Anti-lactose Antibodies Research Articles

Observations of a set of isomeric anti-lactose antibodies directed against a group D streptococcal polysaccharide

Sets of isomeric anti-lactose antibodies with specificity for the lactose units of a cell wall polysaccharide fromStreptococcus faecalis strain N were induced in rabbits immunized with a vaccine of nonviable cells of the organism. Such sets of anti-lactose antibodies were isolated from the serum of immunized animals by affinity chromatography on lactosyl-Sepharose. Gel electrofocusing experiments showed that the preparations consisted of multiprotein components. One preparation of antibodies of 13 isomers was separated into homogeneous components by liquid isoelectrofocusing. The individual isomeric antibodies exhibit specificity for the lactose units of the antigenic polysaccharide, possess isoelectric points in the range of 5.9–8.0, and belong to the IgG class of immunoglobulins, and each member yields one light chain and one heavy chain on dissociation in sodium dodecyl sulfate (SDS) and mercaptoethanol. These results have been interpreted as evidence for the assembly of the chains of isomeric antibodies by a single-chain pairing mechanism.

Purification of anti-lactose antibodies from antisera to Neisseria gonorrhoeae and Neisseria meningitidis.

Lactosyl-sepharose 4B columns were used for purification by affinity chromatography of anti-lactose antibodies from rabbit antisera to the gonococcal strains 8551 and VII, and the serogroup B meningococcal strain M982. Anti-lactose antibodies were obtained from all three antisera. SDS-PAGE of lipopolysaccharide and of bacterial cells and immunoblotting with the anti-lactose antibodies showed that the lipopolysaccharide was the only bacterial component with binding sites for the antibodies.

The pairing of light and heavy chains of isomeric anti-lactose antibodies

Sets of polyclonal isomeric anti-carbohydrate antibodies are present in sera of rabbits immunized with vaccines of non-viable streptococcal cells containing antigenic glycans. Anti-lactose antibodies have been purified by affinity chromatography and have been subjected to dissociation, electrophoretic and agar diffusion analyses. The anti-lactose antibody preparation was found to be composed of 16 isomeric proteins which yield 16 heavy chains and 16 light chains.

Purification and properties of antibodies having specificity for the carbohydrate moieties of synthetic glycoconjugates

Three types of antibodies having specificity for carbohydrate moieties of glycoconjugate antigens have been isolated from the sera of rabbits immunized with vaccines of β- d-glucosyl-bovine serum albumin, β- d-galactosyl-bovine serum albumin, or β-lactosyl-bovine serum albumin. The antibodies were isolated by affinity chromatography on adsorbents bearing appropriate carbohydrate ligands. The antibody types are specific for the β- d-glucosyl, β- d-galactosyl, or β-lactosyl groups of the synthetic glycoconjugates. The specificities have been established by data on hapten-inhibition, agar-diffusion, periodate-oxidation, and affinity-chromatography experiments. Each antibody type is of the IgG class of immunoglobulins and is of uniform molecular size, with molecular weight of 1.5 × 10 5. Data from gel-isoelectrofocusing experiments showed that each preparation of antibodies, although specific for identical determinant groups of the same antigen, nevertheless consisted of a set of different proteins. The anti- d-glucose antibodies consisted of five proteins, the anti- d-galactose antibodies of eleven proteins, and the anti-lactose antibodies of seventeen proteins. The suggestion has been made earlier that the members of such a set of antibodies, in which each member is induced by the same determinant group of an antigen and each member combines with this group in the precipitin reaction, be called isoantibodies.

Specific reactivity of lipid vesicles conjugated with oriented anti-lactose antibody fragments

The method previously described (Sinha, D. and Karush, F. (1979) Biochem. Biophys. Res. Commun. 90, 554–560) for the oriented attachment of immunoglobulins to lipid vesicles has been used to confer specific reactivity on liposomes by their conjugation with anti-lactose Fab′ fragments derived from rabbit IgG antibody. It is estimated that one-third of the Fab′ fragments was irreversibly attached to liposomal membrane, resulting in a membrane concentration of 2 mmol of Fab′ per mol of total lipid. The specific reactivity of the modified liposomes was demonstrated by agglutination with a multivalent, lactose-containing diheteroglycan. The availability of virtually all of the binding sites of the attached antibody for reaction with ligand was established by a fluorescence quenching titration with N-(N ϵ- Dnp- l- lysyl)-p- aminophenyl-β- lactoside . An intrinsic association constant of 8.9 · 10 6 M −1 was found for the attached Fab′ compared to a value of 2.8 · 10 6 M −1 for free anti-lactose Fab′. In addition the maximum values for the quenching by bound ligand of the fluorescence of free and attached antibody were the same. It can be concluded that the chemical procedures used to effect attachment of the antibody to the lipid vesicles allow retention of the original structure of the antibody site and its accessibility to external components.

