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LittleBigPlanet PS Vita
|LittleBigPlanet PS Vita
European cover art
|Double Eleven[1][2]
Tarsier Studios[3]
XDev (Europe)[4]
|Sony Computer Entertainment
|PlayStation Vita
|Release date(s)
|Puzzle, platformer
|Single-player, multiplayer (co-op)
LittleBigPlanet PS Vita is a puzzle platform video game developed by Double Eleven,[1] Tarsier Studios[3] and Sony XDev Europe.[4] for the PlayStation Vita handheld game console. It is the fourth game of the main LittleBigPlanet franchise; a series of puzzle platformers for the PlayStation 3 and PlayStation Portable centered on user-generated content. The game was first announced in January 2011 along with the reveal of the PlayStation Vita console, then known as the Next Generation Portable (NGP),[7] and the first details of the game were revealed on 6 June 2011 at the Electronic Entertainment Expo. It was released on 19 September 2012 in the UK, 20 September 2012 in Japan and Australia, and 25 September 2012 for the North American markets.[5]
As in previous titles in the LittleBigPlanet series, players control a character named Sackboy through a variety of worlds utilising the character's various abilities such as jumping and grabbing objects. It also features various non-platforming mini-games. Numerous multiplayer options are available. As well as up to four-player online competitive or co-op gameplay, the PlayStation Vita's multi-touch display can be used by two players for competitive games. "Pass'n'Play" is also available, enabling turn-based gameplay.[8] The Vita's rear touchpanel is also used for pushing objects toward the player, creating platforms out of parts of the world.[9]
Content creation[edit]
Players are able to create their own levels and share them online using the PlayStation Network. The PlayStation Vita's touch-screen display can be used to directly draw objects and platforms in the game world. As well as unique creation tools such as these, the game also includes all of the tools available in LittleBigPlanet 2 (with the exception of LBP2's DLC tools such as the Worm Hole). Costumes bought from the PSN used to customise the player's character are transferable between the PlayStation 3 and PlayStation Vita games.[8] Content can also be shared over both Wi-Fi and a 3G network.[9]
Additional tools and materials[edit]
|This section needs additional citations for verification. (June 2015)
The PlayStation Vita version of the game has tools from previous versions of LittleBigPlanet that have been adapted to suit the Vita's control system, as well as new tools such as the Motion Recorder, the Touch Sensor, the Touch Tweaker, the Touch Cursor, the Touch Material and the Layer Tool, all of which provide players with touch controls. There are also tools used for other purposes such as the Dephysicalise Tool, the Sticker Scrubber and a tool that makes a jelly like substance that Sackboy can pass through.[10] Another new tool, the Memoriser, can store data between levels and play sessions.[11] Additional Materials include a Waterfall Material.
Additional content[edit]
A bonus with BioShock costumes was included to those who pre-ordered the game. The pack includes a costume of a Big Daddy and a Little Sister. A "Knights Of Old" Pack was also announced, allowing you to dress as a knight, dragon or a princess.[12]
LittleBigPlanet PS Vita received critical acclaim, garnering metascores of 88/100 at Metacritic and 88.68% at GameRankings.[13][14]
|Gamescom Awards
|Best Mobile Game
|TIGA Games Industry Awards
|Game of the Year
|IGN Game Awards
|Vita Game of the Year
|Pocket Gamer Awards
|PS Vita Game of the Year
- ^ a b "Double11 Working Alongside Tarsier On LittleBigPlanet Vita". GamingUnion.net. 27 June 2011. Retrieved 27 June 2011.
- ^ Olly. "Double Eleven » LittleBigAnnouncement". Double11.co.uk. Retrieved 27 June 2011.
- ^ a b "LittleBigPlanet Vita announced at E3". Retrieved 7 June 2011.
- ^ a b "LittleBigPlanet (working title) - PlayStation Vita, PS Vita game". uk.playstation.com. Retrieved 14 December 2011.
- ^ a b c d Simon (2 August 2012). "LittleBigPlanet PS Vita Release Date Announced". Retrieved 5 December 2012.
