title: The influence of spacer-containing enzyme conjugate on the sensitivity and specificity of enzyme immunoassays for hapten.
abstract: BACKGROUND
  In hapten enzyme immunoassays (EIA), there is an increase or decrease of labeled hapten recognition by antibody that affects sensitivity of the assay. We incorporated a spacer between a hapten derivative and enzyme to test its influence on the sensitivity and specificity of enzyme immunoassays.
  
  
  METHODS
  Antibodies were generated against cortisol-3-O-carboxymethyl-oxime-bovine serum albumin (cortisol-3-O-CMO-BSA) and cortisol-21-hemisuccinate-bovine serum albumin (cortisol-21-HS-BSA) as an immunogen. Four cortisol horseradish peroxidase (HRP) enzyme conjugates were prepared using 2 cortisol derivatives (cortisol-3-O-CMO and cortisol-21-HS) with and without adipic acid dihydrazide (ADH) as a spacer. Eight combinations of homologous and heterologous assays were evaluated.
  
  
  RESULTS
  The incorporation of ADH spacer in cortisol-enzyme conjugate improved the sensitivity in heterologous (bridge and site plus bridge) EIA systems. In heterologous assays (site plus bridge), the presence of spacer in enzyme conjugate reduced the cross-reactivity with cross-reacting steroids.
  
  
  CONCLUSIONS
  Spacer in the enzyme conjugate for hapten ELISA can improve the sensitivity of heterologous assay of hapten-like steroids. It may also reduce the cross-reactivity for some assays.
authors.affiliation: Department of Reproductive Biomedicine, National Institute of Health and Family Welfare Munirka, New Delhi, India.
authors.forename: Anupam
authors.initials: A
authors.lastname: Basu
authors.forename: Seema
authors.initials: S
authors.lastname: Nara
authors.forename: Shail K
authors.initials: SK
authors.lastname: Chaube
authors.forename: Kiran
authors.initials: K
authors.lastname: Rangari
authors.forename: Kiran P
authors.initials: KP
authors.lastname: Kariya
authors.forename: Tulsidas G
authors.initials: TG
authors.lastname: Shrivastav
mesh_terms: D000272:Adipates / Q000737:chemistry / Q000276:immunology; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity / Q000276:immunology; D002417:Cattle; D004797:Enzyme-Linked Immunosorbent Assay / Q000295:instrumentation / Q000379:methods*; D006241:Haptens / Q000032:analysis* / Q000276:immunology; D006735:Horseradish Peroxidase / Q000737:chemistry / Q000276:immunology; D006854:Hydrocortisone / Q000737:chemistry / Q000276:immunology; D011817:Rabbits; D015203:Reproducibility of Results; D012710:Serum Albumin, Bovine / Q000737:chemistry / Q000276:immunology
chemical_list: D000272:Adipates; D000911:Antibodies, Monoclonal; D006241:Haptens; D012710:Serum Albumin, Bovine; D006735:Horseradish Peroxidase; C010011:adipic dihydrazide; D006854:Hydrocortisone