scholarly journals Analysis of the Serologic Relationship among San Miguel Sea Lion Virus and Vesicular Exanthema of Swine Virus Isolates. Application of the Western Blot Assay for Detection of Antibodies in Swine Sera to these Virus Types

1995 ◽  
Vol 7 (2) ◽  
pp. 190-195 ◽  
Author(s):  
Bruce S. Seal ◽  
James A. House ◽  
Cecelia A. Whetstone ◽  
John D. Neill
Keyword(s):  
Western Blot ◽  
Capsid Protein ◽  
Negative Control ◽  
Sea Lion ◽  
Western Blot Assay ◽  
Swine Virus ◽  
Polyclonal Antisera ◽  
Gnotobiotic Pigs ◽  
Virus Isolates ◽  
Positive Controls

Caliciviruses are positive-sense single-stranded RNA viruses with a single capsid protein. The serotypes of the marine mammal calicivirus, San Miguel sea lion virus (SMSV), are antigenically related to vesicular exanthema of swine virus (VESV) and are potentially hazardous to swine. Western blot assays using purified SMSV serotypes 1 and 4 were used to further examine the serologic relationship among SMSV and VESV isolates. With the exception of SMSV 8 and SMSV 12, rabbit polyclonal antisera generated against all the available SMSV and VESV isolates reacted positively, as assessed by western blot, with purified capsid protein from SMSV 1 and SMSV 4. Consequently, the SMSV 8 and SMSV 12 virus isolates may not be members of the SMSV/VESV calicivirus group. Using antisera from pigs experimentally inoculated with SMSV and VESV as positive controls, a western blot assay for these virus types was utilized to check for the presence of antibodies to caliciviruses in swine sera. Sera from colostrum-deprived gnotobiotic pigs were used as a negative control in all experiments. Examination of sera from domestic and feral swine collected in Iowa, California, and Florida was completed using this technique. The presence of antibodies to these virus types was not detected in any of the porcine sera tested.

scholarly journals Cloning, Sequencing, and Expression of the Leukotoxin Gene from Fusobacterium necrophorum†

2001 ◽  
Vol 69 (9) ◽  
pp. 5447-5455 ◽  
Author(s):  
Sanjeev Kumar Narayanan ◽  
T. G. Nagaraja ◽  
M. M. Chengappa ◽  
George C. Stewart
Keyword(s):  
Molecular Weight ◽  
Western Blot ◽  
Sequence Similarity ◽  
Blot Hybridization ◽  
Flow Cytometric Analysis ◽  
Southern Blot Hybridization ◽  
Fusobacterium Necrophorum ◽  
Inverse Pcr ◽  
Western Blot Assay ◽  
Polyclonal Antisera

ABSTRACT Fusobacterium necrophorum is a gram-negative, rod-shaped, anaerobic bacterium that is a primary or secondary etiological agent in a variety of necrotic purulent infections in animals and humans. Included are diseases of cattle such as liver abscesses and foot rot, which have economically important consequences for the cattle industry. The major virulence factor of this bacterium is leukotoxin, a secreted protein of high molecular weight active against leukocytes from ruminants. The screening of a genomic DNA library with polyclonal antisera raised against native affinity-purified leukotoxin and further extension of the sequence using inverse PCR led to the cloning of the entire leukotoxin gene. The leukotoxin gene open reading frame (ORF; lktA) consists of 9,726 bp and encodes a protein of 3,241 amino acids with an overall molecular weight of 335,956. The leukotoxin does not have sequence similarity with any other bacterial leukotoxin. Five truncated overlapping polypeptides covering the wholelktA ORF were used to immunize rabbits. In Western blot assays, polyclonal antisera raised against all five truncated polypeptides recognized affinity-purified leukotoxin fromF. necrophorum culture supernatant in a Western blot assay. Antisera directed against two of the five polypeptides had neutralizing activity against the toxin. The entire leukotoxin ORF was expressed in Escherichia coli. Flow-cytometric analysis showed that the recombinant leukotoxin was active against bovine polymorphonuclear leukocytes and was inhibited with antiserum raised against the F. necrophorum leukotoxin. Southern blot hybridization analysis revealed different patterns of lktAhybridizing bands between isolates of the two subspecies ofF. necrophorum.

