers at Malahayati University. Direct competitive chemiluminescence immunoassays were used to measure vitamin D levels, while interviews collected other variables. Logistic regression analysis has been used to analyze the collected data. Results: There was a significant relationship between vitamin D levels and the incidence of COVID-19 infection (p= 0,020; OR= 4,9) & the habit of always wearing long clothes for more than 10 years is significantly related to blood vitamin D levels (p= 0,021; OR= 5,07). In conclusion, the lower the vitamin D level, the greater the risk of being infected with COVID-19, and the longer you wear long clothes, the higher the risk of having low blood vitamin D levels.

Comparison of Direct and Extraction Immunoassay Methods With Liquid Chromatography-Tandem Mass Spectrometry Measurement of Urinary Free Cortisol for the Diagnosis of Cushing's Syndrome.

Twenty-four-hour urinary free cortisol (UFC) measurement is the initial diagnostic test for Cushing's syndrome (CS). We compared UFC determination by both direct and extraction immunoassays using Abbott Architect, Siemens Atellica Solution, and Beckman DxI800 with liquid chromatography-tandem mass spectrometry (LC-MS/MS). In addition, we evaluated the value of 24-hr UFC measured by six methods for diagnosing CS. Residual 24-hr urine samples of 94 CS and 246 non-CS patients were collected. A laboratory-developed LC-MS/MS method was used as reference. UFC was measured by direct assays (D) using Abbott, Siemens, and Beckman platforms and by extraction assays (E) using Siemens and Beckman platforms. Method was compared using Passing-Bablok regression and Bland-Altman plot analyses. Cut-off values for the six assays and corresponding sensitivities and specificities were calculated by ROC analysis. Abbott-D, Beckman-E, Siemens-E, and Siemens-D showed strong correlations with LC-MS/MS (Spearman coefficient r=0.965, 0.922, 0.922, and 0.897, respectively), while Beckman-D showed weaker correlation (r=0.755). All immunoassays showed proportionally positive bias. The areas under the curve were 0.975 for Abbott-D, 0.972 for LC-MS/MS, 0.966 for Siemens-E, 0.948 for Siemens-D, 0.955 for Beckman-E, and 0.877 for Beckman-D. The cut-off values varied significantly (154.8-1,321.5 nmol/24 hrs). Assay sensitivity and specificity ranged from 76.1% to 93.2% and from 93.0% to 97.1%, respectively. Commercially available immunoassays for measuring UFC show different levels of analytical consistency compared to LC-MS/MS. Abbott-D, Siemens-E, and Beckman-E have high diagnostic accuracy for CS.

Development and pathogenicity analysis of full-length infectious cDNA clones of citrus yellow mottle-associated virus in citrus plants

Citrus yellow mottle-associated virus (CiYMaV) belonging to the subgenus Mandarivirus within the genus Potexvirus, was first identified in 2018 from Pakistan (CiYMaV-PK), where it is endemic in several regions. Here, three full-length cDNA clones (pCiYMaV-FL-1, pCiYMaV-FL-18, and pCiYMaV-FL-22) corresponding to the genomic RNA of CiYMaV were constructed and then agroinfiltrated on Chandler pummelo (Citrus grandis) seedlings using the vacuum infiltration method. All the inoculated plants developed severe vein yellowing, leaf mottling, and dwarfing symptoms by 40 days post-infiltration (dpi). The results of a direct tissue blot immunoassay and reverse transcription polymerase chain reaction detection showed 94.7–100% infection rates of pCiYMaV-FL at 60 dpi. Despite there being no observed difference among the three clones in the severity of symptom, pCiYMaV-FL-22 showed the highest accumulation levels of viral RNA and coat proteins. Moreover, pCiYMaV-FL-22 successfully infected seven other citrus varieties and induced symptoms in five of them. Transmission electron microscopy identified the presence of filamentous virus particles in extracts from systemic leaves of the plants infected with pCiYMaV-FL-22 at 6-months post-infiltration. Taken together, the results indicate that Koch's postulates were fulfilled for CiYMaV in citrus plants. This is the first report of full-length infectious cDNA clones of CiYMaV, and thus, the data provide a basis for further study of the molecular mechanisms of virus infection and the development of a viral vector to express foreign genes in citrus plants.

Prediagnostic Hormone Levels and Risk of Testicular Germ Cell Tumors: A Nested Case-Control Study in the Janus Serum Bank.

