Development and standardization of multiplexed antibody microarrays for use in quantitative proteomics.

BACKGROUND: Quantitative proteomics is an emerging field that encompasses multiplexed measurement of many known proteins in groups of experimental samples in order to identify differences between groups. Antibody arrays are a novel technology that is increasingly being used for quantitative proteomics studies due to highly multiplexed content, scalability, matrix flexibility and economy of sample consumption. Key applications of antibody arrays in quantitative proteomics studies are identification of novel diagnostic assays, biomarker discovery in trials of new drugs, and validation of qualitative proteomics discoveries. These applications require performance benchmarking, standardization and specification. RESULTS: Six dual-antibody, sandwich immunoassay arrays that measure 170 serum or plasma proteins were developed and experimental procedures refined in more than thirty quantitative proteomics studies. This report provides detailed information and specification for manufacture, qualification, assay automation, performance, assay validation and data processing for antibody arrays in large scale quantitative proteomics studies. CONCLUSION: The present report describes development of first generation standards for antibody arrays in quantitative proteomics. Specifically, it describes the requirements of a comprehensive validation program to identify and minimize antibody cross reaction under highly multiplexed conditions; provides the rationale for the application of standardized statistical approaches to manage the data output of highly replicated assays; defines design requirements for controls to normalize sample replicate measurements; emphasizes the importance of stringent quality control testing of reagents and antibody microarrays; recommends the use of real-time monitors to evaluate sensitivity, dynamic range and platform precision; and presents survey procedures to reveal the significance of biomarker findings.

Methyleugenol in Ocimum basilicum L. Cv. genovese gigante.

Ocimum basilicum cv. Genovese Gigante is the basil cultivar used the most in the production of a typical Italian sauce called pesto. The aromatic composition of plants at different growth stages was determined. Plants from different areas of northwestern Italy were analyzed at 4 and 6 weeks after sowing and showed methyleugenol and eugenol as the main components. The content of these compounds was correlated with plant height rather than plant age. Particularly, methyleugenol was predominant in plants up to 10 cm in height, whereas eugenol was prevalent in taller plants. These results are important in the evaluation of risk to human health posed by dietary ingestion of methyleugenol contained in pesto.

Inhibition of neutrophil O(2)(-) production by unsymmetrical methylene derivatives of benzopyrans: their use as potential antiinflammatory agents.

Some unsymmetrical derivatives of benzopyrans 9 were synthesized and tested to verify their PKC inhibitory activity. For this purpose, the Mannich bases of 7-hydroxycoumarins 6 were treated with 2-(dialkylamino)benzopyran-4-ones or 3-(dialkylamino)naphtho[2,1-b]pyran-1-ones 8 in the presence of acetic or propionic anhydride, yielding compounds 9. Human neutrophils stimulated with either PMA and f-MLF were used as the cellular model. The efficiency of the compounds 9 was established on their capacity to reduce the O(2)(-) production by activated human neutrophils. Compounds 9d and 9f, bearing an acetoxy group in position 7 of the chromone moiety, seem to counteract the neutrophil activation efficiently.

Disoxaril-related 3-(diethylamino)-5-phenylisoxazoles.

Previous research has shown that 3-(dialkylamino)-5-phenylisoxazoles possessing a compact structure were active against HRV-2 and, consequently, presented a type B activity. In this paper, 3-(diethylamino)-5-phenylisoxazoles, which are structurally more elongated and related to Disoxaril, were synthesized in view to attempt type A activity against HRV-14. Unfortunately, all tested compounds were devoid of activity against HRV-14 (and HIV-1) or exhibited great toxicity.

N,N-dialkylaminosubstituted chromones and isoxazoles as potential anti-inflammatory agents.

The ability of some N,N-dialkylaminosubstituted chromones and isoxazoles to inhibit the protein kinase C (PKC) dependent signal transduction pathway was tested. As a cellular model, human neutrophils stimulated with either phorbol myristate acetate (PMA) or formylmethionine-leucine-phenylalanine (f-MLF) were used. The efficiency of the compounds was established by their capacity to reduce the O2- production by activated human neutrophils. Compounds carrying a 3-bis(2-methoxyethyl)amino group, a substituent found active in previously tested tricyclic compounds, do not show significant anti-PKC activity in this study. On the other hand, substitution with a 1-piperidinyl group leads all tested compounds to a high biological activity against stimulated neutrophils.

Analysis of 1930 bedridden patients in the internal medical sector of the emergency department of a large city hospital: appropriate and non-appropriate admission.

