Determination of the Primary Structure of a Mouse IgG2a Immunoglobulin: Amino‐Acid Sequence of the Fc Fragment

Bourgois, Alain; Fougereau, Michel; Rocca-Serra, José

doi:10.1111/j.1432-1033.1974.tb03428.x

Cited by 26 publications

(10 citation statements)

References 34 publications

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“…These patterned polymer surfaces were used to bind specifically the Fc (to protein A) and Fab (to fluorescein hapten) domains of the known, well-published anti-fluorescein 4-4-20 antibody and its fragments33, 41, 49. This strategy is intended to produce antibody surface pattern formation in adjacent surface lithographed regions, containing both antibody “heads-up” and “tails-up” patterns on protein A- and fluorescein- immobilized regions, respectively.…”

Section: Resultsmentioning

confidence: 99%

Immobilized Antibody Orientation Analysis Using Secondary Ion Mass Spectrometry and Fluorescence Imaging of Affinity-Generated Patterns

et al. 2010

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This study assesses the capability of high-resolution surface analytical tools to distinguish immobilized antibody orientations on patterned surfaces designed for antibody affinity capture. High-fidelity, side-by-side co-patterning of protein A (antibody Fc domain affinity reagent) and fluorescein (antibody Fab domain hapten) was achieved photo-lithographically on commercial amine-reactive hydrogel polymer surfaces. This was verified from fluorescence imaging using fluorescently labeled protein A and intrinsic fluorescence from fluorescein. Subsequently, dyelabeled murine anti-fluorescein antibody (4-4-20), and antibody Fab and Fc fragments were immobilized from solution onto respective protein A-and fluorescein-co-patterned or control surfaces using antibody-ligand affinity interactions. Fluorescence assays support specific immobilization to fluorescein hapten-and protein A-patterned regions through antigen-antibody recognition and natural protein A-Fc domain interactions, respectively. Affinity-based antibody immobilization on the two different co-patterned surfaces generated side-by-side full antibody "heads-up" and "tails-up" oriented surface patterns. Time-of-flight secondary ion mass spectrometry (ToF-SIMS) analysis, sensitive to chemical information from the top 2-3 nm of the surface, provided ion-specific images of these antibody patterned regions, imaging and distinguishing characteristic ions from amino acids enriched in Fab domains for antibodies oriented in "heads-up" regions, and ions from amino acids enriched in Fc domains for antibodies oriented in "tails-up" regions. Principal component analysis (PCA) improved the distinct ToF-SIMS amino acid compositional and ion- NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript specific surface mapping sensitivity for each "heads-up" versus "tails-up" patterned region. Characteristic Fab and Fc fragment immobilized patterns served as controls. This provides first demonstration of pattern-specific, antibody orientation-dependent surface maps based on antibody domain-and structure-specific compositional differences by ToF-SIMS analysis. Since antibody immobilization and orientation are critical to many technologies, orientation characterization using ToF-SIMS could be very useful and convenient for immobilization quality control and understanding methods for improving the performance of antibody-based surface capture assays.

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Section: Resultsmentioning

confidence: 99%

Immobilized Antibody Orientation Analysis Using Secondary Ion Mass Spectrometry and Fluorescence Imaging of Affinity-Generated Patterns

et al. 2010

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“…Other peptides were separated using a combination of high-voltage electrophoresis on paper at pH 6.5 or 3.5 [9,10] and ion-exchange chromatography on Dowex 50x2 (Biorad) or resin P (Technicon), as previously described [4]. Net charge of peptides was determined according to Offord [ll].…”

Section: Methodsmentioning

confidence: 99%

“…From peak B, after decitraconylation, a soluble fraction, separated by centrifugation (4000 rev./min for 10 min) from a pellet, allowed isolation of peptide T l a after ion-exchange chromatography on a column of Dowex 50x2. The pellet was solubilized in the minimal volume of concentrated formic acid, and diluted to 1 ml with the first buffer used for initiating the chromatography on a column of Dowex 50x2 [4], which allowed separation of five fractions (Fig. 2).…”

