Purification of three major forms of β-hCG from urine and production of polyclonal antibodies against them

Abstract

Objectives This study has been conducted to develop a proper and relatively simple method for purification of three major forms of β-hCG from urine to use as clinical calibrators and also to produce anti-β-hCG polyclonal antibodies against these purified forms. Design and methods Major β-hCG forms from urine of pregnant women were purified using an ultrafiltration method followed by three successive steps: 1) affinity chromatography on a Concanavalin A-Sepharose column, 2) ion exchange chromatography on a DEAE-Sephacel column and 3) preparative gel electrophoresis. Specific polyclonal antibodies against the purified forms were produced by immunizing rats. We also extracted β-hCG from urine using trypsin affinity chromatography. Results Three β-hCG forms with the molecular mass (MW) of 28, 32 and 35 kDa were purified and identified by ELISA method using monoclonal antibodies against two distinct epitopes (β2 and β11) on β-hCG. The titer of prepared antibodies was comparable with common standard anti-β-hCG antibodies. Also we could purify a 37 kDa form of β-hCG by trypsin affinity chromatography. Conclusions Here we proposed two different proper methods for purification of major forms of β-hCG from urine. We also found that three prepared β-hCG forms expose the β11 epitope and have an hCGβcf epitope expressed. Specific antibodies against purified β-hCG forms had comparable titers and could be used in diagnostic kits.