IgG subclass distributions in anti-hapten and anti-polysaccharide antibodies induced by haptenated polysaccharides.

Mice were immunized with hapten [NIP, (4-hydroxy-5-iodo-3-nitrophenyl)acetyl or TNP (2,4,6-trinitrophenyl)] conjugates of Ficoll or pneumococcal polysaccharide type 14 (S14), and they were bled on days 10 or 14. Anti-hapten and anti-polysaccharide antibodies were determined from the sera or from fractions (IgM + IgA). IgG1, IgG2a, IgG3 or IgG2b separated by a gradual acid elution from protein A. Approximately one-half of both anti-hapten and anti-polysaccharide antibodies was found in the IgM + IgA fraction. The subclass distribution of the IgG antibodies was dependent on the antigenic determinants. Polysaccharide antibodies were mostly in the IgG3 fraction (36-62%) and in the IgG1 fraction (18-36%). Hapten IgG antibodies were mostly in the IgG1 fraction (38-74%): each of the other three subclasses contributed the average of 13%. These results provide the first evidence that antibodies to different determinants of one antigen have grossly different isotype distributions.

Mouse IgG antibodies have subclass associated affinity differences.

Subclasses of IgG were separated from pools of mouse sera by letting immunoglobulins absorb on protein A-Sepharose and by eluting with buffers of decreasing pH. Most donor mice were immunized with a conjugate of a hapten (NIP) and chicken gamma globulin 20 days previously. The results indicate that concentrations of IgG varied from 5.1 to 8.6 mg/ml in the pools of immune sera and was 3.0 mg/ml in one normal serum tested. One half of this was IgG1, ca. 20% of IgG2a and IgG2b each, and 10% IgG3 in the pools of BALB/c sera. IgG2a and IgG3 could not be separated from C57BL sera (due to allotype b), but their combined share of IgG appears to be higher than in BALB/c. Immune sera contained 0.5-1.6 mg/ml of anti-NIP antibodies. Of this 90-98% was IgG1 and the remainder was split between the other subclasses. Up to one half of the protein in the IgG1 fraction was anti-NIP antibody. This surprising finding was confirmed by demonstrating that nearly 50% of the u.v.-light absorption was specifically removed by a NIP-immunosorbent. Subclass-associated affinity-differences were observed. IgG1 anti-NIP had a greater average affinity than IgG2a anti-NIP antibodies. The difference was ca. 1.5-fold when the equilibrium dialysis was focusing on the high-affinity bracket of the total population (concentration of free hapten 16-200 nM). At higher hapten concentrations the trend was the same but the data are fewer. Antibodies in subclasses IgG2b and IgG3 appear to share the lower affinity of IgG2a.

The range and fine specificity of the anti-hapten immune response: phylogenetic studies.

Heterodontus francisci (horned shark) and Pseudopleuronectes americanus (winter flounder) were immunized with furyl-oxazolone (furyl-Ox) and phenyl-oxazolone (phenyl-Ox) coupled either to bacteria or protein carriers. The antibodies produced were measured by inactivation of furyl- or phenyl-Ox conjugated bacteriophage, and their affinity and fine specificity were estimated by inhibition of phage inactivation with a series of structurally related hapten analogues. In both species, post-immunization peak titres were 100 to 2000 times higher than preimmunization titres. A number of unique features distinguished Heterodontus antibodies from Pseudopleuronectes or mammalian antibodies. Heterondontus antibodies exhibited a lower affinity for the immunizing hapten (furyl-Ox or phenyl-Ox) and a reduced ability to distinguish the homologous immunogenic hapten from its structural analogues. In addition, Heterodontus antibodies exhibited a lower level of inter-individual variation in affinity and fine specificity than did Pseudopleuronectes or mammalian IgM antibodies; this was especially prominent in anti-furyl-Ox responses. Typically the affinity and fine specificity of Heterodontus antibodies did not change over the 146-day period of immunization and were not influenced by the nature of the carrier. The implications of these findings in terms of the phylogenetic origins of antibody diversity are discussed.

An immunological characterization of the basic proteins of rodent sciatic nerve myelin.

Radioimmunoassays (RIA) for the myelin basic protein (MBP) and P2 protein together with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were used to establish the identities of and relationships between the basic proteins (BP) of rodent peripheral nervous system (PNS) myelin. The PNS myelin proteins studied, in order of increasing mobility of SDS-PAGE, are P1, PR (R = rodent) and PB (B = breakdown). The majority of the acid extractable proteins of rodent PNS myelin are MBP related as shown by MBP-RIA. When tested individually, rodents P1, PR and PB were each found to cross-react in the RIA for MBP but not that for P2. The acid extracts of rodent PNS myelin were found to contain P2, although in minute quantities. P2 accounts for approximately 0.05-1.0% of the acid extractable protein in rodent PNS myelin.

Radioimmunoassay for the P2 protein of bovine peripheral nerve myelin.

The P2 protein of bovine nerve root myelin was radiolabeled with 125I in homogeneous solution by the chloramine-T method and purified by gel filtration on Sephadex G-75 to obtain monomeric 125I-P2. Antigen-antibody complexes were isolated by the silica gel, double antibody, or polyethylene glycol techniques by using rabbit antibody to P2. As little as 1 ng/ml P2 could be detected. The RIA was used to measure the P2 content in nerve tissue and isolated myelin. The presence of P2 in spinal cord as well as in the peripheral nervous system was confirmed. Peptides isolated by CNBr digestion of the P2 protein were tested in the RIA. CN-1, comprising 80 to 90 residues from the interior of the molecule displayed complete immunologic cross-reactivity with intact P2. Neither CN-2, representing 18 amino acids from the COOH terminal, nor CN-3, representing 20 amino acids from the NH2 terminal, showed cross-reactivity. Since the major determinant for experimental allergic neuritis in the rabbit is located in peptide CN-2, our present data suggest that the major neuritogen and the major determinant(s) for humoral antibody response in this species may be at different locations within the P2 molecule.