Purification and Characterisation of Polyclonal Anti-hGH Antibodies
Boris Mildner*, Aleksandar Fevr, Vladimir Kasaš
University hospital »Sestre. miIosrdnice«, Vinogradska c. 29, 10 000 Zagreb, Croatia
Received December 4, 1997
Accepted April 7, 1998
human growth hormone (hGH), purification of antisera, antibody preparation, anti-hGH antibodies
Antisera to human growth hormone (hGH) were obtained by immunising rabbits with hGH preparations. These anlisera were further purified by ammonium sulphate precipitation ("unpurified" antibodies) and by DEAE-cellulose chromatography. Antibodies which were eluted with the starling buffer, 10 mM phosphate buffer, pH = 8.0 (antibody preparation I) and antibodies eluted with rising phosphate concentrations, i.e. in the concentration range of 15-40 mM (antibody 11 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" zuas 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 anlisera, by simple ion-excltange chromatography, antibodies with different specificity could be isolated.
*Corresponding author: PLIVA d.d., Research Institute, Prilaz banina Filipovića 25, 10 000 Zagreb, Croatia