Section: Tryptic Peptides Of the H4 Fragmentmentioning

confidence: 99%

“…In one occasion gel filtration on Sephadex G-50 (fine) in 5.0 M guanidine, pH 8.2, was used according to Jaton [8] and followed, after extensive dialysis of high-molecularweight fractions, by ion-exchange chromatography on a column containing 5 ml of DEAE-Sephadex A-25 onto which a gradient of molarity 0.05 M to 1.0 M in ammonium bicarbonate (pH 8.2), was applied. A last step of purification involved a gel filtration on Sephadex G-100 (superfine) in 0.05 M ammonium bicarbonate, pH 8.2.Other peptides were separated using a combination of high-voltage electrophoresis on paper at pH 6.5 or 3.5 [9,10] and ion-exchange chromatography on Dowex 50x2 (Biorad) or resin P (Technicon), as previously described [4]. Net charge of peptides was determined according to Offord [ll].…”

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Determination of the Primary Structure of a Mouse IgG2a Immunoglobulin

Rocca-Serra

Milili

Fougereau

1975

European Journal of Biochemistry

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The complete amino acid sequence of CNBr fragment H 4 of the murine immunoglobulin MOPC 173 (IgG2ap) has been determined, thus completing the sequence determination of the entire heavy chain.The H 4 fragment contains 150 residues, and extends from residue 105 to residue 254 of the heavy chain, which appears thus to be composed of 447 amino acids residues. This fragment contains the end of the V region, the switch peptide, the CHI domain, the hinge region and the beginning of cH2. Sequence comparisons suggest that the C,1 domain is highly conserved in evolution, and allows the definition of two additional isotypic-specific regions.This paper is the last of a series [l-41 which is devoted to the determination of the complete amino acid sequence of the heavy chain of the murine plasmacytoma protein MOPC 173, an IgG2a monoclonal immunoglobulin molecule [ 5 ] . Cleavage of the heavy chain withCNBr leads to the isolation of ten fragments, designated H1 to H10 [I]. We report in this publication the amino acid sequence determination of fragment H4, which covers the end of the V region, the entire C,1 domain, the hinge region and the very beginning of the Fc fragment. The accompanying paper reports the sequence of the MOPC 173 x chain [6]. Co), pepsin (NB Co), carboxypeptidases A and B (Worthington) were performed as previously indicated [3], except that hydrolyses of citraconylated fragment were made in 0.05 M N-ethylmorpholine, pH 8.2, and using various incubation times that will be given in Results. MATERIALS AND METHODS PreparationCitraconylation and decitrdconylation were done according to Gibbons and Perham [7]. Citraconylated peptides were separated by gel filtration on Sephadex G-75 (superfine) or G-25 (superfine) equilibrated in 0.05 M N-ethylmorpholine, pH 8.2. In one occasion gel filtration on Sephadex G-50 (fine) in 5.0 M guanidine, pH 8.2, was used according to Jaton [8] and followed, after extensive dialysis of high-molecularweight fractions, by ion-exchange chromatography on a column containing 5 ml of DEAE-Sephadex A-25 onto which a gradient of molarity 0.05 M to 1.0 M in ammonium bicarbonate (pH 8.2), was applied. A last step of purification involved a gel filtration on Sephadex G-100 (superfine) in 0.05 M ammonium bicarbonate, pH 8.2.Other peptides were separated using a combination of high-voltage electrophoresis on paper at pH 6.5 or 3.5 [9,10] and ion-exchange chromatography on Dowex 50x2 (Biorad) or resin P (Technicon), as previously described [4]. Net charge of peptides was determined according to Offord [ll]. Mobility at pH 3.5 was defined as suggested by Milstein [lo].Labelling of cysteinyl residues with i~do['~C]acetic acid [12], conditions for autoradiography, amino acid analysis [13], tryptophan determination [14],

