scholarly journals Cross Reaction of Haemonchus Contortus ANTIGEN WITH ANTI-Fasciola Gigantica Serum by Using Western Blot Technique

2020 ◽  
Vol 4 (1) ◽  
Author(s):  
Firman Hadi Fanani ◽  
Kusnoto Kusnoto ◽  
Poedji Hastutiek ◽  
Muchammad Yunus ◽  
Setiawan Koesdarto ◽  
...  
Keyword(s):  
Bile Duct ◽  
Gall Bladder ◽  
Western Blot ◽  
Haemonchus Contortus ◽  
Phosphate Buffer ◽  
Fasciola Gigantica ◽  
Cross Reaction ◽  
Blue Dye ◽  
Sds Page ◽  
Blot Technique

The purpose of this research to know cross reaction of Haemonchus contortus protein with anti-Fasciola gigantica serum by using the Western Blot technique. The result can be used as Haemonchosis serologic diagnostic. This research was conducted using Haemonchus contortus worms obtained from abomasum, especially cow worms and Fasciola gigantica worms obtained from the bile duct and gall bladder. The worms were crushed and added with Phosphate Buffer Saline (PBS) to make homogenates and then centrifuged at 3500 rpm for 15 minutes. Homogenate Fasciola gigantica was immunized to mice to get anti-Fasciola gigantica serum at a dose of 200 μg/mice and booster 3 times with a span of two weeks. Homogenates were carried out using the SDS-PAGE technique to analyze proteins using a brilliant blue dye. Cross reaction of Haemonchus contortus protein with anti-Fasciola gigantica serum byusing Western blot technique and obtained 3 protein bands are 94.9, 52.7, 46.7 kDa.

scholarly journals Cross Reaction of Haemonchus contortus Protein with Toxocara vitulorum Anti-L2 Serum Using Western Blot Technique

2021 ◽  
Vol 11 (3) ◽  
pp. 504-509
Author(s):  
Rani Wilujeng Asmorowati ◽  
. Kusnoto ◽  
Hana Eliyani
Keyword(s):  
Western Blot ◽  
Haemonchus Contortus ◽  
Sodium Dodecyl ◽  
Cross Reaction ◽  
Blue Staining ◽  
Toxocara Vitulorum ◽  
Cross Reactions ◽  
Sds Page ◽  
Blot Technique ◽  
The Cross

In the adult stage, Haemonchus contortus worms infect the abomasum host causing anemia and even death in animals. However, identifying the H. contortus protein can be used as a reference for the diagnosis of diseases. The diagnosis is performed by serological cross-reaction between H. contortus protein and anti-L2 Toxocara vitulorum (T. vitulorum) serum using the western blot technique. The main purpose of the current research was to identify the cross-reaction between H. contortus proteins and anti-L2 T. vitulorum serum using the western blot technique. T. vitulorum worms were collected from the intestine of cattle and H. contortus worms were collected from the abomasum of goats. The first step was making antibodies by oral infection of rats with infective eggs (L2) of T. vitulorum. The blood was taken 21 days after infection. Then, the blood was centrifuged at 1500 rpm for 10 minutes to get the serum. The second step was making homogenates from the whole worm extract of H. contortus. After crushing the worms, it was centrifuged at 5000 rpm for 15 minutes and the supernatant was taken. The supernatant was then analyzed using Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE) with coomassie brilliant blue staining. The third step was the analysis of H. contortus protein with serum anti-L2 T. vitulorum using the western blot technique. From the H. contortus homogenates analysis using SDS-PAGE, 16 protein bands were obtained. The cross-reactions were 141.3, 81.3, 64. 6, 51.3, 46.8, and 38 kDa. The data from cross-reactions suggested that the H. contortus protein cannot be used as a diagnostic material. It is serologically Haemonchosis because it caused false positives with diagnostic Toxocariasis.

scholarly journals Western Blot Analysis to Detect Cross-reaction in Toxocara vitulorum Protein with Anti-Mecistocirrus digitatus Serum

2021 ◽  
Vol 11 (1) ◽  
pp. 92-97
Author(s):  
Desy Meta Anggraini ◽  
Ir.H Kusnoto ◽  
Suryanie Sarudji
Keyword(s):  
Western Blot ◽  
Blot Analysis ◽  
Wistar Rats ◽  
Polyclonal Antibodies ◽  
Cross Reaction ◽  
Relative Molecular Mass ◽  
Infected People ◽  
Toxocara Vitulorum ◽  
Blot Technique ◽  
Specific Proteins

