text:
Americans carry a grudge heading into Tuesday's game against Mexico
VANCOUVER, B.C.—Heading into their final group-stage game of the CONCACAF women's soccer Olympic qualifying tournament Tuesday, there are at least two motivations in play for the American team. The first is simple and intrinsic: a draw or a victory against Mexico would give the U.S. top spot in Group B and a (theoretically) easier matchup in the semifinals against whichever of Canada or Costa Rica finishes second in Group A. The second goal has less to do with this particular tournament, but it may be preeminent in the Americans' minds: getting revenge on Mexico.
The U.S. and Mexico have long been rivals in women's soccer, but it's been a mostly one-sided rivalry until recently. Mexico has only made it to the Women's World Cup twice and has never gotten out of the group stage, while the U.S. has two World Cup titles, three Olympic gold medals and six CONCACAF Gold Cup championships. It's in that latter tournament, the North American championship that doubled as qualifying for the 2011 Women's World Cup, where things changed, though. Mexico knocked off the U.S. 2-1 at home in the 2010 Gold Cup semifinals, and that meant that the Americans were held to a third-place finish and had to play Italy for a berth in the World Cup. The U.S. still got into that World Cup and made it all the way to the final before losing to Japan, while Mexico didn't win a game in the World Cup itself, but the stumble against the Mexicans in the qualification round is still very fresh on American minds.
After the U.S.' 13-0 win over Guatemala Sunday, American head coach Pia Sundhage said the team is already locked into preparation for Tuesday's game.
"We will be prepared for Mexico, that's for sure."
Sundhage said things should be different this time around.
"That was 2010; this is 2012," she said. "We have a different personality today, an attacking personality. We're going to concentrate on that and work on our strengths."
Veteran American forward Abby Wambach said after Sunday's game the team will have to be in top form against the 21st-ranked Mexicans, who have won their first two games 5-0 and 7-0 and are a much tougher test on paper than 85th-ranked Guatemala or the 88th-ranked Dominican Republic.
"It's important that everyone be firing on all cylinders when we play Mexico," Wambach said.
Although the U.S. has already qualified for the crucial semifinal round, Wambach said their focus is firmly on Tuesday's game.
"Our team is playing well right now and we have one more important game to get to the semis," she said.
Sundhage said after the Americans' 14-0 win over the Dominican Republic that this team has particularly worked on staying in the moment at all times rather than looking too far ahead. She thinks maintaining that focus will give them an edge.
"We care every single game and every single goal," she said. "If we can keep that feeling, we can win the next game."
American striker Amy Rodriguez, who had five goals in that game against the Dominican Republic, said the memory of the loss in Mexico is still firmly with the U.S. team, motivating them to excel.
"We walked away from Cancun last year very disappointed," Rodriguez said. "We're not going to let that happen again."


text:
New England in the 1630s: William and Katherine lead a devout Christian life with five children, homesteading on the edge of an impassable wilderness. When their newborn son vanishes and crops fail, the family turns on one another. Beyond their worst fears, a supernatural evil lurks in the nearby wood.
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subset: commoncrawl


text:
Sunday 26 August 2012
It was Sunday, under the sign of Virgo. The US president was Barack Obama (Democratic).
In that special week of August people in US were listening to Whistle by Flo Rida.
Lawless, directed by John Hillcoat, was one of the most viewed movies released in 2012
while The Dream Team by Jack McCallum was one of the best selling books.
On TV people were watching Big City.
If you liked videogames you were probably playing Angry Birds Trilogy or Atelier Ayesha: The Alchemist of Dusk.
But much more happened that day: find out below..
You can also have a look at the whole 2012 or at August 26 across the years.
Old Newspapers
Have a look at the old newspapers from 26 August 2012 and get them!
TV Series
Which were the most popular TV series released in the last months ?
Which were the most popular movies released in the last 30 days ?
Find out your future
Get a FREE Numerology report based on the digits of 26 August 2012!
Wish him/her..
Historical Events
Which were the important events of 26 August 2012 ?
- RC saints - St Zephyrinus, Saint Ninian, David Lewis (one of the Forty Martyrs of England and Wales)
- Namibia - Namibia Day or Heroes' Day
- Zanzibar - Sultan's Birthday
Famous Birthdays:See famous people born on 26 August
- Seventeen villagers including two women in Afghanistan's southern Kajaki district are beheaded by unknown organised people. (AP via ''The Australian'')
- Thirty-six people are killed in a bus crash near Yan'an, Shaanxi province of China. (Reuters)
- Tropical Storm Isaac:
- The United States National Hurricane Center issues hurricane warnings for the states of Florida, Alabama, Mississippi and Louisiana. A state of emergency is in effect in these four areas. (Fox News)
- Tropical Storm Isaac has so far caused 10 direct fatalities on Hispaniola and one indirect in Puerto Rico. (Fox News)
- A 7.4 magnitude earthquake occurs in the Pacific Ocean off the coast of El Salvador with tsunami warnings in place for Costa Rica, Nicaragua, El Salvador, Honduras, Guatemala, Panama and Mexico. (AP via Fox News)
- An outbreak of Legionellosis (''Legionnaires' disease'') in Quebec City, Canada, kills 8 people and infects 104. (CBC)
- Women in Togo announce that they will go on sex strike for a week, starting Monday, following the example of women in Liberia. (CNN)
- Several hundred earthquakes hit Southern California, with the largest one measuring 5.5 on the Richter magnitude scale near San Diego. (CNN)
- Heads of state meet at the 16th Summit of the Non-Aligned Movement in Tehran, Iran, until August 31. (Radio Free Europe)
- Two Pussy Riot members, alleged to be the two participants in the band's Cathedral protest on February 21 who remain unarrested, ampquotleaveampquot Russia. (''The Guardian'')
- Libya Abdullah Al-Sha Mausoleum's destruction:
- The president of Libya's newly elected legislative congress, Mohamed Yousef el-Magariaf, calls the prime minister to an emergency meeting, after Saturday's bulldozing of the Sidi el-Sha'ab mausoleum in Tripoli by unnamed organized assailants. (''Wall Street Journal)''
- Libyan interior minister Fawzi Abdelali resigns after harsh criticism due to the Mausoleum attack. An aide says he resigned ampquotto defend the revolutionariesampquot. It becomes known that deputy PM Mustafa A.G. Abushagur on Saturday, referring to Friday's desecrations, sent a tweet saying ampquotI have asked the interior and defence ministers to intervene yesterday but did not do their duty to protect these sites.ampquot (Google Translate)
- 15-year-old amateur Lydia Ko of New Zealand wins the Canadian Women's Open, becoming the youngest ever winner on the LPGA Tour and the first amateur to win on that tour since JoAnne Carner in 1969. (''Vancouver Sun'')
Featured Contents
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Music Charts
Which were the top hits in that special week of August 2012 ?
Top #10 songs in the USA
- Flo Rida - Whistle
- Ellie Goulding - Lights
- Carly Rae Jepsen - Call Me Maybe
- Wide Awake - Katy Perry
- Maroon 5 - Payphone
- Gotye - Somebody That I Used To Know
- Titanium - David Guetta
- Fun. - Some Nights
- Phillip Phillips - Home
- Rihanna - Where Have You Been
Search For Your Ancestors
Sport Games on 26 August 2012
Serie AItalian Football
- Atalanta - Lazio : 0 - 1
- Chievo - Bologna : 2 - 0
- Genoa - Cagliari : 2 - 0
- Milan - Sampdoria : 0 - 1
Jupiler LeagueFootball (Belgium)
- Gent - Lokeren : 2 - 1
Bundesliga 1Football (Germany)
- Hannover - Schalke 04 : 2 - 2
- Stuttgart - Wolfsburg : 0 - 1
Bundesliga 2Football (Germany)
- Bochum - Aalen : 0 - 1
- Ingolstadt - Frankfurt FSV : 0 - 2
- Kaiserslautern - Munich 1860 : 0 - 0
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text:
Option Valuation with Systematic Stochastic Volatility
- Abstract
- Full Text PDF
- Author(s): KAUSHIK I. AMIN, VICTOR K. NG
- Published: Apr 30, 2012
- Pages: 881-910
- DOI: 10.1111/j.1540-6261.1993.tb04023.x
We use an extension of the equilibrium framework of Rubinstein (1976) and Brennan (1979) to derive an option valuation formula when the stock return volatility is both stochastic and systematic. Our formula incorporates a stochastic volatility process as well as a stochastic interest rate process in the valuation of options. If the "mean," volatility, and "covariance" processes for the stock return and the consumption growth are predictable, our option valuation formula can be written in "preference‐free" form. Further, many popular option valuation formulae in the literature can be written as special cases of our general formula.


text:
CodePlexProject Hosting for Open Source Software
tryed to record all current sounds on vista but got only mic record. is it stupid vista twisted all or sample included in rar-file isn't full? anyway thanx for cool project and sorry for my english.
you probably need to open the soundcard control panel to set up recording all current sounds and configure the recording source. WaveOut doesn't offer that choice directly.
i can't do this - in recording settings of sound card i see only mic and line-in.
Are you sure you want to delete this post? You will not be able to recover it later.
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text:
Nicole Kliest, Style Director at Clique Media Group, stayed with us at Rue Albéric Magnard in Auteuil – Passy, Paris. Tucked between the Seine and the Bois de Boulogne, it's a particularly scenic part of the city. It's also a fantastic place for foodies, as Nicole discovered gathering ingredients for her own festive feast.
A little local knowledge goes a long way when you're discovering a new city – which is where Emily comes in. Emily is one of our Meet & Greeters in Paris – when she's not welcoming onefinestay guests to stay in our marvellous homes, she's exploring her adopted city (she's originally from the US). Take a look at her tips for getting to know the City of Lights this winter.
