Comparing the applicability of CGE-on-the-chip and SDS-PAGE for fast pre-screening of mouse serum samples prior to proteomics analysis

2008 ◽  
Vol 29 (21) ◽  
pp. 4332-4340 ◽  
Author(s):  
Tom Grunert ◽  
Martina Marchetti-Deschmann ◽  
Ingrid Miller ◽  
Mathias Müller ◽  
Günter Allmaier
2020 ◽  
Vol 19 (11) ◽  
pp. 1749-1759 ◽  
Author(s):  
Xin Hou ◽  
Xiaomei Zhang ◽  
Xian Wu ◽  
Minya Lu ◽  
Dan Wang ◽  
...  

Coronavirus disease 2019 (COVID-19) is a highly contagious infection and threating the human lives in the world. The elevation of cytokines in blood is crucial to induce cytokine storm and immunosuppression in the transition of severity in COVID-19 patients. However, the comprehensive changes of serum proteins in COVID-19 patients throughout the SARS-CoV-2 infection is unknown. In this work, we developed a high-density antibody microarray and performed an in-depth proteomics analysis of serum samples collected from early COVID-19 (n = 15) and influenza (n = 13) patients. We identified a large set of differentially expressed proteins (n = 132) that participate in a landscape of inflammation and immune signaling related to the SARS-CoV-2 infection. Furthermore, the significant correlations of neutrophil and lymphocyte with the CCL2 and CXCL10 mediated cytokine signaling pathways was identified. These information are valuable for the understanding of COVID-19 pathogenesis, identification of biomarkers and development of the optimal anti-inflammation therapy.


2014 ◽  
Vol 51 (3) ◽  
pp. 181-189 ◽  
Author(s):  
F. Jing ◽  
J. Cui ◽  
R. Liu ◽  
L. Liu ◽  
P. Jiang ◽  
...  

AbstractIn the present study, a sandwich ELISA based on IgY (egg yolk immunoglobulin) was developed for detection of circulating antigens (CAg) in sere of mice experimentally infected with Trichinella spiralis. The IgY-sandwich ELISA assay involved the use of chicken antibody IgY against excretory-secretory (ES) antigens of Trichinella spiralis muscle larvae as a capture antibody and mouse polyclonal antibody IgG to ES antigens as a detecting antibody. This method was able to detect as little as 3 ng/ml of ES antigens added to normal mouse serum. A group of sixteen mice was orally inoculated with 500 T. spiralis muscle larvae per animal. The serum samples from the infected mice were taken during 1–35 days post-infection (dpi). The CAg was detectable as early as 8 dpi in the sera of infected mice. The level of CAg increased dramatically during 13–15 dpi and reached a peak at 22 dpi and remained a plateau for 3 days, then declined gradually. Another peak of CAg occurred at 31 dpi. The anti-Trichinella antibodies was first detected in 14.3 % of the infected mice at 2 weeks post-infection (wpi), and reached a peak positive rate of 100 % at 5 wpi. Moreover, the infected mice were treated with abendazole at 5 wpi and the serum CAg levels increased significantly during 2–6 days posttreatment (dpt) and then declined rapidly during 8–14 dpt. By 42 dpt, the CAg levels decreased to the undetected level, but the detection rate of antibodies was still 100 %. The IgY-sandwich ELISA appears to be a sensitive for detection of antigenemia of T. spiralis and valuable to judge the efficacy of chemotherapy in trichinellosis.


2021 ◽  
Author(s):  
Maryam Hajizadeh ◽  
Fariba Amni ◽  
Maryam Sahlolbei ◽  
Masoumeh Tavakoli-yaraki ◽  
Amirreza Javadi Mamaghani ◽  
...  

Abstract Background: less studies have been done on bone cancers which are complex despite lower incidance. Hydatidosis is a parasitic disease that may influence host immunity by mimicking cancer cells antigens. So, this study aimed to elavuate the similarity of the immunogenic antigens between hydatid cyst and different bone cancers.Method: Cyst wall of hydatid cysts were collected and their antigens were separated with SDS-PAGE gel electrophoresis (SDS-PAGE). Serum samples obtained from patients with bone cancers and the anitigenicity of isolated anitgens were evaluated inwith E. granulosus( Larval form )infection and healthy individuals using western-blot approaches. Results: The crude extract of the laminated layer showed two specific antigens, 53 KDa and 70 KDa, after stainging the membrane with Coomassie blue. Both antigens reacted with the serum of different bone cancers but only the 53 KDa band reacted with all sera.Conclusion: It seems people with bone tumours may have extra antibodies in their serum comparing to healthy and hydatidosis which may be an autoantibodies; and the presence of this antibody against 70 KDa band protein in sera of patients with various types of bone cancers, may be helpful in diagnostic test or designing of preventive approaches in future.


