Characterization of monoclonal antibodies to trichocyst antigens in Paramecium

1988 ◽  
Vol 91 (2) ◽  
pp. 191-199
Author(s):  
A.K. Fok ◽  
R.P. Olegario ◽  
M.S. Ueno ◽  
R.D. Allen
Keyword(s):  
Monoclonal Antibodies ◽  
Small Variation ◽  
Immunogold Labelling ◽  
Immunofluorescence Assay ◽  
Outer Edge ◽  
Thin Sections ◽  
Reducing Conditions ◽  
Sds Page ◽  
The Family

Ten mouse monoclonal antibodies (mAbs) were raised against trichocyst contaminants present in crude or enriched lysosome fractions of Paramecium multimicronucleatum. Using an indirect immunofluorescence assay (IFA) and immunogold labelling on frozen thin sections, epitopes were located on the outer edge, cortex and the core of the trichocyst body, as well as the sheath covering the tip. Except for the two on the tip, epitopes were reactive after SDS-PAGE under non-reducing conditions. Four mAbs (131C1E8, A1-3, A16-2, D7) were directed to a trio of bands of 37, 34 and 29 (x 10(3] Mr from the beaded or meshlike trichocyst body sheath. A fifth mAb (135B9E7), directed to epitopes on the cortex inside the beaded body sheath, reacted strongly with the 37 and 34 bands, but weakly with the 29 × 10(3) Mr band. The last three mAbs (270D5, 22D7F2, D8) were reactive with one or more of three families of antigens found on the trichocyst core. mAb 270D5 reacted mainly with the 34 and 29 (x 10(3] Mr bands of the family containing the above trio, while mAb 22C7F2 reacted consistently with the 47 × 10(3) band of the higher Mr family but variably with both the trio of bands and the 17 × 10(3) band of the lower Mr family. mAb D8, which was directed to epitopes on the trichocyst core and small vesicles in the endoplasm, reacted only with the 29 × 10(3) Mr band. The mAbs were cross-reactive with the trichocysts of P. primaurelia, P. tetraurelia, P. caudatum and P. calkinsi with some small variation in blotting patterns.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

scholarly journals Characterization of a Broadly Reactive Monoclonal Antibody against Norovirus Genogroups I and II: Recognition of a Novel Conformational Epitope

2007 ◽  
Vol 81 (22) ◽  
pp. 12298-12306 ◽  
Author(s):  
Tomoyuki Shiota ◽  
Michio Okame ◽  
Sayaka Takanashi ◽  
Pattara Khamrin ◽  
Makiko Takagi ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Conformational Epitope ◽  
The Novel ◽  
Antigen Specificity ◽  
Virus Like Particle ◽  
The Family ◽  
Immunological Diagnosis ◽  
Further Development

ABSTRACT Norovirus, which belongs to the family Caliciviridae, is one of the major causes of nonbacterial acute gastroenteritis in the world. The main human noroviruses are of genogroup I (GI) and genogroup II (GII), which were subdivided further into at least 15 and 18 genotypes (GI/1 to GI/15 and GII/1 to GII/18), respectively. The development of immunological diagnosis for norovirus had been hindered by the antigen specificity of the polyclonal antibody. Therefore, several laboratories have produced broadly reactive monoclonal antibodies, which recognize the linear GI and GII cross-reactive epitopes or the conformational GI-specific epitope. In this study, we characterized the novel monoclonal antibody 14-1 (MAb14-1) for further development of the rapid immunochromatography test. Our results demonstrated that MAb14-1 could recognize 15 recombinant virus-like particles (GI/1, 4, 8, and 11 and GII/1 to 7 and 12 to 15) and showed weak affinity to the virus-like particle of GI/3. This recognition range is the broadest of the existing monoclonal antibodies. The epitope for MAb14-1 was identified by fragment, sequence, structural, and mutational analyses. Both terminal antigenic regions (amino acid positions 418 to 426 and 526 to 534) on the C-terminal P1 domain formed the conformational epitope and were in the proximity of the insertion region (positions 427 to 525). These regions contained six amino acids responsible for antigenicity that were conserved among genogroup(s), genus, and Caliciviridae. This epitope mapping explained the broad reactivity and different titers among GI and GII. To our knowledge, we are the first group to identify the GI and GII cross-reactive monoclonal antibody, which recognizes the novel conformational epitope. From these data, MAb14-1 could be used further to develop immunochromatography.

scholarly journals Purification, Characterization of an Entomopathogenic Fungal Lectin from Purpureocillium Lilacinum, and its Involvement in Pathogenesis Leading to Mycotic Keratitis

