Figure 5

Specificity of polyclonal antibodies against serum proteins.

(A–D) Polyclonal antibodies were prepared in rabbits as described earlier²⁵. Western blotting was performed in reducing and denaturing conditions as described in Methods. A control specimen containing only mineral NPs was used in lane 1. Reactions against HSA, HSF and human apo-A1 are indicated with a black triangle, a white triangle, or a black circle, respectively. (A) The anti-HSA antibody (α-HSA) reacted with a single band of 85 kDa in whole HS, HS-NPs and purified HSA but produced no reaction against a negative control of mineral NPs or HSF. The anti-HSF antibody (α-HSF) reacted with a 62-kDa band in whole HS and HS-NPs and with two bands of 55 and 62 kDa in HSF ((B) the presence of multiples bands for purified HSF was attributed to glycosylation which may affect protein migration). (B) No reaction was noted for the anti-HSF antibody against the negative NP control or HSA. (C) The anti-apo-A1 antibody (α-h Apo-A1) reacted with a 30-kDa band in whole HS and HS-NPs but produced no reaction against mineral NPs, HSA, or HSF. (D) The polyclonal anti-HS antibodies reacted with three bands of 30, 58 and 75 kDa in whole HS and HS-NPs (these protein bands likely corresponded to HSA, HSF and apo-A1, respectively). In addition, the anti-HS antibody mainly produced a 75-kDa band against HSA and a 55-kDa band in the lane containing HSF. Given that purified proteins were used and that the molecular weights observed for HSA, HSF and apo-A1 correspond to the molecular weight observed for these proteins in previous studies^15,16 (approximately 28, 55 and 72 kDa, respectively), these results suggest that the polyclonal antibodies reacted positively and specifically against the serum proteins used as antigens.