It has been hypothesized that poorly functioning Leydig and/or Sertoli cells of the testes, indicated by higher levels of serum gonadotropins and lower levels of androgens, are related to the development of testicular germ cell tumors (TGCT). To investigate this hypothesis, we conducted a nested case-control study within the Janus Serum Bank cohort. Men who developed TGCT (n = 182) were matched to men who did not (n = 364). Sex steroid hormones were measured using LC/MS. Sex hormone binding globulin, follicle-stimulating hormone (FSH), and luteinizing hormone (LH) were quantified by direct immunoassay. Multivariable logistic regression was used to calculate ORs and 95% confidence intervals (CI) for associations between hormone levels and TGCT risk. Higher FSH levels [tertile (T) 3 vs. T2: OR = 2.89, 95% CI = 1.83-4.57] were associated with TGCT risk, but higher LH levels were not (OR = 1.26, 95% CI = 0.81-1.96). The only sex steroid hormone associated with risk was androstane-3α, 17β-diol-3G (3α-diol-3G; OR = 2.37, 95% CI = 1.46-3.83). Analysis by histology found that increased FSH levels were related to seminoma (OR = 3.55, 95% CI = 2.12-5.95) but not nonseminoma (OR = 1.19, 95% CI = 0.38-3.13). Increased levels of 3α-diol-3G were related to seminoma (OR = 2.29, 95% CI = 1.35-3.89) and nonsignificantly related to nonseminoma (OR = 2.71, 95% CI = 0.82-8.92). Higher FSH levels are consistent with the hypothesis that poorly functioning Sertoli cells are related to the development of TGCT. In contrast, higher levels of 3α-diol-3G do not support the hypothesis that insufficient androgenicity is related to risk of TGCT. Clarifying the role of sex hormones in the development of TGCT may stimulate new research hypotheses.

Methodological considerations in determining sex steroids in children: comparison of conventional immunoassays with liquid chromatography-tandem mass spectrometry.

In laboratory medicine, external quality assessment (EQA) schemes have become versatile tools for detecting analytical flaws. However, EQA schemes are lacking for pediatric sex steroid levels. We aimed to investigate the suitability of different estradiol and testosterone immunoassays in a pediatric setting in comparison with clinical liquid chromatography-tandem mass spectrometry (LC-MS/MS) assays. The study was conducted by staff and the advisory group on endocrinology at Equalis, the Swedish provider of EQA schemes for laboratory medicine. The test material consisted of five pooled serum samples from children who were either prepubertal or in puberty. Clinical laboratories enrolled in Equalis EQA schemes for estradiol and testosterone were invited to participate, as were clinical laboratories using LC-MS/MS-assays. Samples were analyzed by either routine immunoassays (n=18) or in-house LC-MS/MS assays (n=3). For estradiol, LC-MS/MS assays showed a high degree of conformity with interlaboratory coefficients of variation (CV) below 24.2 %. Reported levels were between 4.9±1.2 and 33.9±1.6 pmol/L (group mean±standard deviation). The direct immunoassays had lower precision; their CVs were up to 81.4 %. Reported concentrations were between 25.3±18.1 and 45.7±19.4 pmol/L, an overestimation compared to LC-MS/MS. Testosterone LC-MS/MS also showed a high degree of conformity, CVs were below 13.4 %, and reported concentrations were from 0.06±0.00 to 1.00±0.11 nmol/L. The direct immunoassays had a larger discrepancy between results; CVs were up to 95.8 %. Concentrations were between 0.12±0.11 and 0.85±0.23 nmol/L. For the safe diagnosis and determination of sex steroids in children, analysis with mass spectrometry-based methods is recommended.

Ultrasensitive Electrochemical Immunosensors Using Nanobodies as Biocompatible Sniffer Tools of Agricultural Contaminants and Human Disease Biomarkers.