2021 ◽  
Author(s):  
Narasimhappagari Jagadeesh ◽  
Supreeth Kulkarni ◽  
Vishwanath B Chachadi ◽  
Sanhita Roy ◽  
Shashikala Inamdar
Keyword(s):  
Proinflammatory Response ◽  
Corneal Epithelial ◽  
Purpureocillium Lilacinum ◽  
Ammonium Sulphate Precipitation ◽  
Reducing Conditions ◽  
Sds Page ◽  
Fungal Lectin ◽  
Human Corneal Epithelial Cells ◽  
Ph Range

Abstract A lectin PCL, from Purpureocillium lilacinum a saprophytic, filamentous fungus was purified from the crude extract of the mycelia using 70% ammonium sulphate precipitation followed by affinity chromatography on mucin-Sepharose 4 B column. PCL is a monomer with an apparent molecular mass of 18.5 kDa as revealed by SDS-PAGE under both reducing and non reducing conditions. PCL is a blood group non specific lectin and has highest affinity towards Chitin, Mucin, asialo mucin, Fetuin with a MIC of 0.15µg/mL and also recognizes L-fucose, galactose, lactose, N-acetly galactosamine, Hyaluronic acid. PCL is stable up to 60 ºC and within the pH range 4–8. To understand its role in pathogenesis, effect of PCL was evaluated on Human Corneal Epithelial Cells (HCECs). PCL showed strong glycan mediated binding to HCECsand PCL showed proinflammatory response at lower concentrations by stimulating secretion of IL-6, 8. In contrast PCL at higher concentrations revealed opposite effect of HCECs growth inhibition. All these results collectively support the involvement of PCL in mediating host pathogen interactions possibly leading to pathogenesis. In addition, considering the entomopathogenic effect of Purpureocillium lilacinum, PCL may be attributed for this beneficiary effect, which needs to be explored.

scholarly journals Development of Nanobodies against Mal de Río Cuarto virus major viroplasm protein P9-1 for diagnostic sandwich ELISA and immunodetection

2021 ◽  
Author(s):  
Gabriela Llauger ◽  
Demián Monti ◽  
Matías Adúriz ◽  
Ema Romão ◽  
Analía Delina Dumón ◽  
...  
Keyword(s):  
Sandwich Elisa ◽  
Elisa Test ◽  
Host Proteins ◽  
Maize Breeding ◽  
Thin Sections ◽  
Isolation And Characterization ◽  
The Family ◽  
Fundamental Research ◽  
Nanomolar Range

Abstract Mal de Río Cuarto virus (MRCV) is a member of the genus Fijivirus of the family Reoviridae that causes a devastating disease in maize and is persistently and propagatively transmitted by planthopper vectors. Virus replication and assembly occur within viroplasms formed by viral and host proteins. This work describes the isolation and characterization of llama-derived Nanobodies (Nbs) recognizing the major viral viroplasm component, P9-1. Specific Nbs were selected against recombinant P9-1, with affinities in the nanomolar range as measured by surface plasmon resonance. Three selected Nbs were fused to alkaline phosphatase and eGFP to develop a sandwich ELISA test which showed a high diagnostic sensitivity (99.12%, 95% CI: 95.21–99.98) and specificity (100%, 95% CI: 96.31–100) and a detection limit of 0.236 ng/ml. Interestingly, these Nanobodies recognized different P9-1 conformations and were successfully employed to detect P9-1 in pull-down assays of infected maize extracts. Finally, we demonstrated that fusions of the Nbs to eGFP and RFP allowed the immunodetection of virus present in phloem cells of leaf thin sections. The Nbs developed in this work will aid the study of MRCV epidemiology, assist maize breeding programs, and be valuable tools to boost fundamental research on viroplasm structure and maturation.

Identification of Platelet Antigens by Immunoprecipitation of Unlabelled Platelet Glycoproteins

1990 ◽  
Vol 64 (03) ◽  
pp. 469-472 ◽  
Author(s):  
Werner Stenziger ◽  
Beate Kehrel ◽  
Jürgen van de Loo
Keyword(s):  
Monoclonal Antibodies ◽  
Silver Staining ◽  
Protein G ◽  
Time Saving ◽  
Radioactive Substances ◽  
Reducing Conditions ◽  
Fast Flow ◽  
Murine Monoclonal Antibodies ◽  
Sensitive Method

