The influence of spacer-containing enzyme conjugate on the sensitivity and specificity of enzyme immunoassays for hapten.
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.
Basu
Anupam
A
Department of Reproductive Biomedicine, National Institute of Health and Family Welfare Munirka, New Delhi, India.
Nara
Seema
S
Chaube
Shail K
SK
Rangari
Kiran
K
Kariya
Kiran P
KP
Shrivastav
Tulsidas G
TG
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
D000272:Adipates; D000911:Antibodies, Monoclonal; D006241:Haptens; D012710:Serum Albumin, Bovine; D006735:Horseradish Peroxidase; C010011:adipic dihydrazide; D006854:Hydrocortisone