Worm infections are found in livestock and can be transmitted to humans. Toxocara vitulorum is a worm species which commonly infected people. Cross-reaction among worms can generate false positive to establish helminthiasis diagnosis through antibody inspection. This study aimed to determine specific proteins that caused cross-reaction between Toxocara vitulorum antigen and anti-M. digitatus serum by using the western blot technique. In the present study, the whole worms extracted of T. vitulorum and M. digitatus have been used to make polyclonal antibodies from M. digitatus with Wistar rats as hosts. The cross-reaction between whole worm extract of T. vitulorum protein and anti-M. digitatus serum obtained 12 protein bands that each relative molecular mass (Mr) valued of 176, 124, 92, 68, 59, 47, 31, 29, 26, 16, 12, and 10 kDa. Cross-reaction occurred between T. vitulorum protein and anti-M. digitatus.

scholarly journals Comparative histomorphological study of non affected and affected bile duct and gall bladder by fascioliasis in Black Bengal Goat

2018 ◽  
Vol 4 (4) ◽  
pp. 406-415
Author(s):  
Md Reazul Islam ◽  
Rashida Khaton ◽  
Md Aktharul Alam ◽  
Md Jalal Uddin Sarder ◽  
Md Najmul Hassan Parvez
Keyword(s):  
Bile Duct ◽  
Gall Bladder ◽  
Bile Ducts ◽  
Average Length ◽  
Fasciola Gigantica ◽  
Average Weight ◽  
Bile Pigment ◽  
Tissue Proliferation ◽  
Infected Goat ◽  
Black Bengal Goat

The purpose of this study was to comparative histomorphological investigation of the non affected and affected bile duct and gall bladder by fascioliasis in Black Bengal goat. The average weight of affected gall bladder was 26.10±0.70 gm which was significantly (p<0.001) higher than non affected gall bladder (19.40±0.96 gm). The average length and girth of affected gall bladder were 10.30±0.37 cm and 8.24±0.30 cm, respectively which were also significantly (p<0.001) higher than the length (6.10±0.30 cm) and Girth (5.85±0.25 cm) of non-affected gall bladder of Black Bengal Goat. The gross changes in acute form, thickening of the bile ducts and fibrosis in a portal area due to chronic fascioliasis was found in case of affected bile duct but were not found in case of non affected liver. A brownish exudates and a number of mature Fasciola gigantica were found in the lumen. The adult Fasciola gigantica was noticed in cross section in the lumen of the thickened bile ducts. Acute pathological lesions could only be produced by developing flukes prior to their entry to the bile ducts. Microscopically the epithelial layer of the bile ducts were seen to the partially disintegrated, but simultaneous proliferation of epithelial cells occurred. Thickening of the bile ducts was the result of connective tissue proliferation. Deposition of bile pigment in the tissue space and bile duct in some parts showed periductal cellular infiltrations, mainly neutrophiles, lymphocyte and eosinophiles. No calcification in the wall of the bile ducts in chronic Fascioliasis in goat could be seen in this study. The gall bladder was very dark usually contained blood clots, the consistency of bile was also very dense. Microcopically hyperplasia of the tubuloalveolar glands and numerous eggs were seen in the bile of infected goat which were absence in case of non infected goat. Asian J. Med. Biol. Res. December 2018, 4(4): 406-415

Isolation and Structural Charaderization of a Potent Inhibitor of Coagulation Factor Xa from the Leech Haementeria ghilianii

1989 ◽  
Vol 61 (03) ◽  
pp. 437-441 ◽  
Author(s):  
Cindra Condra ◽  
Elka Nutt ◽  
Christopher J Petroski ◽  
Ellen Simpson ◽  
P A Friedman ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Salivary Gland ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Western Blot ◽  
Potent Inhibitor ◽  
Factor Xa ◽  
Coagulation Factor ◽  
Sds Page ◽  
Bovine Factor

SummaryThe present work reports the discovery and charactenzation of an anticoagulant protein in the salivary gland of the giant bloodsucking leech, H. ghilianii, which is a specific and potent inhibitor of coagulation factor Xa. The inhibitor, purified to homogeneity, displayed subnanomolar inhibition of bovine factor Xa and had a molecular weight of approximately 15,000 as deduced by denaturing SDS-PAGE. The amino acid sequence of the first 43 residues of the H. ghilianii derived inhibitor displayed a striking homology to antistasin, the recently described subnanomolar inhibitor of factor Xa isolated from the Mexican leech, H. officinalis. Antisera prepared to antistasin cross-reacted with the H. ghilianii protein in Western Blot analysis. These data indicate that the giant Amazonian leech, H. ghilianii, and the smaller Mexican leech, H. officinalrs, have similar proteins which disrupt the normal hemostatic clotting mechanisms in their mammalian host’s blood.

