Antigen heterologous enzyme linked immunosorbent assay for the measurement of DHEA in serum

Homologous and heterologous combinations of enzyme conjugate and antibody in steroid enzyme immunoassay (EIA) influences unlabeled steroid recognition by antibody that affects sensitivity of the assay. To develop dehydroepiandrosterone (DHEA) antigen heterologous enzyme linked immunosorbent assay (ELISA), antibodies were generated against DHEA-3-hemisuccinate-bovine serum albumin (DHEA-3-HS-BSA), DHEA-7-carboxymethyloxime-bovine serum albumin (DHEA-7-CMO-BSA), and DHEA-17-carboxymethyloxime-bovine serum albumin (DHEA-17-CMO-BSA). Five horseradish peroxidase (HRP) enzyme conjugates were prepared using five testosterone derivatives [testosterone-3-CMO (T-3-CMO), testosterone-17-HS (T-17-HS), testosterone-17-glucuronoside (T-17-G), testosterone-19-carboxymethylether (T-19-CME), and testosterone-11-HS (T-11-HS)]. Fifteen antigen heterologous combinations of antibody and enzyme conjugates were evaluated in the standard binding assay; only two combinations showed binding. The use of antigen heterologous combination (different antigen in label than the immunogen) resulted in development of a simple, direct, and convenient assay as it permits the direct addition of the serum sample into the assay and it requires only 1.5 h to complete.     

Influence of different length linker containing DHEA-7-CMO-enzyme conjugates on sensitivity and specificity of DHEA-17-CMO-antibody

Introduction of spacers in enzyme conjugates is known to exert an influence on the assay parameters of steroid enzyme immunoassays. We have introduced 3 to 10 atomic length linkers between enzyme and steroid moieties and studied their effects on sensitivity and specificity of dehydroepiandrosterone enzyme immunoassays. Dehydroepiandrosterone-17-carboxymethyloxime-bovine serum albumin (DHEA-17-CMO-BSA) was used as an immunogen to raise the antiserum in New Zealand white rabbits. Five enzyme conjugates were prepared using DHEA-7-CMO as carboxylic derivative of DHEA and horseradish peroxidase (HRP) as 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 with incorporation of linkers, the sensitivity improves, whereas specificity only marginally improves. These differential behaviors of various linkers toward the sensitivity and specificity of assays might be due to the difference in the magnitude of overall forces of attraction between the antibody and the enzyme conjugates.     

Regulation of chemokines and chemokine receptors after experimental closed head injury.

The expression of the chemokines macrophage inflammatory protein (MIP)-2 and MIP-1alpha and of their receptors CXCR2 and CCR5 was assessed in wild type (WT) and TNF/lymphotoxin-alpha knockout (TNF/LT-alpha-/-) mice subjected to closed head injury (CHI). At 4 h after trauma intracerebral MIP-2 and MIP-1alpha levels were increased in both groups with MIP-2 concentrations being significantly higher in WT than in TNF/LT-alpha-/- animals (p < 0.05). Thereafter, MIP-2 production declined rapidly, whereas MIP-1alpha remained elevated for 7 days. Expression of CXCR2 was confined to astrocytes and increased dramatically within 24 h in both mouse types. Contrarily, CCR5 expression remained constitutively low and was mainly localized to microglia. These results show that after CHI, chemokines and their receptors are regulated differentially and with independent kinetics.