title: Influence of different length linker containing DHEA-7-cmo-enzyme conjugates on sensitivity and specificity of DHEA-3-hs-antibody.
abstract: The 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 different homobifunctional spacers having varying atomic length (3 to 10) between enzyme and dehydroepiandrosterone (DHEA) moiety and studied their effects on functional parameters such as sensitivity and specificity of DHEA enzyme immunoassays. DHEA-3-hemisuccinate-bovine serum albumin (DHEA-3-HS-BSA) was used as immunogen to raise the antiserum in New Zealand white rabbits. Five enzyme conjugates were prepared using DHEA-7-carboxymethyloxime (DHEA-7-CMO) as carboxylic derivative of DHEA and horseradish peroxidase (HRP) as an enzyme label. These were DHEA-7-CMO-HRP, DHEA-7-CMO-urea-HRP (DHEA-7-CMO-U-HRP), DHEA-7-CMO-ehylenediamine-HRP (DHEA-7-CMO-EDA-HRP), DHEA-7-CMO-carbohydrazide-HRP (DHEA-7-CMO-CH-HRP), and DHEA-7-CMO-adipic acid dihydrazide-HRP (DHEA-7-CMO-ADH-HRP). The influence of different atomic length linkers on sensitivity and specificity were studied with reference to label without linker. The results of the present investigation revealed that DHEA moiety having a 3-hemisuccinate carboxyl arm that is hydrophilic in nature and spacer arm urea that is also hydrophilic in nature when used for the link to the protein carrier and enzyme for the preparation of immunogen and enzyme conjugate respectively resulted in development of assay having comparable sensitivity and lowest ED(50) as compared to other spacers. Thus sensitivity and ED(50) of the assay depend partly on the nature of the steroid and spacer arm link to the carrier protein and the enzyme.
authors.affiliation: Department of Reproductive Biomedicine, National Institute of Health and Family Welfare, Munirka, New Delhi, India. tgsrivastava@nihfw.org
authors.forename: Tulsidas G
authors.initials: TG
authors.lastname: Shrivastav
authors.forename: Shail K
authors.initials: SK
authors.lastname: Chaube
authors.forename: Kiran P
authors.initials: KP
authors.lastname: Kariya
authors.forename: Rita
authors.initials: R
authors.lastname: Singh
authors.forename: Dinesh
authors.initials: D
authors.lastname: Kumar
authors.forename: Parul
authors.initials: P
authors.lastname: Jain
authors.forename: Suryakant
authors.initials: S
authors.lastname: Karwar
authors.forename: Jyoti
authors.initials: J
authors.lastname: Uyake
authors.forename: Bhavana
authors.initials: B
authors.lastname: Deshmukh
mesh_terms: D000818:Animals; D000906:Antibodies / Q000276:immunology*; D000918:Antibody Specificity / Q000276:immunology*; D002417:Cattle; D003687:Dehydroepiandrosterone / Q000031:analogs & derivatives / Q000737:chemistry* / Q000276:immunology*; D006735:Horseradish Peroxidase / Q000737:chemistry / Q000378:metabolism*; D007124:Immunoenzyme Techniques / Q000379:methods*; D011817:Rabbits; D012680:Sensitivity and Specificity
chemical_list: D000906:Antibodies; D003687:Dehydroepiandrosterone; D006735:Horseradish Peroxidase