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Of 42 purified human myeloma proteins tested, two (IgG3 Her and IgM Mag) were found to possess strong lupus anticoagulant (LA) and anti-cephalin activity, as assessed by a dilute activated partial thromboplastin time (dAPTT) and ELISA test, respectively. For these proteins, we confirmed the observation reported by others that LA activity is present in the antigen-binding (Fab) portion of the immunoglobulin molecule. Rabbit anti-idiotype antibodies against IgG3 Her inhibited the anti-cephalin activity of this protein, suggesting that the anti-cephalin activity of IgG3 Her depends on the hypervariable part of the immunoglobulin and thus most probably is a true antigen-antibody reaction. The anti-Her idiotype antibodies were also able to bind to and inhibit the anti-cephalin activity of IgM Mag. ELISA binding and inhibition experiments showed that the anti-idiotype antiserum contained at least two sets of anti-idiotypes; one set that recognizes a cross-reactive idiotype shared by IgG3 Her and IgM Mag, and another set that seems to be unique to the immunizing protein IgG3 Her. Both sets of anti-idiotype antibodies also bound weakly to polyclonal (patient) IgG, indicating an idiotypic cross reaction.

A number of fragments derived from acid-treated rabbit IgG by digestion with plasmin have been separated by high-resolution gel filtration. Fragments isolated included a dimer and monomer Facb, named F(acb)2 and Facb, respectively and a heterodimer composed of Facb and Fab subunits, named F(acb)(ab). A C gamma 2 fragment was obtained by papain digestion of Facb. A heterodimer composed of Facb and Fab', named F(acb)(ab'), was also prepared by oxidizing a reduced mixture of these fragments. Fragments thus obtained are classified into two groups--those carrying paired C gamma 2 domains, i.e. F(acb)2, and the disulfide-linked dimeric C gamma 2 fragment; and those having a single C gamma 2 domain, i.e. reduced, alkylated Facb and C gamma 2 fragment, F(acb)(ab) and F(acb)(ab'). These fragments exhibited marked differences in their capacity to activate complement in assay systems of hemolysis and complement consumption by immune complexes or aggregates on polystyrene latex. Fragments of the former group could activate complement but with a definitely reduced efficiency (50%) compared to intact IgG, whereas fragments of the latter group were practically inactive. Although it was not determined whether the C1-binding capacity itself is changed by monomerization of the C gamma 2 domain, the results suggested that the intact paired C gamma 2 module is required at least for the activation process of complement.

The present study revealed that the IgG immunoglobulins of normal or non-immune rabbit IgG and anti-bovine serum albumin, anti-ovalbumin, anti-bovine IgG and anti-p-chlorobenzoic acid antibodies could non-specifically bind to bis-(p-chlorophenyl)-acetic acid (DDA) coupled to omega-amino-hexyl Sepharose (AHS) by the use of strengthened hydrophobic interactions dependent on the concentration of NaCl. The main binding site on the adsorbent of DDA-substituted AHS (DDA-AHS) was found to be the DDA ligand. The hydrophobic potency of the DDA ligand was thought to be more effective than that of p-chlorobenzoic acid coupled to AHS. Out study also demonstrated that two different binding sites capable of interacting the DDA ligand were contained in IgG molecules. One was located in the Fc region and the other in the Fab region. The former had an ability to adhere to the DDA-AHS adsorbent in the presence of NaCl of 3 M or over, while the latter showed heterogeneous binding behavior depending upon its antibody specificity. Differences in the chromatographic distribution among the whole IgG immunoglobulins including anti-DDA antibody were found by a hydrophobic salting-out chromatography (HSOC) method on a DDA-AHS column. It was therefore assumed that when whole IgG proteins were subjected to HSOC on a DDA-AHS column, the hydrophobic binding site in the Fc region played a decisive role at high salt concentrations of 3 M or over, while the hydrophobic binding site in the Fab region played a major role at intermediate and low salt concentrations of 2 M or below. Thus, by taking advantage of this HSOC method, whole IgG or its Fab molecules possessing very strongly hydrophobic binding sites to promote high quantum yields of 8-anilinonaphthalene-1-sulfonate (ANS) fluorescence can be easily separated. We concluded that the ligand of DDA is a probe for the hydrophobic regions in IgG immunoglobulins.

An IgGl (lambda) protein which showed a unique susceptibility towards papain digestion was isolated from the serum of a patient (Mot) with multiple myeloma. The Fab fragments of this protein were degraded rapidly into smaller peptides via an Fb fragment [Gall & D'Eustachio (1972), Biochemistry 11, 4621-4628], which corresponded to the constant domains (Cl-Chl). Structural analysis of the isolated Fab fragment, which consisted of the intact L-chain, a 17,000 and a 5000 mol. wt peptide fragment, indicated that the initial cleavage site was located in the vicinity of the second hypervariable region of the Fd fragment. Examination of the partial amino acid sequences of the Mot H-chain suggested that the variable region of the H-chain may be a hitherto unknown hybrid of subgroups I and III. This particular structure seems to have made the Fab fragment highly susceptible to papain. In the course of the present study, we also found in the papain digests of several human IgG proteins an 'intermediate' 5S fragment, which had previously been reported exclusively for the papain digest of rabbit IgG.