Noncompetitive immunoassay detection system for haptens on the basis of antimetatype antibodies.

BACKGROUND: Small molecules classified as haptens are generally measured by competitive immunoassay, which is theoretically inferior to noncompetitive sandwich immunoassay in terms of sensitivity and specificity. We created a method for developing sandwich immunoassays to measure haptens on the basis of antimetatype antibodies. METHODS: We generated antimetatype monoclonal antibodies against a hapten-antibody immunocomplex using an ex vivo antibody development system, the Autonomously Diversifying Library (ADLib) system. We selected 2 haptens, estradiol (E2) and 25-hydroxyvitamin D [25(OH)D], as analytes. Sandwich immunoassays for these 2 haptens were developed by use of a 96-well microtiter plate and a fully automated chemiluminescence analyzer, and the performances of these immunoassays were investigated. RESULTS: The developed assays exhibited sensitivity high enough to detect target haptens in serum samples. The limit of detection of the ELISA for E2 was 3.13 pg/mL, and that of the fully automated chemiluminescent enzyme immunoassay (CLEIA) system was 2.1 ng/mL for 25(OH)D. The cross-reactivity with immunoreactive derivatives was effectively improved compared with the competitive assay. The CVs for the sandwich ELISA for E2 were 4.2%-12.6% (intraassay) and 6.2%-21.8% (total imprecision). The CVs for the sandwich CLEIA for 25(OH)D were 1.0%-2.3% (intraassay) and 1.9%-3.5% (total imprecision). In particular, the sandwich CLEIA for 25(OH)D showed correlations of r = 0.99 with both LC-MS/MS and a commercially available (125)I RIA. CONCLUSIONS: Our method represents a potentially simple and practical approach for routine assays of haptens, including vitamins, hormones, drugs, and toxins.

Cisplatin influences acquisition of resistance to molecular-targeted agents through epithelial-mesenchymal transition-like changes.

Chemotherapy with platinum agents is the standard of care for non-small-cell lung cancer (NSCLC); however, novel molecular-targeted agents like gefitinib have been approved for advanced NSCLCs, including recurrent cases previously treated with platinum-based chemotherapy. Although these agents show antitumor activity through distinct mechanisms and elicit positive initial responses, tumors invariably develop resistance. Recent studies have revealed mechanisms by which both types of agents induce acquired resistance. However, little is known about whether first-line treatment with either type of agent affects cancer cell susceptibility and development of resistance against subsequent treatment with the other. Using in vitro drug-resistant NSCLC cell models, we provide evidence that acquired cisplatin resistance may reduce the sensitivity of cancer cells to subsequent treatment with a molecular-targeted agent. In addition, first-line cisplatin treatment influenced the mechanism by which cancer cells developed resistance to subsequent treatment with a molecular-targeted agent. The influence of cisplatin on acquisition of resistance to a molecular-targeted agent was associated with epithelial-mesenchymal transition (EMT)-like alterations such as increased expression of mesenchymal markers, morphological change, and AXL tyrosine kinase-mediated increased cell motility. Our findings indicate that the influence of platinum-based chemotherapy on molecular-targeted therapies and the involvement of EMT and EMT-related effectors should be considered when developing therapeutic strategies using antitumor agents, especially in the context of sequential therapy.

Overexpressed HER2 in NSCLC is a possible therapeutic target of EGFR inhibitors.

BACKGROUND: "Oncogene addiction" is a concept in which tumor cells exhibit dependence on certain oncogene(s) for their sustained proliferation and survival, thus providing the rationale for molecular targeted therapies. Cancer cells addicted to epidermal growth factor receptor (EGFR) bear activated mutations in the EGFR gene, and these mutations are used as the markers for predicting carcinomas susceptible to EGFR inhibitors such as gefitinib and erlotinib. However, other unknown mechanisms underlying susceptibility to EGFR inhibitors have also been suggested. MATERIALS AND METHODS: The susceptibility of non-small-cell lung cancer (NSCLC) cell lines to EGFR inhibitors and the pattern of their oncogene addiction was examined. The effect of EGFR inhibitors on the activation of the oncogene was analyzed. The possible use of the oncogene protein expression as a biomarker was assessed. RESULTS: HER2 addicted, non-EGFR expressing NSCLC cell line NCI-H2170 was susceptible to EGFR inhibitors. EGFR inhibitor treatment led to markedly decreased phosphorylation levels of activated HER2 and its downstream effector AKT. Furthermore, the soluble form of HER2 was secreted by NCI-H2170 cells and was positively detected in the blood of xenografted mice. CONCLUSION: HER2 seems to be a valid therapeutic target of EGFR inhibitors in HER2-addicted lung carcinomas, and soluble HER2 may be an effective biomarker to guide the appropriate treatment of such cancer cells.

Novel monoclonal antibodies recognizing the active conformation of epidermal growth factor receptor.

The precise regulation of epidermal growth factor receptor (EGFR) is crucial for its function in cellular growth control. Although many antibodies against EGFR have been developed and used to analyze its regulation and function, it is not yet easy to analyze activated EGFR specifically. Activated EGFR has been mainly detected by its phosphorylation state using anti-phospho-EGFR and anti-phosphotyrosine antibodies. In the present study, we have established novel monoclonal antibodies which recognize the activated EGFR independently of its phosphorylation. Our antibodies detected active state of EGFR in immunoprecipitation and immunofluorescence, by recognizing the epitopes which are exposed through the conformational change induced by ligand-binding. Furthermore, we found that our antibodies preferentially detected the conformation of constitutively active EGFR mutants found in lung cancer cell lines. These results indicate that our antibodies may become novel research and diagnostic tools for detecting and analyzing the conformation of active EGFR in various cells and tissues.

Raf1 plays a pivotal role in lipopolysaccharide-induced activation of dendritic cells.

Activation of extracellular-regulated kinases 1/2 (ERK) is involved in lipopolysaccharide (LPS)-induced cellular responses such as the increased production of proinflammatory cytokines. However, mitogen-activated protein kinases (MAPKs) such as p38 are also activated by LPS and have been postulated to be important in the control of these end points. Therefore, establishing the relative contribution of MAPKs in each cell type is important, as is elucidating the molecular mechanisms by which these MAPKs are activated in LPS-induced signaling cascades. We demonstrated in DC2.4 dendritic cells that ERK regulates tyrosine phosphorylation of phosphatidyl-inositol-3-kinase (PI3-K) and the production of TNF-alpha. We also demonstrated that Raf1 is phosphorylated and involved in the production of TNF-alpha and tyrosine phosphorylation of PI3-K via ERK. Raf1 also regulates the activation of NF-kappaB. We propose that Raf1 plays a pivotal role in LPS-induced activation of the dendritic cells.