SummaryA time-saving and sensitive method for the identification of antigens on unlabelled platelet glycoproteins (GP) based on immunoprecipitation has been developed. Platelet solubilisates were incubated with antibodies to form GP-antibody complexes that were precipitated with Protein G Sepharose 4 Fast Flow (PGS). Pcs-bound material was eluted, separated by SDSPAGE under reducing conditions and visualized by silver staining. The method was designed as an alternative to radioimmunoprecipitation for laboratories not equipped to work with radioactive substances. It is proposed as a complementary procedure for the characterization of platelet GP antigens by murine monoclonal antibodies and human alloantibodies.

scholarly journals Characterization of non-covalent oligomers of proteins treated with hypochlorous acid

2003 ◽  
Vol 375 (1) ◽  
pp. 33-40 ◽  
Author(s):  
Anna L. P. CHAPMAN ◽  
Christine C. WINTERBOURN ◽  
Stephen O. BRENNAN ◽  
T. William JORDAN ◽  
Anthony J. KETTLE
Keyword(s):  
Hypochlorous Acid ◽  
Tissue Injury ◽  
Convincing Evidence ◽  
Protein Carbonyls ◽  
Molar Ratio ◽  
Myeloperoxidase Activity ◽  
Reducing Conditions ◽  
Sds Page ◽  
Methionine Residues

Hypochlorous acid (HOCl) is a potent oxidant produced by myeloperoxidase that causes aggregation of many proteins. Treatment of apohaemoglobin and apomyoglobin with HOCl produced a regular series of oligomer bands when the proteins were separated by SDS/PAGE under reducing conditions. Aggregation was detectable at a HOCl/protein molar ratio of 0.5:1 and was maximal at ratios of 10:1–20:1. Dimers formed within 1 min of adding HOCl, and further aggregation occurred over the next 30 min. No convincing evidence for covalent cross-linking was obtained by amino acid analysis, peptide analysis or electrospray ionization-MS of HOCl-modified apomyoglobin. The latter showed an increase in mass consistent with conversion of the two methionine residues into sulphoxides. A 5-fold excess of HOCl generated approximately three chloramines on the apomyoglobin. These underwent slow decay. Protein carbonyls were formed and were almost entirely located only on the polymer bands. Conversion of positively into negatively charged groups on the protein by succinylation caused preformed aggregates to dissociate. Treatment of apomyoglobin with taurine chloramine generated methionine sulphoxides but few protein carbonyls, and did not result in aggregation. We conclude that aggregation was due to strong, non-covalent interactions between protein chains. We propose that formation of protein carbonyls and possibly chloramines, along with methionine oxidation, alters protein folding to expose hydrophobic areas on neighbouring molecules that associate to form dimers and higher-molecular-mass aggregates. This process could lead to the formation of aggregated proteins at sites of myeloperoxidase activity and contribute to inflammatory tissue injury.

Molecular Characterization of South American Bovine Herpesvirus-1 Isolates with Monoclonal Antibodies and SDS-PAGE

1993 ◽  
Vol 40 (1-10) ◽  
pp. 125-130 ◽  
Author(s):  
A. Suarez Heinlein ◽  
A. E. Metzler ◽  
R. Weiblen ◽  
P. Berrios ◽  
A. A. Schudel ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Molecular Characterization ◽  
Bovine Herpesvirus ◽  
South American ◽  
Bovine Herpesvirus 1 ◽  
Sds Page ◽  
Herpesvirus 1

scholarly journals Characterization of the 70 kDa polypeptide of the Na/Ca exchanger

1999 ◽  
Vol 338 (1) ◽  
pp. 139-145 ◽  
Author(s):  
Rami I. SABA ◽  
Alex BOLLEN ◽  
André HERCHUELZ
Keyword(s):  
Wild Type ◽  
Terminal Portion ◽  
Terminal Sequence ◽  
Protein Ratio ◽  
Reducing Conditions ◽  
Sds Page ◽  
Cloned Bovine ◽  
Sarcolemmal Vesicles ◽  
Exchange Activity

The Na/Ca exchanger is associated with 160, 120 and 70 kDa polypeptides whose nature is poorly understood. We have purified and characterized the Na/Ca exchanger from bovine cardiac sarcolemmal vesicles (SLVs) by using ion-exchange and affinity chromatographies. The Na/Ca exchanger-enriched fraction was reconstituted into asolectin liposomes [lipid to protein ratio 10:1 (w/w)] that showed Na/Ca exchange activity. Under non-reducing conditions, SDS/PAGE showed a single 70 kDa polypeptide, which was further characterized by immunoblots with different antibodies: SWant, raised against the purified exchanger protein; NH2-terminus, residues 1–21; NCX1, residues 393–406; and Exon F, residues 622–644. Immunoblots under reducing conditions with SWant, NH2-terminus and NCX1 showed three bands migrating at 160, 120 and 70 kDa for SLV preparations, whereas Exon F reacted only with the 160 and 120 kDa bands. Under non-reducing conditions, immunoblots with purified reconstituted Na/Ca exchanger showed a single band at 70 kDa reacting with SWant, NH2-terminus and NCX1 but not with Exon F. We conclude that the 70 kDa protein is associated with Na/Ca exchange activity, has the same N-terminal sequence as the cloned bovine cardiac exchanger, and has its length decreased by at least 35% from its C-terminal portion as compared with that of the wild-type exchanger.

