Evaluation of methods to reduce background using the Python-based ELISA_QC program.

Almost all immunological approaches [immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), Western blot], that are used to quantitate specific proteins have had to address high backgrounds due to non-specific reactivity. We report here for the first time a quantitative comparison of methods for reduction of the background of commercial biotinylated antibodies using the Python-based ELISA_QC program. This is demonstrated using a recombinant humanized anti-cocaine monoclonal antibody. Several approaches, such as adjustment of the incubation time and the concentration of blocking agent, as well as the dilution of secondary antibodies, have been explored to address this issue. In this report, systematic comparisons of two different methods, contrasted with other more traditional methods to address this problem are provided. Addition of heparin (HP) at 1 µg/ml to the wash buffer prior to addition of the secondary biotinylated antibody reduced the elevated background absorbance values (from a mean of 0.313 ±â€¯0.015 to 0.137 ±â€¯0.002). A novel immunodepletion (ID) method also reduced the background (from a mean of 0.331 ±â€¯0.010 to 0.146 ±â€¯0.013). Overall, the ID method generated more similar results at each concentration of the ELISA standard curve to that using the standard lot 1 than the HP method, as analyzed by the Python-based ELISA_QC program. We conclude that the ID method, while more laborious, provides the best solution to resolve the high background seen with specific lots of biotinylated secondary antibody.

Estrogen and soy isoflavonoids decrease sensitivity of medulloblastoma and central nervous system primitive neuroectodermal tumor cells to chemotherapeutic cytotoxicity.

BACKGROUND: Our previous studies demonstrated that growth and migration of medulloblastoma (MB), the most common malignant brain tumor in children, are stimulated by 17ß-estradiol. The growth stimulating effects of estrogens are mediated through ERß and insulin-like growth factor 1 signaling to inhibit caspase 3 activity and reduce tumor cell apoptosis. The objective of this study was to determine whether estrogens decreased sensitivity of MB cells to cytotoxic actions of chemotherapeutic drugs. METHODS: Using in vitro cell viability and clonogenic survival assays, concentration response analysis was used to determine whether the cytoprotective effects of estradiol protected human D283 Med MB cells from the cytotoxic actions of the MB chemotherapeutic drugs cisplatin, vincristine, or lomustine. Additional experiments were done to determine whether the ER antagonist fulvestrant or the selective ER modulator tamoxifen blocked the cytoprotective actions of estradiol. ER-selective agonists and antagonists were used to define receptor specificity, and the impacts of the soy-derived phytoestrogens genistein, daidzein, and s-equol on chemosensitivity were evaluated. RESULTS: In D283 Med cells the presence of 10 nM estradiol increased the IC50 for cisplatin-induced inhibition of viability 2-fold from ~5 µM to >10 µM. In clonogenic survival assays estradiol decreased the chemosensitivity of D283 Med cells exposed to cisplatin, lomustine and vincristine. The ERß selective agonist DPN and low physiological concentrations of the soy-derived phytoestrogens genistein, daidzein, and s-equol also decreased sensitivity of D283 Med cells to cisplatin. The protective effects of estradiol were blocked by the antiestrogens 4-hydroxytamoxifen, fulvestrant (ICI 182,780) and the ERß selective antagonist PPHTP. Whereas estradiol also decreased chemosensitivity of PFSK-1 cells, estradiol increased sensitivity of Daoy cell to cisplatin, suggesting that ERß mediated effects may vary in different MB celltypes. CONCLUSIONS: These findings demonstrate that E2 and environmental estrogens decrease sensitivity of MB to cytotoxic chemotherapeutics, and that ERß selective and non-selective inhibition of estrogen receptor activity blocks these cytoprotective actions. These findings support the therapeutic potential of antiestrogen adjuvant therapies for MB, and findings that soy phytoestrogens also decrease sensitivity of MB cells to cytotoxic chemotherapeutics suggest that decreased exposure to environmental estrogens may benefit MB patient responses to chemotherapy.

Characterization of a recombinant humanized anti-cocaine monoclonal antibody produced from multiple clones for the selection of a master cell bank candidate.

We have generated a humanized anti-cocaine monoclonal antibody (mAb), which is at an advanced stage of pre-clinical development. We report here in vitro binding affinity studies, and in vivo pharmacokinetic and efficacy studies of the recombinant mAb. The overall aim was to characterize the recombinant antibody from each of the three highest producing transfected clones and to select one to establish a master cell bank. In mAb pharmacokinetic studies, after injection with h2E2 (120 mg/kg iv) blood was collected from the tail tip of mice over 28 days. Antibody concentrations were quantified using ELISA. The h2E2 concentration as a function of time was fit using a two-compartment pharmacokinetic model. To test in vivo efficacy, mice were injected with h2E2 (120 mg/kg iv), then one hour later injected with an equimolar dose of cocaine. Blood and brain were collected 5 min after cocaine administration. Cocaine concentrations were quantified using LC/MS. The affinity of the antibody for cocaine was determined using a [3H] cocaine binding assay. All three antibodies had long elimination half-lives, 2-5 nM Kd for cocaine, and prevented cocaine's entry into the brain by sequestering it in the plasma. Pharmacokinetic and radioligand binding assays supported designation of the highest producing clone 85 as the master cell bank candidate. Overall, the recombinant h2E2 showed favorable binding properties, pharmacokinetics, and in vivo efficacy.