Determination of Leptospiral antigens in naturally infected canine uterus by immunohistochemical immunofluorescence and ELISA methods

Soy proteins are commonly used in the food industry thanks to their technological properties. However, soy is, along with cow’s milk, eggs, wheat, peanuts, tree nuts, fish, crustaceans, and molluscs, responsible for around 90% of food allergies, and is also one of the foodstuffs that can cause anaphylaxis. The aim of this work was to compare the immunofluorescence method for the detection of soy protein in meat products purchased from the retail market with other microscopic methods (immunohistochemical and histochemical), with the ELISA reference method and with the confirmatory results. Within the research, 127 meat products purchased in the retail network were examined using the immunofluorescence method used for the detection of soy protein. The method was compared to Enzyme-Linked ImmunoSorbent Assay (ELISA), immunohistochemical, and histochemical methods. According to McNemar’s test, non-compliance between the immunofluorescence method and immunohistochemical method was low. In addition, a significant difference between the fluorescence method and ELISA (P < 0.05) and a highly significant difference between the fluorescence method and histochemical examination (P < 0.01) was found. The immunofluorescence method was also compared with confirmatory results. According to McNemar’s test, non-compliance between the immunofluorescence method and confirmatory results was low. The results showed the possibilities of this new method to detect the content of soy protein in meat products.

EC1456 is a folate-targeted small molecule drug conjugate (SMDC) of tubulysin B hydrazide (TubBH) that is currently in Phase I clinical studies for treatment of patients with folate receptor (FR)-positive tumors. The ability to detect, quantitate, and localize drug in tissues and biological fluids is particularly important for the characterization of its biodistribution and pharmacodynamic properties. To aid in the biological characterization of our targeted TubBH conjugates, our lab had a rabbit polyclonal antibody produced against TubBH for use in various assays. An IgG-purified fraction of antiserum was used to develop competitive ELISA, immunohistochemical, and flow cytometry methods for analysis of biological samples, such as xenograft tumors from animals dosed with drug. Before assay development, a direct ELISA was performed to determine antibody titer and specificity. The TubBH antibody showed excellent binding to immobilized TubBH, whereas a rabbit IgG negative control antibody had minimal binding. Next, a competitive ELISA was developed in which soluble TubBH competed for antibody binding to immobilized antigen. Multiple experimental parameters were optimized, including coating antigen concentration, antibody dilution, and sample matrix extraction method. Using the optimized conditions, the resulting standard curves for TubBH in acetonitrile-extracted KB tumor sample matrix showed good sensitivity and dose-response. It was determined that, in addition to untargeted TubBH, the antibody could also be used to quantitate intact EC1456, as well as a metabolite of the drug lacking the hydrazide. The utility of the antibody was further demonstrated utilizing a PSMA-targeted version of TubBH. Once optimized, the ELISA method was used to analyze FR-positive KB and FR-negative A549 xenograft tumor homogenates from animals dosed with either EC1456 or untargeted TubBH. The levels of TubBH were higher 4h-post EC1456 dose in the FR-positive KB tumors compared to the FR-negative A549 tumors; however, levels were similar after 24h. Moreover, untargeted TubBH resulted in lower tumor concentrations of the drug compared to EC1456 in KB tumors. An immunohistochemical method developed using the antibody showed strong, focal, cytoplasmic staining in FFPE tumor sections of KB tumors from animals dosed with EC1456, while no staining was observed in tumors from undosed animals. Finally, the antibody was successfully used to detect cell surface FR-bound EC1456 on fixed and unfixed KB cells in vitro by flow cytometry. This experiment confirmed that the antibody is able to recognize the conjugate even when it is situated in the FR binding pocket. Collectively, these results show that this antibody against TubBH can be used as a powerful tool for analysis of tumor drug uptake for folate-targeted TubBH in xenograft models and could hold great potential for use in patient sample analysis as well. Citation Format: Nikki L. Parker, Jonathan M. Shillingford, Melissa Nelson, Joseph A. Reddy, Christopher P. Leamon. Development and characterization of in vitro assays to detect and quantitate tubulysin B hydrazide in biological samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3228. doi:10.1158/1538-7445.AM2017-3228