Love Bloom has a dreamlike quality, evoking romance and intimacy. It's afternoon tea with delicate cakes, strolls through flower gardens on hazy evenings and impromptu picnics. And it's easy to bring it into your home too – furniture in muted pastel-pink or lilac is a great way to make a statement, particularly when paired with light walls. Try experimenting with silk fabrics and floral patterns to add a touch of nature and vibrancy, or set out a dish of macaroons for a little added colour.
Perched on the left bank of the Seine, the Latin Quarter is packed with restaurants, cafés and bars, making it one of Paris' premier spots for nightlife. Nonetheless, the area has a more cerebral, artistic side. The site of the city's first university and home of the Sorbonne, the area has always been a home to students from across Europe. There's still a huge student presence, and the bistros buzz with a carefree joie de vivre.


text:
Beauty queen sentenced for 'insulting' Turkish President on Instagram
Istanbul: A Turkish court found a former Miss Turkey beauty queen guilty of 'insulting' President Recep Tayyip Erdogan on social media, handing her a suspended sentence of over one year in jail.
Merve Buyuksarac. Pic/Getty Images
The Istanbul court sentenced model Merve Buyuksarac (27) to one year and two months in prison for "publicly insulting" Erdogan in a satirical poem she posted on her Instagram account.
The punishment has been suspended, it added, without giving further details. The "Master's Poem" — which was shared by the model while Erdogan was serving as prime minister — criticises the Turkish strongman with verses adapted from the national anthem.
Ironically, Erdogan was himself sent to jail in 1999 for reciting, while mayor of Istanbul, an Islamist-tinged poem that the then authorities deemed was guilty of "inciting religious hatred".
2,000: The number of people who have been prosecuted for insulting Erdogan since he became president


text: Filigree Silk-Jacquard Cocktail Dress 17FN651FLJ Regular Price: $2,990 Special Price $1,495 Out of stock - Notify me when this product becomes available. Add to Wishlist This jewel-tone cocktail dress perfectly captures Oscar de la Renta's timeless elegance. Cut from filigree silk-jacquard, it's designed with elongated short sleeves, a covered front zip and a coordinating self-belt that highlights a small waist. We love how gold jewelry complements the vivid emerald hue. • Front zip closure • Includes self-belt • Two pockets • 23" skirt • 74% polyester / 17% silk / 9% acrylic • Made in Italy • Dry clean • Free ground shipping & returns on full-price U.S. orders Style# 17FN651FLJ Share Contact us


text:
Dean Karnazes will run 3 routes through Arcadia and Messinia to help promote the awareness, and the benefits of the healthy diet of the region. The main focus will be on educating children in Greece, the United States and the UK about the issues of childhood obesity. Dean will essentially be "coming home to Greece" to reconnect with his roots.
His passion is to fight and end childhood obesity. His goal is to show the world (especially children and their parents) an example of how a Messinian diet can help you lead a healthier, longer and more productive life. It should also be pointed out the fact that children in Messinia are not obese and do not have the kinds of health problems, such as diabetes, that you would normally find in the US and UK.


text:
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Reasons Behind the American Bill of Rights and Different Amendments
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text:
Research on Alzheimer's disease has taught us that the disease is complex. There is little overall understanding of how Alzheimer's disease starts and then develops. Despite this lack of understanding, once we observe the decline in cognitive capacity there seems to be a fast and unrelenting pattern of diminished capacity in the patient. The symptoms are real regardless of our lack of knowledge of its causes and development. Very slowly we are moving away from the shock and awe of the disease to looking at patterns and associations.
Although there is closer correlation of Alzheimer's disease among twins that share the same genetic makeup—monozygotic/identical in contrast to dizygotic twins—there is nevertheless still a difference among identical twins. This variance indicates that non-genetic factors are playing a role in the expression of Alzheimer's disease. Aging by itself is associated with epigenetic influences, with younger twins being more epigenetically similar than older twins. Such non-genetic factors might also explain why women are more prone to Alzheimer's disease then men suggesting an important role for external factors.
The interconnections that are being made today across diseases—e.g. amyloidosis—and the connection between genes and geography—e.g. epigenetics—all point towards the need for a unified theory of Alzheimer's disease. There are emerging attempts to do this through a public health approach.[1] But this needs to be broadened further and some of the processes involved in promoting the disease need to be further elucidated. The main theme emerging from the periphery of research is a renewed interest in the importance of emotions in the maintenance of diseases. Although the main reason for this interest might be the observation that emotions have biological repercussions, nevertheless this points to a pivotal change in perception of how we view diseases. We are looking at diseases as involving broader facets—e.g. emotions—as well as longer periods—e.g. development.
The "Barker Hypothesis" complements this view, that early childhood trauma--initial focusing on low birth weight—starts a cascade of negative events that are only expressed in adulthood or late adulthood. [2] The realization that chronic diseases, including Alzheimer's disease, have developmental origins, has started to attract a life-course approach. Diane Miller and James O'Callaghan with the CDC-National Institute for Occupational Safety and Health explored this hypothesis for Alzheimer's disease identifying certain childhood conditions that can contribute to Alzheimer's disease. [3] Some researchers are also examining specific mechanisms for this chronic disease process. Debomoy K. Lahiri with Indiana University School of Medicine and his colleagues have proposed an epigenetic model of chronic disease. In this model, environmental agents (e.g., heavy metals), intrinsic factors (e.g., inflammatory cytokines), and dietary factors (e.g., folate and cholesterol) could alter gene expression through epigenetic influences. These changes that occur during childhood will not be expressed until significantly later in life.[4]
There are other factors that change epigenetics. Adverse childhood experiences—economic hardship, sexual and physical abuse, divorce, homelessness, hunger—initiate a cascade of risk behaviors that are associated with enduring changes in the body and brain—nervous, endocrine, and immune systems. [5] And adverse childhood experiences are common, with rates between 5–35% for physical abuse, 4–9% for severe emotional abuse,15–30% for sexual abuse in girls and 5–15% in boys, and 6–12% for neglect. In a 1997 study of over a thousand Californian elderly community residents the Australian epidemiologist John Lynch with Adelaide University and his colleagues found that self-reported cognitive function was lower than expected for those who had experienced sustained economic hardship, even after adjusting for age, sex, and co-morbidity. [6] Poverty, larger family size and urban residence are also associated with increased Alzheimer's disease risk. [7]
Developmental considerations are important since the brain is an evolving structure and is more vulnerable at certain times than others. In brain development for example, we start to see grey matter loss at age seven which continues throughout older age. In contrast, glial cells in the white matter increase until approximately the mid-40 and peaking at around age 50. [8] Emotional trauma at these vulnerable stages have been shown to have long term effects on the developing brain. There is some support for this view from studies that report that people who engage in mentally stimulating activities at both younger and older ages are less likely to develop Alzheimer's disease.
These epigenetic changes can also be transmitted to the next generation. Some adverse childhood events continue to have a negative effect on later-life cognitive performance on some people. In contrast there are others who seem resilient to this negative effect. Such variance underlie the necessity to consider events individually and not as global test scores.[8] We are still a long way from undertaking experiments that exposes the true nature of Alzheimer's disease, but at least we are starting to define the horizon. A horizon outside of the research cul de sac that has been defined by the federal government's Alzheimer's disease research guidelines.
[1] Garrett MD, Valle R (2015) A New Public Health Paradigm for Alzheimer's Disease Research. SOJ Neurol 2(1), 1-9.
[2] Barker DJP. The origins of the developmental origins theory. J Intern Med 2007;261:412-7. [3] Hall S. Small and thi
[3] Miller, D. B., & O'Callaghan, J. P. (2008). Do early-life insults contribute to the late-life development of Parkinson and Alzheimer diseases? Metabolism, 57, S44-S49.
[4] Maloney, B., Sambamurti, K., Zawia, N., & K Lahiri, D. (2012). Applying Epigenetics to Alzheimer's Disease via the Latent Early–life Associated Regulation (LEARn) Model. Current Alzheimer research, 9(5), 589-599.
[5] Danese A, McEwen BS. Adverse childhood experiences, allostasis, allostatic load, and age-related disease. Physiology & Behavior. 2012;106(1): 29-39. doi: 10.1016/j.physbeh.2011.08.019.
[6] Lynch JW, Kaplan GA, Shema SJ. Cumulative impact of sustained economic hardship on physical, cognitive, psychological, and social functioning. N Engl J Med. 1997;337(26):1889–1895.
[7] Moceri VM, Kukull WA, Emanuel I, et al. Early-life risk factors and the development of Alzheimer's disease. Neurology. 2000;54(2):415–420.
[8] Sowell ER, Thompson PM, Toga AW. Mapping changes in the human cortex
throughout the span of life. Neuroscientist 2004; 10:372–392.
[9] Ritchie K, Jaussent I, Stewart R, Dupuy AM, Courtet P, Malafosse A, et al. Adverse childhood environment and late-life cognitive functioning. International journal of geriatric psychiatry. 2011;26(5):503- 510. doi: 10.1002/gps.2553.