scholarly journals Biological and immunological characterization of ATG and ALG

Blood ◽  
1986 ◽  
Vol 68 (3) ◽  
pp. 712-719
Author(s):  
EL Raefsky ◽  
P Gascon ◽  
A Gratwohl ◽  
B Speck ◽  
NS Young
Keyword(s):  
Clinical Trial ◽  
Flow Cytometry ◽  
Monoclonal Antibodies ◽  
Antithymocyte Globulin ◽  
Antilymphocyte Globulin ◽  
Hla Dr ◽  
Sds Page ◽  
Rabbit Complement ◽  
Leu 7

Antithymocyte globulin (ATG) and antilymphocyte globulin (ALG) are effective therapies in aplastic anemia; their mechanism of action is undefined. We assayed multiple properties of ATG and ALG to address the biological and immunological bases for differences between ATG and ALG and lot variation. In addition, we studied a lot reported to be inactive in an American clinical trial; however in retrospect, this lot appeared to be active in patients treated in Europe. Immunoprecipitation of thymocyte and lymphocyte membrane proteins with ATG and ALG showed between 14 and 18 major bands on SDS-PAGE, but the patterns for ATG and ALG were not identical. The ability of ATG and ALG to block binding of labeled monoclonal antibodies was assessed using flow cytometry and a radioimmunoassay. In general, there was more lot variation among ALGs than ATGs; however, all ALG lots were more potent blockers of binding of anti-HLA-DR and anti-Leu 1 antibodies than was ATG. Both ALG and ATG effectively blocked binding of anti-Leu 2a, anti- Leu 3a, anti-Leu 4, anti-Leu 5b, and anti-IL 2 receptor abs; neither blocked binding of anti-Leu 7. All preparations were capable of inducing T-cell blastogenesis, although there was considerable lot variation. All lots lysed 60% to 75% T cells in a rabbit complement- mediated cytotoxicity assay, with most having a plateau of activity at 5 to 10 ug/mL. Two lots of ALG, including the lot reported to be clinically inactive, showed less toxicity at suboptimal concentrations and did not plateau even at 80 ug/mL. In total, these results indicate important differences between ATG and ALG in general, more lot variation among ALGs than ATGs and only differences in cytotoxicity between an “inactive” lot of ALG and most, but not all, other active ATG and ALG preparations.

scholarly journals Characterization of the Family I inorganic pyrophosphatase fromPyrococcus horikoshiiOT3

Archaea ◽  
2005 ◽  
Vol 1 (6) ◽  
pp. 385-389 ◽  
Author(s):  
Sung-Jong Jeon ◽  
Kazuhiko Ishikawa
Keyword(s):  
Maximum Velocity ◽  
Heat Stability ◽  
Hydrolytic Activity ◽  
Heat Inactivation ◽  
Inorganic Pyrophosphatase ◽  
Gene Encoding ◽  
Sds Page ◽  
The Family ◽  
Hydrolysis Of

A gene encoding for a putative Family inorganic pyrophosphatase (PPase, EC 3.6.1.1) from the hyperthermophilic archaeonPyrococcus horikoshiiOT3 was cloned and the biochemical characteristics of the resulting recombinant protein were examined. The gene (Accession No. 1907) fromP. horikoshiishowed some identity with other Family I inorganic pyrophosphatases from archaea. The recombinant PPase fromP. horikoshii(PhPPase) has a molecular mass of 24.5 kDa, determined by SDS-PAGE. This enzyme specifically catalyzed the hydrolysis of pyrophosphate and was sensitive to NaF. The optimum temperature and pH for PPase activity were 70 °C and 7.5, respectively. The half-life of heat inactivation was about 50 min at 105 °C. The heat stability ofPhPPase was enhanced in the presence of Mg2+. A divalent cation was absolutely required for enzyme activity, Mg2+being most effective; Zn2+, Co2+and Mn2+efficiently supported hydrolytic activity in a narrow range of concentrations (0.05– 0.5 mM). The Kmfor pyrophosphate and Mg2+were 113 and 303 µM, respectively; and maximum velocity,Vmax, was estimated at 930 U mg–1.

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