Diabetes mellitus (DM), a considerable concern for public health, serves as a predisposing factor for numerous diseases. Asprosin and meteorin-like protein (metrnl) are two peptides that play essential roles in glucose and energy metabolism. This study investigated the possible therapeutic potential of vitamin D (Vit D) against diabetes-induced liver damage in terms of asprosin and metrnl proteins. The study comprised four distinct groups: control, DM (60 mg/kg streptozotocin), Vit D (200 IU/kg), and DM+Vit D. Asprosin and metrnl levels were determined using both ELISA and immunohistochemical methods. Additionally, Total Oxidant Status (TOS) and Total Antioxidant Status (TAS) levels were assessed using the ELISA method. Asprosin and TOS levels increased in the DM group, whereas TAS and metrnl levels decreased. In the DM+Vit D group, metrnl levels increased while asprosin and TOS levels decreased significantly. These findings suggest that Vit D had protective effects against diabetes-induced liver damage and improved asprosin and metrnl levels. It has been concluded that Vit D could be considered as an attractive therapeutic agent target for asprosin and metrnl, which have positive effects on diabetes.

Objective. Reveal adenoviruses (AdV) that cause pneumonia in sheep and examine pathologic changes in the pulmonary and mediastinal lymph nodes of naturally infected adenovirus-positive specimens. Material and method. For this purpose, 1459 lungs of sheep slaughtered in a slaughterhouse were macroscopically examined, and pneumonia lesions were detected in 88 (6.03%) of these. The paraffinized tissue sections of these specimens with pneumonia were examined with the immunohistochemical (IHC) and indirect immunofluorescence (IF) methods, whereas their tissue homogenates were examined using the Antigen ELISA and PCR methods for adenovirus positivity. Results. Accordingly, the prevalence of adenoviruses was determined as 19.3% for IHC, 22.7% for IF, 20.5% for ELISA and 13.6% for PCR. Hematoxylin-eosin (HE) staining was performed to examine histopathological changes in the specimens that were naturally infected with adenoviruses. The histopathological examinations of the naturally infected lung specimens revealed mainly interstitial pneumonia, as well as catarrhal and verminous pneumonia findings. Consequently, it was determined that the most effective methods in the detection of adenoviruses in sheep pneumonias were found respectively as IF, ELISA, IHC and PCR. The finding that adenoviruses were observed only in the mediastinal lymph nodes of some specimens in the immunopathological methods suggested that the latency. Conclusions. The presence of adenoviruses in sheep pneumonia cases was determined with the indirect immunofluorescence, antigen ELISA and PCR methods for the first time. The possibility of the latent nature of adenovirus infection in these species was also discussed for the first time.

Candida albicans is a polymorphic fungus that may be observed as both commensal and opportunistic pathogen in humans. As one of the major components of Candida cell wall structure, mannan plays an important role in the fungus-host cell interaction and in virulence. The ability to switch from yeast to hypha form of microorganism is crutial in the development of C.albicans infections. Hyphal form has different antigenic properties compared to yeast form and structural changes occur in the yeast cell wall during transition from yeast to hypha form. Although there are several factors associated with this transition process, sufficient information is not available. The aim of this study was to investigate the change of configuration in mannan structure found in C.albicans cell wall by using monoclonal antibodies. C.albicans (NIHA 207) serotype A strains were used as test strains throughout the study, together with Salmonella choleraesuis 211 and Salmonella infantis as controls with similar cell wall structures to that of C.albicans. Cultures were maintained on YPD-agar medium by incubating at 28°C for yeast forms, and on YPD-broth medium in a shaking incubator at 37°C for 3-4 hours for the growth of hyphal forms. Cells were harvested in the exponential phase, and after being washed, the mannan content from C.albicans were extracted from pellet by heating in 20 mM sodium citrate buffer for 90 minutes at 125°C. Hybridoma technique was used for the production of monoclonal antibodies. After immunizing the Balb/C mice with antigen, the splenocytes were harvested and fusion was performed between spleen cells and F0 myeloma cells. The clones grown in HAT medium were screened for the presence of antibody producing hybrid cells by ELISA method. The antibody isotypes were determined by using a commercial kit (Pierce Biotechnology, ABD). The culture supernatants which contained monoclonal antibodies were collected and purified according to the ammonium sulphate method. Sandwich ELISA and immunofluorescence (IF) methods have been used to detect the experimental reactions. In our study, highly specific class IgM murine monoclonal antibodies (mAb-2B7) against C.albicans yeast cell wall were obtained from clone 2B7. These antibodies cross-reacted with S.choleraesuis 211 and S.infantis bacteria sharing similar cell wall structure of C.albicans. The existence of mannan β-1,2 bonds on the surface of C.albicans yeast form was confirmed with a commercial monoclonal antibody (mAb-ACMK-1; Matriks Biotek(®), Turkey) specific for those bonds. Besides, mAb-ACMK-1 interacted with C.albicans yeast form and gave intense fluorescence (high positive reaction) in IF method, but no fluorescence (negative) was detected with hyphal form. This data, obtained for the first time with this study, indicates that the mannan β-1,2 bonds are either found infrequently or none in the fungal hyphal wall. Although both monoclonal antibodies recognize the mannan antigen, mAb-2B7 reacted with S.choleraesuis 211, while mAb-ACMK-1 did not, due to the difference of epitope specificity. In conclusion, monoclonal antibodies may facilitate the characterization of antigenic structures of Candida, which will lead for the identification of new determinants that may increase the sensitivity and specificity of commercial tests used for mannan detection in serum.