Nanobodies (Nbs) are known as camelid single-domain fragments or variable heavy chain antibodies (VHH) that in vitro recognize the antigens (Ag) similar to full-size antibodies (Abs) and in vivo allow immunoreactions with biomolecule cavities inaccessible to conventional Abs. Currently, Nbs are widely used for clinical treatments due to their remarkably improved performance, ease of production, thermal robustness, superior physical and chemical properties. Interestingly, Nbs are also very promising bioreceptors for future rapid and portable immunoassays, compared to those using unstable full-size antibodies. For all these reasons, Nbs are excellent candidates in ecological risk assessments and advanced medicine, enabling the development of ultrasensitive biosensing platforms. In this review, immobilization strategies of Nbs on conductive supports for enhanced electrochemical immune detection of food contaminants (Fcont) and human biomarkers (Hbio) are discussed. In the case of Fcont, the direct competitive immunoassay detection using coating antigen solid surface is the most commonly used approach for efficient Nbs capture which was characterized with cyclic voltammetry (CV) and differential pulse voltammetry (DPV) when the signal decays for increasing concentrations of free antigen prepared in aqueous solutions. In contrast, for the Hbio investigations on thiolated gold electrodes, increases in amperometric and electrochemical impedance spectroscopy (EIS) signals were recorded, with increases in the antigen concentrations prepared in PBS or spiked real human samples.

Development of Magnetic Lateral Flow and Direct Competitive Immunoassays for Sensitive and Specific Detection of Halosulfuron-Methyl Using a Novel Hapten and Monoclonal Antibody.

Halosulfuron-methyl (HM) is widely used for the removal of noxious weeds in corn, sugarcane, wheat, rice, and tomato fields. Despite its high efficiency and low toxicity, drift to nontarget crops and leaching of its metabolites to groundwater pose potential risks. Considering the instability of HM, the pyrazole sulfonamide of HM was used to generate a hapten and antigen to raise a high-quality monoclonal antibody (Mab, designated 1A91H11) against HM. A direct competitive immunoassay (dcELISA) using Mab 1A91H11 achieved a half-maximal inhibitory concentration (IC50) of 1.5 × 10-3 mg/kg and a linear range of 0.7 × 10-3 mg/kg-10.7 × 10-3 mg/kg, which was 10 times more sensitive than a comparable indirect competitive ELISA (icELISA) and more simple to operate. A spiking recovery experiment performed in tomato and maize matrices with 0.01, 0.05, and 0.1 mg/kg HM had average recoveries within 78.9-87.9% and 103.0-107.4% and coefficients of variation from 1.1-6.8% and 2.7-6.4% in tomato and maize, respectively. In addition, a magnetic lateral flow immunoassay (MLFIA) was developed for quantitative detection of low concentrations of HM in paddy water. Compared with dcELISA, the MLFIA exhibited 3.3- to 50-fold higher sensitivity (IC50 0.21 × 10-3 mg/kg). The average recovery and RSD of the developed MLFIA ranged from 81.5 to 92.5% and 5.4 to 9.7%. The results of this study demonstrated that the developed dcELISA and MLFIA are suitable for rapid detection of HM residues in tomato and maize matrices and paddy water, respectively, with acceptable accuracy and precision.

The content of sEMMPRIN/CD147 in the blood serum of patients with bone tumors and its relationship with the clinical and morphological characteristics of the disease

Introduction. Enzyme-linked immunosorbent assay of biochemical markers is one of the most important methods for diagnosing tumors. One of these markers is an inducer of expression of matrix metalloproteases EMMPRIN/CD147. Changes in its expression are associated with the progression of some tumors. This study is the first work devoted to the study of the content of the soluble form of the transmembrane glycoprotein EMMPRIN (sEMMPRIN) in the blood serum of patients with various bone tumors.Aim. To study the content of sEMMPRIN in the blood serum of patients with malignant bone tumors, its relationship with the clinical and morphological characteristics of neoplasms and prognosis.Materials and methods. The study included 88 patients with malignant tumors (osteosarcoma – 37 cases, chondrosarcoma – 39, chordoma – 5, Ewing’s sarcoma – 7) and borderline (11 cases) bone neoplasms, of which 14 patients were under the age of 18 years. The control group consisted of 29 healthy donors, 8 of which were under the age of 18 years. The concentration of EMMPRIN was determined in the serum of patients and donors with reagents for direct enzyme immunoassay “Human EMMPRIN” (R&D, USA) in accordance with the manufacturer’s instructions and expressed in nanograms (ng) per 1 ml of blood serum. The obtained data were processed using the GraphPad Prizm 9.4 program. When comparing indicators and analyzing their relationships, we used the nonparametric Mann–Whitney and Kruskal–Wallis tests. Overall survival was analyzed using the Kaplan–Meier method.Results. Our analysis of the sEMMPRIN content in the blood serum of patients with bone tumors did not reveal statistically significant differences between the control group and patients with borderline and malignant tumors, both in adults and in children. At the same time, a trend towards a decrease in the level of sEMMPRIN in the blood serum was noted in the presence of a malignant neoplasm of the bone compared with the corresponding control group. Additionally, we found that the content of sEMMPRIN is associated with age and higher in the group of patients under 18 years of age, both among healthy donors and oncological patients. An analysis of the association of sEMMPRIN content with clinical and morphological characteristics did not reveal statistically significant patterns, however, a trend towards an increase in the level of the marker with disease progression in both studied age groups was observed, which is consistent with other studies conducted on other solid tumors.Conclusion. ELISA revealed the marker sEMMPRIN in the blood serum of all examined children and adults with borderline malignant bone tumors and healthy donors. At the same time, the levels of sEMMPRIN did not differ between the above groups, however, there was a tendency for a decrease in the concentration of the marker in patients with bone sarcomas compared with the control group, regardless of age.

