scholarly journals The Production and characterization of Polyclonal Antibodies Against Interferon Alpha in Mice

2021 ◽  
Vol 3 (4) ◽  
Author(s):  
Mehreen Fatima ◽  
Fatima Khalid ◽  
Azra Quraishi
Keyword(s):  
Immune Response ◽  
Autoimmune Diseases ◽  
Interferon Alpha ◽  
Polyclonal Antibodies ◽  
Mouse Serum ◽  
Interferon Production ◽  
Dot Blot ◽  
Western Blots ◽  
Response Activation

The polyclonal antibodies are used extensively for research purposes in many areas of biology, such as immunoprecipitation, histochemistry, enzyme linked immunosorbent assays (ELISA), diagnosis of disease and western blots. Typically, an animal’s immune system will generate a large group of antibodies that recognize several epitopes of a particular antigen. Interferon alpha plays an important role in immune response activation and therefore is of interest in studies related to autoimmune diseases. In this paper the production of antibodies against interferon was studied in order to quantify interferon production to analyze interferon levels in autoimmune disorders in the future. For the antibody production, one month old laboratory grade mice were injected with interferon alpha in combination with a Freund’s complete adjuvant for a course of five weeks after which the antibodies were obtained in mouse serum. Confirmation of the production of anti-interferon alpha antibodies was carried out by the Elisa, immune dot blot and western blot analysis. An interferon alpha of approximately 20.5-21.5 KDa was detected in immunedot blot test.  These antibodies may be produced in these mouse models commercially and could be used in future for treatment of autoimmune diseases by managing the interferon levels in the patients. Copyright(c) The Authors

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 Dissection of the anti-Candida albicans mannan immune response using synthetic oligomannosides reveals unique properties of β-1,2 mannotriose protective epitopes

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Boualem Sendid ◽  
Karine Lecointe ◽  
Mayeul Collot ◽  
Pierre-Marie Danzé ◽  
Sébastien Damiens ◽  
...  
Keyword(s):  
Immune Response ◽  
Candida Albicans ◽  
Polyclonal Antibodies ◽  
Basic Knowledge ◽  
Western Blots ◽  
Protective Epitope ◽  
Human Sera ◽  
Antibody Specificities ◽  
Chain Lengths ◽  
Antibody Levels

AbstractCandida albicans mannan consists of a large repertoire of oligomannosides with different types of mannose linkages and chain lengths, which act as individual epitopes with more or less overlapping antibody specificities. Although anti-C. albicans mannan antibody levels are monitored for diagnostic purposes nothing is known about the qualitative distribution of these antibodies in terms of epitope specificity. We addressed this question using a bank of previously synthesized biotin sulfone tagged oligomannosides (BSTOs) of α and β anomery complemented with a synthetic β-mannotriose described as a protective epitope. The reactivity of these BSTOs was analyzed with IgM isotype monoclonal antibodies (MAbs) of known specificity, polyclonal sera from patients colonized or infected with C. albicans, and mannose binding lectin (MBL). Surface plasmon resonance (SPR) and multiple analyte profiling (MAP) were used. Both methods confirmed the usual reactivity of MAbs against either α or β linkages, excepted for MAb B6.1 (protective epitope) reacting with β-Man whereas the corresponding BSTO reacted with anti-α-Man. These results were confirmed in western blots with native C. albicans antigens. Using patients’ sera in MAP, a significant correlation was observed between the detection of anti-mannan antibodies recognizing β- and α-Man epitopes and detection of antibodies against β-linked mannotriose suggesting that this epitope also reacts with human polyclonal antibodies of both specificities. By contrast, the reactivity of human sera with other α- and β-linked BSTOs clearly differed according to their colonized or infected status. In these cases, the establishment of an α/β ratio was extremely discriminant. Finally SPR with MBL, an important lectin of innate immunity to C. albicans, classically known to interact with α-mannose, also interacted in an unexpected way with the protective epitope. These cumulative data suggest that structure/activity investigations of the finely tuned C. albicans anti-mannose immune response are worthwhile to increase our basic knowledge and for translation in medicine.