Restriction in IgM expression. III. Affinity analysis of monoclonal anti-lactose antibodies.

A clonal analysis has been made of the murine BALB/C response to lactose-containing immunogens with respect to the affinity restriction in IgM expression. Monoclonal IgM and IgG antibodies were prepared from antilactosyl hybridomas generated from mice immunized with p-aminophenyl-beta-lactoside (PAPL) coupled to BGG or with a vaccine of Streptococcus faecalis (Strain N). Association constants for the binding of monovalent derivatives of PAPL were measured by quenching fluorescence. These derivatives carried a probe (2,4-dinitrophenyl or 1-dimethylaminonaphthalene-5-sulfonyl) that served to quench the protein fluorescence when the ligand was complexed with the protein. The central finding was that with both immunogens a restriction in the affinity of IgM was demonstrated since the highest values exhibited by IgG antibody exceeded by at least a factor of 50 the highest comparable values of the association constants for IgM antibody. It is suggested that the hypothesis of germ-line restriction, previously proposed as the basis for the affinity restriction of IgM, may also be applicable to the T cell receptor since its distinctive properties parallel those of IgM antibody.

Restriction in IgM expression—II. The V H regions of murine anti-lactose antibodies

A comparison of the anti-lactose IgM response among individual animals of five inbred strains of mice has been made with a view to possible germ-line restriction in IgM diversity. The V H regions of the antibodies were isolated and the individual preparations examined by isoelectrical focusing (IEF) in 7 M urea. The main findings were the complexity of the IEF patterns as well as their similarity among individuals of the same strain and their near-identity to the patterns for the V H regions derived from normal IgM. These observations parallel results found earlier with V H preparations of equine anti-lactose IgM. This homology of behavior between the two systems suggests a cautious generalization about IgM responses to the effect that limited longevity of the B cell clones and germ-line restriction of V H in IgM expression remain tenable notions.

Restriction in IgM expression—1: The V H regions of equine anti-lactose antibodies

The phenomenon of affinity maturation, characteristic of the IgG response, is generally limited or absent in the induced IgM response. This observation has led to the proposal that IgM is restricted to the expression of germ-line V region genes whereas IgG utilizes both germ-line genes and their somatic variants. In this study the V H regions prepared from purified anti-lactose IgM antibody of six horses were compared by analytical isoelectric focusing (IEF). The six patterns were virtually identical with respect to their complexity (about 30 bands), the isoelectric points of the bands and their relative intensities. This finding is predicted from the assumption of germ-line restriction. The V H regions from normal equine IgM and antiSIII IgM exhibited a similar complexity but different intensity distributions. The parallel complexity is attributed to the degeneracy of the number of distinguishable isoelectric points observable by IEF for a large number (e.g. 1350) of different compositions of charged amino acids. A cellular model is proposed for the germ-line restriction in the expression of IgM antibody based on the assumption that secondary (memory) B cells are not involved in IgM production. The consequent limitation to a primary response for IgM precludes the utilization of the expanded repertoire generated by somatic variants because these variants are unable to undergo sufficient clonal expansion to yield adequate numbers of IgM-producing plasma cells.

Interaction of a bivalent ligand with IgM anti-lactose antibody

Interaction of a bivalent ligand with IgM anti-lactose antibody

Antibody to an artificial disaccharide antigen cross-reactive with Neisseria gonorrhoeae lipopolysaccharide.

An artificial antigen was prepared from 4-O-beta-I-galactopyranosyl-D-glucose (lactose) and 8-ethoxycarbonyloctanol. Covalent attachment to bovine serum albumin provided an antigen that elicited antilactose antibody in rabbits and goat. These antibodies were active against Neisseria gonorrhoeae lipopolysaccharide in passive hemagglutination tests. The same antibody agglutinated cells of Streptococcus faecalis, strain N, and precipitated the lactose-containing cell wall diheteroglycan of this organism. Fractionation of rabbit and goat antibody raised against the synthetic antigen of S. faecalis vaccine provided two antibody fractions only one of which, eluted from the immunoadsorbent by galactose, was active against N. gonorrhoeae lipopolysaccharide.

Anti-glycosyl antibodies. Two sets of isoantibodies with specificity for different carbohydrate moieties of the same glycosyl antigen.

Two sets of anti-glycosyl antibodies have been isolated by affinity chromatography methods from the antisera of rabbits immunized with a vaccine of nonviable cells of Streptococcus faecalis, strain N. Both types of antibodies are directed against a dineteroglycan of glucose and galactose present in the cell wall of this organism. The members of one set, anti-galactose antibodies, combine with the terminal lactose residues of the glycan and the member of the other set, anti-lactose antibodies, combine with terminal lactose residues of the same glycan. Each set of antibodies is composed of multiprotein components. The electrofocusing method had been used to isolate the individual antibody proteins in homogeneous states as shown by both electrophoresis and ultracentrifugation techniques. Since the components of each set combine with the same structural unit of the antigen, they have been designated as isoantibodies. The sedimentation constants, electrophoretic properties, carbohydrate constituents, and amino acid compositions of the two sets of antibodies are recorded.

