Alkaline phosphatase interference in immuno-enzymatic assays.

Background: Alkaline phosphatase (ALP) enzymes are widely used as signal amplifiers in immunoenzymatic methods. Conditions that cause ALP elevations, such as bone or liver diseases can cause interference in immunoenzymatic methods. Objective: We aimed to examine ALP's effect on immunoenzymatic assay by adding isolated pure ALP to the prepared serum pool. Material and Methods: We prepared a serum pool and divided into 4 groups. By adding isolated pure ALP at different concentrations to each group, we obtained sample groups containing ALP enzyme at concentrations of 85 U/L, 340 U/L, 870 U/L and 1570 U/L. In each group, 20-repetition of βhCG, Ferritin, FT4, TSH, Troponin I and Vit B12 tests were performed. Coefficient of variation, bias, and total error were calculated. All groups were compared by using Friedman test for paired samples. Result: After ALP addition, the calculated total error values of FT4, βhCG and troponin I tests were found to be above the acceptable error limits. There were statistically significant differences in βhCG ,FT4, troponin I and Vit B12 tests when compared to the baseline ALP level (P<0,0125).Conclusion: Isolated ALP elevations can be a source of interference for immunoenzymatic methods.KeywordsAlkaline phosphatase, ALP, bias, immunoenzymatic, total error

Steroid hormonal endpoints in goats carrying single or twin fetuses reared in semi-extensive systems

The study provides baseline data regarding 17-β-estradiol (E2), progesterone (P4), and cortisol profile of 30 Nicastrese goats during different physiological periods. Animals were evaluated monthly from the pre-mating period (non-pregnant), during pregnancy, and from 30 to 105 d of lactation. The effects of single or twin births and the kid's sex were also considered. Serum E2, P4, and cortisol concentrations were measured using immunoenzymatic assay kits. The highest concentrations of E2 and P4 (P<0.0001) were found during pregnancy and their lowest values (P<0.0001) in the non-pregnant period. E2 was negatively correlated with P4 (r=-0.41; P<0.01) during lactation. The mothers with twin kids showed the highest concentration of P4 (P<0.04) at > 95–115 d of gestation and the lowest of E2 (P<0.04) at > 50–70 d of lactation. Pregnant goats carrying male kid(s) presented the highest E2 concentrations (P<0.02) at > 130–150 d of gestation. Different physiological conditions induced a temporal relationship with the endocrine profile in Nicastrese goats. Understanding the effects of single or twin fetuses on the gestation and lactation will also be helpful to improve the managemental approach for the health of mothers and their kids.

Eotaxins and Their Receptor as Biomarkers of Colorectal Cancer

Colorectal cancer (CRC) is one of the most common malignancies. Despite the availability of diagnostic tests, an increasing number of new cases is observed. That is why it is very important to search new markers that would show high diagnostic utility. Therefore, we made an attempt to assess the usefulness of eotaxins, as there are few studies that investigate their significance, in patients with CRC. The study included 80 subjects (CRC patients and healthy volunteers). Serum concentrations of all eotaxins were measured using a multiplexing method (Luminex), while CCR3 was measured by immunoenzymatic assay (ELISA). CRP levels were determined by immunoturbidimetry and classical tumor marker levels (CEA and CA 19-9) and were measured using chemiluminescent microparticle immunoassay (CMIA). The highest usefulness among the proteins tested showed CCR3. Its concentrations were significantly higher in the CRC group than in healthy controls. The diagnostic sensitivity, specificity, positive and negative predictive value, and the area under the ROC curve (AUC) of CCR3 were higher than those of CA 19-9. The maximum values for sensitivity, negative predictive value, and AUC were obtained for a combination of CCR3 and CRP. Our findings suggest the potential usefulness of CCR3 in the diagnosis of CRC, especially in combination with CRP or CEA.

