Purification of cystic fluid antigen of Taenia solium metacestodes by affinity chromatography using monoclonal antibody and its antigenic characterization

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.

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Biochemical characterization including amino acid and carbohydrate compositions of canine and human brain Thy-1 antigen

Biochemical Journal ◽

10.1042/bj1970629 ◽

1981 ◽

Vol 197 (3) ◽

pp. 629-636 ◽

Cited By ~ 19

Author(s):

J L McKenzie ◽

A K Allen ◽

J W Fabre

Keyword(s):

Monoclonal Antibody ◽

Amino Acid ◽

Human Brain ◽

Affinity Chromatography ◽

Gel Electrophoresis ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Gel Filtration ◽

Antibody Affinity ◽

Lentil Lectin

Human and canine brain Thy-1 antigens were solubilized in deoxycholate and antigen activity was followed both by conventional absorbed anti-brain xenosera of proven specificity and by mouse monoclonal antibodies to canine and human Thy-1. It is shown that greater than 80% of Thy-1 activity in the dog and man binds to lentil lectin, that the mobility on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of canine and human Thy-1 is identical with that of rat Thy-1 and that the Stokes radius in deoxycholate of canine and human brain Thy-1 is 3.0 nm and 3.25 nm respectively. Both lentil lectin affinity chromatography followed by gel-filtration chromatography on the one hand and monoclonal antibody affinity chromatography on the other gave high degrees of purification of the brain Thy-1 molecule in the dog and man, resulting in single bands staining for both protein and carbohydrate on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis (except for a slight contaminant of higher molecular weight staining for protein but not carbohydrate with human Thy-1 purified by lentil lectin and gel-filtration chromatography). Analysis of canine and human brain Thy-1 purified by monoclonal antibody affinity chromatography with additional gel filtration through Sephadex G-200 showed that these molecules had respectively 38% and 36% carbohydrate. The amino acid and carbohydrate compositions were similar to those previously reported for Thy-1 of the rat and mouse, the main point of interest being the presence in canine and human brain Thy-1 of N-acetylgalactosamine, which has been reported in rat and mouse brain Thy-1 but not in Thy-1 from other tissues.

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Protein A affinity chromatography of Chinese hamster ovary (CHO) cell culture broths containing biopharmaceutical monoclonal antibody (mAb): Experiments and mechanistic transport, binding and equilibrium modeling

Journal of Chromatography B ◽

10.1016/j.jchromb.2018.02.032 ◽

2018 ◽

Vol 1083 ◽

pp. 44-56 ◽

Cited By ~ 9

Author(s):

Matic Grom ◽

Mirijam Kozorog ◽

Simon Caserman ◽

Andrej Pohar ◽

Blaž Likozar

Keyword(s):

Cell Culture ◽

Monoclonal Antibody ◽

Affinity Chromatography ◽

Chinese Hamster Ovary ◽

Protein A ◽

Chinese Hamster ◽

Cho Cell ◽

Equilibrium Modeling ◽

Cho Cell Culture

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Evaluation of a monoclonal antibody affinity purified antigen for zymodeme specific serological diagnosis of Trypanosoma cruzi infection

Memórias do Instituto Oswaldo Cruz ◽

10.1590/s0074-02761987000100014 ◽

1987 ◽

Vol 82 (1) ◽

pp. 81-85 ◽

Cited By ~ 1

Author(s):

Mauro Schechter

Keyword(s):

Monoclonal Antibody ◽

Trypanosoma Cruzi ◽

Affinity Chromatography ◽

Immunosorbent Assay ◽

Enzyme Linked Immunosorbent Assay ◽

Specific Monoclonal Antibody ◽

Antibody Affinity ◽

Specific Diagnosis ◽

Purified Antigen ◽

Cruzi Infection

Theoretically, serological assays with affinity purified marker antigens can allow strain-specific diagnosis even when parasites cannot be retrieved from and infected host. A Trypanosoma cruzi antigen was purified by affinity chromatography using a zymodeme (Z) 2 specific monoclonal antibody (2E2C11). An indirect enzyme-linked immunosorbent assay (ELISA) based on the purified antigen could discriminate between sera from rabbits immunized with T. cruzi zymodeme clones but could not discriminate between sera from mice infected with different zymodemes.

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Purification of Porcine Circovirus Type 2 Using an Affinity Chromatography Based on a Neutralizing Monoclonal Antibody against Viral Capsid Protein

Pathogens ◽

10.3390/pathogens10121564 ◽

2021 ◽

Vol 10 (12) ◽

pp. 1564

Author(s):

Haiqiao Bian ◽

Chong Yu ◽

Yanwu Wei ◽

Li Feng ◽

Changming Liu ◽

...

Keyword(s):

Electron Microscopy ◽

Monoclonal Antibody ◽

Affinity Chromatography ◽

Porcine Circovirus Type ◽

Porcine Circovirus Type 2 ◽

Porcine Circovirus ◽

Enzyme Linked Immunosorbent Assay ◽

Chromatography Method ◽

Viral Adsorption

Porcine circovirus type 2 (PCV2) is a DNA virus without an envelope. The viral particle is icosahedral and has a diameter of approximately 17 nm. In order to obtain the purified virus, a broad-spectrum monoclonal antibody 3A5 against PCV2 was coupled to CNBr-activated SepharoseTM 4B, and an affinity chromatography was established for PCV2 purification. A total of 6.5 mg of purified PCV2a/LG with 97% purity was obtained from 120 mL of the viral culture medium, and only PCV2 was detected by electron microscopy. No significant changes in the antigenic characteristics of the purified virus were detected by a capture enzyme-linked immunosorbent assay (ELISA). Furthermore, the titer of the purified PCV2 was 100 times higher than that of the unpurified virus. This affinity chromatography method was also used to purify PCV2b/LN590516 and PCV2d/SD446F16, and the purified viruses were detected by electron microscopy, capture ELISA, and virus titration, respectively. The results showed that these two strains can be successfully purified, but the yield is lower than that of the PCV2a strain. In addition, the purified virus could be used to study the viral adsorption and invasion of PK15 cells using indirect immunofluorescence assays. A large number of PCV2 signals were detected to transfer from the cellular surface to the periphery of the nucleus of the PK15 cells after 30 min of adsorption of the PCV2 to the PK15 cells. The affinity chromatography is a simple and convenient tool to obtain PCV2 with high purity. It could be applied for virus structure analysis, antibody preparation, and viral adsorption and invasion research.

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