scholarly journals Conjugation of horseradish peroxidase to staphylococcal protein A with benzoquinone, glutaraldehyde, or periodate as cross-linking reagents.

1981 ◽  
Vol 29 (2) ◽  
pp. 266-270 ◽  
Author(s):  
H Nygren ◽  
H A Hansson
Keyword(s):  
Horseradish Peroxidase ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Protein A ◽  
Molecular Size ◽  
High Yield ◽  
Second Step ◽  
Cross Linking ◽  
Staphylococcal Protein A ◽  
Staphylococcal Protein

Horseradish peroxidase was conjugated to Staphylococcal protein A by three different two-step procedures using an increasing excess of peroxidase in the second step reaction. The yield of conjugated protein A was analyzed by SDS-polyacrylamide gel electrophoresis. Conjugation of peroxidase to protein A with benzoquinone or glutaraldehyde as cross-linking reagents at a 3- to 4-fold molar excess of peroxidase resulted in a high yield of coupled protein A with conjugates of low molecular size. Conjugation of peroxidase to protein A by the periodate method resulted in a high yield of coupled protein A with polymeric conjugates of large molecular size. Based on these results, conjugates produced with glutaraldehyde as cross-linking reagents were further analyzed. The capacity of the conjugates to precipitate human immunoglobulin evaluated by radial immunodiffusion was found to be reduced to about 50% of that of native protein A. Conjugates produced with glutaraldehyde as cross-linking reagent retained 70% of the enzyme activity of native peroxidase.

scholarly journals The molecular size of N-methylglutamate dehydrogenase of Pseudomonas aminovorans

1977 ◽  
Vol 167 (2) ◽  
pp. 509-512 ◽  
Author(s):  
C W Bamforth ◽  
P J Large
Keyword(s):  
Molecular Weight ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Protein A ◽  
Sodium Dodecyl ◽  
Molecular Size ◽  
Partial Specific Volume ◽  
Triton X

N-Methylglutamate dehydrogenase, purified to a specific activity of 0.29 unit/mg of protein, gave one band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, corresponding to a molecular weight of 130 000. Enzyme-Triton complexes were found to have a partial specific volume of 0.73 cm3/g, suggesting that the protein binds less than 0.1 g of Triton/g of protein. A molecular weight for the intact enzyme in the presence of 1% (w/v) Triton X-100 of 550 000 suggested that the enzyme may be a tetramer.

scholarly journals Comparative study of conjugation between horseradish peroxidase and D-cytochrome b5. Incorporation into subcellular membranes.

1985 ◽  
Vol 33 (6) ◽  
pp. 523-530
Author(s):  
M Collot ◽  
M Kalff ◽  
E Delaive ◽  
J Remacle
Keyword(s):  
Horseradish Peroxidase ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Cytochrome B5 ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Molecular Size ◽  
Molar Ratios ◽  
Microsomal Membranes

Horseradish peroxidase was conjugated to D-cytochrome b5 by three different two-step methods. The yield of conjugates based on the peroxidase enzymatic activity recovered after gel filtration was very low in the glutaraldehyde method, but higher in the N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) and periodate methods. The molecular size of the conjugates was analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Monomeric conjugates were mostly formed via the glutaraldehyde and SPDP methods in the presence of appropriate molar ratios of proteins. Most of the conjugates formed via the periodate method were polymers. The conjugate preparations of the three methods could be incorporated into microsomal membranes. Conjugate polymers, however, appeared less able to be incorporated then monomers. There was a nonpreferential incorporation of free or conjugated D-cytochrome b5 contained in the conjugate preparation of the glutaraldehyde method. In conclusion, this study gives preference to the glutaraldehyde method for the preparation of conjugates that will subsequently be used as an in vivo marker of the D-cytochrome b5 incorporation into membranes.

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.

scholarly journals Quantification of platelet-bound IgG by 125I-Staphylococcal protein A in immune thrombocytopenic purpura and other thrombocytopenic disorders

Blood ◽  
1984 ◽  
Vol 63 (1) ◽  
pp. 154-161 ◽  
Author(s):  
GM Shaw ◽  
J Axelson ◽  
JG Maglott ◽  
AF LoBuglio
Keyword(s):  
Immune Thrombocytopenic Purpura ◽  
Protein A ◽  
Normal Subjects ◽  
Unknown Etiology ◽  
Staphylococcal Protein A ◽  
Thrombocytopenic Purpura ◽  
Clinical Criteria ◽  
Staphylococcal Protein ◽  
Wide Range ◽  
Immune Hemolytic Anemia

Abstract In this report we describe the use of an 125I-Staphylococcal protein A (SPA) assay to measure platelet-bound IgG in the evaluation of 62 thrombocytopenic patients. Platelets from 150 normal subjects were found to bind 146 +/- 112 molecules of SPA per platelet (mean +/- 2 SD). Nineteen of 20 patients with untreated immune thrombocytopenia had platelet IgG values above this range, with 15 of 20 having values above 1,000 molecules of SPA per platelet. Patients with immune thrombocytopenic purpura by clinical criteria, but who had failed conventional therapy (corticosteroids or splenectomy), had a wide range of platelet IgG levels: 4 of 20 had normal values, 6 of 20 had minimally elevated levels in the range seen with nonimmune thrombocytopenia, and 10 of 20 had much higher values. Fifteen patients with thrombocytopenia of apparent nonimmune origin and 7 others with chronic stable thrombocytopenia of unknown etiology were found to have platelet IgG levels within or only slightly above the normal range. Because of its simplicity, accuracy, and clinical correlation, the 125I- SPA assay provides an important new approach for studying platelet IgG in thrombocytopenic states. The data obtained with this technique are similar to those found in immune hemolytic anemia and suggest that the platelet-bound IgG so measured has pathophysiologic relevance in immune thrombocytopenic purpura.

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