anti-ARS Antibodies Research Articles

Affinity labeling of residues within Hv2 of guinea pig anti-azobenzenearsonate antibodies of different isotypes and from different strains.

Anti-p-azobenzenearsonate (ARS) antibodies of IgG1 and IgG2 isotypes produced in inbred strain 13 and strain 2 guinea pigs were affinity labeled with N-(bromoacetyl)-3-[(p-arsonophenyl)azo]-L-tyrosine (BAAT) or N-(bromoacetyl)-p-arsanilic acid (BAA). BAAT was shown to modify approximately 50% of the binding sites specifically and BAA approximately 30%. Both reagents preferentially modified residues in the heavy (H) chain to the extent that it contained over 80% of the affinity label associated with the native molecule. At least 80% of label borne by the variable domain of the H chain (VH) was found in the second hypervariable region (Hv2). BAAT labeled all anti-ARS antibodies exclusively at position N-59, which contains a lysyl residue. BAA labeled predominantly tyrosine at N-57 and, to a lesser extent, lysine-59 and tyrosine-50. Comparison of Hv2 sequences in anti-ARS and in antibodies reactive with other haptens has shown that tyrosine at N-50 and N-57 as well as lysine at N-59 is distinctive of antibodies with anti-ARS specificity, thus implying their involvement in antigen binding. The predominant sequence of Hv2 was identical in anti-ARS IgG1 and IgG2 molecules induced in either inbred guinea pig strain following either carrier priming or conventional immunization. Although limited variability does occur among the various populations of anti-ARS antibodies in certain residue positions in Hv2, no significant differences in the binding affinities or in the indexes of heterogeneity were seen among the various kinds of anti-ARS antibodies.

Two structurally distinct and independently regulated idiotypic families associated with the A/J response to azophenylarsonate.

At least two distinct subpopulations of A/J anti-azophenylarsonate (Ars) antibodies can be defined on the basis of serological cross-reactivity and sequence homology of heavy and light chains. The most frequently occurring idiotype family, IdCR, was initially identified by a rabbit antiserum and is present only in A/J mice. Examination of several randomly selected hybridoma proteins which failed to express IdCR-associated determinants revealed a second set of closely related molecules. The second idiotype family, Id36-60, is recognized by a rabbit antiserum prepared against the hybridoma protein 36-60. It is present in practically all A/J and BALB/c, but not B10. A Ars immune sera. On average, A/J immune sera contain approximately one-fifth as much Id36-60 as IdCR. A/J mice can be suppressed for subsequent expression of either idiotype by neonatal injection of anti-IdCR or anti-Id36-60 antisera. Suppression of IdCR does not affect the production of Id36-60 and vice versa. Together, these idiotype systems provide two independent markers for monitoring network regulation of the anti-Ars antibody response in A/J mice.

Unique determinants associated with hybridoma proteins expressing a cross-reactive idiotype: frequency among individual immune sera.

It is possible to prepare antisera that recognize individual members of a family of hybridoma proteins in which all members of the family express the cross-reactive idiotype (IdCR) characteristic of the A/J anti-Ars response. These hybridoma-specific reagents also recognize a small percentage of the IdCR+ anti-Ars antibodies, which constitute the normal A/J anti-Ars response. Thus, the previously reported sequence heterogeneity of our IdCR+ hybridoma proteins actually represents components of the normal A/J anti-Ars response. Determinants associated with one particular IdCR+ hybridoma protein, 36-71 (Id36-71), were found on approximately 0.01 to 5.0% of the IdCR+ antibodies in all of the A/J hyperimmune sera that were examined. Determinants associated with 2 other IdCR+ hybridoma proteins, 16-46-4-8 (Id16-46) and 44-10 (Id44-10) were also found in almost all A/J immune sera, again at various concentrations. These results indicate that the heritable trait (IdCR) is expressed as a large number of antibody sequences that probably arise from the somatic diversification of a limited number of germ line genes. The pattern of diversification is qualitatively similar among individual mice but varies with regard to the relative proportions of the individual antibody components.

Detection of mouse cells producing antibodies against the azophenylarsonic group by haemolytic plaque assay

For detection of mouse cells producing anti-ARS antibody a modified method using ARS-SRBC is described. For optimal results a suitable source of SRBC must be selected. The number of ARS groups per SRBC required for optimal lysis in PFC assay varies, and ARS-SRBC prepared by the method of Ingraham which are suitable for detection of rabbit-PFC give negative results with the mouse system. ARS-SRBC prepared for the PFC assay are unsuitable for the haemagglutination test and vice versa.