Development and Validation of an Immunoenzymatic Assay for Pig IgG Quantification in Serum Samples

This research describes the development and validation of a sandwich-type Enzyme-Linked-Immunosorbent Assay (ELISA) assay based on a sheep polyclonal antibody developed to determine porcine immunoglobulin G (IgG) concentration in serum samples. The coating and blocking conditions were established. The immunoassay demonstrated the ability to specifically quantify pig IgG in serum samples without reacts with rabbit or mouse IgG, evaluated in serum and ascites, respectively. Given its precision, specificity and accuracy, this ELISA is a tool for the measurement of IgG in porcine serum samples, to evaluate the humoral immune status of pigs.

Levels of Advanced Oxidation Protein Products in Blood Plasma of Peri- and Postmenopausal Women with Insomnia

Background. Insomnia occurs in more than half of menopausal women. These disorders can contribute to a change in the prooxidant-antioxidant balance, causing the damage to structural cellular elements. Currently, there is a lack of research on this issue.Aim. To carry out a comparative analysis of the level of advanced oxidation protein products in in periand postmenopausal women with insomnia.Materials and methods. The study included peri(n = 30) and postmenopausal (n = 60) women, who were divided into 2 groups (control and main groups) in each menopausal phase after being questioned using special sleep questionnaires: Insomnia Severity Index; Epworth Sleepiness Scale; Munich Chronotype Questionnaire. The advanced oxidation protein products (AOPP) levels was determined by immunoenzymatic assay using ImmunDiagnostik (German) kits on a BioTek EL×808 (USA) analyzer. Statistical analysis was performed using Mann – Whitney test.Results. Comparative analysis of the AOPP levels in control groups, depending on the menopausal periods, showed an increase in their levels in the postmenopausal period as compared to perimenopause (p < 0.05). When comparing the AOPP levels between the control and the main group in different menopausal periods, statistically significant differences were revealed only in the perimenopausal period towards a higher content in women with insomnia (p < 0.05). The presence of insomnia in postmenopausal women is accompanied by a higher AOPP levels as compared to the perimenopausal women (p < 0.05).Conclusion. The obtained results indicate the association between insomnia and oxidative proteins modification only in the perimenopausal period.

Effect of postoperative corticosteroids on surgical outcome and aqueous autotaxin following combined cataract and microhook ab interno trabeculotomy

To evaluate the effect of postoperative corticosteroids on surgical outcome and autotaxin (ATX) levels after microhook ab interno trabeculotomy combined with cataract surgery (μLOT-CS), prospective, consecutive non-randomized case series comparing outcomes of 30 eyes with primary open angle glaucoma was performed. The aqueous ATX, intraocular pressure (IOP) and glaucoma medications were monitored for 3 months postoperatively. An in-vivo mouse μLOT model was generated. In vitro, ATX and fibrotic changes induced by dexamethasone (Dex) treatment following scratch (S) in cultured human trabecular meshwork (hTM) cells were assessed by immunofluorescence, immunoenzymatic assay, and RT-qPCR. Postoperative ATX at 1 week and the number of antiglaucoma medications at 3 months were significantly lower in non-steroid group, and steroid use was the only variable significantly associated with postoperative medications at 3 months in multiregression analyses. In vitro, ATX activity was significantly upregulated in the Dex + S group, and αSMA was significantly upregulated in the Dex and Dex + S groups. Fibronectin and COL1A1 were significantly upregulated in the S group. μLOT-CS decreased IOP and medications in the overall cohort, and non-use of postoperative steroids resulted in a smaller number of postoperative medications. Limiting postoperative steroids in μLOT may minimize IOP elevation and postoperative fibrosis.