IgM antibody—III: The role of light chains in equine anti-lactose Fabμ

The diversity and role of the L chains in equine IgM anti-lactose antibody were investigated by examination of Fab fragments which were fractionated by preparative isoelectric focusing to provide subpopulations of restricted isoelectric range. Analysis by analytical isoelectric focusing revealed that the diversity of L chains associated with each of these subpopulations was comparable to that associated with the parent IgM pool. In addition the pattern of diversity was largely invariant. The extensive degeneracy in L chain utilization indicated by these results suggests a minor role for the L chain in the structure of the combining site. This view is supported by the recovery of binding activity in recombination experiments with Fab dissociated into Fd fragments and L chains. Both homologous and heterologous recombination yielded about 30% recovery of binding sites with no loss of affinity. The apparent degeneracy of the L chain population has led to an hypothesis regarding somatic diversification of the V L gene pool. If the μ chain provides the binding specificity and is controlled by germ-line genes, then proliferative stimulation of B cells with anti-lactose receptors would include B cells in which a somatic variant of the germ-line V L gene had occurred. The result would be the expansion of clones of cells carrying somatic derivatives of this germ-line gene. This expansion would, in turn, provide a multiplicity of V L regions which could be utilized by γ chains, for example, associated with somatic variants of V H genes. The proposed model for the expansion of clones with somatic variants of V L genes provides an explanation for the use of a common pool of L chains by all classes of immunoglobulins.

IgM antibody-I: Heterogeneity of the component chains of equine anti-lactose antibody

The heterogeneity of the IgM response has been studied with anti-lactose antibody purified from the sera of seven horses. The IgM antibody was induced with a bacterial vaccine and the sera were obtained during a one-year period of immunization. L and H chain preparations were derived from separate bleedings of each horse and examined by analytical isoelectric focusing. All of the L chain preparations were complex and similar and, under optimum conditions, exhibited about 45 bands. Their similarity included almost identical concentration distributions over the entire pH gradient. Isoelectric bands due to J chains could also be identified at the acidic end of the gradient. The H chain preparations also showed very similar patterns which were limited to the relatively narrow pH range of 5.4–6.2. Only 10 distinct bands could be recognized. The striking similarity within the H chain populations and the L chain populations, including preparations from IgM without anti-lactose specificity, suggest that the isoelectric pattern is primarily due to the isoelectric distribution of the major subgroups of both μ and L chains. The implications of these findings for the notion that IgM is specified by structural genes acquired by inheritance is examined. It is pointed out that the system described here provides an experimental approach to this proposal.

58] Labeling of antilactose antibody

Publisher Summary Affinity labeling studies with anticarbohydrate antibodies have been very limited. In earlier studies, diazoniumphenyl glycosides were employed as affinity labeling reagents for rabbit and equine anti-p-azophenyl-β-lactoside and p-azophenyl- β-galactoside antibodies. Although these antibodies were heterogeneous, it was possible to identify the labeled residues in the heavy or light chains since the modified residues had characteristic absorption spectra. With the discovery of bacterial cell walls of Streptococcus groups A and C induced antipolysaccharide antibodies of restricted heterogeneityp such antibodies have been used for affinity labeling studies. This chapter describes the production of antilactose antibodies, synthesis of affinity labeling reagents containing lactose, the labeling conditions, and the distribution of label in the polypeptide chains of the antibodies. It explains the antilactose antibodies, synthesis of affinity labeling reagents, affinity labeling conditions, and distribution of label in the polypeptide Chains of the antibodies.

Anti-glycosyl antibodies: Preparation and characterization of rabbit anti-galactose and anti-lactose antibodies

Anti-galactose and anti-lactose antibodies have been isolated from the antisera of rabbits immunized with non-viable cells of Streptococcus faecalis , strain N containing an antigenic diheteroglycan of glucose and galactose in the cell wall. The anti-galactose antibodies are specific for the galactosyl moiety while the anti-lactose antibodies are specific for the lactosyl moiety of the diheteroglycan. Hapten inhibitions with galactose and lactose, the sedimentation constant, the immunoglobulin type, the carbohydrate content, the electrophoretic mobility and the amino acid composition have been determined for the two new types of anti-glycosyl antibodies.