The immunoglobulin-like T-cell receptor. III. Binding of the arsonate hapten by idiotype-bearing T cells and antibody is blocked by avian antibody to (Fab′) 2

Chicken antibody to the (Fab′) 2 fragment of mouse IgG inhibited binding of antigen by the idiotype-bearing A/J antibody to the azobenzenearsonate (ARS) hapten. Purified peripheral T cells from ARS-primed mice were shown to bind the ARS hapten, and this binding was also inhibitable both by a rabbit anti-ARS idiotype antibody and by a chickpn anti-(Fab′) 2 antibody.

Structural Studies on Induced Antibodies with Defined Idiotypic Specificities

Abstract The murine VH genetic map consists of two different subloci of VH genes. Seven idiotypes have been allocated to these two different subloci, one of which contains genes coding for the A5A idiotype, while the second contains the genes encoding for six other idiotypic markers. Previous studies from this laboratory have shown that anti-Ars antibodies are best classified in the murine VHII subgroup. Studies from other laboratories indicate that the J558 idiotype could be classified in a similar fashion. The sequence of the T15 myeloma protein VH region is most characteristic of the murine VHIII subgroup. These studies were undertaken in order to study the VH subgroup of two additional murine VH markers. Since one of these markers has been allocated to a separate sublocus, comparisons can be made for the first time between the “framework” residues (VH subgroup) and the murine genetic map. The data indicate that within a single presently defined sublocus proteins exist which contain remarkably different “framework” residues.

An Anti-Benzene-Arsonate Antibody of Restricted Heterogeneity with Light Chains of b5 Allotype: Amino Acid Sequences of Amino-Terminal Regions of Light Chains from Antibodies and Normal Immunoglobulins of Various Allotypes

Abstract A rabbit which lived for over 3.5 years produced anti-benzene-arsonate antibody at various times over a 33-month period. The animal was partially suppressed for expression of b5-κ-type light chains when it was first immunized with benzene-arsonate coupled to edestin (Ed-Ars). The purified antibodies from January, 1968, October, 1969 and May 1970 were S-sulfitolysed and an LII fraction of light chains prepared. In October, 1969, the L chains were of a single V-region subgroup and gave a unique sequence for the first 21 residues. Light chains of earlier and later anti-Ars were very similar at the first 21 positions but additional residues were detectable at several positions in lesser amounts. We found an unusual residue, Ala, at position 8 which has only been found in human λ-type L chains of subgroup II. The major sequence of the anti-Ars antibody L chains was of the KII-length (Hood) but some of the heterogeneity could be explained as due to a similar chain, of KI length.

Studies on chicken γ-globulins and hapten-specific chicken antibodies

1. 1. Chickens were injected with a mixture of Ars-BSA and R 4N-BSA, i.e. azoproteins prepared by coupling BSA with diazotized arsanilic acid or diazotized p-aminophenyl-N-trimethylammonium chloride, or with the doubly labeled antigen Ars-R 4N-BSA. Serotologically pure hapten-specific anti-Ars and anti-R 4N antibodies were prepared by means of immunosorbents; they were free of anti-BSA and other γ-globulins. 2. 2. Reduction of the hapten-specific antibodies with mercaptoethanol and alkylation with iodoacetamide yielded a mixture of peptide chains which on starch gel electrophoresis in urea at pH 8·2 was resolved into a diffuse band which migrated toward the cathode and 7–8 anodically migrating narrow bands. The cathodically migrating material was identified with the heavy A chains, the anodically migrating bands with the light B chains of chicken γ-globulin. No consistent significant difference was found when the band pattern of A and B chains from anti-Ars antibody was compared with the analogous bands from anti-R 4N antibody. 3. 3. Ultracentrifugation of the hapten-specific antibodies isolated from hyperimmune chickens revealed the presence of some 18 S antibodies in the 7 S population. Digestion of the isolated hapten-specific anti-Ars and anti-R 4N antibodies with papain yielded a product which on ultracentrifugation sedimented as a single peak of 3·7 S; material of the same sedimentation coefficient was obtained by the papain digestion of normal chicken γ-globulin. 4. 4. The peptide maps of the tryptic digests of anti-Ars and anti-R 4N antibodies were extremely similar if not identical. No distinct and consistent difference was found. However, distinct differences were found between fragments I and II from normal chicken γ-globulin, and also between peptide maps of anti-R 4N (or anti-Ars) antibodies from chicken and rabbit serum. 5. 5. The identity of the peptide maps of anti-Ars on the one hand and anti-R 4N on the other can be reconciled with the clear-cut serological differences between these two antibodies by the assumption that the combining sites of the antibody molecules are highly heterogeneous, that the distinct spots of the peptide maps, which are identical in anti-Ars and anti-R 4N are those of amino acid sequences which are common to both hapten-specific antibodies, and that the specific combining sites of the antibody molecules are formed by variable amino acid sequences, each of these occurring in amounts too small to produce distinct spots in the peptide maps.