Deoxynivalenol reduction through the processing of whole grain cookies

Whole-grain products are increasingly integrated into consumers diets due to the presence of the bran fraction that concentrates phenolic and antioxidant compounds. However, the bran may also contain mycotoxins, toxic compounds, harmful to health and undesirable in food. For wheat, the mycotoxin deoxynivalenol (DON) is the most prevalent. The objective of this work was to evaluate the reduction of DON after the processing of whole wheat cookies. Five commercial wheat, harvest 2017/18, naturally contaminated by Fusarium spp. with DON content higher or as established to Brazilian legislation (1000 ppb), were provided by Embrapa Trigo from Passo Fundo/RS. The cookies were baking mostly using flour, sugar and fat. The detection of DON was performed using the ELISA immunoenzymatic assay. Cookie production reduced the contamination of DON in the samples, and two products were in compliance with Brazilian legislation. The reduction of contamination by DON may have occurred due to the dilution effect by the ingredients in samples 1 and 3. The reduction of contamination by DON of the other samples (2, 4 and 5) was attributed to thermal degradation due to the baking temperature. Cookie processing is a complementary strategy to reduce the DON content in wheat products.

ASSESSMENT OF THE DEPENDENCE OF THE CLINICAL MANIFESTATION OF ACUTE GASTROENTERITIS CAUSED BY ROTAVIRUS ON ITS GENOTYPES

The leading cause of acute gastroenteritis (AGE) in children is rotavirus. In different countries, different rotavirus genotypes prevail and are associated with different severity of disease. The purpose of our study was to identify the distribution of rotavirus genotypes in Kyiv, Ukraine, and to determine the correlation between the genotypes and course of disease. Materials and methods. 978 children under 5 years of age were examined with АGE symptoms and not vaccinated against rotavirus. Determination of rotavirus antigen and genotype were performed using the immunoenzymatic assay and real-time RT-PCR. We assessed the demographics, clinical manifestations of AGE, the Vesikari scale AGE severity. Results. The G4P[8] genotype prevailed in Ukraine during 2014-2018. The G1P[8] was the second most common. G9P8 was the third, the fourth place was shared by G2P[4] and G3P[8]. Fever, as a manifestation, was more pronounced in G1P[8] and G3P[9]. The highest number of vomiting episodes per day occurred in the G1P[8] and G4P[8]-related cases. Maximum of diarrhea episodes per day was observed in genotypes G1P[8], G3P[8], G4P[8] and G9P[8]. Mucus and blood in stool were found in genotypes G3P[8] (1/33.33 %), G4P[8] – blood (1/2.27 %). The children with genotypes G1P[8] and G4P[8] had catarrhal symptoms. More cases of moderate and severe dehydration, occurred in the G4P[8]. The Vesikari scale analysis showed that only G1P[8] led to mild cases(3.57 %). The most widespread genotypes, G1P[8] and G4P[8], led to a moderate illness in 14.29 % and 13.56 % cases, respectively, and to a severe illness in 82.14 % and 86.44 % cases, respectively. Conclusions. G4P[8] was associated with the most severe disease due to more frequent and prolonged vomiting, febrile fever and bloody diarrhea. G1P[8] and G4P[8] were associated with catarrh.

Production and Easy One-Step Purification of Bluetongue Recombinant VP7 from Infected Sf9 Supernatant for an Immunoenzymatic Assay (ELISA)

Bluetongue (BT) is non-contagious, vector-borne viral disease of domestic and wild ruminants, transmitted by midges (Culicoides spp.) and is caused by Bluetongue virus (BTV). BTV is the type species of the Orbivirus genus within the Reoviridae family and possesses a genome consisting of 10 double-stranded RNA segments encoding 7 structural and 4 nonstructural proteins. Viral Protein 7 (VP7) is the major sera group-specific protein and is a good antigen candidate for immunoenzymatic assays for the BT diagnosis. In our work, BTV-2 recombinant VP7 (BTV-2 recVP7), expressed in Spodoptera frugiperda (Sf9) cells using a baculovirus system, was produced and purified by affinity chromatography from the supernatant of infected cell culture. The use of the supernatant allowed us to obtain a high quantity of recombinant protein with high purity level by an easy one-step procedure, rather than the multistep purification from the pellet. RecVP7-BTV2 was detected using a MAb anti-BTV in Western blot and it was used to develop an immunoenzymatic assay.