Antibodies against purified epithelial sodium channel protein from bovine renal papilla

1988 ◽  
Vol 255 (6) ◽  
pp. C835-C843 ◽  
Author(s):  
E. J. Sorscher ◽  
M. A. Accavitti ◽  
D. Keeton ◽  
E. Steadman ◽  
R. A. Frizzell ◽  
...  
Keyword(s):  
Sodium Channel ◽  
Polyclonal Antibodies ◽  
High Yield ◽  
Enzyme Linked Immunosorbent Assay ◽  
Epithelial Cell Line ◽  
Renal Papilla ◽  
Dot Blot ◽  
Renal Epithelial Cell ◽  
Western Blots ◽  
Channel Protein

Monoclonal and polyclonal antibodies against amiloride-sensitive sodium channel protein purified from bovine renal papilla have been produced. These antibodies show considerable specificity in enzyme-linked immunosorbent assay (ELISA) and dot-blot assays and can immunoprecipitate radiolabeled channel protein. The polyclonal antibodies bind two sodium channel subunits on Western blots, namely, the 300- and 110-kDa polypeptides, and cross react with channel protein isolated from the amphibian A6 renal epithelial cell line. They can be used to immunoaffinity purify the channel in relatively high yield from a crude, detergent-solubilized bovine kidney homogenate. These antibodies should be useful in isolating the sodium channel gene, in studying the channel protein structure, in studying immunocytochemical localization, and in allowing purification of the channel from other tissue sources.

scholarly journals Molecular Characterization of Annexin B2, B3 and B12 in Taenia multiceps

Genes ◽  
2018 ◽  
Vol 9 (11) ◽  
pp. 559 ◽  
Author(s):  
Cheng Guo ◽  
Yue Xie ◽  
Yuchen Liu ◽  
Ning Wang ◽  
Jiafei Zhan ◽  
...  
Keyword(s):  
Developmental Stages ◽  
Polyclonal Antibodies ◽  
Parasite Development ◽  
Economic Losses ◽  
Mrna Levels ◽  
Western Blots ◽  
Taenia Multiceps ◽  
Homologous Genes ◽  
Coenurus Cerebralis

Coenurus cerebralis, the metacestode of Taenia multiceps, causes coenurosis, a disease severely affecting goat, sheep, cattle and yak farming and resulting in huge economic losses annually. Annexins bind calcium ions and play an important role in flatworm parasite development. To explore potential functions of annexins in T. multiceps, three homologous genes, namely, TmAnxB2, TmAnxB3 and TmAnxB12, were screened from the transcriptome dataset, amplified from C. cerebralis cDNA and subjected to bioinformatics analysis. Then, polyclonal antibodies recognizing the recombinant TmAnxB2 (rTmAnxB2) and rTmAnxB3 were prepared for localization of TmAnxB2 and TmAnxB3 in different tissues and developmental stages by immunofluorescence. The transcription of all three genes was also measured by relative fluorescent quantitative PCR. The sizes of rTmAnxB2, rTmAnxB3 and rTmAnxB12 were 58.00, 53.06 and 53.51 kDa, respectively, and rTmAnxB12 was unstable. Both rTmAnxB2 and rTmAnxB3 were recognized by goat-positive T. multiceps sera in Western blots. Immunofluorescence revealed that TmAnxB2 and TmAnxB3 were localized in the protoscolex and cyst wall and TmAnxB3 was also detected in adult cortex. TmAnxB2 and TmAnxB12 mRNA levels were determined to be highest in oncospheres and protoscolex, whereas transcription of TmAnxB3 was highest in scolex and immature segments. Taken together, these findings indicate that TmAnxB2 and TmAnxB12 may play critical roles in T. multiceps larvae, while TmAnxB3 may have important functions in adults. These results will lay the foundation for functional research of annexins in T. multiceps.

Molecular characterization of mitofilin (HMP), a mitochondria-associated protein with predicted coiled coil and intermembrane space targeting domains

1996 ◽  
Vol 109 (9) ◽  
pp. 2253-2264 ◽  
Author(s):  
P.R. Odgren ◽  
G. Toukatly ◽  
P.L. Bangs ◽  
R. Gilmore ◽  
E.G. Fey
Keyword(s):  
Polyclonal Antibodies ◽  
Limited Proteolysis ◽  
Coiled Coil ◽  
Cell Types ◽  
Cdna Clones ◽  
Intermembrane Space ◽  
Western Blots ◽  
Double Label ◽  
Helical Region

We have identified and characterized a human protein of the mitochondria which we call mitofilin. Using monoclonal and polyclonal antibodies, we have isolated cDNA clones and characterized mitofilin biochemically. It appears as a 90 and 91 kDa doublet in western blots and is translated from a single 2.7 kb mRNA. Antibodies raised against cellular and bacterially-expressed protein given identical cytoplasmic immunofluorescence and immunoblot results. Mitofilin co-localizes with mitochondria in immunofluorescence experiments and co-purifies with mitochondria. Double label studies show co-localization only with mitochondria and not with Golgi or endoplasmic reticulum. Co-localization with mitochondria is retained when actin or tubulin are de-polymerized, and mitofilin is expressed in all human cell types tested. The cDNA encodes a polypeptide with a central alpha-helical region with predicted coiled coil domains flanked by globular amino and carboxy termini. Unlike coiled coil motor proteins, mitofilin is resistant to detergent extraction. The presence of mitochondrial targeting and stop-transfer sequences, along with the accessibility of mitofilin to limited proteolysis suggests that it resides predominantly in the intermembrane space, consistent with immuno-electron micrographs which show mitofilin mainly at the mitochondrial periphery. The cDNA sequence of mitofilin is identical to that recently reported by Icho et al. (1994; Gene 144, 301–306) for a mRNA preferentially expressed in heart muscle (HMP), consistent with the high levels of mitochondria in cardiac myocytes.