Differentiation of Yam Virus Isolates Using Symptomatology, Western Blot Assay, and Monoclonal Antibodies

1996 ◽  
Vol 144 (5) ◽  
pp. 235-240 ◽  
Author(s):  
C. Goudou-Urbino ◽  
L. Givord ◽  
J. B. Quiot ◽  
M. Boeglin ◽  
G. Konate ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Western Blot ◽  
Western Blot Assay ◽  
Virus Isolates

scholarly journals The presence of Chlamydia phage PhiCPG1 capsid protein VP1 genes and antibodies in patients infected with Chlamydia trachomatis

2016 ◽  
Vol 63 (3) ◽  
Author(s):  
Jingyue Ma ◽  
Yuan Liu ◽  
Yuanjun Liu ◽  
Lingjie Li ◽  
Shuping Hou ◽  
...  
Keyword(s):  
Chlamydia Trachomatis ◽  
Western Blot ◽  
Capsid Protein ◽  
Pcr Amplification ◽  
Antibody Test ◽  
Vp1 Gene ◽  
Western Blot Assay ◽  
Phage Capsid ◽  
Capsid Protein Vp1 ◽  
Guinea Pig Model

Chlamydia phage PhiCPG1 has been found in Chlamydia caviae in a guinea pig model for inclusion conjunctivitis, raising the possibility that Chlamydia phage is also present in patients infected with C. trachomatis (Ct). In the present study, we assayed for presence of Chlamydia phage capsid protein VP1 genes and antibodies in 84 non-Ct controls and 206 Ct patients using an enzyme-linked immunoassay (ELISA), followed by verification with Western blot. None of the subjects were exposed to an antibiotic treatment or had a C. pneumoniae infection. The VP1 antibody test was positive in both, the ELISA and Western blot assay, in 4 Ct patients. PCR amplification experiments revealed presence of the VP1 gene in 5 Ct patients. The results suggest that Chlamydia phage capsid protein VP1 may exist in some Ct patients.

scholarly journals Serological Detection of Grapevine Associated Closteroviruses in Infected Grapevine Cultivars

Plant Disease ◽  
1997 ◽  
Vol 81 (7) ◽  
pp. 802-808 ◽  
Author(s):  
Judit Monis ◽  
Richard K. Bestwick
Keyword(s):  
Western Blot ◽  
Polyclonal Antiserum ◽  
Enzyme Linked Immunosorbent Assay ◽  
Mixed Infections ◽  
Serological Detection ◽  
Western Blot Assay ◽  
Disease Symptoms ◽  
Polyclonal Antisera ◽  
Infected Grapevine ◽  
Grapevine Disease

Western blot immunoassay and enzyme-linked immunosorbent assay using different monoclonal antibodies (MAb) and polyclonal antisera (PA) revealed mixed infections of serologically related and unrelated grapevine leafroll associated viruses (GLRaVs) and grapevine corky bark associated virus (GCBaV) in symptomatic grapevines. A PA designated rootstock-scion incompatibility (RSI)-24 kDa, grapevine corky bark PA, and GLRaV-2b MAb reacted to polypeptides of approximately 24 kDa isolated from grapevines exhibiting rootstock-scion incompatibility, leafroll, and corky bark disease symptoms, suggesting that these isolates are infected with closely related viruses. A PA designated GLRaV-2 US detected virus specific polypeptides of 38, 37, 36, and 24 kDa, while a polyclonal antiserum designated GLRaV-2 FR detected a single virus-specific polypeptide of approximately 24 kDa. The reactivity of GLRaV-2 US to various polypeptides suggests that the immunogen used to produce this antiserum was a mixture of viruses. Apical meristems were excised and cultured to eliminate the infection of viruses in the grapevines showing RSI symptoms and in the cultivar French Colombard infected with GLRaV-1. The elimination of these viruses was confirmed by Western blot assay. These studies show that the Western blot assay can be used to detect and differentiate grapevine disease-associated closteroviruses.

scholarly journals Western Blot Assay Using Recombinant p26 Antigen for Detection of Equine Infectious Anemia Virus-Specific Antibodies