Western Blot Analyses of Prekallikrein and its Activation Products in Human Plasma

1991 ◽  
Vol 65 (04) ◽  
pp. 382-388 ◽  
Author(s):  
Dulce Veloso ◽  
Robert W Colman
Keyword(s):  
Complex Formation ◽  
Western Blot ◽  
Human Plasma ◽  
Contact System ◽  
Plasma Activation ◽  
Normal Plasma ◽  
Sds Page ◽  
Activation Products ◽  
Major Antigen ◽  
Formation Of Complexes

SummaryPrekallikrein (PK), a zymogen of the contact system, and its activation products, kallikrein (KAL), KAl-inhibitor complexes and fragments containing KAL epitope(s) have been detected in human plasma by immunoblotting with a monoclonal anti-human plasma PK antibody, MAb 13G1L. Detection of antigen-MAb 13G11 complexes with peroxidase-conjugated anti-IgG showed that the two variants of PK (85- and 88-kDa) are the only major antigen species in normal, non-activated plasma. Upon plasma activation with kaolin, the intensity of the PK bands decreased with formation of complexes of KAL with CL inhibitor (C1 INH) and α2-rrtzcroglobulin (α2M) identical to those formed by the purified proteins. Immunoblots of normal plasma showed good correlation between the PK detected and the amount of plasma assayed. Increasing amounts of KAL incubated with a constant volume of PK-deficient plasma showed increasing amounts of KAL and of KAL-C1 INH and KAL-α2M complexes. Complexes of KALantithrombin III (ATIII) and the ratio of KALα2M/ KAL-CL INH were higher in activated CL INH-deficient plasmas than in activated normal plasmas. Protein resolution by 3-12% gradient SDS-PAGE and epitope detection with [125I]MAb 13G11 showed four KALα2M species and a 45-kDa fragment(s) in both surface-activated normal plasma and complexes formed by purified KAL and α2M. Immunoblots of activated plasma also showed bands at the position of KALCL INH and KALATIII complexes. When α1-antitrypsin Pittsburgh (cα1-AT, Pitts) was added to plasma before activation, KAL-α1-ALPitts was the main complex. The non-activated normal plasma revealed only an overloaded PK band. This is the first report of an antibody that recognizes KAL epitope(s) in KAL-α2M, KALATIII and KALa1-α1Pitts complexes and in the 45-kDa fragment(s). Therefore, MAb 13G11 should be useful for studying the structure of these complexes as well as the mechanism of complex formation. In addition, immunoblotting with MAb 13G11 would allow detection of KAl-inhibitor complexes in patient plasmas as indicators of activation of the contact system.

scholarly journals OP0054 NEW ROLE FOR PROTEINASE 3 IN IL-16 BIOACTIVITY CONTROL IN GRANULOMATOSIS WITH POLYANGIITIS

2021 ◽  
Vol 80 (Suppl 1) ◽  
pp. 28.1-29
Author(s):  
A. Kerstein-Staehle ◽  
C. Alarcin ◽  
J. Luo ◽  
G. Riemekasten ◽  
P. Lamprecht ◽  
...  
Keyword(s):  
Cell Death ◽  
Western Blot ◽  
Disease Activity ◽  
Chronic Inflammation ◽  
Immune Cells ◽  
Peripheral Blood ◽  
Granulomatosis With Polyangiitis ◽  
Granulomatous Inflammation ◽  
Proteinase 3 ◽  
Sds Page