Affinity-labeling of anti-lactose antibody

Purified anti-lactose antibodies induced in the horse and rabbit with a bacterial vaccine have been subjected to affinity-labeling with two bromoacetyl derivatives of lactose-containing ligands. The synthesis of these new radioactive reagents is described and the optimum conditions for the labeling reaction given. The specificity of the reaction has been demonstrated by inhibition with lactose. The maximum extent of specific labeling observed was 1·5 mole of label per mole of 7S antibody. The distribution of the label between heavy and light chains was measured with the separated chains fractionated by acrylamide gel electrophoresis. Except for one antibody preparation the heavy chain carried between 77 and 90 per cent of the total label. It is concluded that bromoacetyl derivatives of appropriate ligands can be used to label the hypervariable regions of the heavy chains present in antibody with anti-lactose specificity.

Antibody Affinity

Abstract The method of fluorescence quenching by bound ligand for the measurement of antibody affinity has been extended to anti-lactose antibodies. The basis for this extension is the use of a lactosyl ligand covalently linked to the 2,4-dinitrophenyl (DNP) group. This group serves as a sensor of the bound state by causing fluorescence quenching similar to that observed with anti-DNP antibody. The synthesis and characterization of this new ligand, N-(Nα-acetyl, Nε -DNP-L-lysyl)-p-aminophenyl-β-lactoside, are described. The affinity of its interaction with three rabbit IgG and one equine IgM antilactose antibody preparations has been measured. The Qmax values range from 24 to 47% and the calculated association constants from 2.5 × 105 M-1 to 7.2 × 105 M-1. For one IgG preparation, measurement of the association constant by equilibrium dialysis with the tritiated ligand gave a value of 6.75 × 105 M-1 compared to the value of 7.2 × 105M-1 obtained by fluorescence quenching. It is evident that with monoclonal anti-lactose antibody the accurate determination of Qmax, which is possible in this system, provides another structurally dependent clonal characterization of the variable region of the antibody. This optical probe along with others can be used for the identification of individual clones of antibody-producing cells through time and inheritance.

Affinity Labeling of Anti-Lactose Antibodies with Bromoacetylaminolac Dye in Dark

Affinity labeling of rabbit IgG anti-lactose antibodies of restricted heterogeneity by bromacetylaminolac dye was carried out in dark and the label on the isolated chains determined by SDS gel electrophoresis. The pattern of labeling was compared with that obtained when the reaction was carried out in light using the same reagent. Differences were observed in the mode of labeling of chains. Possible use of mapping the active site of the antibody by the same labeling reagent which can exist in two geometrical forms under dark and light conditions is discussed.

Equine Anti-Hapten Antibody

Abstract The immune response to a bacterial vaccine of Streptococcus faecalis (strain N) was characterized in all of the seven horses studied by the sustained production of about 90% IgM anti-lactose antibody over a period of 44 weeks with maximum values of the total antibody ranging from 4 mg/ml of serum to 12 mg/ml of serum. With respect to the binding of a lactose-containing ligand the association constants of the antibodies purified from sera obtained between 5 and 44 weeks fell in the range of 1 × 105 M-1 to 2 × 105 M-1. Not only was there no significant indication of maturation of affinity in this period but there was a selective limitation of affinity compared to that of 7S antibodies. It was inferred that the synthesis of IgM antibody involves the selective utilization of VH and/or VL genes.

Chain Interactions and Idiotypic Specificities of Homogeneous Rabbit Anti-Lactose Antibodies

Abstract Functionally homogeneous fractions of rabbit anti-lactose IgG antibody have been characterized with respect to their idiotypic specificity and the effect of homologous recombination of isolated chains on the recovery of functional homogeneity and idiotypic determinants. Dimeric heavy chains and monomeric light chains were obtained from homogeneous antibody fractions and used for the preparation of reconstituted IgG molecules in high yield. The isolated chains were also evaluated with respect to binding affinity and inhibition of anti-idiotype antibody. The most significant observation was the recovery of idiotypic specificity in the homologous recombinants as well as binding affinity and functional homogeneity. These results provide strong evidence that the homogeneous antibody fractions are monoclonal products. The isolated chains exhibited little or no specific activity. With respect to binding, the dimeric heavy chain bound a lactosyl-containing ligand with an association constant about 100-fold less than that of the parent antibody. Although the binding was homogeneous only one binding site per dimer was expressed. The heavy chain was also found to inhibit slightly the anti-idiotype reaction. The isolated monomeric light chain, on the other hand, showed no reactivity in either kind of experiment. The anti-idiotype reaction was subject to inhibition with lactose and p-amino-phenyl-β-lactoside with some antisera, depending on the form in which the homogeneous antibody was presented for immunization. It is suggested that the major portion of the idiotypic determinant is constituted of amino acid residues located along the periphery of the binding site rather than the contact residues in the groove or cleft of the site. This proposal would account for the inhibition by ligands as the result of the inability of ligand and anti-idiotype antibody to be accommodated simultaneously at contiguous sites because of overlapping volumes. Those cases in which ligand inhibition is not observed could be attributed to unusual geometric relationships between the contiguous sites.