To evaluate ANA specificity using the fully automated multiplexed fluorescent microsphere immunoassay in patients affected either by rheumatoid arthritis or ankylosing spondylitis who developed strong positivity for ANA as assessed by indirect immunofluorescent method on HEp-2 cells during infliximab treatment. Three men affected by ankylosing spondylitis and 12 women affected by rheumatoid arthritis who developed ANA positivity at high titres during infliximab treatment underwent the identification of ANA specificity by multiplexed fluorescent microsphere immunoassay; moreover anti-DNA and anti-ENA antibodies were tested by indirect immunofluorescence and ELISA method, respectively. In 4 out of 15 cases, the determination of ANA reactivity by multiplexed fluorescent microsphere immunoassay was also performed on the serum collected before infliximab administration. One patient affected by rheumatoid arthritis showed multiple ANA reactivities against SS-A, SS-B, RNP, Sm, Jo-1 and histones; one patient affected by ankylosing spondylitis resulted positive for the same autoantibodies, except for anti-Sm antibody. Moreover, two patients, one with rheumatoid arthritis and one with ankylosing spondylitis, showed single antibody specificity to SS-B and RNP, respectively. The remaining 11 cases did not show any positivity. Instead, all the patients resulted negative for anti-ENA antibodies by the ELISA method. In the four cases tested for ANA specificity by multiplexed fluorescent microsphere immunoassay before and after infliximab administration no difference was found. The search for anti-DNA antibody always resulted negative by both the traditional immunofluorescent assay and the novel technique. The use of multiplexed fluorescent microsphere immunoassay in patients treated with infliximab with ANA positivity at high titres allowed to find some ANA specificities which were not revealed by ELISA method. Nevertheless, the majority of patients resulted negative in spite of ANA positivity at high titres; the molecular target of ANA which develop after infliximab administration still remains to be identified.

Aim. Investigate the promoter methylation of the Thrombospondin-1 (TSP1) gene in gastric cardia adenocarcinoma (GCA). Methods. MSP approach, immunohistochemistry method, and RT-PCR were used respectively to examine the promoter methylation of TSP1, its protein and mRNA expression in tumors and corresponding normal tissues. The expression and concentration of TGF-β1 were examined respectively by immunohistochemistry and ELISA method. The status of T cell immunity was examined by Flow cytometry analysis. Results. TSP1 was methylated in 34/96 (35.4%) tumor specimens, which was significantly higher than that in corresponding normal tissues (P < .001). Protein and mRNA expression of TSP1 in GCA tumor tissues were reduced significantly and were associated with TSP1 methylation. The protein expression of TGF-β1 was significantly higher in tumor tissues (P < .001) and was associated with TNM stage and histological differentiation. The concentration of active and total TGF-β1 did not show significant difference between the GCA patients with hypermethylation of TSP1 and without methylation of TSP1 (P > .05). The function of T cell immunity was significantly different between the GCA patients with hypermethylation of TSP1 and without methylation of TSP1. Conclusions. Epigenetic silencing of TSP1 gene by promoter hypermethylation may play an important role in GCA.

