Blocking and binding type antibodies against all major vitamin B12-binders in a pernicious anaemia serum

Abstract

The simultaneous occurrence of blocking and binding antibodies to intrinsic factor (IF), transcobalamin (TC- II, TC I) and other R type vitamin B12 proteins in the serum of a patient with treated pernicious anaemia (PA) is reported here for the first time. The dialysed and purified immunoglobulin-G (IgG), but not the immunoglobulin-M (IgM), from a PA patient neutralized the total unsaturated vitamin B12 binding capacity (UB12BC) of human gastric juice, saliva and serum and also of rabbit serum, suggesting that the PA IgG contained blocking antibodies against, IF, TC II, TC I and other R-binders. In addition, the PA IgG and IgM preparations contained binding antibodies since they could form macromolecular complexes with 57Co-B12 bound to IF, TC I or TC II so that each one of the latter was totally excluded from Sephadex G-200. The presence of the binding antibodies was further confirmed by the formation of radioactive precipitation lines on agarose with each one of the vitamin B12-binders bound to 58Co-B12. The PA serum did not exhibit any measurable UB12BC after dialysis against 7.5 M guanidine-HCl followed by renaturation with phosphate buffer (pH 7.4). But it did form TC I and TC II complexes with 57Co-B12 when the latter was added during the renaturation step indicating that the serum contained circulating immunoglobulin-TC complexes. The blocking antibodies should be distinguished from the previously described binding antibodies. The blocking of the binding of vitamin B12 to TCs resulted in relatively lower serum vitamin B12 levels in the present case in contrast to the presence of binding antibodies where high serum vitamin B12 levels have been reported. In addition, the binding antibodies form immunocomplexes with TCs which can easily be detected because they can bind radioactive vitamin B12 while the corresponding immunocomplexes of blocking antibodies are hidden because they prevent the binding of the vitamin to TCs.