title: Influence of hydrophobic and hydrophilic spacer-containing enzyme conjugates on functional parameters of steroid immunoassay.
abstract: Introduction of spacers in coating steroid antigen or enzyme conjugates or immunogen is known to exert an influence on the sensitivity of steroid enzyme immunoassays. We have introduced hydrophobic and hydrophilic spacers between enzyme and steroid moieties and studied their effects on functional parameters of enzyme immunoassays, using cortisol as a model steroid. Cortisol-3-O-carboxymethyloxime-bovine serum albumin (F-3-O-CMO-BSA) was used as immunogen to raise the antiserum in New Zealand white rabbits. Three enzyme conjugates were prepared using cortisol-21-hemisuccinate (F-21-HS) as carboxylic derivative of cortisol and horseradish peroxidase (HRP) as an enzyme label. These were F-21-HS-HRP (without spacer), F-21-HS-adipic acid dihydrazide-HRP (adipic acid dihydrazide as hydrophobic spacer), and F-21-HS-urea-HRP (urea as hydrophilic spacer). The influence of hydrophobic and hydrophilic spacers on the functional parameters of assays such as lower detection limit, ED50, and specificity was studied with reference to enzyme conjugate without spacer. The results of the present investigation revealed that the presence of a hydrophilic spacer in the enzyme conjugate decreases the lower detection limit, decreases the ED50, and marginally improves the specificity of assays. These improvements in functional parameters of assays may be due to the decreased magnitude of the overall hydrophobic interactions existing between the spacer in enzyme conjugate and the antigen binding site of the antibody.
authors.affiliation: Department of Reproductive Biomedicine, National Institute of Health and Family Welfare, Munirka, New Delhi 110067, India.
authors.forename: Seema
authors.initials: S
authors.lastname: Nara
authors.forename: Vinay
authors.initials: V
authors.lastname: Tripathi
authors.forename: Shail K
authors.initials: SK
authors.lastname: Chaube
authors.forename: Kiran
authors.initials: K
authors.lastname: Rangari
authors.forename: Harpal
authors.initials: H
authors.lastname: Singh
authors.forename: Kiran P
authors.initials: KP
authors.lastname: Kariya
authors.forename: Tulsidas G
authors.initials: TG
authors.lastname: Shrivastav
mesh_terms: D000818:Animals; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D011817:Rabbits; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013256:Steroids / Q000378:metabolism*
chemical_list: D013256:Steroids