© USA Copyrighted 2016 Mario D. Garrett


text:
|Use this URL to cite or link to this record in EThOS:
|The approximation of Cartesian coordinate data by parametric orthogonal distance regression.
|Turner, David Andrew.
|Awarding Body:
|University of Huddersfield
|Current Institution:
|University of Huddersfield
|Date of Award:
|Availability of Full Text:
This thesis is concerned with the approximation of Cartesian coordinate data by parametric curves and surfaces, with an emphasis upon a technique known as parametric orthogonal distance regression (parametric ODR). The technique has become increasingly popular in the literature over the past decade and has applications in a wide range of fields, including metrology-the science of measurement, and computer aided design (CAD) modelling. Typically, the data are obtained by recording points measured in the surface of some physical artefact, such as a manufactured part. Parametric ODR involves minimizing the shortest distances from the data to the curve or surface in some norm. Under moderate assumptions, these shortest distances are orthogonal projections from the data onto the approximant, hence the nomenclature ODR. The motivation behind this type of approximation is that, by using a distance-based measure, the resulting best fit curve or surface is independent of the position or orientation of the physical artefact from which the data is obtained. The thesis predominately concerns itself with parametric ODR in a least squares setting, although it is indicated how the techniques described can be extended to other error measures in a fairly straightforward manner. The parametric ODR problem is formulated mathematically, and a detailed survey of the existing algorithms for solving it is given. These algorithms are then used as the basis for developing new techniques, with an emphasis placed upon their efficiency and reliability. The algorithms (old and new) detailed in this thesis are illustrated by problems involving well-known geometric elements such as lines, circles, ellipse and ellipsoids, as well as spline curves and surfaces. Numerical considerations specific to these individual elements, including ones not previously reported in the literature, are addressed. We also consider a sub-problem of parametric ODR known as template matching, which involves mapping in an optimal way a set of data into the same frame of reference as a fixed curve or surface.
|Not available
|Not available
|Qualification Name:
|Thesis (Ph.D.)
|Qualification Level:
|EThOS ID:
|Not available
|ODR; Projections; Template matching; Curves


text:
|Publication number
|Publication type
|Application number
|PCT number
|Publication date
|12 Jul 2005
|Filing date
|30 Dec 1998
|Priority date
|30 Dec 1997
|Fee status
|Also published as
|Publication number
|Ib Mendel-Hartvig, Jörgen Gustafsson
|Original Assignee
|Pharmacia Diagnostics Ag
|Export Citation
|BiBTeX, EndNote, RefMan
|Patent Citations (25), Referenced by (25), Classifications (15), Legal Events (8)
|External Links: USPTO, USPTO Assignment, Espacenet
The invention relates to a method associated with a process for the determination of an analyte in a sample, which process involves utilizing biospecific affinity reactions. The method includes the steps of:
Analyte′ is the analyte as such (in the sample) or an analyte related reactant, i.e. an added biospecific affinity reactant, included in the complex in an amount which is related to the amount of analyte in the sample. Reactant* and Reactant I can bind Analyte′ at the same time. This means that they bind to spatially separated binding sites.
This type of analytical methods has been carried out in so-called flow matrices, whereby reactants including analyte are transported in a process flow through the matrix (=flow methodology) to a detection zone (DZ) where Reactant* is captured in an amount related to the amount of analyte in the sample. Capture occurs via a reactant (Capturer) which is firmly anchored to the matrix in DZ. That is, Capturer is bound via bonds which are stable under the conditions used to capture Reactant* in the detection zone. The Capturer may be Reactant I or a reactant which has biospecific affinity to Reactant I or to another reactant, which in turn, optionally via one or more additional reactants, has biospecific affinity to Reactant I.
By reactants (including analyte) exhibiting biospecific affinity (bioaffine reactants) is meant individual members of the reactant pairs: antigen/hapten-antibody; biotin-avidin/streptavidin; two complementary single chains of nucleic acid etc. As antibodies, antigen binding antibody fragments such as Fab, F(ab)2′, single chain Fv antibodies (scFv) etc. are considered. The reactants in question need not be naturally occurring but can also be synthetically prepared molecules/binders.
The type of test methods in question has previously been used primarily for biospecific affinity reactants where at least one part of an employed reactant pair has exhibited protein structure, in particular in connection with so-called immunochemical determination procedures.
The biospecific affinity reactions are primarily performed in aqueous media (such as water).
Previously used calibrators
Conventionally, the calibrator and analyte have often both been able to bind to Reactant*. The binding sites in question on the calibrator for binding to Reactant* often having binding properties equivalent to corresponding binding sites on the analyte. In practice this means that the calibrator and the analyte have had binding sites which are structurally equal or similar, and cross-react with each other with respect to Reactant*. Binding sites which cross-react with each other for/about a given reactant are equivalent.
Calibrator amount has in the prior art commonly been equated with standard amount.
Calibrator values, corresponding to different analyte amounts/concentrations (standard amounts), have often been compiled to a dose-response curve (calibration curve) or an algorithm.
The expression "to compare a sample value with calibrator value(s)" has also encompassed that the comparison may take place with a calibration curve and/or algorithm, corresponding to several calibrator values.
The calibrator and the analyte have often been the same substance. There are exceptions. In antibody determination one and the same calibrator has often been operational for several antibody specificities, provided that the calibrator substance has been selected such that it exhibits a constant domain of the antibody to be determined. See for example Abbott WO 97/27486.
Disadvantages of the prior art
The prior art has usually involved determination of several calibrator values in parallel with samples by running known amounts of analyte (standard amounts) in a way corresponding to samples. This has in turn led to 5-20% of all runs having been calibrator runs. By reducing the number of calibrator runs, possibly also by reducing the number of reaction steps in each calibrator run, time and consumption of reagent could be saved.
Often problems occur depending on calibrator and sample solutions having different properties and contents. This is particularly pronounced in immunological tests where the calibrator often is measured in a buffer, and the analysis of sample is performed on serum or plasma samples. A difference in contents and viscosity yields different responses (i.a. measured as "recovery" and parallelity). In addition the viscosity in a flow method becomes extra important since it influences the migration/flow velocity. This difference can be compensated for but at the same time it renders the systems more sensitive to disturbances, and thus increased inter assay variation. Other problems with tests utilizing flows are possible flow variations depending on temperature and moisture fluctuations etc.
The above problems have to some extent been overcome by the assay method disclosed in EP-A-253,464 and which uses a test zone and a reference zone on a solid phase.
A first object of the invention is to improve the calibration methods presently used in tests of the kind initially mentioned.
Another object of the invention is to simplify the use of calibrators, primarily by reducing the necessary consumption of reagents needed and/or reducing the number of measurements for obtaining calibrator values.
A third object of the invention, in particular in connection with flow methods, is to enable compensation for the differences that may exist between calibrator and sample solution and between runs performed at different times and/or at different places.
We have now realized that these objects can be achieved if the calibrator is bound to the matrix before beginning the determination of calibrator value in accordance with a relevant protocol. This type of calibrator will be referred to below as a matrix calibrator. The first main aspect of the invention is therefore a method in accordance with the procedure mentioned initially, and which has the characterizing feature that the calibrator, or a reactant capable of binding to the calibrator, has been bound to a matrix which is insoluble in the liquid medium in which binding of Reactant* to the calibrator occurs, before beginning the determination of a calibrator value. This means that the calibrator or the calibrator binder, respectively, usually has been bound to the matrix already by the manufacturer, such that the matrix calibrator is delivered as a ready component in a kit. The binding between calibrator and matrix normally is of another kind than that obtained between Analyte′ and Reactant I when running a sample.
Matrix calibrators provide great advantages, if transport of Reactant* to the calibrator occurs by means of a flow (process flow) in a so-called flow matrix to a zone in the matrix, which contains the matrix calibrator or the calibrator binder (calibrator zone, CZ).
When a calibrator binder is bound to the matrix, the calibrator may be either movably (diffusively) pre-deposited in the matrix in a zone separated from the detection zone, or it may be added together with or separately from the sample.
The calibrator binder is usually one member of a specific binding pair (reactant pair), the other member of the binding pair being coupled or conjugated to the calibrator substance. Such specific binding pairs are well-known to a person skilled in the art, and as examples may be mentioned: immunological binding pairs, such as antigen-antibody and hapten antibody, biotin-avidin or -streptavidin, lectin-sugar, hormone-hormone receptor, nucleic acid duplex.
Flow matrices
The flow matrix defines the space in which the reactants are transported. Thus, the matrix may be the inner surface of a single flow channel (such as a capillary), the inner surface of a porous matrix having a system of flow channels (porous matrix) etc. extending through. This type of matrices is called flow matrices. The matrices may exist in the form of monoliths, sheets, columns, membranes, single flow channels having capillary dimensions, or aggregated systems of such flow channels etc. They may also exist in the form of particles packed in column casings, compressed fibers etc. The inner surface of the matrix, i.e. the surface of the flow channels, should be hydrophilic, such that aqueous media (primarily water) may be absorbed and transported through the matrix. The minimum inner dimension of the flow channels (measured as a diameter for channels having a circular cross section) should be sufficiently large for allowing transport through the matrix of the reactants being used. The rule of thumb is that suitable matrices are selectable among those having flow channels with the smallest inner dimension in the interval 0.4-1000 μm, preferably 0.4-100 μm if the matrix has a system of mutually communicating flow channels. Flow channels having a smallest inner dimension in the upper part of the broad interval (up to 1000 μm) are primarily of interest for flows driven by an externally imposed pressure/suction.
Matrices of interest are often built up from a polymer, e.g. nitrocellulose, nylon etc. The material in the matrix as well as the physical and geometrical design of the flow channels may vary along the flow, depending on what a certain part of the matrix is to be used for (Pharmacia AB WO 96/22532; Medix WO 94/15215).
Along the flow in the matrix there may be one or more defined zones for application of sample, reactants, buffer etc. (ASZ, ARZ, ABz etc.), and one or more zones for calibrator and/or detection (CZ and DZ, respectively).
Various flow matrices that may be used in the type of tests in question are described in previous patent publications. See e.g. Behringwerke U.S. Pat. No. 4,861,711, Unilever 88/08534, Abbott U.S. Pat. Nos. 5,120,643 and U.S. Pat. No. 4,740,468, Becton Dickinson EP 284,232 and U.S. Pat. No. 4,855,240; Pharmacia AB WO 96/22532.
Process flow
The direction of the flow is from a zone of application of sample and/or reactant and towards existing calibrator and detection zones (CZ and DZ, respectively). Precisely which zones the process flow is to pass is determined by the test protocol in question. A process flow may start from a point with a radial spread and a flow front in the form of a circular periphery or a part thereof. A process flow may also start from a zone in the form of a band and may have a straight flow front perpendicular to the direction of flow.