Magnetic Iron Oxide Nanoparticles as a Tool for the Advancement of Biomedical and Environmental Application: A Review

Recently, a number of studies have been devoted for the exploitation of magnetic nanoparticles with potential applications in medicine and environment. Among the oxide of magnetic nanoparticles, iron oxide has been emerged as essential tool in nanotechnology particularly, biotechnology. This is attributed to its exceptional properties such as size, shape, supermagnetism and biocompatibility. The iron oxide nanoparticle is less toxic and more biocompatible than other metal nanoparticles, making it an ideal substrate for biosensing, direct drug delivery, catalysis, (CT/MRI) dual-modality, photothermal therapy, and immunoassays applications. In this review, various synthetic methods of iron oxide nanoparticles have been discussed in detail. The potential applications of these iron oxide nanoparticles in the field of drug delivery, hyperthermia and MRI have also been reviewed in detail. These nanoparticles enhance the mechanical properties of a restorative material and improve the overall bonding between dentin and biomaterials, thus affecting the bond strength.

Evaluation of testosterone, estradiol andprogesterone immunoassay calibrators byliquid chromatography mass spectrometry.

In clinical practice, steroid measurements are performed mainly by direct, non-extraction immunoassays adapted to high throughput, automated immunoassay platforms and employing secondary calibrators. The accuracy of such steroid immunoassays is limited by cross-reactivity with structurally related steroids and nonspecific matrix interference as well as the metrological traceability of manufacturer supplied calibrators. The accuracy of steroid immunoassay calibrators has been little investigated by independent chemical methods. Steroid concentrations of 41 calibrators (4-6 replicates per calibrator) supplied by four manufacturers for use in testosterone (T), estradiol (E2), and progesterone (P4) commercial immunoassays were measured by ultra-pressure liquid chromatography-mass spectrometry (UPLC-MS). Among 14 non-zero T calibrators, six (43 %) deviated significantly from the label concentration with 29 % outside 20 % of it. Among 14 E2 calibrators, eight (57 %) deviated significantly, whereas seven (50 %) were outside 20 % of the label concentration. Among 11 P4 calibrators, eight (73 %) deviated significantly whereas four (36 %) were outside within 20 % of the label concentration. We conclude that inaccurate calibration of manufacturer's supplied standards may contribute to inaccuracy of commercial direct steroid immunoassays.

Calibration of a serum reference standard for Group B streptococcal polysaccharide conjugate vaccine development using surface plasmon resonance

Group B streptococcus (GBS) is a leading cause of neonatal morbidity and mortality worldwide. Development of a maternal vaccine to protect newborns through placentally transferred antibody is considered feasible based on the well-established relationship between anti-GBS capsular polysaccharide (CPS) IgG levels at birth and reduced risk of neonatal invasive GBS. An accurately calibrated serum reference standard that can be used to measure anti-CPS concentrations is critical for estimation of protective antibody levels across serotypes and potential vaccine performance. For this, precise weight-based measurement of anti-CPS IgG in sera is required. Here, we report an improved approach for determining serum anti-CPS IgG levels using surface plasmon resonance with monoclonal antibody standards, coupled with a direct Luminex-based immunoassay. This technique was used to quantify serotype-specific anti-CPS IgG levels in a human serum reference pool derived from subjects immunized with an investigational six-valent GBS glycoconjugate vaccine.

