Hydroxyapatite chromatography antibodies

Two monoclonal antibodies, one highly specific for human albumin (HSA-1) and one of broader specificity for several primates (HSA-2), were selected for subsequent use. These antibodies were purified by hydroxyapatite chromatography to single heavy- and light-chain bands (Figure 4) and then biotinylated. 

Figure 4. SDS-PAGE profile of monoclonal antibodies HSA-1 and HSA-2. After purification by hydroxyapatite chromatography, HSA-1 (lane 2) and HSA-2 (lane 3) were electrophoresed on a 10% polyacrylamide gel in the presence of 0.1% SDS. Approximately 1 (xg of HSA-1 and HSA-2 was loaded in each lane. The gel shows two peptide bands corresponding to heavy and light (56-and 24-kilodalton [KD], respectively) immunoglobulin chains and no evidence of other contaminating proteins. Lane 1 was loaded with molecular weight markers.

Hydroxyapatite chromatography (HAC) has gained wider popularity as a purification tool for antibodies because of the development of uniform ceramic media that provide excellent physical strength, high flow properties, and scale-up... 

Poiesi, C., Tamanini, A., Ghielmi, S., and Albertini, A. (1989). Protein A, hydroxyapatite and diethylaminoethyl Evaluation of three procedures for the preparative purification of monoclonal antibodies by high-performance liquid chromatography. ]. Chromatogr. 465, 101-111. 

Aoyama, K., and Chiba, J. (1993). Separation of different molecular forms of mouse IgA and IgM monoclonal antibodies by high-performance liquid chromatography on spherical hydroxyapatite beads. J. Immunol. Methods 162, 201-210. 

An Fe-S protein has been found to be present also in the b /f complex of chloroplasts and cyanobacteria. This protein has been dissociated from the complex with Triton X-100 and hydroxyapatite column chromatography, and was shown to be associated with the 20000 Da subunit in chloroplasts [111] and the 22000 Da one in A. variabilis [128]. In all cases the dissociation resulted in an irreversible loss of activity the involvement of the Fe-S protein in electron transport was also proved by the inhibition by an antibody raised against the Triton isolated protein (but not by one against the SDS denatured subunit) [129,130]. An oxidoreduction potential of 0.290 V was measured in intact chloroplast membranes and in the complex or in the isolated homogeneous preparation the potential was pH independent below pH 8 [111]. 

Ceramic hydroxyapatite (CHT) is a spherical, macroporous form of hydroxyapatite. Compared to most other chromatography adsorbents, CHT is unusual in that it is both the ligand and the support matrix. Two types of CHT ceramic hydroxyapatite. Type 1 and Type 11, have elution characteristics similar to crystalline hydroxyapatite but also have some important differences. CHT Type 1 has a higher protein-binding capacity and better capacity for acidic proteins, while CHT Type 11 has a lower overall protein-binding capacity but has better resolution for nucleic acids and certain proteins. The Type 11 material also has a very low affinity for albumin and is especially suitable for the purification of many species and classes of antibodies. 

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