This paper discusses criterion for the appropriateness of admission to the hospitalization ward in the internal medical sector of the emergency department, and analyses the bedridden patients in the emergency department of the major hospital in the city of Genoa. The analysis covers 1930 patients, for which considerations are made, globally and separately in two different age groups, as to the appropriateness of admission to the hospitalization ward of the emergency department, the occurrence of subjective urgencies and objective instabilities, and progression subsequent to hospitalization (discharge, transfer into other hospital wards, decease). The most significant results of the analysis were the following: (1) no significant difference was found between younger and older patients regarding appropriateness of admission; (2) in cases of appropriate admission subjective urgency was clearly prevalent in relation to objective instability, the latter being much more frequent in the older age group; (3) a lack of self-sufficiency and the absence of adequate family support were important factors regarding inappropriate admission of older patients; (4) the greater frequency of objective instability in the older patients-as well as a lack of self-sufficiency-was the major factor in their greater length of stay in the emergency department. These results challenge the misconceived but diffused conviction that there is widespread mishandling of the elderly regarding admission to the emergency department, while at the same time stresses the need for alternative services and structures concerning hospital admission of older patients.

Detection and measurement of inflammatory cytokines in implant crevicular fluid: a pilot study.

Peri-implantitis has been shown to possess clinical characteristics similar to those of periodontitis. This pilot study was conducted to determine levels of inflammatory cytokines in crevicular fluid from healthy implants and those implants affected by peri-implantitis. Fifty implants from 13 patients were examined. A clinical examination was performed, and gingival crevicular fluid samples were collected and analyzed for cytokines. Implants were categorized clinically as healthy, early peri-implantitis, or advanced peri-implantitis. Interleukin-1 beta was detected in the crevicular fluid of implants in all three groups (healthy = 59.47 +/- 15.55 pg/site; early peri-implantitis = 460.77 +/- 35.67 pg/site; and advanced peri-implantitis = 191.10 +/- 21.60 pg/site [mean +/- SEM]). These results indicate that interleukin-1 beta is present in implant gingival crevicular fluid and may be modulating attachment loss in implants suffering from peri-implantitis. Thus, interleukin-1 beta may be used to monitor disease progression.

Monoclonal-antibody-based enzyme-linked immunosorbent assay for Trichomonas vaginalis.

Trichomonas vaginalis is estimated to infect 4 million women per year in the United States. The diagnosis of trichomoniasis is predominantly achieved by direct microscopic examination of vaginal exudates. This subjective diagnostic procedure is reported to be 75% sensitive under ideal circumstances. We have developed an enzyme-linked immunosorbent assay (ELISA) for the detection of T. vaginalis directly from vaginal exudates. The ELISA employs a monoclonal antibody specific for a 65-kilodalton surface polypeptide of T. vaginalis as the capture antibody in a sandwich format. A polyclonal rabbit anti-T. vaginalis antibody labeled with horseradish peroxidase serves as the probe. An evaluation of vaginal specimens from women attending clinics revealed a sensitivity and specificity of the ELISA of 89 and 97%, respectively, versus the culture technique. These results indicate the usefulness of this ELISA as an alternative to microscopic and culture methods for the detection of T. vaginalis in vaginal exudates.

A highly sensitive immunoenzymometric assay involving "common-capture" particles and membrane filtration.

This highly sensitive immunoenzymometric method involves monoclonal antibodies, a common-capture microsphere, and a rapid, membrane-filtration separation step. The common-capture solid phase is monoclonal anti-fluorescein antibody convalently attached to 6.5 micron-diameter latex particles. In sandwich-type assays for large-molecule analytes, the capture antibody is conjugated with fluorescein isothiocyanate and the probe antibody is conjugated with beta-galactosidase (EC 3.2.1.23). In competitive assays for small analytes, the analyte-beta-galactosidase conjugate competes with the analyte in the clinical samples for the fluoresceinated capture antibody. After simultaneous incubation of the reagents for 2 h, the bound and unbound reagents are separated by filtration through the bottom of each well of a 96-well plate. Substrate (4-methylumbelliferyl-beta-D-galactopyranoside) is then added to the wells, and the rate of product formation is determined kinetically for 12 min. The rate is proportional to the concentration of analyte in the sandwich assays and inversely proportional in the competitive assays. The assay results for choriogonadotropin, thyrotropin, digoxin, and thyroxin show the assay to be sensitive, rapid, and applicable to any size analyte. With this system, several different sandwich and (or) competitive-type assays can be performed simultaneously on the same plate.