scholarly journals Dirofilaria immitis proteins recognized by antibodies from individuals living with microfilaremic dogs

2020 ◽  
Vol 14 (12) ◽  
pp. 1442-1447
Author(s):  
Oswaldo M Torres-Chable ◽  
Ligia G Brito-Argaez ◽  
Ignacio R Islas-Flores ◽  
Claudia V Zaragoza-Vera ◽  
Maritza Zaragoza-Vera ◽  
...  

Introduction: Dirofilaria immitis is a nematode that affects human health in several countries of the world. This study was conducted to examine whether serum samples from the owners of microfilaremic dogs present immunoreactivity to parasite proteins. Methodology: Eight serum samples from the owners of microfilaremic dogs were examined. Total proteins were extracted from adult worms and 12% SDS-PAGE was performed. The gel was electroblotted to a nitrocellulose membrane, and a Western blot (WB) was performed. Reactive bands of 22, 33, 39, 49, and 63 kDa in WB were excised from the gel and analyzed by mass spectrometry (MS). Results: The MS results showed the presence of 10 different proteins of D. immitis recognized by the human serum samples. Conclusions: These results indicate that in endemic areas of D. immitis, owners of infected dogs recognize specific proteins of the parasite, suggesting a possible infection.

2016 ◽  
Vol 78 (11-3) ◽  
Author(s):  
Tan Wee Yee ◽  
Darah Ibrahim ◽  
Khoo Boon Yin ◽  
Chew Ai Lan

D6 and DARC are decoy chemokine binding receptors. They had been studied extensively in recent years for their role in breast cancer studies. The purpose of this study was to improve some critical steps in Western Blot in order to detect and identify recombinant D6 and DARC which has been expressed intracellularly by Pichia-GS115. Total proteins of Pichia-GS115 were obtained by breaking the yeast cells using acid-washed glass beads. Then, the lysates were treated with treatment buffer before electrophoresed on SDS-polyacrylamide gel. Western Blot was carried out after SDS PAGE to detect and identify the presence of recombinant D6 and DARC. Several parameters of Western Blot were studied and improved to enhance the specificity of results obtained. It was found that blocking of nitrocellulose membrane with 3% (w/v) skimmed milk at room temperature for 1 hour, incubation of primary antibody at the dilution of 1: 300 at room temperature for 16 hours and incubation of secondary antibody at the dilution of 1: 1000 at room temperature for 1 hour were able to enhance the sensitivity and specificity of Western Blot results. With the aid of prestained protein ladder as a marker, recombinant D6 and also DARC were shown as clear and precise bands at the range of molecular weight of 50-60 kDa on developed films using ECL substrate. The film exposure time was found to be 5 minutes for D6 and 10 minutes for DARC. As a result, the studies showed that improved Western Blot condition detected and identified the presence of recombinant D6 and DARC in the cell pellets of Pichia-GS115


2020 ◽  
Vol 103 (6) ◽  
pp. 1461-1468
Author(s):  
Guru Prasad Katuri ◽  
Xinghua Fan ◽  
Shabana Siddique ◽  
Cariton Kubwabo ◽  
Ivana Kosarac ◽  
...  

Abstract Background Synthetic musk compounds are widely used as fragrances in many consumer products; however, information on human exposure and health effects is limited. Also, analytical methods for their quantification in biological matrices are limited. Objective In this study, an integrated method was developed and validated for the analysis of selected synthetic musk compounds in human serum. Method The method is based on liquid-liquid extraction (LLE), sample clean-up by solid-phase extraction (SPE), and separation and detection by gas chromatography coupled with tandem mass spectrometry (GC-MS/MS). Results The method demonstrated good recoveries (86–105%) and high sensitivity, with low method detection limits (MDLs) ranging from 0.04 to 0.17 µg/L. The method was applied to the analysis of 10 synthetic musk compounds in 40 serum samples collected from Canadian women aged 20–44 years (20 individual samples collected in 2014 and 20 pooled samples collected in 2006). The most commonly detected compound was Galaxolide (HHCB), with median concentrations of 0.59 µg/L in samples collected in 2006, and 0.34 µg/L for samples collected in 2014. Musk ketone (MK) was not detected in any of the samples collected in 2006, but was detected in 60% of the samples collected in 2014 with a median concentration of 0.29 µg/L. Tonalide (AHTN) was detected in only one sample above its MDL (0.12 µg/L). Conclusions This is the first study in Canada to report levels of synthetic musks in human. The data generated from this study has been used in risk screening assessment by Environment and Climate Change Canada and Health Canada.