Background:The immunomodulatory cytokine IL-16 is increased in several inflammatory and autoimmune diseases1. IL-16 recruits and activates CD4+ immune cells such as T cells, dendritic cells, or monocytes. IL-16 is produced by various immune and non-immune cells, but synthesis and storage of IL-16 is regulated differentially depending on the cell type and stimulation. For its biological activity, IL-16 cleavage by caspase-3 is required1. Necrotizing granulomatous inflammation is a hallmark of granulomatosis with polyangiitis (GPA) with neutrophil dysregulation as a central driver of chronic inflammation and autoimmunity2. Earlier studies showed a correlation between increased serum IL-16 and disease parameters in AAV, including GPA3, but functional evidence for a direct link between IL-16 and neutrophils in granulomatous inflammation is missing so far.Objectives:In this study we aim to identify a functional link between increased IL-16, neutrophils, and the autoantigen proteinase 3 (PR3) with regard to chronic inflammation and autoimmunity in GPA.Methods:IL-16 was measured in sera of GPA patients (n = 40) and healthy controls (HC, n = 50) by ELISA and correlated with clinical features, such as disease activity (BVAS), creatinine, GFR, VDI and PR3-ANCA status. IL-16 protein expression was analyzed in peripheral blood mononuclear cells (PBMC) and polymorphonuclear cells (PMN) from GPA patients and HC (n = 5, each) by SDS-PAGE and western blot. Binding affinity of recombinant pro-IL-16 to native human PR3 was assessed by microscale thermophoresis. Cleavage of pro-IL-16 by active human PR3 was performed at various time points at 37°C. Cleavage products were analyzed by SDS-PAGE and western blot.Results:Circulating IL-16 was significantly increased in GPA patients compared to HC. Elevated IL-16 positively correlated with BVAS, creatinine, VDI and PR3-ANCA status and negatively correlated with GFR. In PMBC and PMN from GPA and HC we identified different expression patters of precursor and active forms of IL-16. In healthy PBMC we found high amounts of precursor (80kD), pro-IL-16 (55kD) and active IL-16 (17kD). In contrast, PBMC from GPA patients had lower amounts of pro-IL-16 and no active IL-16, indicating activation and secretion of IL-16 due to inflammatory stimulation, as shown earlier5. In GPA PMN we detected no precursor IL-16, but pro-IL-16 and its active form, in contrast to very low amounts of all IL-16 forms in healthy PMN. Processing and release of IL-16 in neutrophils has been linked to apoptosis and secondary necrosis5. By interaction studies we demonstrated direct binding of pro-IL-16 to PR3 with a Kd of 10 nM. In a subsequent cleavage assay we confirmed IL-16 processing by PR3 in a time-dependent manner.Conclusion:Correlation of serum IL-16 with clinical features of GPA suggests that IL-16 is associated with markers of disease activity, tissue damage and autoreactivity. We showed that PBMC and PMN represent a source of IL-16 in GPA. By the identification of PR3 as an additional IL-16-activating enzyme we could demonstrate a potential link between excessive PR3 expression, cell death and IL-16-dependent mechanisms, contributing to chronic granulomatous inflammation and autoimmunity in GPA.References:[1]Glass, W. G. et al. Not-so-sweet sixteen: The role of IL-16 in infectious and immune-mediated inflammatory diseases. J. Interf. Cytokine Res. 26, 511–520 (2006).[2]Millet, A. et al. Proteinase 3 on apoptotic cells disrupts immune silencing in autoimmune vasculitis. J. Clin. Invest. 125, 4107–4121 (2015).[3]Yoon, T. et al. Serum interleukin-16 significantly correlates with the Vasculitis Damage Index in antineutrophil cytoplasmic antibody-associated vasculitis. Arthritis Res. Ther. 22, 1–6 (2020).[4]Elssner, A. et al. IL-16 Is Constitutively Present in Peripheral Blood Monocytes and Spontaneously Released During Apoptosis. J. Immunol. 172, 7721–7725 (2004).[5]Roth, S. et al. Secondary necrotic neutrophils release interleukin-16C and macrophage migration inhibitory factor from stores in the cytosol. Cell Death Discov. 1, 15056 (2015).Disclosure of Interests:None declared

scholarly journals AB0050 A NOVEL METHOD FOR ISOLATION OF EXOSOMES FROM SYNOVIAL FLUID

2021 ◽  
Vol 80 (Suppl 1) ◽  
pp. 1057.2-1057
Author(s):  
Y. Liu ◽  
Y. Huang ◽  
Q. Huang ◽  
S. Sun ◽  
Z. Ji ◽  
...  
Keyword(s):  
Synovial Fluid ◽  
Western Blot ◽  
Biological Fluid ◽  
High Sensitivity ◽  
Protein Distribution ◽  
Sds Page ◽  
Close Relationship ◽  
Novel Method ◽  
Low Levels