In a less preferred variant, the process flow proceeds from an application zone for Reactant*, which at the same time is a calibrator zone or a detection zone. In this variant the flow is preferably radially spread from the zone of application, and may pass additional calibrator zones and/or detection zones.
Flow through the matrices may be achieved by influence from capillary forces, e.g. by starting off with a substantially dry matrix. A sucking body may be placed at the very end of the flow as an aid. By means of an imposed electrical field, dissolved components may be transported from the zone of application to a detection/calibrator zone.
The utilized flow is preferably lateral, i.e. parallel with the upper surface of the matrix. Also other types of flows, such as in depth in the matrix, may be used.
Calibrator and detection zones in flow matrices
The flow matrix used in the preferred embodiment exhibits one or more distinct zones with calibrator (calibrator zones, CZ1, CZ2, CZ3 etc.). Each calibrator zone contains matrix calibrator in an amount such that the measurement signal from Reactant* (calibrator value), detected in the zone when a flow passes, distinctly corresponds to a certain amount of analyte in the sample (standard amount).
The calibrator may be selected in the same way as previously was the case for the types of tests in question. Using flow methodology and arranging for sample (the analyte) to be transported through a calibrator zone, the calibrator should be selected such that it does not bind to the analyte. If the calibrator is able to bind analyte it imposes special requirements on the position of the calibrator zone in relation to the zone of application of sample. See below.
The amount of calibrator that has bound to a calibrator zone does not need to be the same as the corresponding standard amount. This is because the binding activity in relation to Reactant* often is changed, when the sample substance is bound to a matrix.
If it is desirable to determine antibodies with different specificity but from the same species, of the same Ig class or Ig subclass, it is preferred that the calibrator exhibits a binding site which is unique for the species, the class, or subclass. As a rule this means that a calibrator for determination of antibodies exhibits an epitope which is present in a constant domain of the antibodies in question, for mammal antibodies primarily a part of Ig(Fc).
One and the same matrix may exhibit one or more detection zones (DZ1, DZ2, DZ3 etc.) together with one or more calibrator zones. In the detection zone, complexes containing Analyte′ and Reactant* bind to the matrix via the initially mentioned Capturer, which is firmly fixed in a DZ. If Reactant I binds to the matrix via the Capturer, Reactant I need not be immobilized in the matrix from the start but may either be movably (diffusively) pre-deposited in the matrix in an area or zone separated from the detection zone, or it may be added together with or separately from the sample.
If there are several calibrator and/or detection zones in the same flow matrix, the greatest advantages with the invention are achieved if several of the zones are located along the same process flow.
If there are several detection zones (DZ1, DZ2, DZ3 etc.) in one and the same matrix, these may correspond to different analytes. One can utilize the same calibrator for analytes having equivalent binding sites. If the analytes lack equivalent binding sites one calibrator is required for each analyte. If all analytes have the same equivalent binding site the simpliest condition will be at hand. The same calibrator, the same calibrator zones and the same Reactant* may then be utilized for all analytes.
Calibrator zones and detection zones may be geometrically designed in various manners (rectangular, circular, linear, dot-shaped etc.). The zones may have different configurations relative to each other. Good configurations are such wherein a common flow consecutively or simultaneously penetrates several zones, in particular zones of different kinds (DZ and CZ). An example of consecutive penetration is parallel zones located after each other in the same process flow. An example of simultaneous penetration is zones located next to each other on the same circular periphery, where the process flow is radially spread from the centre of the corresponding circle. Combinations of these variants may be used, i.e. apart from zones on a circular periphery there are also zones on the periphery of circles which are concentric with the first-mentioned circular periphery. Simultaneous penetration may also be achieved with a straight flow front having detection and calibrator zones located next to each other at the same distance from the starting point of the process flow.
If several detection zones and/or calibrator zones are located in the same process flow, a measurement signal for these zones may be obtained in one and the same test run/reagent application. If there are several calibrator zones in the same process flow, a dose-response curve (calibration curve) or algorithm may be set up for the values obtained for the same application of Reactant*. A calibrator zone that exists together with a detection zone in the same flow may function as a positive internal calibrator (PIC).
In one variant a matrix is utilized exhibiting at least one calibrator zone (CZ1, CZ2, etc. (positive internal calibrators)) and at least one detection zone (DZ1, DZ2, etc.) in combination with one or more separately obtained calibrator values. The separately obtained calibrator values need not refer to the same conditions under which the sample is to be run. To the extent separate matrix values, calibration curve and algorithm are intended to be used during a longer period of time, reference is made to master values, master curve and master algorithm, respectively.
The use of separately obtained calibrator values involves:
Alternatively, one may adapt the separate calibrator values to deviations in conditions and then directly compare a measured sample value with adapted calibrator values. This is equivalent to the steps (iv) and (v) above (called vice versa in claims). In the steps (iv) and (v) it is, of course, included as an alternative to adapt the corresponding calibration curve or algorithm in order to calculate the level of analyte by comparing the sample value with either of these.
What has been said above, applies, of course, also to the case that a binder for the calibrator has been bound to the calibration zone(s) of the matrix.
A calibrator and detection zone in the same process flow will reduce previous sources of error having been caused by differences in sample and calibrator. A positive internal calibrator and several calibrator zones in the same process flow will completely or partly compensate for variations in flows between separate runs. The spread in the measurement result should be lower while internal as well as external factors may be compensated for completely or partly. The problem with sample and calibrator having different compositions is eliminated. For near patient tests, the internal calibrator will be able to provide a well defined limit as to what constitutes a positive response, and to provide the quality assurance which today is missing for these types of tests.
The anchoring of the calibrator to the matrix may take place via covalent bonding or via physical adsorption, biospecific affinity etc. Like prior art in this field the invention may utilize combinations of binding types, such as covalent bonding to the matrix of a biospecific affinity reactant directed to the calibrator. Specifically, physically adsorbed or covalently bonded streptavidin in combination with a biotinylated calibrator may be mentioned, or a similarly bound antibody directed to the calibrator. Anchoring of the calibrator to the matrix may take place via particles having been deposited in/on the matrix, and to which the calibrator is covalently, physically adsorptively or biospecifically etc. bound. The particles attach to the matrix either because their size has been selected such that they cannot be transported through the matrix, or via physical adsorption. See i.a. Abbott/Syntex U.S. Pat. No. 4,740,468; Abbott EP 472,376; Hybritech EP 437,287 and EP 200,381; Grace & Co EP 420,053; Fuji Photo Film U.S. Pat. No. 4,657,739; Boehringer Mannheim WO 94/06012.
The Capturer may be bound to a detection zone according to the same principles as those applying to a calibrator. In one and the same process flow a calibrator and Capturer may be bound to their respective zones in the same way or in different ways. What has been said above concerning the anchoring of the calibrator and the Capturer, is, of course, also applicable to the anchoring of a binder for the calibrator substance. For example, the above mentioned combination of a biotinylated calibrator substance and physically or covalently bound streptavidin may be used.
Zone of application of sample (ASZ)
The zone of application of sample may be located upstream or downstream in relation to calibrator zones, preferably upstream. In case the matrix calibrator has been selected such that it binds analyte, the zone of application of sample must be located downstream of the matrix calibrator. In relation to detection zones the zone of application of sample should always be located upstream in useful embodiments.
In certain less preferred embodiments it is conceivable to apply sample in a calibrator or detection zone.
Zone of application of Reactant* (AR*Z) and other biospecific affinity reactants (ARZ)
An application zone for Reactant* (AR*Z) should always be located upstream of the calibrator zones.
If there is a detection zone in the process flow, the order of the zones of application of biospecific affinity reactants should ensure that Analyte′ is transported into its detection zone before or simultaneously with Reactant*. One or more reactants may be added in the same zone of application. If the zone of application is common to sample and at least one reactant, let us say Reactant*, application may occur simultaneously, e.g. by having mixed a sample and a reactant before they are applied in the zone. If desired, the mixture may be preincubated such that the reactant will bind to the analyte or to other components in the sample, as intended, before application of the sample. Having knowledge of various protocols, the skilled person will be able to easily determine which zones he needs and the possible order thereof.
If Reactant I is present in dissolved form, the matrix has a zone of application for it at the same time as there is a Capturer firmly fixed in the detection zone. If the Capturer requires additional biospecific affinity reactants in order to bind Reactant I (see under "Technical field"), there are zones of application for these reactants. Zones of application for Reactant I, when it is not a Capturer, and any additional reactants must be positioned such that Reactant I reaches the detection zone before or at the same time as Analyte′. If Reactant I is in soluble form, the Capturer may preferably be one member of a specific binding pair, the other member of which is coupled or conjugated to Reactant I. Exemplary specific binding pairs are immunological binding pairs, such as antigen-antibody and hapten-antibody, biotin-avidin or -streptavidin, lectin-sugar, hormone-hormone receptor, nucleic acid duplex.
If both the calibrator and Reactant I are in soluble form to then bind to the matrix via specific binding pairs, these two binding pairs are, of course, different.
In certain less preferred embodiments biospecific affinity reactants (inclusive Reactant*) may be applied in a calibrator or detection zone. See under the heading "Process flow".
Reactants utilized in the method may be predeposited in their respective zone or may be added in connection with performing the method of determination. Predepositing involves application of the reactant in question in advance in such a way that it will not spread outside its zone of application until a flow of liquid is initiated in or passes the zone.