The Analyst ◽  
2015 ◽  
Vol 140 (21) ◽  
pp. 7443-7450 ◽  
Author(s):  
Xuan Zhang ◽  
Wenjuan Zhou ◽  
Zhiqin Yuan ◽  
Chao Lu

Fluorosurfactant functionalized gold nanorods can act as selective colorimetric nanoprobes for H2S. These proposed nanoprobes can accurately detect biological H2S in human and mouse serum samples as well evaluate the activity of H2S synthetase.


Endocrinology ◽  
2011 ◽  
Vol 152 (11) ◽  
pp. 4443-4447 ◽  
Author(s):  
Daniel J. Haisenleder ◽  
Aleisha H. Schoenfelder ◽  
Elizabeth S. Marcinko ◽  
Lisa M. Geddis ◽  
John C. Marshall

The University of Virginia Center for Research in Reproduction Ligand Core performed an evaluation of nine commercial estradiol (E2) immunoassays for use with mouse serum. The evaluation had two components. 1) Recovery Studies: a mouse pool was spiked with E2 concentrations across the assay range, and percent recovery and parallelism to the assay standard curve were determined. 2) Correlation Studies: serum pools were collected from intact females, ovariectomized (OVX) and OVX-E2 treated mice and E2 assayed, then measured by gas chromatography/tandem mass spectrometry (GC/MSMS) for comparison to a gold standard method. Recovery results showed that E2 recovery from spiked mouse pools varied greatly (from <18% to >640%) among kits tested. However, three kits (DiaSorin Radioimmunoassay, Siemens Double Antibody RIA, and CalBiotech Enzyme Immunoassay) showed reasonable recoveries and parallelism. Data collected from the Correlation Study showed that values from intact, OVX and OVX-E2-treated mouse pools varied by several fold vs. GC/MSMS for most of the kits tested. The DiaSorin RIA and CalBiotech Enzyme Immunoassay Kits showed the best correlation to GC/MSMS. Unfortunately, while this evaluation was ongoing, the DiaSorin Kit was discontinued. In summary, the CalBiotech Kit was the only available assay tested that demonstrated good E2 parallelism to the assay standard curve and accuracy vs. a gold standard method (i.e. GC/MSMS). Also of note, the CalBiotech assay is sensitive and requires minimal sample volume. Therefore, based on these findings the CalBiotech E2 assay has been implemented for use in mouse serum samples within the Ligand Core.


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):  
Noriko Tosa ◽  
Tomoko Ishida ◽  
Kumiko Yoshimatsu ◽  
Nobuhito Hayashimoto ◽  
Kanae Shiokawa ◽  
...  

Serologic monitoring of infectious diseases is important for microbial control in colonies of laboratory mice. Rapid and simple tests that do not require killing animals are valuable for this purpose. In this study, we developed a multiplex immunochromatographic assay (ICA) for detection of antibodies to mouse hepatitis virus (MHV), Sendai virus (also known as hemagglutinating virus of Japan [HVJ]), and Clostridium piliforme (The pathogen that causes Tyzzer disease), which are major infectious diseases in mice. For this assay, an ICA strip was put into a microtube containing 150 μL PBS and either 0.75 μL mouse serum or 1.5 μL whole blood. Binding antibodies were visualized by using protein A-conjugated colloidal gold. Under these conditions, multiplex ICA simultaneously and specifically detected antibodies to multiple antigens. To evaluate the sensitivity and specificity of multiplex ICA, positive serum samples for each infectious disease were used. Sensitivities of the multiplex ICA test for MHV, HVJ, and C. piliforme were 100%, 100%, and 90%, respectively. No nonspecific reaction was observed in any of the 30 positive sera. In addition, 10 samples of uninfected sera did not show any bands except for the control line. These observations indicate high specificity of the multiplex ICA test. Moreover, the multiplex ICA could be applied to diluted blood. These results indicate that the multiplex ICA is appropriate for rapid, simple, and safe serologic testing of laboratory mice.


PLoS ONE ◽  
2021 ◽  
Vol 16 (4) ◽  
pp. e0250516
Author(s):  
Kelly Bohning ◽  
Stephanie Sonnberg ◽  
Hui-Ling Chen ◽  
Melissa Zahralban-Steele ◽  
Timothy Powell ◽  
...  

