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.

Homologous ELISA for detection of prednisolone in human serum

“…The immunoglobulins (IgG) of non-immunized rabbits were used for generating antirabbit gamma globulin (ARGG) by immunizing the goat Shrivastav, Chaube, Kariya, Kumari, et al, 2011;Shrivastav, Chaube, Kariya, Singh, et al, 2012). Following, the booster injection, blood was collected from the goat and centrifuged.…”

“…Coating of antibody to microtitre plates. The 96 well microtitre plates were coated using the immunobridge technique for primary antibody immobilization according to the method of Shrivastav et al (Shrivastav, Chaube, Kariya, Kumari, et al, 2011;Shrivastav, Chaube, Kariya, Singh, et al, 2012;Shrivastav, Kariya, Prasad, Chaube, & Kumar, 2014). Briefly, 250 μL of normal rabbit serum (NRS) diluted (1:250) in water was dispensed into each well and incubated at 37°C overnight.…”

“…A QBASIC language-based program developed in house has been used to compute the values of unknown samples that uses the logit-log regression method . The affinity constant of PSL antibody and sensitivity of the developed assay were calculated by the Feldman and Rodbard method (Shrivastav, Chaube, Kariya, Singh, et al, 2012).…”

“…Cross-reactivity of analogous steroid compounds with PSL-21-HS-BSA-antibody using PSL-21-HS-HRP-enzyme conjugate assay was calculated. The % cross-reaction was calculated using the following formula (Shrivastav, Chaube, Kariya, Kumari, et al, 2011;Shrivastav, Chaube, Kariya, Singh, et al, 2012;Tao et al, 2014):…”

Development of Enzyme-Linked Immunosorbent Assay for Estimation of Urinary Albumin

“…[21] The sensitivity of the developed method was determined according to the method described elsewhere. [22] The statistical analyses-such as mean, standard deviations, and logit-log transformation-of the data were done using the Microsoft Excel computer program.…”