title: Influence of different length spacers containing enzyme conjugate on functional parameters of progesterone ELISA.
abstract: In steroid enzyme immunoassay (EIA), there is an increase or decrease of labeled steroid recognition by antibody due to homologous and heterologous combinations of enzyme conjugate with immunogen that affects sensitivity of the assay. We have introduced three to 18 atomic length linkers between enzyme and steroid moieties and studied their effects on functional parameters such as sensitivity, ED(50), and specificity of progesterone enzyme immunoassays. Progesterone-3-carboxymethyloxime-bovine serum albumin (P-3-CMO-BSA) was used as an immunogen to raise the antiserum in New Zealand white rabbits. Five enzyme conjugates were prepared using 17-α-hydroxy-progesterone-3-carboxymethyloxime (17-α-OH-P-3-CMO) as carboxylic derivative of 17-α-hydroxy-progesterone and horseradish peroxidase (HRP) as label. These were 17-α-OH-P-3-CMO-HRP, 17-α-OH-P-3-CMO-urea-HRP (17-α-OH-P-3-CMO-U-HRP), 17-α-OH-P-3-CMO-ehylenediamine-HRP (17-α-OH-P-3-CMO-EDA-HRP), 17-α-OH-P-3-CMO-carbohydrazide-HRP (17-α-OH-P-3-CMO-CH-HRP), and 17-α-OH-P-3-CMO-adipic acid dihydrazide-6-aminocaproic acid-HRP (17-α-OH-P-3-CMO-ADH-6ACA-HRP). The influence of different atomic length linkers on sensitivity, ED(50), and specificity were studied with reference to label without linker. The results of the present investigation revealed that the incorporation of ADH-6ACA spacer in 17-α-hydroxy-progesterone-enzyme conjugate improved the sensitivity in antigen plus bridge heterologous EIA system. The presence of spacer in enzyme conjugate improved the sensitivity and specificity (cross-reactivity) in some antigen plus bridge heterologous assay of progesterone.
authors: Department of Reproductive Biomedicine, National Institute of Health and Family Welfare, Munirka, New Delhi, India. tgsrivastava@nihfw.org
authors: Tulsidas G
authors: TG
authors: Shrivastav
authors: Shail K
authors: SK
authors: Chaube
authors: Kiran P
authors: KP
authors: Kariya
authors: Pramod K V
authors: PK
authors: Prasad
authors: Dinesh
authors: D
authors: Kumar
mesh_terms: D019326:17-alpha-Hydroxyprogesterone / Q000737:chemistry; D000272:Adipates / Q000737:chemistry; D015119:Aminocaproic Acid / Q000737:chemistry; D000818:Animals; D000941:Antigens / Q000737:chemistry / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005029:Ethylenediamines / Q000737:chemistry; D006735:Horseradish Peroxidase / Q000737:chemistry*; D007074:Immunoglobulin G / Q000276:immunology; D011374:Progesterone / Q000031:analogs & derivatives* / Q000737:chemistry / Q000276:immunology; D011817:Rabbits; D012710:Serum Albumin, Bovine / Q000737:chemistry* / Q000276:immunology*; D014508:Urea / Q000737:chemistry
chemical_list: D000272:Adipates; D000941:Antigens; D005029:Ethylenediamines; D007074:Immunoglobulin G; C075151:progesterone 3-(O-carboxymethyl)oxime-bovine serum albumin; C016965:adipic dialdehyde; D012710:Serum Albumin, Bovine; D011374:Progesterone; C031234:ethylenediamine; D019326:17-alpha-Hydroxyprogesterone; D014508:Urea; D006735:Horseradish Peroxidase; D015119:Aminocaproic Acid