[33] Preparation of monoclonal antibodies and site-directed polyclonal antibodies against the lac permease of Escherichia coli

1986 ◽  
pp. 453-467 ◽  
Author(s):  
Nancy Carrasco ◽  
Doris Herzlinger ◽  
Waleed Danho ◽  
H. Ronald Kaback
Keyword(s):  
Escherichia Coli ◽  
Monoclonal Antibodies ◽  
Polyclonal Antibodies

scholarly journals Assembly of Colicin A in the Outer Membrane of Producing Escherichia coli Cells Requires both Phospholipase A and One Porin, but Phospholipase A Is Sufficient for Secretion

2002 ◽  
Vol 184 (13) ◽  
pp. 3723-3733 ◽  
Author(s):  
Daniele Cavard
Keyword(s):  
Escherichia Coli ◽  
Monoclonal Antibodies ◽  
Outer Membrane ◽  
Polyclonal Antibodies ◽  
Sodium Dodecyl ◽  
Phospholipase A ◽  
Cell Fractionation ◽  
Heat Labile ◽  
Molecular Masses ◽  
Outer Membrane Phospholipase A

ABSTRACT Three oligomeric forms of colicin A with apparent molecular masses of about 95 to 98 kDa were detected on sodium dodecyl sulfate (SDS)-polyacrylamide gels loaded with unheated samples from colicin A-producing cells of Escherichia coli. These heat-labile forms, called colicins Au, were visualized both on immunoblots probed with monoclonal antibodies against colicin A and by radiolabeling. Cell fractionation studies show that these forms of colicin A were localized in the outer membrane whether or not the producing cells contained the cal gene, which encodes the colicin A lysis protein responsible for colicin A release in the medium. Pulse-chase experiments indicated that their assembly into the outer membrane, as measured by their heat modifiable migration in SDS gels, was an efficient process. Colicins Au were produced in various null mutant strains, each devoid of one major outer membrane protein, except in a mutant devoid of both OmpC and OmpF porins. In cells devoid of outer membrane phospholipase A (OMPLA), colicin A was not expressed. Colicins Au were detected on immunoblots of induced cells probed with either polyclonal antibodies to OmpF or monoclonal antibodies to OMPLA, indicating that they were associated with both OmpF and OMPLA. Similar heat-labile forms were obtained with various colicin A derivatives, demonstrating that the C-terminal domain of colicin A, but not the hydrophobic hairpin present in this domain, was involved in their formation.

An Immunoradiometric Assay for Factor VIII Related Antigen (VIIIRAg) Using Two Monoclonal Antibodies-Comparison with Polyclonal Rabbit Antibodies for Use in von Willebrand’s Disease Diagnosis

1984 ◽  
Vol 52 (03) ◽  
pp. 250-252 ◽  
Author(s):  
Y Sultan ◽  
Ph Avner ◽  
P Maisonneuve ◽  
D Arnaud ◽  
Ch Jeanneau
Keyword(s):  
Monoclonal Antibodies ◽  
Structural Changes ◽  
Polyclonal Antibodies ◽  
Disease Diagnosis ◽  
Immunoradiometric Assay ◽  
Von Willebrand's Disease ◽  
Von Willebrand’S Disease ◽  
Structural Abnormalities ◽  
Antigenic Material ◽  
Von Willebrand

SummaryTwo monoclonal antibodies raised against FVIII/von Willebrand protein were used in an immunoradiometric assay (IRMA) to measure this antigen in normal plasma and plasma of patients with different forms of von Willebrand’s disease. The first antibody, an IgG1 was used to coat polystyrene tubes, the second one, an IgG2a, iodinated and used in the second step. Both antibodies inhibit ristocetin induced platelet agglutination and react strongly with platelets, megacaryocytes and endothelial cells. The IRMA test using these antibodies showed greater sensitivity than that using rabbit polyclonal anti VIIIRAg antibodies. A good correlation between the two tests was nevertheless found when VIIIRAg was measured in the majority of patient’s plasma. However 5 patients from 3 different families showed more antigenic material in the rabbit antibody IRMA than in the monoclonal antibody IRMA. It is suggested therefore that the monoclonal antibodies identify part of the VIIIR:Ag molecule showing structural abnormalities in these vWd patients, these structural changes remaining undetected by the polyclonal antibodies.

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 Development and characterization of a novel mAb against bilitranslocase - a new biomarker of renal carcinoma

2013 ◽  
Vol 47 (2) ◽  
pp. 128-137 ◽  
Author(s):  
Sendi Montanic ◽  
Michela Terdoslavich ◽  
Uros Rajcevic ◽  
Luigina De Leo ◽  
Serena Department of Medical Sciences, Uni ◽  
...  
Keyword(s):  
Renal Cell Carcinoma ◽  
Monoclonal Antibodies ◽  
Cell Carcinoma ◽  
Renal Cell ◽  
Renal Carcinoma ◽  
Vascular Endothelial Cells ◽  
Polyclonal Antibodies ◽  
Functional Characterization ◽  
Immunological Approach

Background. Bilitranslocase (TC 2.A.65.1.1) is a bilirubin-specific membrane transporter, found on absorptive (stomach and intestine) and excretory (kidney and liver) epithelia and in vascular endothelium. Polyclonal antibodies have been raised in rabbits in the past, using a synthetic peptide corresponding to AA65-77 of rat liver bilitranslocase, as an antigen. Affinity-purified antibodies from immune sera have been found to inhibit various membrane transport functions, including the bilirubin uptake into human hepatocytes and the uptake of some flavonoids into human vascular endothelial cells. It was described by means of immunohistochemistry using polyclonal antibodies that bilitranslocase expression is severely down-regulated in clear cell renal carcinoma. The aim of our work was development and characterization of high-affinity, specific mAbs against bilitranslocase, which can be used as a potential diagnostic tool in renal cell carcinoma as well as in a wide variety of biological assays on different human tissues. Materials and methods. Mice were immunized with a multi-antigen peptide corresponding to segment 65-75 of predicted primary structure of the bilitranslocase protein. By a sequence of cloning, immune- and functional tests, we aimed at obtaining a specific monoclonal antibody which recognizes a 37 kDa membrane protein, and influences the transport activity of bilitranslocase. Results. On the basis of previous results, specific IgM monoclonal antibodies were produced in BALB/c mice, in order to further improve and extend the immunological approach to the study of bilitranslocase in renal cancer cells as well as to develop its potential diagnostics use. Conclusions. In this article we show an immunological approach, based on newly developed monoclonal antibodies, to a detailed biochemical and functional characterization of a protein whose gene and protein structure is still unknown. We were able to demonstrate our novel mAb as a tumor marker candidate of renal cell carcinoma, which may prove useful in the diagnostic procedures.

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