Author(s):  
Tuom Thi Tinh Truong ◽  
Trang Thi Phuong Phan ◽  
Hoang Duc Nguyen

p24 protein is a component of the HIV particle capsid. It plays an essential role in HIV to infect into the host cell and in the cycle life of virus. Therefore, this protein can be used in the orientative study “to create and produce HIV’s vaccine”. This study created the new Bacillus subtilis strain which expressed p24 protein. B. subtilis a safety and non-toxic bacteria strain for humans and animals, has system expression to allow over expression recombinant protein up to 10-30 % of total proteins. Plasmid pHT1537 was cloned successfully, containing lysSN-6his-gagp24 gene to encode p24 protein fused with LysSN protein and to allow the expression of p24 protein in B. subtilis by IPTG inducer. The target protein in the cell was cheked by SDS-PAGE. The p24 fused protein was parified from His Trap column which contained Ni2+. Evaluation of the ability to produce antibody against p24 protein in mice by ELISA and Western blot was caried out.


2016 ◽  
Vol 83 (1) ◽  
Author(s):  
. Siswanto

AbstrakLateks alam maupun produk jadi yang berasal dari karet alam diketahui mengandung protein alergen. Namun demikian identifikasi jenis protein allergen belum banyak dilaporkan. Penelitian ini bertujuan untuk mendeteksi protein alergen dari sarung tangan dan lateks karet alam menggunakan metode immuno-chemiluminescense. Protein di-ekstrak dari tiga fraksi sentrifugasi lateks (serum B, serum C dan partikel karet) serta tujuh jenis sarung tangan komersial, kemudian dipisahkan berdasarkan berat molekulnya melalui Gel elektroforesis 1-D (SDS PAGE) dan 2-D. Selanjutnya untuk deteksi protein allergen secara immuno-chemiluminescense dilaku-kan imunobloting menggunakan serum Ig_E tiga pasien yang terbukti positif alergi terhadap protein asal sarung tangan lateks, kemudian diwarnai dengan Sypro Ruby protein blot fluorescence. Hasil penelitian menunjukkan bahwa  berdasarkan hasil analisis Western blot one-DE sampel protein lateks menggunakan serum tiga orang tenaga medis yang terbukti positif alergi terhadap protein lateks, maka dapat diidentifikasi 14 jenis protein alergen pada sarung tangan lateks, empat diantaranya merupakan pita major yaitu Berat Molekul (BM) 35, 38, 46 dan 56 kDa. Protein allergen pada sarung tangan tersebut kemungkinan berasal dari bagian C-serum terutama protein  BM 46 dan 56 kDa ataupun campuran antara C-serum dan B-serum dari lateks karet alam. Hal ini dibuktikan bahwa dari sampel C-serum lateks dapat teridentifikasi 12 protein alergen,  empat diantaranya merupakan pita major yaitu BM 42, 46, 51 dan        56 kDa. Sedangkan dari sampel B-serum teridenti-fikasi tiga pita major dengan BM 14, 16 and 51 kDa. Hasil analisis Western blot 2-DE ekstrak protein sarung tangan menggunakan serum tiga orang tenaga medis yang terbukti positif alergi terhadap protein lateks, maka dapat diidentifikasi 12 - 13 spot protein alergen dengan pI at 4.0 to 7.0 dan yang paling dominan adalah dengan BM 23, 35, 38, 42, 45, 46 kDa.Abstract  Natural rubber latex and finished products derived from natural rubber is known to contain allergenic proteins. Nevertheless identification of allergenic protein has not been widely reported. This study aims to detect the protein allergens from the glove of hands and natural rubber latex using immuno-chemiluminescense. Proteins extracted from the latex centrifugation three fractions (serum B, serum C and rubber particles) as well as seven types of commercial gloves, then separated by molecular weight through 1-D gel electrophoresis (SDS PAGE) and 2-D. Furthermore, for the detection of allergen proteins in immuno-chemiluminescense performed immunoblotting using the serum IgE three patients who tested positive for allergy to latex gloves native protein, and then stained with fluorescence Sypro Ruby protein blot. The results showed that based on the results of Western blot analysis of one-DE latex proteins using serum samples three medical personnels who tested positive for allergy to latex proteins, we can identify 14 types of protein allergens in latex gloves, four of which are major bands that having Molecular Weight (MW) 35, 38, 46 and 56 kDa. Protein allergen on the gloves are likely to come from the C-serum protein mainly MW 46 and 56 kDa, or a mixture of C-serum and B-serum of natural rubber latex. It was proved that from C-serum samples could be identified as many as 12 protein latex allergens, four of which were major bands that MW 42, 46, 51 and 56 kDa. While the B-serum samples identified three major bands with MW 14, 16 and 51 kDa. Results of Western blot analysis of 2-DE protein extracts glove using the serum three medical personnel who tested positive for allergy to latex proteins, it could be identified 12-13 allergen protein spot with pI at 4.0 to 7.0 and most dominant is the MW 23, 35, 38, 42, 45, 46kDa.