2007 ◽  
Vol 14 (12) ◽  
pp. 1646-1648 ◽  
Author(s):  
I. Alvarez ◽  
G. Gutierrez ◽  
E. Ostlund ◽  
M. Barrandeguy ◽  
K. Trono
Keyword(s):  
Western Blot ◽  
Capsid Protein ◽  
Diagnostic Tool ◽  
Equine Infectious Anemia Virus ◽  
Enzyme Linked Immunosorbent Assay ◽  
Agar Gel ◽  
Western Blot Assay ◽  
Specific Antibodies ◽  
Equine Infectious Anemia ◽  
Anemia Virus

ABSTRACT We analyzed the performance of a single-band Western blot (WB) test using recombinant p26 (rp26) capsid protein of equine infectious anemia virus. According to the results obtained, the rp26 WB test is a reliable confirmatory diagnostic tool to be used as a complementary test after an enzyme-linked immunosorbent assay or agar gel immunodiffusion test yielding doubtful results.

The Anti-tumor Activity and Mechanisms of rLj-RGD3 on Human Laryngeal Squamous Carcinoma Hep2 Cells

2020 ◽  
Vol 19 (17) ◽  
pp. 2108-2119
Author(s):  
Yang Jin ◽  
Li Lv ◽  
Shu-Xiang Ning ◽  
Ji-Hong Wang ◽  
Rong Xiao
Keyword(s):  
Wound Healing ◽  
Western Blot ◽  
Morphological Changes ◽  
In Vivo Studies ◽  
Migration And Invasion ◽  
Tunel Staining ◽  
Dna Ladder ◽  
Western Blot Assay

Background: Laryngeal Squamous Cell Carcinoma (LSCC) is a malignant epithelial tumor with poor prognosis and its incidence rate increased recently. rLj-RGD3, a recombinant protein cloned from the buccal gland of Lampetra japonica, contains three RGD motifs that could bind to integrins on the tumor cells. Methods: MTT assay was used to detect the inhibitory rate of viability. Giemsa’s staining assay was used to observe the morphological changes of cells. Hoechst 33258 and TUNEL staining assay, DNA ladder assay were used to examine the apoptotic. Western blot assay was applied to detect the change of the integrin signal pathway. Wound-healing assay, migration, and invasion assay were used to detect the mobility of Hep2 cells. H&E staining assay was used to show the arrangement of the Hep2 cells in the solid tumor tissues. Results: In the present study, rLj-RGD3 was shown to inhibit the viability of LSCC Hep2 cells in vitro by inducing apoptosis with an IC50 of 1.23µM. Western blot showed that the apoptosis of Hep2 cells induced by rLj- RGD3 was dependent on the integrin-FAK-Akt pathway. Wound healing, transwells, and western blot assays in vitro showed that rLj-RGD3 suppressed the migration and invasion of Hep2 cells by integrin-FAKpaxillin/ PLC pathway which could also affect the cytoskeleton arrangement in Hep2 cells. In in vivo studies, rLj-RGD3 inhibited the growth, tumor volume, and weight, as well as disturbed the tissue structure of the solid tumors in xenograft models of BALB/c nude mice without reducing their body weights. Conclusion: hese results suggested that rLj-RGD3 is an effective and safe suppressor on the growth and metastasis of LSCC Hep2 cells from both in vitro and in vivo experiments. rLj-RGD3 might be expected to become a novel anti-tumor drug to treat LSCC patients in the near future.

Use of monoclonal and polyclonal antibodies to detect prostatic acid phosphatase by immunoblotting.

1986 ◽  
Vol 32 (10) ◽  
pp. 1832-1835 ◽  
Author(s):  
P C Patel ◽  
L Aubin ◽  
J Côte
Keyword(s):  
Monoclonal Antibodies ◽  
Acid Phosphatase ◽  
Western Blot ◽  
Polyclonal Antibodies ◽  
Prostatic Acid Phosphatase ◽  
The Other ◽  
Dot Blot ◽  
Western Blots ◽  
Dot Blot Assay ◽  
Polyclonal Antisera

Abstract We investigated two techniques of immunoblotting--the Western blot and the dot blot--for use in detecting prostatic acid phosphatase (PAP, EC 3.1.3.2). We used polyclonal antisera to human PAP, produced in rabbits by hyperimmunization with purified PAP, and PAP-specific monoclonal antibodies in the immunoenzymatic protocols. We conclude that PAP can be readily detected by Western blots with use of polyclonal antisera, but not with monoclonal antibodies. On the other hand, using a dot blot assay, we could easily detect PAP with both polyclonal and monoclonal antibodies.

