engleski

Separation and characterization of polyclonal hGH antibodies

Antisera to hGH (human growth hormone) were raised in rabbit and 3 different antibodies were separated by DEAE-cellulose ion exchange chromatography, i.e. Ab-1 was eluted with 0.01M phosphate buffer, pH 8.0, while Ab-2 and Ab-3 were eluted with 0.024M and 0.048M, respectively, phosphate buffer pH 8.0. The purity of separated antibodies was checked by HPLC gel filtration chromatography. It was found that antibodies belong to IgG class, since their Mr's were in the range of 160000 and all were able to bind protein A. Purified antibodies showed different physico-chemical properties since binding of 125I-hGH was differently influenced by pH and ionic strength of the reaction milleu. From Scatchard plots affinity constants were determined and they were 3.0xE12 M-1 for Ab-1, 4.2xE12 M-1 for Ab-2 and 5.2xE12 M-1 for Ab-3. From binding studies, where different combinations of antibodies were allowed to react with hGH, it was concluded that separated antibodies showed different epitope specificities. The most pronounced epitope binding differences were found between Ab-1 and Ab-3.

anti-hGH antibodies; affinity constants; antibody purification

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