Author(s):  
Carl Jenkinson ◽  
Reena Desai ◽  
Andrzej T. Slominski ◽  
Robert C. Tuckey ◽  
Martin Hewison ◽  
...  

Abstract Objectives Clinical evaluation of vitamin D status is conventionally performed by measuring serum levels of a single vitamin D metabolite, 25-hydroxyvitamin D predominantly by immunoassay methodology. However, this neglects the complex metabolic pathways involved in vitamin D bioactivity, including two canonical forms D3 and D2, bioactive 1,25-dihydroxy metabolites and inactive 24-hydroxy and other metabolites. Methods Liquid chromatography-tandem mass spectrometry (LC-MS/MS) can measure multiple analytes in a sample during a single run with high sensitivity and reference level specificity. We therefore aimed to develop and validate a LC-MS/MS method to measure simultaneously 13 circulating vitamin D metabolites and apply it to 103 human serum samples. Results The LC-MS/MS method using a Cookson-type derivatization reagent phenyl-1,2,4-triazoline-3,5-dione (PTAD) quantifies 13 vitamin D metabolites, including mono and dihydroxy-metabolites, as well as CYP11A1-derived D3 and D2 metabolites in a single run. The lower limit of quantitation was 12.5 pg/mL for 1,25(OH)2D3 with accuracy verified by analysis of National Institute of Standards and Technology (NIST) 972a standards. Quantification of seven metabolites (25(OH)D3, 25(OH)D2, 3-epi-25(OH)D3, 20(OH)D3, 24,25(OH)2D3, 1,25(OH)2D3 and 1,20S(OH)2D3) was consistently achieved in human serum samples. Conclusions This profiling method can provide new insight into circulating vitamin D metabolite pathways forming the basis for improved understanding of the role of vitamin D in health and disease.


2020 ◽  
Vol 32 (1) ◽  
pp. 10-15
Author(s):  
Takeshi Saito ◽  
Akira Namera ◽  
Tomoatsu Tsuji ◽  
Wataru Noguchi ◽  
Sadaki Inokuchi

We developed and validated an assay for determination of glyphosate (GLYP) and glufosinate (GLUF) in human serum. Serum samples were extracted by using a MonoSpin® TiO column and analyzed by liquid chromatography–tandem mass spectrometry (LC–MS/MS). MonoSpin® TiO tends to specifically bind to phosphate groups. The assay was linear over a concentration range of 1–250 μg/mL. The recoveries for the 2 compounds were 1.6%–2.3%. The intra- and inter-day variations were <15%. Precision and accuracy were 5.6%–12.7% and 97.0%–103.9%, respectively. The validated method was applied to quantify the GLYP and GLUF content in the serum of GLYP and GLUF-poisoned patients. In conclusion, the method was successfully applied for accurate determination of GLYP and GLUF in serum obtained from patients with GLYP and GLUF poisoning.