Predeposition of reactants may take place by methods known per se. See for example (Behringwerke U.S. Pat. No. 4,861,711; Unilever WO 88/08534; Abbott U.S. Pat. No. 5,120,643; Becton Dickinson EP 284,232). It is important to take into consideration the fact that a predeposited reactant should easily dissolve when liquid passes through the zone of application in question. In order to achieve quick dissolution it is common to incorporate/codeposit reactants in/with substances that quickly dissolve. This type of substances are often hydrophilic having polar and/or charged groups, such as hydroxy, carboxy, amino, sulphonate etc. In particular there may be mentioned hydrophilic quickly soluble polymers, e.g. having carbohydrate structure, simple sugars including mono-, di- and oligosaccharides and corresponding sugar alcohols (mannitol, sorbitol etc.). It is common practice to first coat the zone of application in question with a layer of the quickly soluble substance, whereupon the reactant is applied, possibly followed by one additional layer of quickly soluble substance. An alternative way is by incorporating the reactant in particles of quickly soluble material, which then is deposited in the zone in question of the matrix.
Zones for buffer (ABZ)
Buffer systems that are required may be included in solutions added simultaneously with samples and reactants. In conventional techniques addition of buffer takes place in the zone of application that is located upstream of all other zones of application. This has often been equal to the sample application zone. In the present invention buffer may in principle be added in an optional position along the flow of transport. See below.
In a co-pending PCT application "Analytical method comprising addition in two or more positions and a device and test kit therefor" (based on SE 9704934-0) there is disclosed an invention, which in one variant provides a preferred embodiment of the present invention. This application is hereby incorporated by reference in the present text. The invention in this separate patent application is based on the discovery that liquid from two subsequent zones (AZ2 and AZ1) in a flow matrix may migrate after each other without mixing. This will be achieved if liquid is applied to the zone (AZ1) located downstream before or essentially simultaneously with application of liquid to the zone (AZ2) located upstream. This discovery has led to the ability to achieve zonewise migration of any reactants present in the liquids, towards a detection zone. If the zone of application of sample (ASZ) is located downstream of the zone of application of Reactant* (AR*Z), and if liquid is applied to AR*Z and sample to ASZ, the analyte may migrate into the detection zone before the liquid containing Reactant* does. If there is one zone of application for liquid alone (buffer) (ABZ) between AR*Z and ASZ, a wash of the detection zone DZ is obtained between capture of analyte and Reactant*. Such an intermediate buffer zone (ABZ) may also ensure that a reactant (including analyte), that is applied in a zone located downstream, reaches DZ before a reactant, starting from a zone of application located upstream. The latter may be important if the matrix as such retards the reactant that has been applied in the zone located downstream.
Reactants may be included in the liquid that is applied to a zone. Alternatively they may be pre-deposited in the zone where the corresponding liquid is to be applied, or in a zone that is located between this and the nearest zone that is located downstream, for application of liquid. Sample (the analyte) normally is applied in the form of liquid.
This embodiment of the invention is particularly interesting for sequential methods of the type is question in flow matrices, i.e. methods wherein the matrix in addition to a calibrator zone also contains a detection zone, and where the sample/analyte is to be transported into the detection zone before liquid containing Reactant*.
Analytically detectable reactant (Reactant*)
Usually analytical detectability of a reactant is obtained because it comprises an analytically detectable group. Well-known examples of often used groups are enzymatically active groups (enzyme, cofactor, coenzyme, enzyme substrate etc.), fluorophore, chromophore, chemiluminescent, radioactive groups etc. Groups being detected by means of a biospecific affinity reactant are also usually referred to this category, e.g. biotin, hapten, Ig-class, Ig-subclass and Ig-species specific determinants etc. In this invention, particles the surfaces of which have been coated with a biospecific affinity reactant have proved to be particularly good. The particles may contain any of the previously mentioned detectable groups, such as fluorophore groups, or they may be coloured (=containing chromogenic groups). Useful particles often have a size in the interval 0.001-5 μm, preferably 0.01-5 μm. The particles may be spherical and/or monodisperse or polydisperse. They may have colloidal dimensions, so-called sol (i.e. usually spherical and monodisperse having a size in the interval 0.001-1 μm). Well-known particulate label groups are metal particles (such as gold sol), non-metal particles (such as SiO2, carbon, latex and killed erythrocytes and bacteria). In certain cases it has been emphasized that the particles should be non-sedimentable under the utilized conditions. (See Pharmacia AB, WO 96/22532).
See also Unilever, WO 88/08534; Abbott, U.S. Pat. No. 5,120,643; Becton Dickinson, EP 284,232.
In connection with the development of matrix calibrators we have surprisingly found that good results may be obtained if one simultaneously utilizes:
We have achieved a functioning system wherein label particles and anchoring particles have had substantially the same dimensions, which means that in all probability the label particles may be larger than the anchoring particles and vice versa, as long as they remain smaller than the flow channels defined by the matrix. The system may function with as well as without predeposition of Reactant*. This embodiment is described in more detail in a co-pending PCT-application "Analytical method using particles and test kit for performing the method" (based on SE 9704935-7). Also this latter application is incorporated by reference. Applied to the present invention this means that Reactant* has particles as an analytically detectable group according to a above, and that the calibrator and/or the Capturer binds to the matrix via particles according to b above.
Relevant test protocols
The invention may primarily be applied to non-competitive (non-inhibition) test variants, but also to competitive (inhibition) test variants, if these involve that a complex is formed with an analyte-related reactant bound between Reactant I and Reactant*. The protocols may be run as simultaneous or sequential variants. By simultaneous methods is meant that Reactant* and Analyte′ are co-transported during at least a part of the transport towards the detection zone, and preferably reach the latter simultaneously. By sequential method is means that Analyte′ during at least a part of the transport towards the detection zone migrates in front of Reactant*, and preferably reaches the detection zone before Reactant*. Illustrative examples are given below. "-" relates to firm anchoring to the matrix from the start. " - - - " relates to binding via biospecific affinity. It has been assumed that the reactants are monofunctional with regard to the binding sites being utilized.
A. Sandwich protocol: Reactant I (=Capturer) and Reactant* have biospecific affinity to the analyte (=Analyte′). x is the number of moles of Reactant I on the matrix. y is the number of moles of Analyte′ (=moles of Reactant*) that has been captured on the matrix via Reactant I.
B. Sandwich protocol: Reactant II (=Capturer) has biospecific affinity to Reactant I, which in turn has biospecific affinity to the analyte (=Analyte′). Reactant* has biospecific affinity to the analyte. x is the number of moles of Reactant II on the matrix. y is the number of moles of Analyte′ (=moles of Reactant*) that has been captured on the matrix via Reactant II - - - Reactant I. z+y is the number of moles of Reactant I that has been captured on the matrix via Reactant II.
C. Protocol of inhibition type: Reactant I is an analyte analogue (=Capturer) and has binding sites that are equivalent with the binding sites on the analyte. Analyte′ is a reactant that has biospecific affinity to the analyte and to Reactant I. Reactant* has biospecific affinity to Analyte′. Analyte′ is included in the formed complex in an amount that is related to the amount of analyte in the sample. x is the number of moles of Reactant I on the matrix. y is the number of moles of Analyte′ (=number of moles of Reactant*) that has been captured on the matrix via Reactant I.
The invention is primarily adapted for determination of bio-specific affinity reactants (analytes) of the types mentioned initially. Great advantages are obtained for analytes occurring in multiple forms, which have as a common denominator at least one binding site with equivalent binding properties.
For non-competitive methods (sandwich) the analyte may be an antibody directed to an antigen (including allergen), or hapten (Test protocols A and B above). Reactant I in this case is the antigen or the hapten to which the antibody is directed, and Reactant* is an antibody directed to the analyte. Alternatively Reactant* is the antigen or the hapten, and Reactant I is an antibody directed to the analyte. For non-competitive methods the analyte may also be an antigen, Reactant* and Reactant I being antibodies directed to the antigen. As examples of analyte-antigen may be mentioned immunoglobulin, possibly of a particular Ig class or Ig subclass. When the analyte is an antibody or an immunoglobulin, Reactant* and Reactant I, respectively, may exhibit biospecific affinity towards an Ig determinant that is specific for an Ig class such as IgA, IgD, IgE, IgG or IgM and/or for a subclass if present (e.g. IgG1, IgG2, IgG3 or IgG4), and/or for a certain species. This means that Reactant* and Reactant I, respectively, normally is an antibody exhibiting some of these specificities when the analyte is an antibody or an immunoglobulin.
Competitive variants are primarily applicable to low molecular analytes. In the test protocol C above the analyte may be an antigen/hapten, in which case Reactant I is the antigen/hapten bound to the matrix, Analyte′ is an antibody directed to the antigen/hapten, and Reactant* is an antibody directed to Analyte′.
It has been particularly interesting for the inventors to be able to measure analytes the occurrence and/or amount of which being related to autoimmune diseases and allergy. It is particularly interesting to measure anti-allergen antibodies of IgE of IgG class, for the latter preferably with emphasis on some of the mentioned subclasses. Measurement of allergen specific antibodies may be employed in connection with diagnosing of IgE mediated allergy.
Relevant samples may be of biological origin, e.g. from different body fluids (whole blood, serum, plasma, saliva, urine, tear liquid, cerebrospinal fluid etc.), extracts from biological tissue, from cell culture media, processing procedures in biotechnology, from foodstuff, from the environment (environmental analysis samples) etc. The samples may be pretreated in order to fit e.g. the matrix, the test protocol involved etc.
A second aspect of the invention
This aspect of the invention relates to a test device where the matrix calibrator constitutes a central point. The matrix calibrator is used in analytical methods for transferring measured signal values (sample values) for a complexed, analytically detectable reactant (=Reactant*) to real amounts of analyte in a sample, in connection with performing an analytical method utilizing biospecific affinity reactions. As in the method aspect Reactant* is complexed in an amount that is related to the amount of analyte in a sample. The most important type of analytical methods for which the device may be used are those for which the method of the invention is used, that is methods where one forms complexes comprising Reactant I - - - Analyte′ - - - Reactant*. Reactant I, Analyte′, Reactant* and - - - have the same meanings as in the method aspect.