scholarly journals Salidroside promoted osteogenic differentiation of adipose-derived stromal cells through Wnt/β-catenin signaling pathway

2021 ◽  
Vol 16 (1) ◽  
Author(s):  
Xiao-hua Li ◽  
Fu-ling Chen ◽  
Hong-lin Shen
Keyword(s):  
Western Blot ◽  
Osteogenic Differentiation ◽  
Signaling Pathway ◽  
Differentially Expressed Genes ◽  
Stromal Cells ◽  
Differentially Expressed ◽  
Calcium Deposition ◽  
Western Blot Assay ◽  
Adipose Derived Stromal Cells ◽  
Interfering Rna

Abstract Background Bone disease causes short-term or long-term physical pain and disability. It is necessary to explore new drug for bone-related disease. This study aimed to explore the role and mechanism of Salidroside in promoting osteogenic differentiation of adipose-derived stromal cells (ADSCs). Methods ADSCs were isolated and treated with different dose of Salidroside. Cell count kit-8 (CCK-8) assay was performed to assess the cell viability of ADSCs. Then, ALP and ARS staining were conducted to assess the early and late osteogenic capacity of ADSCs, respectively. Then, differentially expressed genes were obtained by R software. Then, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of the differentially expressed genes were further analyzed. The expression of OCN, COL1A1, RUNX2, WNT3A, and β-catenin were measured by real-time PCR and Western blot analysis. Last, β-catenin was silenced by small interfering RNA. Results Salidroside significantly increased the ADSCs viability at a dose-response manner. Moreover, Salidroside enhanced osteogenic capacity of ADSCs, which are identified by enhanced ALP activity and calcium deposition. A total of 543 differentially expressed genes were identified between normal and Salidroside-treated ADSCs. Among these differentially expressed genes, 345 genes were upregulated and 198 genes were downregulated. Differentially expressed genes enriched in the Wnt/β-catenin signaling pathway. Western blot assay indicated that Salidroside enhanced the WNT3A and β-catenin expression. Silencing β-catenin partially reversed the promotion effects of Salidroside. PCR and Western blot results further confirmed these results. Conclusion Salidroside promoted osteogenic differentiation of ADSCs through Wnt/β-catenin signaling pathway.

Ginkgolic acid induces apoptosis and autophagy of endometrial carcinoma cells via inhibiting PI3K/Akt/mTOR pathway in vivo and in vitro

2021 ◽  
pp. 096032712110237
Author(s):  
L Zhou ◽  
S Li ◽  
J Sun
Keyword(s):  
Endometrial Cancer ◽  
B Cells ◽  
Western Blot ◽  
Signal Pathway ◽  
Mtor Pathway ◽  
Related Protein ◽  
Western Blot Assay ◽  
Ginkgolic Acid

Endometrial cancer (EC) is the fourth most common malignancy in women in developed countries. The prognosis of EC is extremely poor, and it is an important factor that contributes to the death of patients. Therefore, studying EC pathogenesis and therapeutic targets, and exploring effective drugs are the primary tasks to improve the prognosis of EC. In the present study, we aimed to explore the function of ginkgolic acid (GA) in EC cell apoptosis and autophagy through PI3K/Akt/mTOR signal pathway in vitro and in vivo. Firstly, MTT assay and clone formation assay were employed to analyze the Ishikawa and HEC-1-B cell viabilities and proliferation after treatment with GA. The results showed that GA inhibited endometrial cancer cell survival. Flow cytometry assay and western blot assay were applied to examine the apoptosis and apoptosis related protein Bcl-2, Bax, Cleaved caspase-3 expression levels of Ishikawa and HEC-1-B cells after treatment with GA. Next, we applied western blot assay to analyze the autophagy associated proteins LC3I, LC3II, p62 and Beclin-1 in GA treated Ishikawa and HEC-1-B cells. We found that GA promoted apoptosis and induced autophagy of endometrial cancer cells. Meanwhile, western blot assay was also used to determine the expression levels of the PI3K/Akt/mTOR signal pathway related protein and the results revealed that GA inhibited the activity of PI3K/Akt/mTOR pathway. Finally, we found that GA inhibited tumor growth in vivo through immunohistochemistry assay. In conclusion, GA induces apoptosis and autophagy of EC cells via inhibiting PI3K/Akt/mTOR pathway in vivo and vitro.

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