Purification of three major forms of beta-hCG from urine and production of polyclonal antibodies against them.
OBJECTIVE
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.


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.
Khademi
Fatemeh
F
Department of Biochemistry, Faculty of Science, Payame Noor University, Tehran, Iran.
Hamzehee
Koorosh
K
Mostafaie
Ali
A
Hajihossaini
Reza
R
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000302:isolation & purification*; D000918:Antibody Specificity / Q000276:immunology; D018997:Chorionic Gonadotropin, beta Subunit, Human / Q000276:immunology / Q000302:isolation & purification* / Q000652:urine*; D002846:Chromatography, Affinity; D002848:Chromatography, DEAE-Cellulose; D002852:Chromatography, Ion Exchange; D003208:Concanavalin A; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000276:immunology; D005260:Female; D006801:Humans; D007114:Immunization; D011247:Pregnancy; D051381:Rats; D012685:Sepharose
D000911:Antibodies, Monoclonal; D018997:Chorionic Gonadotropin, beta Subunit, Human; D000939:Epitopes; D003208:Concanavalin A; D012685:Sepharose