Characterization of putative membrane protein genes of the ‘Candidatus Phytoplasma asteris’, chrysanthemum yellows isolate

2008 ◽  
Vol 54 (5) ◽  
pp. 341-351 ◽  
Author(s):  
Luciana Galetto ◽  
Jacqueline Fletcher ◽  
Domenico Bosco ◽  
Massimo Turina ◽  
Astri Wayadande ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Expression Vector ◽  
Polyclonal Antibodies ◽  
Western Blots ◽  
Protein Fragments ◽  
Aster Yellows ◽  
Euscelidius Variegatus ◽  
Macrosteles Quadripunctulatus ◽  
Aster Yellows Phytoplasma

To characterize potentially important surface-exposed proteins of the phytoplasma causing chrysanthemum yellows (CY), new primers were designed based on the conserved regions of 3 membrane protein genes of the completely sequenced onion yellows and aster yellows witches’ broom phytoplasmas and were used to amplify CY DNA. The CY genes secY, amp, and artI, encoding the protein translocase subunit SecY, the antigenic membrane protein Amp and the arginine transporter ArtI, respectively, were cloned and completely sequenced. Alignment of CY-specific secY sequences with the corresponding genes of other phytoplasmas confirmed the 16S rDNA-based classification, while amp sequences were highly variable within the ‘Candidatus Phytoplasma asteris’. Five CY partial sequences were cloned into the pRSetC expression vector, and 3 of the encoded protein fragments (Amp 64/651, Amp 64/224, ArtI 131/512) were expressed as fusion antigens for the production of CY-specific polyclonal antibodies (A416 against Amp 64/224; A407 against ArtI 131/512). A416 recognized, in Western blots, the full-length Amp from CY-infected plants (periwinkle, daisy) and insect vectors ( Euscelidius variegatus , Macrosteles quadripunctulatus ). A416 also reacted to European aster yellows, to primula yellows phytoplasmas, to northern Italian strains of ‘Ca. Phytoplasma asteris’ from lettuce and gladiolus, but it did not react to American aster yellows phytoplasma.

scholarly journals Characterization of an IgM-like immunoglobulin from silver catfish (Rhamdia quelen) serum and its use for the production of polyclonal antibodies and development of immunoassays

2016 ◽  
Vol 36 (9) ◽  
pp. 819-825 ◽  
Author(s):  
Luiz C. Kreutz ◽  
Raíssa Canova ◽  
Cristian O. Nied ◽  
Márcia Bortoluzzi ◽  
Rafael Frandoloso
Keyword(s):  
Immune Response ◽  
Fish Species ◽  
Humoral Immune Response ◽  
Polyclonal Antibodies ◽  
Cross Reactivity ◽  
Dot Blot ◽  
Silver Catfish ◽  
Humoral Immune ◽  
Abstract Knowledge ◽  
Wide Range

Abstract: Knowledge on fish immunoglobulin (Ig) characteristics and the availability of monoclonal or polyclonal antibodies to fish Igs are essential to evaluate the humoral immune response and the Ig distribution on leukocyte cells. We demonstrated that silver catfish serum Ig is composed of one immunodominant H chain with approximately 75k Da and one L chain with approximately 28 kDa, similar to human IgM. Rabbit polyclonal antibodies to the catfish IgM-like Ig recognized both the H and L chain and were useful in developing an indirect ELISA to measure the production of antibodies in fish immunized with bovine serum albumin. Dot blot and western blot cross-reactivity studies indicated a wide degree of epitope sharing amongst Ig from several Siluriformes and Characiformes fish indigenous to Brazilian rivers. In these fish species, polyclonal antibodies reacted mostly with the H chain. The results presented here are central to the development of tools and strategies to investigate the antibody production to inoculated antigens and tissue distribution of Ig molecules in native fish species. Furthermore, because of the wide range of cross-reactivity, polyclonal antibodies to silver catfish IgM-like Ig might be used to develop immunoassays to measure the humoral immune response in other fish species.

scholarly journals Characterization of a Pepper Vein Banding Virus from Chili Pepper in India

Plant Disease ◽  
1997 ◽  
Vol 81 (6) ◽  
pp. 673-676 ◽  
Author(s):  
K. S. Ravi ◽  
J. Joseph ◽  
N. Nagaraju ◽  
S. Krishna Prasad ◽  
H. R. Reddy ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Mosaic Virus ◽  
Polyclonal Antibodies ◽  
Cross Reactivity ◽  
Mottle Virus ◽  
Western Blots ◽  
Peanut Stripe Virus ◽  
Karnataka State ◽  
Distinct Member