The device is characterized by exhibiting:
If the calibrator zone/zones instead of the calibrator contains a binder for calibrator substance, the device preferably also contains:
The process flow may also contain (a) a detection zone (DZ) located downstream or coinciding with AR*Z, and in which there is a firmly fixed Capturer via which Reactant* may bind to DZ, and (b) a zone of application for sample (ASZ) located upstream or coinciding with said DZ. AR*Z may be located upstream or downstream or coincide with ASZ (if present), preferably upstream or downstream. If ASZ and DZ are present in the same process flow as the calibrator zone, ASZ is preferably located upstream and DZ preferably downstream of existing calibrator zones.
In preferred embodiments the firmly anchored reactant (Capturer) has biospecific affinity to the analyte or to an analyte-related reactant that may be analytically detectable. Analyte related reactant is primarily relevant to competitive test variants.
Calibrator substance is selected in the same way as in the method aspect of the invention. In those cases where the selected calibrator substance exhibits biospecific affinity to the analyte, the corresponding calibrator zone shall be located upstream of ASZ.
Additional details regarding calibrators, zones, reactants, matrices, process flows, test protocols, samples etc. are apparent from the description of the method aspect of the invention.
The invention will now be illustrated with a number of examples showing various preferred embodiments thereof. The invention is defined by the attached claims and what is disclosed in the description.
Methods and materials
Adsorption of phenyldextran to polystyrene particles: Phenyldextran (degree of substitution: 1 phenyl group on each fifth monosaccharide unit=20%, Mw dextran 40,000, Pharmacia Biotech AB, Uppsala, Sweden) was adsorbed to polystyrene particles (0.49 μm Bangs Laboratories, USA) by incubations under stirring with phenyldextran dissolved in deionized water to 1) 5 mg/ml, 10% particle suspension, RT 1 h, 2) 5 mg/ml, 5% particle suspension, RT 1 h, 3) 20 mg/ml, 1% particle suspension, RT overnight 15 h. The particles were subsequently washed twice with deionized water. The particle suspensions were centrifuged between each incubation and wash (12,100×g, 25 min, Beckman, J-21, JA-20, 10,000 rpm). The particle suspension was finally sonicated (Ultrasonic bath, Branson 5210, 5 min).
Coupling of human IgE (hIgE) to polystyrene particle (=hIgE particles): Human IgE was coupled to phenyldextran coated polystyrene particles with CDAP (1-cyano-4-dimethylamino-pyridinium bromide (Kohn J and Wilchek M, FEBS Letters 154(1), (1983) 209-210).
Desalting and change of buffer of hIgE were performed by gel filtration (PD-10, Pharmacia Biotech AB, Sweden) in NaHCO3, 0.1 M, pH 8.5. 278 mg of polystyrene particles (as above) in 2% solution in 30% (by volume) acetone were activated with 4.2 ml CDAP (0.44 M) and 3.4 ml TEA (0.2 M triethylamine, Riedel-de Haen, Germany). CDAP was added during stirring for 60 s and TEA during 120 s. The particles were washed with 30% (by volume) acetone and centrifuged at 12,100×g (25 min, Beckman, J-21, JA-20, 10,000 rpm). 25 mg of hIgE were coupled to the activated particles in incubation with stirring overnight at +4° C. Then the particles were centrifuged before deactivating with glutamic acid 0.05 M and aspartic acid 0.05 M in NaHCO3 buffer. Incubation was performed with stirring overnight at +4° C. Coupled particles were washed with 0.1 M NaHCO3 and twice with 20 mM borate buffer pH 8.5. The particle concentration was determined spectrophotometrically at A600 nm with untreated particles as reference. Concentration of coupled protein was determined by having radioactive human IgE present during coupling.
Extraction of t3 (birch pollen, Betula verrucosa): 1 part (weight) of birch pollen (Allergon, Sweden) was extracted with 10 parts (volume) 0.1 M of phosphate buffer (denoted 1/10), pH 7.4. The extraction lasted for 2 hours on a shaker table at +4° C. The extract was centrifuged at 4000 rpm for 1.75 h. After filtering the solution was applied to a PD-10 column and eluted in 0.1 M NaHCO3, pH 8.5 (denoted 1/14).
Coupling of t3 extract to a polystyrene particle (t3 particles: t3 extract (1/14) was coupled with CDAP (Kohn and Wilchek, FEBS Letters 154(1) (1983) 209-210) to polystyrene particles coated with phenyldextran. Polystyrene particles (400 mg, coated with phenyldextran as above) in 30% (by volume) acetone, 2% particle suspension, were activated with 60 mg of CDAP (100 mg/ml in 30% acetone) and 0.48 ml 0.2 M triethylamine (Riedel-de Haen, Germany). CDAP was added with stirring and TEA was added dropwise during 90 seconds and stirring for 120 s in total. The reaction was quenched by the addition of 30% acetone (4 times volume) and centrifuging at 12,400×g for 35 min. The particles were washed once with deionized water. 32 ml of t3 extract in 0.1 M NaHCO3, pH 8.5, were added to 80 mg of the activated particles and coupling was continued for 1 hour on a shaker table. Then the particles were centrifuged before they were deactivated with 0.05 M aspartic acid och 0.05 M glutamic acid in 0.1 M NaHCO3, pH 8.5. Incubation on shaker table overnight at +4° C. The particles were washed by centrifuging in 1) 0.1 M NaHCO3, 0.3 M NaCl, pH 8.5; 2) 0.1 M Na acetate, 0.3 M NaCl, pH 5; 3) 0.1 M NaHCO3, pH 8.5; and 4) 20 mM Na borate, pH 8.5.
The particle concentration was determined spectrophotometrically at 600 nm with uncoated polystyrene particles as reference.
Adsorption of anti-human IgE antibody to carbon particles (carbon particle conjugate=Reactant*): Monoclonal anti-hIgE was adsorbed to carbon particles (sp100 from Degussa, Germany). See Pharmacia AB, WO 96/22532. The ready suspension was diluted with buffer to 400 μg of carbon particles per ml.
Deposition of t3 particles on membrane in a detection zone: One sheets on nitrocellulose with polyester backing (Whatman, 8 μm, 5 cm wide) 4% of the above-mentioned t3-coupled particles were applied with Linear Striper (IVEK Corporation) with a flow of 1 μl/s and 1 μl/cm as a straight zone. The sheets were dried for 1 hour, 30° C., whereupon the sheets were cut at right angles relative to the zone to 0.5 cm wide strips (Matrix 1201 Membrane Cutter, Kinematics Automation).
Deposition of hIgE particles in calibrator zones: On nitrocellulose sheets with polyester backing (Whatman, 8 μm, 5 cm wide) hIgE particles were deposited as parallel calibrator zones with Linear Striper (IVEK Corporation, USA). The flow was 1 μl/s and 1 μl/cm. Sheets intended for strips having only calibrator zones were coated with six parallel zones. hIgE concentrations in the zones were 0, 0.84; 3.4; 14; 54.2 and 217 ng hIgE/0.5 cm. Before performing the deposition the hIgE particles were diluted in borate buffer (20 mM, pH 8.5, Dextran T5000 4.2% w/w, sorbitol 5.8% w/w). All zones also comprised 1% phenyldextran-coated particles in order to yield the same amount of particles in each zone. On a separate nitrocellulose sheet there was deposited a zone with hIgE particles (14 ng hIgE/0.5 cm, PIC=positive internal calibrator), and in a parallel zone t3 particles as above (detection zone). The deposition took place with the same parameter as for hIgE particles. The sheets were dried 1 h, 30° C., and were then cut, perpendicularly relative to the zones, to strips 0.5 cm wide (Singulator: Matrix 1201 membrane cutter, Kinematic automation, USA).
Test procedure: Strips were mounted on a plane plastic surface. At the top (0.5 cm) on the strip a sucking membrane was placed (width 3 cm, Whatman, 17 Chr). To obtain a constant pressure metal weights were put on the sucking membranes. 10 mm from the lower edge a 2 mm wide Inplastor strip was mounted (preglued polyester film). The Inplastor strip should prevent applied liquids from flowing out over too large a portion of the membrane. To the lower end of the strip there was applied 30 μl of sample or buffer in the alternative. After suction of the sample volume the following components were applied in the given order: 15 μl buffer, 15 μl carbon particle conjugate as above and 30+30 μl buffer. The buffer was: NaPO4 0.1 M, BSA 3%, NaN3 0.05%, sucrose 3%, NaCl 0.5%, phenyldextran 0.25%, bovine gammaglobulin 0.05%, pH 7.5. The degree of blackening of the reaction zones was measured as absorbance with ultroscan (Ultroscan XL, Enhanced Laser Densitometer, LKB).
A) Activity determination on deposited IgE calibrator curve against IgE calibrators (24° C.) run as samples on separate strips with anti-hIgE antibody in the binding zone.
amount IgE
Abs (x1000)*
266 used below as positive
internal calibrator
*= absorbance on a reaction zone after carbon particle conjugate having bound.
B) Determination of birch specific IgE antibody in patient samples run at 18, 24 and 37° C., with and without positive internal calibrator (PIC) in order to adjust the standard curve (run at 24° C.).
Results (KU/L) with and without corrected calibrator curve:
Not corrected
Sample 1
Sample 2
The results show that it is possible to compensate for the variation in the separate runs by using positive internal calibrators. In addition the results show that it is possible to use predeposited calibrators.
The embodiment shown in this example may be modified such that one or more of the following criteria are met, (a) has predeposited Reactant* in a zone of application and/or (b) has a zone of application of sample located downstream or upstream of the zone of application of Reactant*, (c) has zones allowing simultaneous addition of Reactant* and sample.