2011 ◽  
Vol 2011 ◽  
pp. 1-9 ◽  
Author(s):  
Aleksandar Rakovic ◽  
Anne Grünewald ◽  
Lisa Voges ◽  
Sarah Hofmann ◽  
Slobodanka Orolicki ◽  
...  

Recent publications suggest that the Parkinson's disease- (PD-) related PINK1/Parkin pathway promotes elimination of dysfunctional mitochondria by autophagy. We used tandem affinity purification (TAP), SDS-PAGE, and mass spectrometry as a first step towards identification of possible substrates for PINK1. The cellular abundance of selected identified interactors was investigated by Western blotting. Furthermore, one candidate gene was sequenced in 46 patients with atypical PD. In addition to two known binding partners (HSP90, CDC37), 12 proteins were identified using the TAP assay; four of which are mitochondrially localized (GRP75, HSP60, LRPPRC, and TUFM). Western blot analysis showed no differences in cellular abundance of these proteins comparingPINK1mutant and control fibroblasts. When sequencingLRPPRC, four exonic synonymous changes and 20 polymorphisms in noncoding regions were detected. Our study provides a list of putative PINK1 binding partners, confirming previously described interactions, but also introducing novel mitochondrial proteins as potential components of the PINK1/Parkin mitophagy pathway.


RADIOISOTOPES ◽  
2013 ◽  
Vol 62 (8) ◽  
pp. 517-523 ◽  
Author(s):  
Takeshi KOBAYASHI ◽  
Teiko TOYOGUCHI ◽  
Kazuhiro KATO ◽  
Fuyuki TOKANAI ◽  
Tadashi SHIRAISHI

2021 ◽  
Author(s):  
Michele Iannone ◽  
Anna Pia Dima ◽  
Francesca Sciarra ◽  
Francesco Botrè ◽  
Andrea M. Isidori

Adrenal and gonadal disorders are very often coupled, due to common etiology or pathophysiology. We present the development, validation and application of a liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the simultaneous analysis of androgens (androstenedione (A4), testosterone (T), dihydrotestosterone (DHT), and dehydroepiandrosterone sulfate (DHEA-S)), estrogens (estrone (E1), estradiol (E2), estriol (E3)), glucocorticoids (cortisol (F), cortisone (E), corticosterone (B), 11-deoxycortisol (S), 21-deoxycortisol (21DF), 11-deoxycorticosterone (11DB)), and progestagens (progesterone (P4), 17α-hydroxyprogesterone (17OHP4) and 17α-hydroxypregnenolone (17OHP5)) in human serum for clinical use. Samples (250 μL of matrix) spiked with isotopic labelled internal standards were extracted with tert-butylmethyl ether (TBME) prior to LC-MS/MS analysis. The chromatographic separation of the underivatized endogenous steroids was achieved on a reversed-phase column (C18 Zorbax Eclipse Plus) using a methanol-water gradient. The LC column was coupled to a triple quadrupole mass spectrometer equipped with an electrospray (ESI) source operating both in positive and in negative mode, with acquisition in multiple reaction mode. The method was validated using surrogated matrices and human serum samples. The proposed method was proven to be specific for all the considered steroids; and linearity was also assessed (R2 > 0.99) in the ranges of quantification investigated. The lower limits of quantification (LLOQs) were in the range of 10 - 400 pg/mL depending on the target steroid. Accuracy was in the range 80 - 120% for all the target compounds, the extraction recovery was higher than 65% for all the steroids considered and no remarkable matrix effect, expressed in terms of ion enhancement and ion suppression, was observed. To test the reliability of the developed and validated method, the analysis of serum samples collected from ten healthy subjects (5M/5F) was performed. In the clinical settings there is a growing need to develop accessible methods for full steroid hormone profiling. The dynamic link between steroidogenic glands and liver enzymatic processing (activation and clearance) attributes to the profile a much greater clinical meaning than a set of individually measured hormones. The presented method can be used to identify trajectories of deviation from the concentration normality ranges applied to disorders of the gonadal and adrenal axes.


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