Properties of Whole Human IgG Molecules Produced by the Expression of Cloned Anti-DNA Antibody cDNA in Mammalian Cells

Rahman, Mohamad A.; Kettleborough, Catherine A.; Latchman, David S.; Isenberg, David

doi:10.1006/jaut.1998.0241

Cited by 17 publications

(23 citation statements)

References 34 publications

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“…The wild-type heavy and light chain constructs, the chimeric light chain constructs, and the point mutant B3(R27aS) V L were prepared as described in detail in previous reports (30,31). Plasmid neSLE122H45.4, which encodes a human V H 3-23, D6-25, J H 5 IgH chain was a kind gift from Dr. Hedda Wardemann (Rockefeller University, New York, NY).…”

Section: Methodsmentioning

confidence: 99%

“…The whole IgG molecules were expressed in COS-7 cells as described previously (30). Equal quantities (10 g) of recombinant heavy chain and recombinant light chain vector were transfected into 10 7 COS-7 cells in 700 l phosphate buffered saline, by electroporation (1.9 kV, 25 F).…”

Section: Methodsmentioning

confidence: 99%

“…Human IgG was purified from the supernatant using a protein A column, and the product was analyzed for purity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and quantified by spectrophotometry. Purified human IgG was sent back to our institution, where the amount of whole IgG antibody was quantified by ELISA as previously described (20,25,30).…”

Section: Lambrianides Et Almentioning

confidence: 99%

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Arginine mutation alters binding of a human monoclonal antibody to antigens linked to systemic lupus erythematosus and the antiphospholipid syndrome

Lambrianides¹,

Giles²,

Ioannou³

et al. 2007

Arthritis & Rheumatism

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Objective. Previous studies have shown the importance of somatic mutations and arginine residues in the complementarity-determining regions (CDRs) of pathogenic anti-double-stranded DNA (anti-dsDNA) antibodies in human and murine lupus, and in studies of murine antibodies, a role of mutations at position 53 in V H CDR2 has been demonstrated. We previously demonstrated in vitro expression and mutagenesis of the human IgG1 monoclonal antibody B3. The present study was undertaken to investigate, using this expression system, the importance of the arginine residue at position 53 (R53) in B3 V H .Methods. R53 was altered, by site-directed mutagenesis, to serine, asparagine, or lysine, to create 3 expressed variants of V H . In addition, the germline sequence of the V H 3-23 gene (from which B3 V H is derived) was expressed either with or without arginine at position 53. These 5 new heavy chains, as well as wild-type B3 V H , were expressed with 4 different light chains, and the resulting antibodies were assessed for their ability to bind to nucleosomes, ␣-actinin, cardiolipin, ovalbumin, ␤ 2 -glycoprotein I (␤ 2 GPI), and the N-terminal domain of ␤ 2 GPI (domain I), using direct binding assays.Results. The presence of R53 was essential but not sufficient for binding to dsDNA and nucleosomes. Conversely, the presence of R53 reduced binding to ␣-actinin, ovalbumin, ␤ 2 GPI, and domain I of ␤ 2 GPI. The combination B3 (R53S) V H /B3 V L bound human, but not bovine, ␤ 2 GPI.Conclusion. The fact that the R53S substitution significantly alters binding of B3 to different clinically relevant antigens, but that the alteration is in opposite directions depending on the antigen, implies that this arginine residue plays a critical role in the affinity maturation of antibody B3.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Lambrianides Et Almentioning

confidence: 99%

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Arginine mutation alters binding of a human monoclonal antibody to antigens linked to systemic lupus erythematosus and the antiphospholipid syndrome

Lambrianides¹,

Giles²,

Ioannou³

et al. 2007

Arthritis & Rheumatism

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show abstract

“…These L chains differ in nucleotide sequence (although only by Յ7%) as a result of their different patterns of somatic mutation (25). Statistical analysis has shown that the observed patterns of replacement mutations in the CDRs of these sequences are consistent with Ag-driven selection (33,37). There are five Arg residues in IS4VHCDR3.…”