Background:Exosomes in synovial fluid (SF) has a close relationship with the pathogenesis of rheumatiod arthritis. As a complex biological fluid, SF presents challenges for exosomes isolation using standard methods, such as ExoquickTM kit and ultracentrifugation.Objectives:The study aims to compared the quality of exosomes separated by ExoquickTM kit (TM), ExoquickTM kit+ExoquickTC kit (TM-TC), ultracentrifugation (UC) and TM-TC+UC(TM-TC-UC) from SF.Methods:Exosomes was separated by TM, TM-TC, UC and TM-TC-UC respectively. The size and concentrations of exosomes were detected by high sensitivity flow cytometry for nanoparticle analysis. Total protein and RNA were extracted from exosomes. SDS-PAGE was used to detect the protein distribution of exosomes. Western blot was used to examine the level of albumin and exosomes marker (TSG101 and CD81).Results:There was no statistic difference in the diameters of exosomes separated by the four methods. The concentrations of exosomes in TM, TM-TC, TM-TC-UC and UC were (5.65±0.93), (3.02±1.19), (1.67±0.25) and (4.61±0.73) *109Particles/mL. The protein concentrations of exosomes separated by the four methods were consistent with the concentrations of exosomes. SDS-PAGE showed that the protein distribution of exosomes separated by the four methods were different. Low levels of albumin were detected in TM-TC and TM-TC-UC, while high levels of albumin in TM and UC. Total RNA concentrations from exosomes in TM-TC was higher than other groups.Conclusion:TM-TC can be used to obtain higher quality exosomes from SF for the study of exosome-enriched components.References:[1]Helwa I, et al, A Comparative Study of Serum Exosome Isolation Using Differential Ultracentrifugation and Three Commercial Reagents. PloS one, 2017. 12(1): p. e0170628-e0170628.Figure 1.A: SDS-PAGE showed the protein distribution of exosomes; B: the detection of albumin, TSG101 and CD81 by western blot.Disclosure of Interests:None declared

scholarly journals Trypanosoma cruzi: identification of specific epimastigote antigens by human immune sera

1989 ◽  
Vol 84 (3) ◽  
pp. 309-314 ◽  
Author(s):  
M. G. Morgado ◽  
J. Ivo-dos-Santos ◽  
R. T. Pinho ◽  
E. Argüelles ◽  
J. M. Rezende ◽  
...  
Keyword(s):  
Visceral Leishmaniasis ◽  
Trypanosoma Cruzi ◽  
Western Blot ◽  
Chagas Disease ◽  
Cross Reactivity ◽  
Cross Reaction ◽  
The Other ◽  
Molecular Weights ◽  
Human Visceral Leishmaniasis ◽  
Human Immune

Soluble antigens from epimastigotes of Trypanosoma cruzi were analyzed by western blot in terms of their reactivity with sera from patients with Chagas' disease. In addition, sera from patients with visceral (AVL) and tegumentar leishmaniasis (ATL) were also tested in order to identify cross-reactivities with Trypanosoma cruzy antigens. Twenty eight polypeptides with molecular weights ranging from 14 kDa to 113 kDa were identified with sera from Chagas' disease patients. An extensive cross-reactivity was observed when sera from human visceral leishmaniasis were used, while only a slight cross-reaction was observed with sera from tegumentar leishmaniasis. On the other hand, 10 polypeptidesspecifically reacting with sera from Chagas' disease patients were identified. Among them, the antigens with molecular weights of 46 kDa and 25 kDa reacted with all sera teste and may be good candidates for specific immunodiagnosis of Chagas' disease.

scholarly journals Production and characterization of monoclonal antibodies to the edta extract of Leptospira interrogans, serovar icterohaemorrhagiae

1996 ◽  
Vol 29 (5) ◽  
pp. 483-489
Author(s):  
Lilian Terezinha de Queiroz Leite ◽  
Mauricio Resende ◽  
Wanderley de Souza ◽  
Elizabeth R.S. Camargos ◽  
Matilde Cota Koury
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Western Blot ◽  
Silver Staining ◽  
Colloidal Gold ◽  
Leptospira Interrogans ◽  
Outer Envelope ◽  
Lethal Challenge ◽  
Sds Page

Monoclonal antibodies (MABs) ivere produced against an etbylenediaminetetraacetate (EDTA) extract of Leptospira interrogans serovar icterohaemorrhagiae being characterized by gel precipitation as IgM and IgG (IgGl and IgG2b). The EDTA extract was detected as several bands by silver staining in SDS-PAGE. In the Western blot the bands around 20 KDa reacted with a monoclonal antibody, 47B4D6, and was oxidized by periodate and was not digested by pronase, suggesting that the determinant is of carbohydrate nature, lmmunocytochemistry, using colloidal gold labeling, showed that an EDTA extract determinant recognized by monoclonal antibody 47B4D6, is localized under the outer envelope of serovar icterohaemorrhagiae. Hoe AIAB raised against the EDTA extract was not able to protect hamsters from lethal challenge with virulent homologous leptospires.

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