A Direct Immunoassay Based on Surface-Enhanced Spectroscopy Using AuNP/PS-b-P2VP Nanocomposites

A biosensor was developed for directly detecting human immunoglobulin G (IgG) and adenosine triphosphate (ATP) based on stable and reproducible gold nanoparticles/polystyrene-b-poly(2-vinylpyridine) (AuNP/PS-b-P2VP) nanocomposites. The substrates were functionalized with carboxylic acid groups for the covalent binding of anti-IgG and anti-ATP and the detection of IgG and ATP (1 to 150 μg/mL). SEM images of the nanocomposite show 17 ± 2 nm AuNP clusters adsorbed over a continuous porous PS-b-P2VP thin film. UV–VIS and SERS were used to characterize each step of the substrate functionalization and the specific interaction between anti-IgG and the targeted IgG analyte. The UV–VIS results show a redshift of the LSPR band as the AuNP surface was functionalized and SERS measurements showed consistent changes in the spectral features. Principal component analysis (PCA) was used to discriminate between samples before and after the affinity tests. Moreover, the designed biosensor proved to be sensitive to different concentrations of IgG with a limit-of-detection (LOD) down to 1 μg/mL. Moreover, the selectivity to IgG was confirmed using standard solutions of IgM as a control. Finally, ATP direct immunoassay (LOD = 1 μg/mL) has demonstrated that this nanocomposite platform can be used to detect different types of biomolecules after proper functionalization.

Galectin-3 and matrix metalloproteinases 2 and 9 in peripheral blood of gastric cancer patients

Background: High incidence of gastric cancer (GC), its aggressive clinical course, rapid tumor dissemination, low sensitivity to chemotherapy and lack of reliable laboratory diagnostic criteria urgently require a search for the most informative markers associated with key biologic properties of the tumors.
 Aim: Comparative analysis of galectin-3, matrix metalloproteinase (MMP)-2, and MMP-9 levels in peripheral blood of GC patients and healthy donors, assessment of association of these markers with clinical morphological characteristics of the disease, and prognosis of overall and relapse-free survival.
 Materials and methods: Sixty (60) primary treatment-nave GC patients (38 men, 22 women) aged 29 to 81 years and 90 healthy donors compatible with their age and sex were included into the study. Galectin-3 was measured in EDTA plasma, MMP-2 and MMP-9 in serum with standard direct enzyme immunoassay kits "Human MMP-2 (total)", "Human MMP-9 (total)", "Human Galectin-3" (RD Systems, USA).
 Results: Plasma galectin-3 concentration in the GC patients was significantly higher than in the healthy controls (median 12.9 and 10.6 ng/ml, respectively; p 0.0001). No difference in serum MMP-9 levels between GC patients and control subjects were found, while MMP-2 level in the control group was significantly higher, than in the GC patients (p = 0.039). No association between galectin-3, MMP-2, and MMP-9 blood levels in the GC patients could be identified. In contrast to GC patients, there was a positive correlation of plasma galectin-3 with age in the control group (rs = 0.51, p 0.005). No associations between the biomarkers levels in blood and clinical and morphological characteristics of GC were established, except MMP-9 being higher at Т4а invasion depth as compared to the earlier Т2 level. Marked differences in the overall survival depending on plasma galectin-3 levels were found, with the cut-off level of 12.9 ng/ml: the 5-year overall survival in the patients with low galectin-3 was better, than in those with its higher level (50 and 43%, respectively; however, the difference was non-significant, р 0.1). Both overall and relapse-free survival of the GC patients was higher in those with low ( 212 ng/ml) serum MMP-2: the 5-year overall survival in this group comprised 60% versus 23% in the patients with higher MMP-2 (p = 0.018). The difference in relapse-free survival was non-significant. Serum MMP-9 levels had no significant impact on the survival of GC patients.
 Conclusion: The ambiguous data on the clinical role of galectin-3, MMP-2, MMP-9 in GC obtained in this study indicate the necessity of further investigation of their possible utility for the diagnostics and prognosis of treatment results.

Reversed-phase allergen microarrays on optical discs for multiplexed diagnostics of food allergies

A high percentage of the population suffers from multiple food allergies justifying the importance of reliable diagnostic methods. Single-analyte solutions based on the determination of specific immunoglobulins E (sIgE) are safe and fast but are generally time-consuming and expensive. Thus sustainable microanalytical methods that provide multianalyte profiling information are highly demanded. This work presents the in vitro biosensing of specific IgE levels based on a reversed-phase allergen array. The approach consists of optical biosensing supported by direct multiplex immunoassays and on-disc technology. It identifies 12 sIgE associated with food allergies in a single analysis with a low serum sample volume (25 µL). After processing captured images, specific signals for each target biomarker correlate to their concentration. The assay analytically performs well with 0.3 IU/mL and 0.41 IU/mL as the detection and quantification limits in serum, respectively. This novel method achieves excellent clinical specificity (100%) and high sensitivity (91.1%), considering the diagnosis obtained by clinical history and ImmunoCAP analysis. The results demonstrate that microanalytical systems based on allergen arrays can potentially diagnose multiple food allergies and are easily implemented in primary care laboratory settings.Graphical

