scholarly journals Distinct populations of high-Mr mucins secreted by different human salivary glands discriminated by density-gradient electrophoresis

1995 ◽  
Vol 309 (3) ◽  
pp. 801-806 ◽  
Author(s):  
J Bolscher ◽  
E Veerman ◽  
A Van Nieuw Amerongen ◽  
A Tulp ◽  
D Verwoerd
Keyword(s):  
Monoclonal Antibodies ◽  
Sialic Acid ◽  
Salivary Glands ◽  
Density Gradient ◽  
Human Saliva ◽  
Exchange Chromatography ◽  
Dot Blot ◽  
Cell Organelles ◽  
Sulphate Content ◽  
Gradient Electrophoresis

High-M(r) mucins [mucin glycoprotein 1 (MG1)] isolated from human saliva from the individual salivary glands were chemically characterized. The carbohydrate content of MG1 derived from palatal (PAL), submandibular (SM) and sublingual (SL) saliva was typical of mucins but showed heterogeneity, especially in the amount of sialic acid and sulphated sugar residues. The physicochemical properties of native MG1s make conventional SDS/PAGE and ion-exchange chromatography unsuitable for investigating differences between individual samples. Recently a density-gradient electrophoresis (DGE) device has been developed, primarily for separation based on the charge of entire cells or cell organelles [Tulp, Verwoerd and Pieters (1993) Electrophoresis 14, 1295-1301]. We have used this apparatus to study the high-M(r) salivary mucins. Using DGE, the MG1s of individual glands were seen to have clearly distinct electrophoretic mobilities, as monitored by ELISA using MG1-specific monoclonal antibodies. Even within a particular MG1 preparation, subpopulations could be distinguished. DGE analysis of a chemically and enzymically modified MG1 series, followed by ELISA and dot-blot detection using specific monoclonal antibodies, lectins and high-iron diamine staining, suggests that the high electrophoretic mobility of PAL-MG1 is mainly the result of a high sulphate content, whereas the SL subpopulations differ mainly in binding type and amount of sialic acid. SM-MG1 most resembles the low-mobility subpopulation of SL-MG1, except that it has a lower sulphate content. In conclusion, DGE appears to be a powerful method for analysis of native mucin; it has been used to demonstrate that MG1s from the various salivary glands are biochemically much more diverse than was previously assumed.

Evaluation of the Efficiency of Protein A Affinity Chromatography to Purify a Monoclonal Antibody for Cancer Treatment and its Purity Analysis

2020 ◽  
Vol 7 (2) ◽  
pp. 121-133
Author(s):  
Ayesha Akhtar ◽  
Shivakumar Arumugam ◽  
Shoaib Alam
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Affinity Chromatography ◽  
Protein A ◽  
Exchange Chromatography ◽  
Size Exclusion ◽  
High Yield ◽  
Staphylococcal Protein A ◽  
Purification Step ◽  
Purity Analysis

Background:: Protein A affinity chromatography is often employed as the most crucial purification step for monoclonal antibodies to achieve high yield with purity and throughput requirements. Introduction:: Protein A, also known as Staphylococcal protein A (SPA) is found in the cell wall of the bacteria staphylococcus aureus. It is one of the first discovered immunoglobulin binding molecules and has been extensively studied since the past few decades. The efficiency of Protein A affinity chromatography to purify a recombinant monoclonal antibody in a cell culture sample has been evaluated, which removes 99.0% of feed stream impurities. Materials and Method:: We have systematically evaluated the purification performance by using a battery of analytical methods SDS-PAGE (non-reduced and reduced sample), Cation Exchange Chromatography (CEX), Size-exclusion chromatography (SEC), and Reversed phased-Reduced Chromatography for a CHO-derived monoclonal antibody. Results and Discussion:: The analytical test was conducted to determine the impurity parameter, Host Cell Contaminating Proteins (HCP). It was evaluated to be 0.015ng/ml after the purification step; while initially, it was found to be 24.431ng/ml. Conclusion:: The tests showed a distinct decrease in the level of different impurities after the chromatography step. It can be concluded that Protein A chromatography is an efficient step in the purification of monoclonal 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.

Prenatal development of human major salivary glands and immunohistochemical detection of keratins using monoclonal antibodies

1990 ◽  
Vol 89 (2) ◽  
pp. 213-235 ◽  
Author(s):  
Suk-Keun Lee ◽  
Chang Yun Lim ◽  
Je Geun Chi ◽  
Kazuto Yamada ◽  
Kouji Hashimura ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Salivary Glands ◽  
Prenatal Development ◽  
Immunohistochemical Detection ◽  
Major Salivary Glands

Separation of human lymphocyte subpopulations by density gradient electrophoresis

1980 ◽  
Vol 51 (2) ◽  
pp. 238-249 ◽  
Author(s):  
Chris D. Platsoucas ◽  
Robert A. Good ◽  
Sudhir Gupta
Keyword(s):  
Density Gradient ◽  
Human Lymphocyte ◽  
Lymphocyte Subpopulations ◽  
Gradient Electrophoresis

Density Gradient Electrophoresis of Mouse Spleen Lymphocytes

1980 ◽  
Vol 63 (3) ◽  
pp. 258-265 ◽  
Author(s):  
C.D. Platsoucas ◽  
S.G. Robbins ◽  
N. Catsimpoolas
Keyword(s):  
Density Gradient ◽  
Mouse Spleen ◽  
Gradient Electrophoresis ◽  
Spleen Lymphocytes

scholarly journals Purification and characterization of prolixin S (nitrophorin 2), the salivary anticoagulant of the blood-sucking bug Rhodnius prolixus

1995 ◽  
Vol 308 (1) ◽  
pp. 243-249 ◽  
Author(s):  
J M C Ribeiro ◽  
M Schneider ◽  
J A Guimarães
Keyword(s):  
Cation Exchange ◽  
Salivary Glands ◽  
Coagulation Factor ◽  
Rhodnius Prolixus ◽  
Exchange Chromatography ◽  
Factor X ◽  
Blood Sucking ◽  
Weak Cation Exchange ◽  
Haem Protein ◽  
Strong Cation Exchange Chromatography

The salivary anticoagulant of the blood-sucking bug Rhodnius prolixus was purified to homogeneity using a protocol consisting of weak cation-exchange, DEAE, hydrophobic-interaction and octadecyl reverse-phase chromatography, yielding a protein with the same N-terminal sequence as nitrophorin 2, one of the four NO haem protein carriers present in the salivary glands of Rhodnius with a molecular mass of 19689 Da [D. Champagne, R.H. Nussenzveig and J.M.C. Ribeiro, (1995) J. Biol. Chem. 270, in the press]. To exclude the possibility of the nitrophorin being a contaminant, another chromatographic protocol was performed, consisting of chromatofocusing followed by strong-cation-exchange chromatography. Again the anticoagulant was eluted with nitrophorin 2. Nitrophorin 2 inhibits coagulation Factor VIII-mediated activation of Factor X and accounts for all the anti-clotting activity observed in Rhodnius salivary glands.

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