Purification of labeled antibody by minicolumn gel centrifugation

Parkinson, Alan J.; Scott, E. N.; Muchmore, H. G.

doi:10.1016/0003-2697(81)90602-3

Cited by 10 publications

(6 citation statements)

References 8 publications

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“…The pepsin was freshly dissolved at 2 mg/mL in 0.1 M sodium acetate buffer (pH 3.9) and added to the mAbs to give a final concentration of 60 μg/mL. The mixture was incubated for 16 h at 37° C. The F(ab') 2 fragments were purified from the mixture by centrifugal gel filtration (CGF) (Parkinson et al, 1981; Brennan et al, 1985).…”

Section: Methodsmentioning

confidence: 99%

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Preparation and Characterization of Bifunctional Biopolymers for Receptor‐Based Liposomal Immunosensing

Yu¹,

Wie²,

Koch³

et al. 1998

Biotechnology Progress

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In this study, we prepared bifunctional biopolymers for development of a novel liposomal immunosensing element. These biopolymers were produced such that a rat monoclonal antibody fragment Fab' was linked to a cardiac protein Troponin I (TnI) peptide by a cross-linking reagent, o-phenylenedimaleimide (o-PDM) or N-sucinimidyl 3-(2-pyridyldithio)propionate (SPDP). The biopolymer formation yields were approximately 10% for Fab-TnIMal and 30% for Fab-TnISPDP. Molar ratios of Fab' to SPDP or o-PDM and conjugated Fab' to TnI peptide and conjugation pH have considerable effects on the biopolymer yield. Purification of these biopolymers was achieved by employing size-exclusion HPLC. These biopolymers can bind to receptor channels on one end, while the peptide end can be recognized by an anti-TnI antibody serving as a protein linker to block the channels in the immunosensing element. Then reactions may be used where free analyte competes for cross-linker binding sites whereby channels are rendered active. Characterization of purified biopolymers was performed using gel electrophoresis, ELISAs, and a BIAcore instrument. Furthermore, results of real-time biospecific interaction experiments with use of the BIAcore show that competition binding reactions of free TnI peptide occurred in this new immunosensing design. The binding activities of these two biopolymers are slightly different.

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Section: Methodsmentioning

confidence: 99%

“…The mixture was incubated for 16 h at 37°C. The F(ab′) 2 fragments were purified from the mixture by centrifugal gel filtration (CGF) (Parkinson et al, 1981;Brennan et al, 1985).…”

Section: Introductionmentioning

confidence: 99%

Preparation and Characterization of Bifunctional Biopolymers for Receptor‐Based Liposomal Immunosensing

Yu¹,

Wie²,

Koch³

et al. 1998

Biotechnology Progress

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“…ras p21 immunoassay Affinity-purified pan-reactive rat monoclonal antibody Y13-259 (Furth et al, 1982) was radiolabeled by using chloramine-T and [ 1251]-sodium iodide. Free iodide was separated from IgG by microcolumn gel centrifugation over Sephadex G-25 (fine) (Parkinson et al, 1981). Specific activity of the mAb was 3,000-8,000 cpmtng.…”

Section: Inositol Phosphate (Ip) Productionmentioning

confidence: 99%

Regulation of hepatocyte growth: Alpha‐1 adrenergic receptor and ras p21 changes in liver regeneration

Cruise

Muga

Lee

et al. 1989

Journal Cellular Physiology

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Catecholamines, acting via the alpha-1 adrenergic receptor, have been demonstrated to influence adult rat hepatocyte DNA synthesis in primary culture and in vivo during liver regeneration following partial hepatectomy (PHX). Earlier investigations have suggested that the alpha-1 effect on DNA synthesis is significant only during the first day following PHX. We examined receptor binding at several early and late time points after surgery, and we observed a significant loss of specific [3H]-prazosin binding to cells isolated from rat livers 48 and 72 hr after PHX. In contrast, the ability of norepinephrine to stimulate inositol phosphate production in isolated cells prelabeled with [3H]-myo-inositol was transiently reduced between 8 and 16 hr, when alpha-1 binding capacity was virtually unchanged. This uncoupling of phosphoinositide turnover from binding was preceded by a drop in hepatic membrane ras p21 content, as assayed by liquid competition radioimmunoassay. The loss of immunoreactive p21 from membranes was significant by 2 hr after PHX. These findings suggest a role for alpha-1 receptors and ras protein in the early events of liver regeneration.

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“…MAb,, (2.76 mg/ml) or non-immune IgG (2.55 mglml) was brought to PH 6 by centrifugal gel filtration (Parkinson et al, 1981) on Sephadex G25 in [50 mM phosphate (Na), PH 6/100 m~ NaCI]. A stock solution of 0.5 M NaIO, (Fluka, Buchs, Switzerland) in water was added to a final concentration of 22 m~ and the mixture was incubated in the dark, on ice, for 75 min (method adapted from Rodwell et al, 1986).…”

Section: Oxidationmentioning

confidence: 99%

A novel derivative of the chelon desferrioxamine for site‐specific conjugation to antibodies

Pochon¹,

Buchegger

Pèlegrin

et al. 1989

Intl Journal of Cancer

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We describe the preparation of the modified chelator aminooxyacetyl-ferrioxamine, and the replacement of its iron atom by 67Ga at high specific activity. The aminooxy function of this compound was allowed to react with the aldehyde groups generated by the periodate oxidation of the oligosaccharide of a mouse IgG1 monoclonal antibody (MAb) directed against carcino-embryonic antigen (CEA). The use of the aminooxy group allowed a stable bond to be formed between the chelon and the antibody with no need for reduction. Iron was removed from the ferrioxamine moiety and replaced by 67Ga either before or after conjugation of the chelon to the antibody. In either case the labelled antibody was injected into nude mice bearing a human colon carcinoma having the appropriate antigenicity. Unoxidized antibody, labelled with 125I by conventional methods, was co-injected as an internal control. Additional control experiments were carried out with a non-immune IgG using the same 67Ga-labelled modified chelon as above. The in vivo distribution of the modified antibodies was evaluated at various times between 24 and 96 hr after injection. The methods used were gamma-camera imaging and, more quantitatively, gamma-counting of the various organs after dissection. Interestingly, with the metal-chelon-labelled antibody, the intensity and specificity of tumor labelling was comparable and in some cases superior to the results obtained with radio-iodinated antibody. In particular, there was almost no increase in liver and spleen uptake of radioactive metal relative to radio-iodine, contrary to what has been observed with most antibodies labelled with 111In after conjugation with DTPA.

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Purification of labeled antibody by minicolumn gel centrifugation

Cited by 10 publications

References 8 publications

Preparation and Characterization of Bifunctional Biopolymers for Receptor‐Based Liposomal Immunosensing

Preparation and Characterization of Bifunctional Biopolymers for Receptor‐Based Liposomal Immunosensing

Regulation of hepatocyte growth: Alpha‐1 adrenergic receptor and ras p21 changes in liver regeneration

A novel derivative of the chelon desferrioxamine for site‐specific conjugation to antibodies

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