Affinity and temporal variation of isoelectric fractions of rabbit antilactose antibody

Ghose, A.M.; Karush, Fred

doi:10.1021/bi00737a011

Cited by 29 publications

(15 citation statements)

References 18 publications

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“…It is interesting that the IgM antibodies produced by BSVS are the highest in average avidity of the strains tested . Multiple hypotheses (2,14,38) have been advanced to account for the large amounts of restricted antipolysaccharide antibody, but none is totally satisfactory . In some antipolysaccharide responses, such as that of mice to the phosphorylcholine moiety of pneumococcal C carbohydrate, all mice within an inbred strain make antibodies with the same binding site specificity and the same idiotype (39,40) .…”

Section: Discussionmentioning

confidence: 99%

“…It has been suggested that affinity maturation may in fact be responsible for the restricted antibody response to polysaccharides (2,14,38) . By this mechanism, which has been shown to operate in the antibody response to proteins and hapten-protein conjugates (10)(11)(12)(13), the initial antibody response is heterogeneous and includes both low and high affinity antibodies .…”

Section: Discussionmentioning

confidence: 99%

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Clonal dominance. I. Restricted nature of the IgM antibody response to group A streptococcal carbohydrate in mice.

Briles¹,

Davie²

1975

The Journal of Experimental Medicine

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The IgM antibody response of mice to the streptococcal group A carbohydrate (GAC) was measured. With most strains tested, large amounts of IgM antibody were produced; in AKR mice, over 1% of the total nucleated spleen cells secreted IgM anti-GAC antibody after hyperimmunization. The relative avidity of the antibody was extimated by a modification of the Jerne plaque assay where spleen cells from individual mice were tested against erythrocytes with varying GAC epitope densitymthese studies showed that the earliest, as well as latest, IgM antibodies produced were highly restricted in avidity heterogeneity. No evidence of affinity maturation was seen upon hyperimmunization. These data favor the conclusion that the restricted IgG response seen in mice hyperimmunized to GAC is not the result of affinity driven competition for antigen among precursor cells.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Clonal dominance. I. Restricted nature of the IgM antibody response to group A streptococcal carbohydrate in mice.

Briles¹,

Davie²

1975

The Journal of Experimental Medicine

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“…Normal rabbit IgG (Pentex) was stored as a lyophilized powder at -20 °C. Rabbit anti-lactose IgG was obtained from New Zealand white rabbits injected with Streptococcus faecalis (strain N) as described previously (Ghose & Karush, 1973). Immune serum was chromatographed on a p-aminophenyl-/3-lactoside (PAPL) conjugated Sepharose 4B column (Lac-Sepharose).…”

Section: Methodsmentioning

confidence: 99%

Proximity relationships within the Fc segment of rabbit immunoglobulin G analyzed by resonance energy transfer

Luedtke¹,

Owen²,

Vanderkooi³

et al. 1981

Biochemistry

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Resonance energy transfer analysis has been carried out with a noncovalent rabbit hybrid of immunoglobulin G (IgG) composed of normal rabbit IgG and rabbit anti-lactose IgG. The hybrid IgG was prepared from proteins in which the single inter-heavy-chain disulfide linkage was specifically reduced and alkylated. Normal rabbit IgG was alkylated with the iodoacetyl derivative of N-(aminoethyl)-5-naphthylamine-1-sulfonic acid while the rabbit anti-lactose IgG was alkylated with either iodoacetamide or the iodoacetyl derivative of p-[[p-(dimethylamino)phenyl]azo]aniline. Fractionation with an anti-lactose-specific immunoadsorbent yielded a population in which each fluorescent donor [N-[(acetylamino)ethyl]-5-naphthylamine-1-sulfonic acid] was adjacent to a nonfluorescent acceptor [N-acetyl-p[[p-dimethylamino)phenyl]. Data on fluorescence quantum yield, excited-state lifetime, time-resolved emission anisotropy, and steady-state fluorescence polarization revealed a distribution of distances between the donor and acceptor. In the native molecule, the hinge regions are known to be covalently liked by a single disulfide bond. In the absence of this linkage, the hinge regions were separated such that for a majority of the molecules in solution (approximately 60%) the separation was 50-60 A. In the context of current knowledge of the IgG molecule, it is evident that the principle forces maintaining the integrity of the native, functional Fc segment are the strong noncovalent interactions of the CH3 domains and the single inter-heavy-chain disulfide bond.

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“…The absorption spectrum of the p-[p-(dimethylamino)benzeneazo]phenyl /3-lactoside (Lac-Dye) in PBS at pH 7.2, shown in Figure 1, is characterized by a maximum at 444 to 445 nm with a shoulder around 400 nm (Karush, 1957). Ghose & Karush (1974) compared the difference spectra of free Lac-Dye and bound Lac-Dye for several homogeneous rabbit antilactose IgG's purified by preparative isoelectric focusing of antibody from rabbits with restricted antilactose responses. They found hyperchromicity as a general feature although each antibody exhibited a distinctive difference spectrum.…”

Section: Resultsmentioning

confidence: 99%

“…Antilactose antibody was elicited in female white New Zealand rabbits immunized with both Streptococcus faecalis (strain N) vaccine (Ghose & Karush, 1973), which contains a cell wall diheteroglycan component possessing multiple lactosyl moieties (Pazur et al, 1973), and PAPL diazotized to bovine -globulin (BGG), as previously described (Utsumi & Karush, 1964). The antiserum used in these experiments was obtained after the third course, each course consisting of three intravenous injections per week for three consecutive weeks, at 0.5, 1.0, and 2.0 mg of dry weight per injection for each succeeding week.…”

Section: Methodsmentioning

confidence: 99%

Proximity of antibody binding sites studied by fluorescence energy transfer

Luedtke¹,

Owen²,

Karush³

1980

Biochemistry

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Fluorescence energy transfer experiments by steady-state and nanosecond monophoton techniques were carried out with a covalently linked hybrid rabbit IgG antibody containing one antilactose site and one anti-Dns [5-(dimethylamino)-1-naphthalenesulfonyl] site. The hybrid antibody was prepared from antilactose and anti-Dns antibody by mild reduction, dissociation into half-molecules in acid, and random reassociation with re-formation, to the extent of 80%, of the single disulfide bond between the heavy chains. Fractionation with an antilactose-specific immunoadsorbent yielded a population in which each IgG molecule contained no more than one anti-Dns site per antibody. The acceptor molecules used for intramolecular energy transfer were derivatives of p-aminophenyl beta-lactoside (PAPL): (dimethyl-amino)benzeneazo-PAPL and N-fluorescyl-PAPL. The fluorescence lifetime (24 ns) and quantum yield (0.57) of the bound Dns group were unaffected by the presence of the acceptor in the adjacent site. Three models were used to calculate the minimum distance between the adjacent sites of the IgG antibody based on the overlap in the emission and absorption spectra of the donor-acceptor pairs and the segmental flexibility of the immunoglobulin molecule. The calculations yielded values in the range of 5.5-7.0 nm for the minimum distance of separation between the antibody sites in solution and demonstrated a substantial energy barrier to the closer approach of the sites.

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Affinity and temporal variation of isoelectric fractions of rabbit antilactose antibody

Cited by 29 publications

References 18 publications

Clonal dominance. I. Restricted nature of the IgM antibody response to group A streptococcal carbohydrate in mice.

Clonal dominance. I. Restricted nature of the IgM antibody response to group A streptococcal carbohydrate in mice.

Proximity relationships within the Fc segment of rabbit immunoglobulin G analyzed by resonance energy transfer

Proximity of antibody binding sites studied by fluorescence energy transfer

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