A survey conducted in pepper-growing tracts of Karnataka State, covering 165 fields in 33 villages, revealed the occurrence of many pepper mosaic diseases. Based on reactions on selected test plants, the viruses were identified as pepper vein banding virus (PVBV), pepper veinal mottle virus, potato virus Y, cucumber mosaic virus, and tobacco mosaic virus. Among these, PVBV was the most prevalent. PVBV was purified from infected leaves of Capsicum annuum cv. California Wonder. Electron microscopy revealed flexuous rod-shaped particles in the purified preparations. The coat protein (CP) molecular weight was 35,000, which is similar to members of the Potyvirus group. As in other potyviruses, the CP underwent proteolytic degradation to a fragment with a molecular weight of 31,000. Both of these bands cross-reacted with antibodies against tobacco etch virus in Western blots. Polyclonal antibodies were produced against PVBV. Cross-reactivity studies with other potyviral antisera showed that PVBV is serologically closer to peanut mottle virus than to peanut stripe virus or sorghum potyvirus. N-terminal sequence analysis of the intact CP and trypsin-resistant core revealed that PVBV is a distinct member of the Potyvirus group.

Functional Characterization of PM6/13, a β3-specific (GPIIIa/CD61) Monoclonal Antibody that Shows Preferential Inhibition of Fibrinogen Binding over Fibronectin Binding to Activated Human Platelets

1998 ◽  
Vol 79 (01) ◽  
pp. 177-185 ◽  
Author(s):  
Ashia Siddiqua ◽  
Michael Wilkinson ◽  
Vijay Kakkar ◽  
Yatin Patel ◽  
Salman Rahman ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Platelet Aggregation ◽  
Platelet Rich Plasma ◽  
Functional Characterization ◽  
Human Platelets ◽  
Western Blots ◽  
Activated Platelets ◽  
Reducing Conditions ◽  
Fibronectin Binding

SummaryWe report the characterization of a monoclonal antibody (MAb) PM6/13 which recognises glycoprotein IIIa (GPIIIa) on platelet membranes and in functional studies inhibits platelet aggregation induced by all agonists examined. In platelet-rich plasma, inhibition of aggregation induced by ADP or low concentrations of collagen was accompanied by inhibition of 5-hydroxytryptamine secretion. EC50 values were 10 and 9 [H9262]g/ml antibody against ADP and collagen induced responses respectively. In washed platelets treated with the cyclooxygenase inhibitor, indomethacin, PM6/13 inhibited platelet aggregation induced by thrombin (0.2 U/ml), collagen (10 [H9262]g/ml) and U46619 (3 [H9262]M) with EC50 = 4, 8 and 4 [H9262]g/ml respectively, without affecting [14C]5-hydroxytryptamine secretion or [3H]arachidonate release in appropriately labelled cells. Studies in Fura 2-labelled platelets revealed that elevation of intracellular calcium by ADP, thrombin or U46619 was unaffected by PM6/13 suggesting that the epitope recognised by the antibody did not influence Ca2+ regulation. In agreement with the results from the platelet aggregation studies, PM6/13 was found to potently inhibit binding of 125I-fibrinogen to ADP activated platelets. Binding of this ligand was also inhibited by two other MAbs tested, namely SZ-21 (also to GPIIIa) and PM6/248 (to the GPIIb-IIIa complex). However when tested against binding of 125I-fibronectin to thrombin stimulated platelets, PM6/13 was ineffective in contrast with SZ-21 and PM6/248, that were both potent inhibitors. This suggested that the epitopes recognised by PM6/13 and SZ-21 on GPIIIa were distinct. Studies employing proteolytic dissection of 125I-labelled GPIIIa by trypsin followed by immunoprecipitation with PM6/13 and analysis by SDS-PAGE, revealed the presence of four fragments at 70, 55, 30 and 28 kDa. PM6/13 did not recognize any protein bands on Western blots performed under reducing conditions. However Western blotting analysis with PM6/13 under non-reducing conditions revealed strong detection of the parent GP IIIa molecule, of trypsin treated samples revealed recognition of an 80 kDa fragment at 1 min, faint recognition of a 60 kDa fragment at 60 min and no recognition of any product at 18 h treatment. Under similar conditions, SZ-21 recognized fragments at 80, 75 and 55 kDa with the 55kDa species persisting even after 18 h trypsin treatment. These studies confirm the epitopes recognised by PM6/13 and SZ-21 to be distinct and that PM6/13 represents a useful tool to differentiate the characteristics of fibrinogen and fibronectin binding to the GPIIb-IIIa complex on activated platelets.

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