Background Mongolian medical warm acupuncture is efficacious in treatment of insomnia but the mechanism still remains unclear. There is no research on the effect on the changes in expression of mRNA in the receptors of 5HT, DA, 5HT1α, 5HT2α, and DRD2 and the levels of hormones related to the central HPA axis in the stress response. Objective To further explain the mechanism of Mongolian medical warm acupuncture in insomnia treatment and its role in protecting cerebral neurons, elaborate the effect of Mongolian medical warm acupuncture on the changes in expression of 5HT, DA, 5HT1α, and 5HT2α at different sites in the brains of the PCPA-induced insomnia model rats and the levels of hormones related to the central HPA axis in the stress response. Method 72 Wistar rats were randomly divided into blank group, model group, Mongolian medical warm acupuncture group, and diazepam group of 18 each. The rats were intraperitoneally injected with PCPA (400 mg/kg) for 2 consecutive days to induce insomnia. The sleep latency and sleep duration before and after treatment were observed. The content of 5HT in the hypothalamus and hippocampus was measured with the ELISA method. The content of mRNA in the 5HT1A and 5HT2A receptors in the hypothalamus and hippocampus with the qPCR technique. The content of DA in the hypothalamus and corpus striatum was determined with the ELISA method. The content of ELISA in the hypothalamus and the content of ACTH in the serum of the insomnia rats were determined with the ELISA method. The changes in content of the BCL-2 and BAX proteins in the hippocampus were observed with the immunohistochemical method. Result PCPA intraperitoneal injections were able to prolong the sleep latency of the rats and shorten the sleep duration (P&lt;0.05) when compared with the blank group. Mongolian medical warm acupuncture was able to alleviate the tendency of decreasing weight gain arising from insomnia (P&lt;0.05). Mongolian medical warm acupuncture was able to decrease the levels of CRH and ACTH in the hormones related to central HPA axis in the stress response (P=0.017, P=0.015) and alleviate the overexciting state of the HPA axis arising from insomnia. Mongolian medical warm acupuncture group was able to up-regulate the expression of the BCL-2 protein in the hippocampus, down-regulate the expression of the BAX protein, and increase the BCL-2/BAX ratio (P=0.011). Mongolian medical warm acupuncture was able to protect the hippocampal neurons by changing the content of the apoptosis factors (P=0.024). Conclusion Mongolian medical warm acupuncture decreases the levels of CRH and ACTH in the hormone related to the HPA axis, up-regulates the expression of the BCL-2 protein in the hippocampus, and down-regulates the expression of the BAX protein for improving rat insomnia by influencing the expression of 5HT1A and 5HT2A receptors in the hypothalamus and hippocampus.

Di-n-butyl phthalate induces oversecretion of vascular endothelium-derived NAP-2 and promotes epithelial-mesenchymal transition of urothelial cells in newborn hypospadias rats

ABSTRACTGliadin is a major allergen causing allergies occurring also in meat products. Since wheat protein is used as a meat substitute to reduce cost of meat products. Sensitive consumers of these products are really threatened by food allergies in different allergic reaction. The objective of the study was to compare the histochemical, immunochemical (ALERT gliadin screening test) and immunofluorescence methods for the detection of wheat protein in model meat samples and meat products. The limit of detection for the ALERT gliadin screening test was 10 × 104 mg kg−1 of addition, while the histochemical method demonstrated concentration of wheat protein already from 10 × 103 mg kg−1, and the immunofluorescence method from the concentration of 20 mg kg−1. Comparison of the methods using McNemar’s test shows a statistically highly significant difference (p = .01) between the immunofluorescence method and ELISA and a statistically highly significant difference (p = .01) between the immunofluorescence and histochemical methods.

LGK974 suppresses the formation of deep vein thrombosis in mice with sepsis

Investigation of leptospiral antigen with immunohistochemical and immunofluorescence methods in cattle kidney

This work compares the commonly used immunochemical methods for soya protein detection and alternative microscopic methods. Immunochemical methods were represented by the competitive ELISA method. Histochemical and immunohistochemical methods were used for microscopical examination. From a group of 252 meat products, each sample was examined for soya proteins by ELISA, histochemical, and immunohistochemical methods. The products came from the following categories: cooked sausages, ham, dry cooked sausages, and fermented sausages. The results showed that the highest accuracy was achieved by immunohistochemical examination. However, in the category of cooked sausages, this result was not statistically significant. Since the results in the individual categories differed, our results demonstrate that one single method does not always provide reliable and completely objective results. Immunohistochemical methods seem to be the most suitable for the verification of the reference immunochemical method results and prevention of false results.

Effects of combined transplantation of rat Schwann cells and fibroblasts on nerve regeneration of denervated perforator flaps in rats and the mechanism

Since gluten can induce coeliac symptoms in hypersensitive consumers with coeliac disease, it is necessary to label foodstuffs containing it. In order to label foodstuffs, it is essential to find reliable methods to accurately determine the amount of wheat protein in food. The objective of this study was to compare the quantitative detection of wheat protein in model sausages by ELISA and immunohistochemical methods. Immunohistochemistry was combined with stereology to achieve quantitative results. High correlation between addition of wheat protein and compared methods was confirmed. For ELISA method the determined values were r = 0.98, P &lt; 0.01; for stereologythe determined values were r = 0.94, P &lt; 0.01. Although ELISA is an accredited method, it was not reliable, unlike immunohistochemical methods (stereology SD = 3.1).