Expression, characterisation and immunoassay of recombinant marmoset chorionic gonadotrophin dimer and beta-subunit.
A specific and sensitive ELISA for measuring marmoset chorionic gonadotrophin (mCG) in culture medium, urine and plasma was developed using a polyclonal antibody raised against recombinant mCG, tagged with six histidine molecules (rmCG-6His), as the capture antibody. A well-characterised monoclonal antibody (518B7), which was generated against bovine luteinising hormone (bLH) and has been shown to detect CG and LH in Callithrichid monkeys, was biotinylated and used as the secondary antibody. Purified rmCG, calibrated against human CG (hCG; CR127) by bioassay, or the beta-subunit (rmCGbeta), quantified from amino acid analysis and carbohydrate analysis, was used as the standard. The assay was able to detect CG activity in medium collected from cultured marmoset embryos before attachment and through to the trophoblastic vesicle stage, plasma and urine collected from pregnant marmosets, marmoset placenta and pituitary homogenates. The assay was validated and its performance compared with a bioassay based on MA10 cell response to CG, with hCG as the standard. The sensitivity was 103 pg/ml (5 pg/well) of rmCGbeta and 476 pg/ml (24 pg/well) of the heterodimer rmCG. The mean recovery of standard added to embryo culture medium, marmoset urine and plasma was 104, 112 and 92% respectively. The intra- and interassay variation was less than 10 and 16% respectively. The low cross-reactivity with cynomolgus monkey and baboon LH, their beta-subunits, cynomolgus monkey and baboon follicle-stimulating hormone and hCG suggests that the assay is specific for mCG.
Amato
F
F
Reproductive Medicine Unit, Department of Obstetrics and Gynaecology, University of Adelaide, The Queen Elizabeth Hospital, Adelaide, South Australia, Australia.
Simula
A P
AP
Gameau
L J
LJ
Norman
R J
RJ
D000818:Animals; D000906:Antibodies; D016466:CHO Cells; D002144:Callithrix / Q000378:metabolism*; D002478:Cells, Cultured; D006063:Chorionic Gonadotropin / Q000032:analysis* / Q000235:genetics; D018997:Chorionic Gonadotropin, beta Subunit, Human / Q000032:analysis / Q000235:genetics; D006224:Cricetinae; D019281:Dimerization; D004622:Embryo, Mammalian / Q000737:chemistry; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005260:Female; D010902:Pituitary Gland / Q000737:chemistry; D010920:Placenta / Q000737:chemistry; D011247:Pregnancy; D011270:Pregnancy, Animal / Q000378:metabolism*; D011994:Recombinant Proteins / Q000276:immunology
D000906:Antibodies; D006063:Chorionic Gonadotropin; D018997:Chorionic Gonadotropin, beta Subunit, Human; D011994:Recombinant Proteins