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“…Samples loaded on the column never exceeded 1/5 of the overall bed volume. Ion-exchange chromatography was performed on Dowex 50x2 (Biorad) and resin P (Technicon), as previously described [6].…”

Section: Gel Filtration and Ion-exchange Chromatographymentioning

confidence: 99%

Determination of the Primary Structure of a Mouse IgG2a Immunoglobulin

Schiff¹,

Fougereau²

1975

European Journal of Biochemistry

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The amino acid sequence of the light chain of the mouse monoclonal MOPC 173 immunoglobulin molecule (IgG2a,x) is presented. This kappa chain contains 214 residues. Comparisons of this sequence with murine kappa chains already published by other workers bring a confirmation of the large size of the murine Vx chain pool. A complete identity was found with the constant region of the light chain of MOPC 21 from residue 97 to residue 214.We have undertaken the determination of the complete amino acid sequence of the murine MOPC 173 immunoglobulin molecule, which is a monoclonal protein of the IgG2a (x) class [I]. A topological model of the molecule has been proposed, on the grounds of results derived from the isolation and characterization of the CNBr fragments of the entire molecule [2], and from the identification of the interchain and intrachain disulfide bridges [3,4]. The sequence of the CNBr fragments H1, H2, H3 (covering most of the V, region), and that of fragments H5 to H10, accounting for the hinge region and the Fc fragment have already been published [5 -71.We report in the present paper the amino acid sequence of the light chain of this molecule. MATERIALS AND METHODS Isolation of the MOPC 173 ProteinPlasmacytomas which originated from Dr Potter's laboratory, were serially transplanted in Balb/c mice, in solid form, by subcutaneous injection of small tumor fragments. On the average, mice were bled three weeks after the transplantation. The immunoglobulin fraction of pooled sera was precipitated with ammonium sulfate (33 % saturation, final concentration), and was further purified by chromatography on DEAE cellulose (DE 52, Whatman) equilibrated with 0.017 M phosphate buffer (pH 7.3). Purity was checked by immunoelectrophoresis [8], using a rabbit antiserum against whole Balb/c mouse serum. Isolation of the Light ChainsThe MOPC 173 immunoglobulin molecule (500mg) was mildly reduced in 0.1 M Tris buffer, 0.15 M NaC1, 0.002 M EDTA, pH 8.2, with dithiothreitol (Calbiochem), according to Frangione et al. [9] (except that the molarity of the reagent was 8 mM) and alkylated with a 2.5 molar excess of iodoacetamide. Chains were then separated by gel-filtration on a Sephadex G-100 superfine column ( 5 x 100 cm) equilibrated in 1.0 M propionic acid. Purity of the light chains was tested by polyacrylamide (7.5 %) gel electrophoresis according to Shapiro and Maize1 [lo].Complete reduction and alkylation of the isolated light chains was made according to Waxdal et al.[ 1 1 1.except that 8 M urea was used instead of guanidine. Whenever labelled peptides were required, alkylation was performed, according to Frangione et al. [9], by making the reduced solution 0.02 M with iodo['"C]acetic acid (Amersham), using a 0.1 M stock solution (specific activity 83 pCi/ml). Radioactivity was counted in a Packard spectrometer 3380. Isolation of the CNBr FragmentsCNBr fragments were prepared by direct attack of the whole MOPC 173 molecule. Fragments L1 and L3 were isolated by successive steps of gel filtration, as previously descri...

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Determination of the Primary Structure of a Mouse IgG2a Immunoglobulin: Amino‐Acid Sequence of the Fc Fragment

Cited by 26 publications

References 34 publications

Immobilized Antibody Orientation Analysis Using Secondary Ion Mass Spectrometry and Fluorescence Imaging of Affinity-Generated Patterns

Immobilized Antibody Orientation Analysis Using Secondary Ion Mass Spectrometry and Fluorescence Imaging of Affinity-Generated Patterns

Determination of the Primary Structure of a Mouse IgG2a Immunoglobulin

Determination of the Primary Structure of a Mouse IgG2a Immunoglobulin

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