engleski

Purification and Characterisation of Polyclonal Anti-hGH Antibodies

Antisera to human growth hormone (hGH) were obtained by immunising rabbits with hGH preparations. These antisera were further purified by ammonium sulphate precipitation ("unpurified" antibodies) and by DEAE-cellulose chromatography. Antibodies which were eluted with the starting buffer, 10 mM phosphate buffer, pH=8.0 (antibody preparation I) and antibodies eluted with the rising phosphate concentrations, i.e. in the concentration range 15-40 mM (antibody II preparation) and in the concentration range 45-60 mM of phosphate buffer (antibody III preparation) were further characterised. By gel-filtration it was found that all purified anti-hGH antibodies belong to the IgG class, since the majority of "immunoactivity" was eluted in Mr range 165-158 000. That anti-hGH antibodies belong to IgG was further confirmed by precipitation with Staphylococcus aureus cells (Pansorbin cells). pH optima, influence of various salts, as well as affinity constants were determined for each antibody preparation. Further, each antibody preparation was tested for specificity to various hormones. It was found that different antibody preparations react differently with hGH related hormones such as prolactin (hPRL) and placental lactogen (hPL). In further experiments it was found that antibody I preparation reacted with different epitopes of hGH than antibody II and antibody III preparations. From these studies it can be concluded that by purification of antisera, by simple ion-exchange chromatography, antibodies with different specificity could be isolated.

human growth hormone (hGH); purification of antisera; antibody preparation; anti-hGH antibodies

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