Improved procedure for preparation of F(ab′)2 fragments of mouse IgGs by papain digestion

Boguslawski, Sophie; Ledden, David J.; Fredrickson, R. A.

doi:10.1016/0022-1759(89)90288-3

Cited by 14 publications

(7 citation statements)

References 10 publications

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“…Purity of IgG was confirmed on a 4-15% gradient SDS-PAGE gel (Bio-Rad) quantitated by nano drop (Thermo fisher) before using it for IgG Fab preparation. IgG Fab fragments were prepared as published earlier with slight modification [21]. Papain (2mg/ml, Sigma) was pre-activated in Fab Digestion Buffer (Pierce) + 20mM L-cysteine, for 10 minutes at 37°C.…”

Section: Methodsmentioning

confidence: 99%

PcpA of Streptococcus pneumoniae mediates adherence to nasopharyngeal and lung epithelial cells and elicits functional antibodies in humans

Khan

Sharma

Filkins

et al. 2012

Microbes and Infection

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Section: Methodsmentioning

confidence: 99%

PcpA of Streptococcus pneumoniae mediates adherence to nasopharyngeal and lung epithelial cells and elicits functional antibodies in humans

Khan

Sharma

Filkins

et al. 2012

Microbes and Infection

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“…On the other hand, digestion with proteolytic enzymes is not applicable to all mAbs. If active fragments can be produced, this technique can be limited by problems such as low yield, contamination with enzyme, and others (14,15). Therefore, our aim was to generate a mutant antibody with characteristics of a F(ab')2 fragment that is useful for tumor imaging.…”

mentioning

confidence: 99%

Serum half-life and tumor localization of a chimeric antibody deleted of the CH2 domain and directed against the disialoganglioside GD2.

Mueller

Reisfeld

Gillies

1990

Proc. Natl. Acad. Sci. U.S.A.

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Recombinant techniques allow one to engineer an antibody molecule and, in this way, manipulate its properties and functions. We engineered a chimeric human/ mouse antibody to the tumor-associated antigen ganglioside GD2, with the aim ofdecreasing its serum half-life, maintaining its full antigen-binding capacity, and deleting its effector functions, thus making it a potentially useful reagent for the radioimaging of tumors. To this end, the constant region of the human yl chain was mutated by deleting the second domain (CH2). Here we show that the Cn2-deleted antibody (ch14.18-ACH2) was cleared from the blood of athymic (nu/nu) mice bearing human melanoma tumors with the same kinetics as human IgG F(ab')2. At a 3 t1/, of 12 hr, 0.9% of the inected dose of 'MI-labeled chl4.18-ACH2 was found per milliliter of blood 24 hr after i.v. injection. In biodistribution experiments, '25I-labeled chl4.18-ACH2 targeted specifically to melanoma xenografts, achieving optimal tumor-to-tissue ratios 12-16 hr after i.v. injection. chl4.18-ACH2 was localized to the melanoma tumors more rapidly and with better localization ratios than the intact chimeric antibody chl4.18. Sixteen hours after i.v. injection, the tumor-to-blood and tumor-to-liver ratios of chl4.18-ACH2 were 5 and 12, respectively, while optimal localization ratios obtained for chl4.18 were 1 and 5, respectively, but 96 hr after injection. A reagent such as ch14.18-ACH2 should be useful for radioimmunodetection of human tumors because of reduced immunogenicity, increased targeting specificity, and rapid clearance from circulation.Radiolabeled monoclonal antibodies (mAbs) against tumorassociated antigens have been shown to localize specifically to melanoma and other solid tumors and can be used in radioimmunoimaging for the identification of occult lesions (1-3). For tumor imaging, it is advantageous to use antibody fragments instead of intact antibodies, because fragments clear faster from circulation and have a better tumor accessibility due to their smaller size. This has been found in experimental animals (4-6) as well as in cancer patients (7). Recombinant DNA techniques make it possible to engineer the antibody molecule and to obtain antibodies with novel properties and functions (8,9 (17)], we were able to express an antibody that was deleted of CH2. The mutant antibody deleted of CH2 (chl4.18-ACH2) was characterized and compared with the intact chimeric antibody. By gel electrophoresis on SDS gels and by high-pressure exclusion chromatography under nondenaturing conditions, chl4.18-ACH2 appeared to have a molecular mass of 120 kDa. chl4.18-ACH2 bound to purified ganglioside GD2; however, unusual binding characteristics of chl4.18-ACH2 were observed in that it competes much more efficiently with chl4.18 for antigen than does chl4.18 with itself, especially at short incubation times. This is thought to reflect some conformational changes in the overall structure of the molecule rather than in binding affinity (17). In contrast to chl4.18, chl4.18-ACH2 did...

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“…Papain and ficin are used for the preparation of Fab fragments from IgG to be employed in assay systems where the presence of the Fc region may cause problems (Mariani et al 1991;Boguslawski et al 1989). Papain is also used to digest IgM (Newkirk et al 1987) as well as in red cell serology to modify the red cell surface to enhance or destroy the reactivity of many red cell antigens as an adjunct to grouping, antibody screening or antibody identification procedures.…”

Section: Use Of Cysteine Proteases In Pharmacy and Medicinementioning

confidence: 99%

Native and Biotechnologically Engineered Plant Proteases with Industrial Applications

Feijoo-Siota

Villa

2010

Food Bioprocess Technol

130

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Proteases occupy the most relevant position among industrial enzymes. Plant proteases have been used in medicine, detergent manufacturing, and food science for many years, but their production is diminishing in favor of those of microbial origin because lower production costs. Papain, bromelain, and ficin are the most frequently employed plant proteases, although new proteases with new and more appealing physicochemical properties for industry are still emerging. DNA technology and genetic engineering shall play, without a doubt, an important role for the production of these proteases at the industrial level. The present review focuses on the applications of traditional plant proteases as well as new proteases discovered during the last 20 years, some of which have already been genetically engineered either to increase production or to strengthen some of their physicochemical properties. The review also refers to the protease classification, action pattern, and main characteristics.

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Improved procedure for preparation of F(ab′)2 fragments of mouse IgGs by papain digestion

Cited by 14 publications

References 10 publications

PcpA of Streptococcus pneumoniae mediates adherence to nasopharyngeal and lung epithelial cells and elicits functional antibodies in humans

PcpA of Streptococcus pneumoniae mediates adherence to nasopharyngeal and lung epithelial cells and elicits functional antibodies in humans

Serum half-life and tumor localization of a chimeric antibody deleted of the CH2 domain and directed against the disialoganglioside GD2.

Native and Biotechnologically Engineered Plant Proteases with Industrial Applications

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