Association between 25(OH)D3 Levels and the Presence of COVID-19 Symptoms

Background: Recently, there have been studies reporting a relationship between vitamin D levels in the blood and the immune system. This study aimed to analyze the association between vitamin D levels and symptoms of coronavirus disease 2019 (COVID-19).Materials and methods: This study was an analytical survey study with a cross-sectional approach, with lecturers at Universitas Malahayati that have been infected with COVID-19 in 2022 as subjects. Total 47 subjects were included. Subjects were fasted overnight, then blood samples were taken from subjects in the next morning. The blood was centrifuged, then the serum was separated for examination by the direct competitive chemiluminescence immunoassay (CLIA) method using Architect 25-OH Vitamin D Reagent. Data was analyzed using logistic regression.Results: None of the subjects had normal blood levels of 25(OH)D3 and almost half (48.9%) of the subjects had symptoms when infected with COVID-19. There was a significant relationship between the level of 25(OH)D3 (p=0.001) and the status of the COVID-19 vaccine (p=0.013) with the presence of symptoms in COVID-19 patients.Conclusion: The lower the level of 25(OH)D3 in the blood and the more incomplete the COVID-19 vaccine, the greater the onset potential of COVID-19 symptoms. It is necessary to maintain vitamin D intake and increase the coverage of the COVID-19 vaccine, especially at booster doses.Keywords: vitamin D, COVID-19, vaccination, health protocol

Recent development of microfluidics-based platforms for respiratory virus detection.

With the global outbreak of SARS-CoV-2, the inadequacies of current detection technology for respiratory viruses have been recognized. Rapid, portable, accurate, and sensitive assays are needed to expedite diagnosis and early intervention. Conventional methods for detection of respiratory viruses include cell culture-based assays, serological tests, nucleic acid detection (e.g., RT-PCR), and direct immunoassays. However, these traditional methods are often time-consuming, labor-intensive, and require laboratory facilities, which cannot meet the testing needs, especially during pandemics of respiratory diseases, such as COVID-19. Microfluidics-based techniques can overcome these demerits and provide simple, rapid, accurate, and cost-effective analysis of intact virus, viral antigen/antibody, and viral nucleic acids. This review aims to summarize the recent development of microfluidics-based techniques for detection of respiratory viruses. Recent advances in different types of microfluidic devices for respiratory virus diagnostics are highlighted, including paper-based microfluidics, continuous-flow microfluidics, and droplet-based microfluidics. Finally, the future development of microfluidic technologies for respiratory virus diagnostics is discussed.

Measurement of aldosterone by LCMS/MS and immunoassay before and after dichloromethane extraction in post seated saline suppression test samples

Background: The seated saline suppression test (SSST) is a confirmatory test for diagnosis of primary aldosteronism. Discordant results have been recently observed for LC-MS/MS compared to direct immunoassay in SSST patients.1 This study investigates the concordance of LC-MS/MS and immunoassay results after dichloromethane extraction for the SSST.

Nanobody-Based Blocking of Binding ELISA for the Detection of Anti-NS1 Zika-Virus-Specific Antibodies in Convalescent Patients.

Zika virus has spread around the world with rapid pace in the last five years. Although symptoms are typically mild and unspecific, Zika's major impact occurs during pregnancy, generating a congenital syndrome. Serology plays a key role in its diagnosis. However, its use is limited due to the uncertainty caused by the cross-reaction of antibodies elicited in response to other flavivirus infections when tested in direct immunoassays. Using a panel of previously generated anti-Zika non-structural protein 1 (NS1) nanobodies, a set was selected that only recognizes epitopes present in Zika and is immunogenic to humans. A proper arrangement of these nanobodies was made and conditions were optimized in order to develop a novel serology assay. This new ELISA relies on the inhibition of the binding of a set of selected nanobodies to Zika-immobilized NS1 when previously incubated with Zika convalescent sera. Using the developed blocking of binding assay, it was possible to discriminate between Zika-specific and cross-reactive antibodies in serum samples from infections with Zika and other flaviviruses.