Zika virus is a Flavivirus, transmitted via Aedes mosquitos, that causes a range of symptoms including Zika congenital syndrome. Zika has posed a challenging situation for health, public and economic sectors of affected countries. To quantitate Zika virus neutralizing antibody titers in serum samples, we developed a high throughput plate based Zika virus reporter virus particle (RVP) assay that uses an infective, non-replicating particle encoding Zika virus surface proteins and capsid (CprME) and a reporter gene (Renilla luciferase). This is the first characterization of a Zika virus RVP assay in 384-well format using a Dengue replicon Renilla reporter construct. Serially diluted test sera were incubated with RVPs, followed by incubation with Vero cells. RVPs that have not been neutralized by antibodies in the test sera entered the cells and expressed Renilla luciferase. Quantitative measurements of neutralizing activity were determined using a plate-based assay and commercially available substrate. The principle of limiting the infection to a single round increases the precision of the assay measurements. RVP log10EC50 titers correlated closely with titers determined using a plaque reduction neutralization test (PRNT) (R2>95%). The plate-based Zika virus RVP assay also demonstrated high levels of precision, reproducibility and throughput. The assay employs identical reagents for human, rhesus macaque and mouse serum matrices. Spiking studies indicated that the assay performs equally well in different species, producing comparable titers irrespective of the serum species. The assay is conducted in 384-well plates and can be automated to simultaneously achieve high throughput and high reproducibility.


2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Zhuan-zhuan Liu ◽  
Xue-yan Li ◽  
Lin-lin Fu ◽  
Fei Yuan ◽  
Ren-xian Tang ◽  
...  

Abstract Background Toxoplasma gondii infection endangers human health and affects animal husbandry. Serological detection is the main method used for epidemiological investigations and diagnosis of toxoplasmosis. The key to effective diagnosis of toxoplasmosis is the use of a standardized antigen and a specific and sensitive detection method. Peroxiredoxin is an antigenic protein and vaccine candidate antigen of T. gondii that has not yet been exploited for diagnostic application. Methods In this study, recombinant T. gondii peroxiredoxin protein (rTgPrx) was prepared and used in dot-immunogold-silver staining (Dot-IGSS) to detect IgG antibodies in serum from mice and pregnant women. The rTgPrx-Dot-IGSS method was established and optimized using mouse serum. Furthermore, serum samples from pregnant women were analyzed by rTgPrx-Dot-IGSS. Results Forty serum samples from mice infected with T. gondii and twenty negative serum samples were analyzed. The sensitivity and specificity of rTgPrx-Dot-IGSS were 97.5 and 100%, respectively, equivalent to those of a commercial ELISA kit for anti-Toxoplasma IgG antibody. Furthermore, 540 serum samples from pregnant women were screened with a commercial ELISA kit. Eighty-three positive and 60 negative serum samples were analyzed by rTgPrx-Dot-IGSS. The positive rate was 95.18%, comparable to that obtained with the commercial ELISA kit. Conclusions The Dot-IGSS method with rTgPrx as an antigen might be useful for diagnosing T. gondii infection in individuals.


2006 ◽  
Vol 18 (2) ◽  
pp. 183 ◽  
Author(s):  
D. I. Jin ◽  
H. R. Lee ◽  
H. R. Kim ◽  
H. J. Lee ◽  
J. T. Yoon ◽  
...  

To identify early pregnancy-specific serum proteins in bovine, we performed proteomics analysis using blood serum samples of pregnant and non-pregnant Holstein dairy cattle Days 21 and 35 after AI. A total of eight pregnant and eight non-pregnant cattle were used for collection of the blood samples. The global proteomics approach was exploited by the use of 2-D gel electrophoresis and mass spectrometry to sort out pregnancy-specific proteins. Serum proteins within isoelectric point ranges of 4.0 to 7.0, 6.0 to 9.0, and 5.5 to 6.7 were analyzed separately by 2-D electrophoresis with three replications of each sample. The stained gels were scanned and calibrated at an optical resolution of 63.5 �m/pixel using a GS-710 imaging densitometer (Bio-Rad Laboratories, Philadelphia, PA, USA). A total of approximately 1200 spots were detected in 2-D gels stained with Coomassie-blue. In the comparison of serum samples from pregnant and non-pregnant cattle, nine pregnancy-specific spots were detected unanimously in Day 21 and Day 35 serum samples. Pregnancy-specific proteins were identified as transferrin, albumin, IgG2a heavy chain constant region, and immunoglobulin gamma heavy chain variable region by means of MALDI-TOF-MS (PerSeptive Biosystems, Framingham, MA, USA). Even though the identified spots were abundant serum proteins, their molecular weights and pI values were different from those of the main serum proteins. Most proteins identified in this analysis appeared to be related with pregnancy-specific subunits or fragments of transferrin, albumin, and IgG. One of the pregnancy-specific proteins, transferrin, is known to be related to iron transport during pregnancy. Western blot analysis using polyclonal anti-transferrin antibody revealed specific transferrin expression in the serum samples from the pregnant cattle but no detectable expression in the serum samples from the non-pregnant cattle. Our results revealed composite profiles of key proteins involved in early pregnancy and suggest the potential use of identified proteins to detect early pregnancy in bovine.


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