Section: Sequences Of Expressed Ab Variable Regionsmentioning

confidence: 95%

“…The expression vectors containing both H and L chain cDNA were adapted from the single chain expression vectors previously used in our transient expression experiments (25,33). The original vectors (pLN10, pLN100, pG1D1, and pG1D210) were all gifts from Drs.…”

Section: Preparation Of Ab Expression Constructsmentioning

confidence: 99%

Arginine Residues Are Important in Determining the Binding of Human Monoclonal Antiphospholipid Antibodies to Clinically Relevant Antigens

Giles

Lambrianides

Pattni

et al. 2006

The Journal of Immunology

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In the antiphospholipid syndrome (APS), antiphospholipid Abs (aPL) bind to anionic phospholipids (PL) and various associated proteins, especially β2-glycoprotein I (β2GPI) and prothrombin. In the present study, we show that altering specific Arg residues in the H chain of a human pathogenic β2GPI-dependent aPL, IS4, has major effects on its ability to bind these clinically important Ags. We expressed whole human IgG in vitro by stable transfection of Chinese hamster ovary cells with expression plasmids containing different VH and VL sequences. VH sequences were derived from IS4 by altering the number of Arg residues in CDR3. VL sequences were those of IS4, B3 (anti-nucleosome Ab), and UK4 (β2GPI-independent aPL). Binding of the expressed H/L chain combinations to a range of anionic, neutral, and zwitterionic PL, as well as prothrombin, β2GPI, dsDNA, and chicken OVA, was determined by ELISA. Of four Arg residues in IS4VH CDR3 substituted to Ser, two at positions 100 and 100g, reduced binding to all Ags, while two at positions 96 and 97 reduced binding to β2GPI but increased or decreased binding to different PL. Eleven of 14 H/L chain combinations displayed weak binding to OVA with Arg to Ser replacements of all four Arg residues enhancing binding to this Ag. Only one H/L chain combination bound neutral PL and none bound dsDNA; hence, these effects are particularly relevant to Ags important in antiphospholipid syndrome. We hypothesize that these four Arg residues have developed as a result of somatic mutations driven by an Ag containing both PL and β2GPI.

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Preparation and characterization of organic–inorganic hybrid composites based on multiepoxy silsesquioxane and cyanate resin

Liang

Guo

2006

J of Applied Polymer Sci

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A multiepoxy cubic silsesquioxane was prepared by the hydrolysis and polycondensation of trifunctional monomer (␥-glycidoxypropyl)trimethoxysilane in a solvent mixture of methyl isobutyl ketone and anhydrous ethanol with a tetraethyl ammonium hydroxide aqueous solution acting as the catalyst, and it was successfully introduced into a cyanate resin and formed highly crosslinked organic-inorganic hybrid composites on a molecular level via a mutual cure reaction. The properties of the multiepoxy cubic silsesquioxane/bisphenol A dicyanate ester resin composites were investigated, and the results showed that introducing the cubic silsesquioxane unit into the cyanate resin successfully modified the local structure of the molecule, made the chain more rigid, restricted the chain mobility, and eventually improved the thermal stability and flame retardancy of the resin.

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Properties of Whole Human IgG Molecules Produced by the Expression of Cloned Anti-DNA Antibody cDNA in Mammalian Cells

Cited by 17 publications

References 34 publications

Arginine mutation alters binding of a human monoclonal antibody to antigens linked to systemic lupus erythematosus and the antiphospholipid syndrome

Arginine mutation alters binding of a human monoclonal antibody to antigens linked to systemic lupus erythematosus and the antiphospholipid syndrome

Arginine Residues Are Important in Determining the Binding of Human Monoclonal Antiphospholipid Antibodies to Clinically Relevant Antigens

Preparation and characterization of organic–inorganic hybrid composites based on multiepoxy silsesquioxane and cyanate resin

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