Methods and materials
Coupling of streptavidin to polystyrene particles: Streptavidin (Amersham Pharmacia Biotech AB, Sweden) were covalently coupled to phenyldextran-adsorbed polystyrene particles with CDAP (1-cyano-4-dimethylaminopyridinium bromide) (Kohn J and Wilchek M, FEBS Letters 154 (1) (1983) 209-210), according to the description in Example 1 above for hIgE. The coupled particles were washed three times with 50 mM NaPO4, 0.05% NaN3, pH 7.4. The particle concentration was determined spectrophotometrically at A600 nm with untreated particles as reference.
Deposition of streptavidin-coupled particles on nitrocellulose membranes: To nitrocellulose sheets with polyester backing (Whatman, 8 μm, 5 cm wide) were applied with Linear Striper (IVEK Corporation) zones of:
The deposits were dried for 1 hour at 30° C., and the sheets were cut to 0.5 cm wide strips (Matrix 1201 Membrane Cutter, Kinematics Automation).
Coupling of anti-hIgE antibodies to detection particles: Antibodies to hIgE which had been cleaved with pepsin to fab′2 fragments were coupled to fluorescent polystyrene particles having aldehyde groups on their surface (Molecular Probes C-17177 TransFluoSpheres, aldehyde-sulphate microspheres, 0.1 μm, 633/720, 2% solids). 23 mg of antibody were then coupled to 66 mg of particles in 50 mM NaPO4 buffer, pH 6.5, overnight at room temperature, whereupon 205 μL of NaCNBH4 (5 M) were added to reduce the coupling for 3 hours at room temperature. Centrifugation was performed at 20,800×g (50 min in Eppendorf 5417R, 14,000 rpm), and deactivation in glutamic acid 0.05 M and aspartic acid 0.05 M in deionized water, pH 6.5, was then carried out overnight with stirring at room temperature. After centrifugation at 20,800×g for 50 min, blocking was performed with 0.2% BSA in 50 mM NaPO4, pH 7.4, with 0.05% NaN3, and incubation took place at +4° C. Centrifugation was then performed again at 20,800×g for 50 min followed by two washes with blocking buffer which was then also used for storage. The particle concentration was determined in a fluorimeter (Perkin-Elmer LS50B) with a standard curve prepared with the original particle. The coupled protein during the coupling was determined by having radioactive anti-hIgE present during the coupling.
Biotinylation of hIgE: Biotinylation of hIgE was performed according to the conditions recommended by the supplier (Pierce). hIgE was desalted by gel filtration with PD-10 (Amersham Pharmacia Biotech AB) in 0.15 M KPO4, 0.15 M NaCl, pH 7.8. To 0.95 mL (0.59 mg) hIgE were added 0.010 mL biotin-LC-Sulfo-NHS (3.59 mM, Pierce). Incubation then took place at room temperature for 1 hour, whereupon the coupling reaction was quenched by the addition of 40 μL of 2 M glycine. The mixture was then applied to a PD-10 gel filtration column equilibrated with 50 mM NaPO4, 0.15 M NaCl, pH 7.4. Yields and final concentration were calculated from the radioactivity as I-125-labelled hIgE was included in the coupling. The concentration of hIgE was analyzed by immunochromatography and UniCAP tIgE (Pharmacia & Upjohn Diagnostics AB, Sweden).
Deposition of biotinylated IgE on application filter: To application filters 5×5 mm (Whatman F075-14) were dispensed 0.006 mL of biotinylated IgE (1.6 ng) diluted in 50 mM NaPO4, 0.15 M NaCl, 6% BSA, 5% lactose, 5% dextran T5000, pH 7.4. The filters were dried at 30° C. for 1 hour.
Test procedure: Strips were mounted to a surface inclined about 16° from the bench plane. Sucking membranes (3.5 cm wide, Schleicher & Schuell, GB004) were placed 0.5 cm into the upper end of the membrane. To obtain constant pressure, metal weights were placed on the sucking membranes. Samples and reagents were then pipetted successively as described below. Each sample and reagent volume was sucked into the membrane before the following volume was pipetted.
1) 30 μL of 50 mM NaPO4, 0.15 M NaCl, pH 7.4.
2) A filter with predeposited biotinylated IgE was placed at the bottom of the strip.
3) 30 μL of patient serum were pipetted to each filter.
4) 20 μL of test buffer (0.1 Na—PO4, 0.15 M NaCl, 10% sucrose, 3% BSA, 0.05% bovine gammaglobulin, 0.05% NaN3, pH 7.4) were added to the filter.
5) The application filter was removed.
6) 20 μL of detection conjugate (75 μg/ml) diluted in test buffer.
7) 2×30 μL of test buffer.
8) The fluorescence of the detection zone was measured as a response area (Vmm) with a scanning red laser fluorometer (635 nm).
Three positive t3-sera were selected and analysed in triplicate with three different conjugate batches. Signal areas obtained with nitrocellulose coated with different IgE particle concentrations (described in Example 1 above) run with conjugate 2 were used as a stored calibration curve.
PIC correction meant that the signal for the reaction zone for t3 was multiplied by PICexp/PICobs before reading against the stored calibration curve (master curve). PICexp was defined as the average of the PIC signals obtained in the run with conjugate 2.
Conc (KU/L) Average of triplicate
Conjugate 1
Conjugate 2
Conjugate 3
Reading against Master curve for uncorrected signal
Reading against master curve for PIC-corrected signal
Between CV (%) is calculated as the variation of the three averages obtained for the different conjugates. The results show that the idea of a predeposited calibrator substance in the application zone is functional and that the use thereof as a PIC additionally gives a reduced between-assay-variation.
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|International Classification
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Owner name: PHARMACIA & UPJOHN DIAGNOSTICS AB, SWEDEN
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Owner name: PHADIA AB, SWEDEN
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text: Montana Drug Courts Enter Title On-Line Training Opportunities The New York City-based Center for Court Innovation, with support from the Bureau of Justice Assistance, has developed a series of free online training courses through its new National Drug Court Online Learning System (www.drugcourtonline.org/). Most drug court professionals learn on the job or through conferences and training. But that's changing. "Remote learning has become a much more common method of learning and training," says Valerie Raine, Director of the Center. "They can go to this site on their own time and at their own pace and get a pretty good handle on drug court." The Center has also gone to great lengths to assure that these course offerings are relevant to rural drug court personnel. The Center's online training offerings include video lessons from national experts on the following topics: Understanding Drug Use and Addiction, Treatment Modalities, Cultural Competency, Essential Components of a Successful Drug Testing Program, Sanctions and Incentives, Confidentiality, Trauma Informed Care Responses for Drug Courts, Legal Representation of the Non-Citizen, Maximizing Participant Interactions: "Transference" Revealed, Prescription Medication Abuse: Knowledge and Skills for Drug Court Practitioners, Changing the Direction of Methamphetamine Addiction. Courses are continually being updated and new ones developed. There are also interviews with practitioners and guided tours of drug courts, as well as a resource library of documents and reference tools. Additionally, there are numerous webinars covering a wide range of drug court-relevant topics posted on the websites of the National Drug Court Institute (NDCI.org) and the BJA Drug Court Technical Assistance Project at American University (www.american.edu/justice) and the Tribal Law and Policy (www.tribal-institute.org). On-site technical assistance is also available through the BJA Drug Court Technical Assistance Collaborative (American University, National Association of Drug Court Professionals, Tribal Law and Policy Institute, and Center for Court Innovation). Additional resources include: the National Rural Institute on Alcohol and Drug Abuse, Children and Family Futures, and other organizations. Other resources include: Treatment Improvement Protocols (monograph publications from the Substance Abuse and Mental Health Services Administration-Center for Substance Abuse Treatment), regional Addiction Treatment and Training Centers, state drug court associations, local colleges and universities. Veteran Drug Court Training Webinar Series


text: Hi, Thanks for the info. I need to ask one for thing - Is LVS possible to implement and test on Virtual Machines[I mean Vmware workstation -Desktop] ?? Regards, Sagar Shimpi, Senior Technical Specialist, OSS Labs Tieto email sagar shimpi tieto com, Wing 1, Cluster D, EON Free Zone, Plot No. 1, Survery # 77, MIDC Kharadi Knowledge Park, Pune 411014, India, www.tieto.com www.tieto.in TIETO. Knowledge. Passion. Results. -----Original Message----- From: linux-cluster-bounces redhat com [mailto:linux-cluster-bounces redhat com] On Behalf Of Rajagopal Swaminathan Sent: Sunday, October 16, 2011 9:51 AM To: linux clustering Subject: Re: [Linux-cluster] Apache active-active cluster Greetings, On Fri, Oct 14, 2011 at 1:47 PM, <Sagar Shimpi tieto com> wrote: > Hi, > > Can I configure Apache Active -Active cluster using Redhat Cluster Suit in > RHEL6? > > If yes, can someone please pass me the link for the same. > If you are using htpasswd for authentication, perhaps you may not be able to build it. Look at LVS at the same time. That might solve your problem. and oh! never forget that without fencing (storage/in-band/out-of-band) -- Regards, Rajagopal +91 9930 633 852 -- Linux-cluster mailing list Linux-cluster redhat com https://www.redhat.com/mailman/listinfo/linux-cluster


text:
Josepha by Carlos and Miguel
This album has been an incredible discovery.
The song "Caricia del cielo" is so beautiful that it almost makes me cry sometimes. An incredible combination of soft flamenco style and a wonderful and powerful melody.
Instrumental acoustic, guitar music, that simply speaks to my heart, so beautiful, passionately, emotionally, lovingly without thought or words but touching every part of my heart and soul, as this outstanding, graceful, fantastic, amazing, master piece of wonderful music, lifts my heart and soul to another level and carried me away with it from within


text:
Thursday, November 10, 2016
Wednesday, November 9, 2016
November Special - It's All About Screenwriting
Please join Marsha Cook on November 9th at 9 PM PST 10 PM MT 11PM CST 12 EST - when she welcomes Sammy Montana. Sammy is someone you should know. He has been in the business of finding great stories that make wonderful movies for years. You won't want to miss this show.
