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Purification of three major forms of beta-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 beta-hCG from urine to use as clinical calibrators and also to produce anti-beta-hCG polyclonal antibodies against these purified forms. DESIGN AND METHODS:Major beta-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 beta-hCG from urine using trypsin affinity chromatography. RESULTS:Three beta-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 (beta2 and beta11) on beta-hCG. The titer of prepared antibodies was comparable with common standard anti-beta-hCG antibodies. Also we could purify a 37 kDa form of beta-hCG by trypsin affinity chromatography. CONCLUSIONS:Here we proposed two different proper methods for purification of major forms of beta-hCG from urine. We also found that three prepared beta-hCG forms expose the beta11 epitope and have an hCGbetacf epitope expressed. Specific antibodies against purified beta-hCG forms had comparable titers and could be used in diagnostic kits.

journal_name

Clin Biochem

journal_title

Clinical biochemistry

authors

Khademi F,Hamzehee K,Mostafaie A,Hajihossaini R

doi

10.1016/j.clinbiochem.2009.05.019

subject

Has Abstract

pub_date

2009-09-01 00:00:00

pages

1476-82

issue

13-14

eissn

0009-9120

issn

1873-2933

pii

S0009-9120(09)00245-8

journal_volume

42

pub_type

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