Sammy has worked in several capacities in the film industry. He served as the VP of Production and Development for the Producer of the HALLOWEEN remakes. Then he ran his own Film/TV literary management company, Anarchy Management and had an active list of clientele, including writers and directors. He co-wrote a script that placed in the Nicholl Quarterfinal and semifinals 2016. Currently, he is a script consultant drawing on his years of experience in the film industry. Under the Anarchy Consulting banner, he is developing in-house projects.
To get free screenwriting advice subscribe to https://www.youtube.com/watch?v=C7lKeQFHrEk For high quality script coverage with effective notes to improve your script visit http://anarchymanagement.com/cgi/wp/?page_id=77
Marsha has more than 20 years of experience in the writing industry. Overall, there are 10 books and 11 feature-length screenplays,two that were optioned for movies.
Stay up late and have some fun - live show but will be on demand right after the show is over.
How to find Sammy
Marsha info
Sammy Montana is the Special guest on World Of Ink
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LiveCycle Designer Specialist
This course is designed for form and template designers and developers who are interested in learning proper LiveCycle Designer techniques and want to prepare for the Adobe LiveCycle Form Developer exam to become an Adobe Certified Expert.
This is an advanced, three-day, instructor-led workshop designed for developers who are familiar with design environments and wish to create dynamic PDF templates. The workshop focuses on creating complex, dynamic PDFs using LiveCycle Designer and is geared towards developers who build front-end applications and forms. Through a series of lectures, demonstrations and exercises, you will develop a dynamic form template that utilizes some of the more advanced features of creating dynamic forms. Over the course of the first two days, you will build a dynamic account information form. Topics covered include working with multiple master pages, scripting, subform containers, floating fields and Designer's dynamic properties. On the third day, there is a self-paced lab where you will build a solution that meets a specific set of requirements. The instructor will assess the solution you build. The workshop uses Adobe LiveCycle Designer and may use LiveCycle Workbench, which is the integrated development environment (IDE) for creating LiveCycle applications. Most of the workshop is spent in the Designer environment.
Course versions are available for all versions of Adobe LiveCycle Enterprise Suite including: Adobe LiveCycle ES2, Adobe LiveCycle ES2.5, Adobe LiveCycle ES3, Adobe LiveCycle ES3.5, Adobe LiveCycle ES4.
3 days
The student will be able to develop a multi-page, data capture PDF form using LiveCycle Designer. This form will make use of multiple master pages and be dynamic in nature, both in response to merging data as well as the user interacting with the form.
Introduction: LiveCycle: Designer Specialist
Lesson 1: Using Master Pages
Exercise 1: Creating and Configuring Master Pages
Lesson 2: Data Connections and Designer
Exercise 2: Using Data Connections
Lesson 3: Working with Form objects
Exercise 3: Defining and Designing Form Page
Lesson 4: Designing with Fragments
Exercise 4: Creating and Editing Fragments
Lesson 5: Scripting in Designer
Exercise 5: Adding Script to your Form
Lesson 6: Advance Scripting Techniques
Exercise 6: Scripting with XFA and Acrobat Object Models
Lesson 7: Designing User Driven Dynamic Forms
Exercise 7: Adding User Driven Dynamic Features
Lesson 8: Designing Data Driven Dynamic Forms
Exercise 8: Adding Data Driven Dynamic Features
Lesson 9: Submitting Data
Exercise 9: Submitting XDP Data
Optional Lesson 10: Extending Form Capabilities
Optional Exercise 10: Adding a Barcode and Signature Field
Optional Lesson 11: Designing for Accessibility
Optional Exercise 11: Adding Accessibility Feature
Contact us to learn more about this course


text:
Photographer's Note
Sunrise at Berry Pond, Gros Morne National Park. This was our first sunrise in Newfounland, and we were lucky because it was the only one where the sun actually DID appear!
Berry Pond is easily accessible on a short hike from the Berry Hill campsite near Rocky Harbour. The hike around the pond is flat and takes about 45 minutes at a leisurely pace.
In the background to the right is the shoulder of Gros Morne itself.
I did not have a graduated filter, and I wish I had to capture the foreground a little bit better.
Nobody has marked this note useful
Photo Information
- Copyright: Ro Co (Fiddlee) (216)
- Genre: Places
- Medium: Color
- Date Taken: 2009-08-14
- Camera: Canon 350D (Rebel XT), Canon EF 17-85mm f4-5.6 IS USM, UV
- Exposure: f/10.0, 1/13 seconds
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- Date Submitted: 2009-10-12 7:52
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Where to Find Online Freelance Job Opportunities
Are you self-employed or thinking of doing some freelance work? Are you looking for ways to grow your part-time workload and find new clients? Depending on your skillset, there are various online job boards where you can find new projects and build your business. Let's look at some good online freelance job opportunities for Canadians.
Elance.com is one of the top freelance websites. With job categories such as Website Design, Graphic Design, Content Writing and Data Entry, there is a variety of work available. To start, create a free profile and list your skills and work experience. You may also build a portfolio to showcase some of your past work. Once your profile is ready, you can search through the job postings and apply to those that interest you. You may also be contacted by potential clients asking you to submit proposals for their new job postings. Elance takes care of billing and payments, helping to protect both clients and freelancers.
Similar to Elance, oDesk.com allows you to build your profile and apply to jobs. If you work hourly, they track your time automatically and guarantee payment, eliminating the need for freelancers to issue invoices. It's free to sign up and apply to jobs. Popular job categories include Web Development, Administrative Support, Customer Service and Marketing.
Like other job sites, Freelancer.ca makes it easy to browse listings and apply to projects. They are currently the world's biggest online marketplace for freelancing and outsourcing. With over 8 million employers and freelancers, it's a busy website with projects in categories such as Writing, Accounting, Software and Legal Services.
One of the smaller online freelance websites, Guru.com offers many projects in the fields of Ecommerce, Websites, Programming and Databases. Guru users like the work platform and SafePay, ensuring payment for their work is protected.
The smallest of the online freelance marketplaces, PeoplePerHour.com works much like the others. They do have an innovative "Hourlie" feature, which allows freelancers to create a fixed price service offer. This lets you set your pay rate and offer potential clients a quick and easy way to hire you.
Fiverr.com is a unique take on the online marketplace. Freelancers can list a specific service they are willing to complete for $5. With categories such as Fun & Bizarre, Video & Animation, Gifts and Advertising, there's something for everyone. If you're looking for a break from your usual professional workload, Fiverr may be the place for you.
When looking for new clients, another avenue is through online classifieds sites. Craigslist has an extensive Canadian site which includes a job board. Simply pick the city nearest you to bring up your local opportunities. With categories such as Customer Service, Legal and Health Services, it appeals to a wide range of job seekers. When using online classified sites, it's very much buyers beware – don't give out your personal information before doing your research and never pay for the chance to work. Look for reputable companies and local businesses that are well established, and verify they are indeed the ones posting the job ad.
Much like Craigslist, Kijiji offers a job board letting you browse through local postings. They also have a services board where you can post a free ad describing your skills. There are no payment guarantees when using online classified site to find work, so take the time to ask potential employers questions about their business, work expectations, payment details and contact information. Companies and business owners posting legitimate jobs will understand your need for information and will expect job seekers to ask relevant questions.
Before applying to online job postings, here are a few guidelines to consider.
- If you are considering an online marketplace site, do some research to see if it is the best fit for you. Make sure you read and understand their policies in regards to personal information, payment and work obligations
- Take the time to build your profile. Potential clients want to know who they are hiring. Providing your education, qualifications, employment experience, skills and examples of your work can help you get that first project
- Be realistic and upfront about the workload you can handle. Clients are looking for reliable workers who complete their work as described and on time
- Build your reputation. With each completed project, you slowly build an online reputation which help build your business and client base
- Protect yourself. Follow the necessary steps to ensure you get paid for your completed work
The hardest part of finding online freelance work is starting. It will require patience and time to look through job postings, submit proposals, answer questions from potential clients and build your online profile. It will also take time to build up your workload to the level you need. Luckily, there are many online opportunities available as companies worldwide are turning to freelancers to help them build their businesses and meet their needs.
Have you had success with an online freelance site? Which one do you prefer?


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Item description for Names of Jesus Pamphlet (Pack of 5) (Package of 5) by Rose Publishing...Overview
Names of Jesus package of 10 pamphlets
There are many names for Jesus given in the Bible and each has a special blessing for us that will enrich your worship and prayer life. This fold-out pamphlet shows more than 50 names for Jesus, each with the meaning and Bible reference:
? King of Kings, Lord of Lords ? Good Shepherd ? Chief Cornerstone ? The Way, The Truth, and the Life
? Advocate ? I AM ? Wonderful Counselor ? High Priest ? Holy One ? Alpha and Omega ? Mighty God
? Morning Star ? Lion of Judah ? Son of God ? Son of Man ? Rock ? Immanuel ? Beloved
? and many more
This beautiful chart gives the definition, a scripture reference, and shows the spiritual meaning for today. Fold-Out Pamphlet size: 5.5" wide x 8.5" tall. Fits inside most Bible covers. Unfolds to 33" long.
Publishers Description
There are many names for Jesus given in the Bible, and each will enrich your worship and prayer life. The Names of Jesus chart and pamphlet give the definition, a scripture reference, and show how to apply each name to our lives today. Pamphlet is 8.5" x 5.5" and unfolds to 33" long. Also available as a PowerPoint presentation (p. 43) or a wall chart (p. 36)
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Books > Bible Study > Reference > General
Books > General Interest > General Topic > Travel
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