- ^ "LittleBigPlanet PlayStation Vita". Retrieved 5 December 2012.
- ^ Proven28. "Sony NGP Includes Uncharted, Killzone, Resist". Shacknews.com. Retrieved 27 June 2011.
- ^ a b Schramm, Mike (7 June 2011). "LittleBigPlanet (PS Vita) preview: Touched by a Sackboy". Joystiq. Retrieved 27 June 2011.
- ^ a b "EB Games Expo 2011: Little Big Planet Vita Stage Demo". GameSpot. 14 October 2011. Retrieved 5 December 2012.
- ^ StevenI (13 February 2012). "New Tools, Features, Tweaks and More!". Retrieved 5 December 2012.
- ^ a b Miller, Greg. "Sackboy's Definitive Adventure". IGN. IGN Entertainment. Retrieved 23 April 2013.
- ^ Moriarty, Colin (25 June 2012). "Get BioShocked by LittleBigPlanet Vita". IGN. Retrieved 5 December 2012.
- ^ a b "LittleBigPlanet PS Vita for Playstation Vita". GameRankings. CBS Interactive. Retrieved 23 April 2013.
- ^ a b "LittleBigPlanet PS Vita for PlayStation Vita Reviews". Metacritic. CBS Interactive. Retrieved 23 April 2013.
- ^ Parkin, Simon (20 September 2012). "Handmade, Handheld". Eurogamer. Gamer Network. Retrieved 23 April 2013.
- ^ "リトルビッグプラネット PlayStation Vita". Famitsu. Retrieved 2013-05-14.
- ^ Helgeson, Matt (12 September 2012). "Sackboy Conquers Another Sony System". Game Informer. GameStop. Retrieved 23 April 2013.
- ^ Calvert, Justin (18 September 2012). "LittleBigPlanet PS Vita Review". GameSpot. CBS Interactive. Retrieved 23 April 2013.
- ^ Tong, Sophia (12 September 2012). "Another Joyous Adventure for Sackboy Packed onto the PS Vita". GamesRadar. Future Publishing. Retrieved 23 April 2013.
- ^ "LittleBigPlanet Vita Review". GameTrailers. Viacom Entertainment Group. 24 September 2012. Retrieved 23 April 2013.
- ^ Davies, Emma (12 September 2012). "PlayStation's Cuddliest Scales Down Wonderfully". Official PlayStation Magazine (UK). Future Publishing. Retrieved 23 April 2013.
- ^ "And the winners of the gamescom 2012 awards are…". Retrieved 23 August 2012.
- ^ "THE WINNERS HAVE BEEN ANNOUNCED!". Retrieved 11 October 2012.
- ^ "Best PS Vita Game". Retrieved 23 December 2012.
- ^ http://www.pocketgamer.co.uk/pgawards2013
External links[edit]
- Official website
- Official community site
- Double Eleven Limited official website
- Tarsier Studios official website


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Development and Evaluation of an Isospora Suis Sporozoite Neutralizing Antibody Assay in Swine Testicular Cells
Steger, Ann M.
MetadataShow full item record
An in vitro Isospora suis sporozoite neutralizing antibody assay has been developed in swine testicular (ST) cells. This assay measures the titer of neutralizing antibodies present in sera, milk and monoclonal antibody culture fluids by determining both the amount of intracellular sporozoites by immunofluorescence and the cytopathic effect in ST cells. A monoclonal antibody against the apical complex of I. suis sporozoites inhibited the infection of sporozoites in vitro. Hyperimmune sera produced in goats and guinea pigs inhibited sporozoite infection in ST cells. Milk samples from gilts vaccinated with I. suis contained neutralizing antibodies, however the titers did not correlate with protection observed in nursing pigs after challenge.
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subset: commoncrawl


text:
Originally written on Fox Sports Arizona | Last updated 7/18/12
Trevor Bauer is headed back to the minor leagues for additional seasoning -- and hopefully improved control. The Diamondbacks announced Wednesday that they have optioned Bauer to Triple-A Reno and recalled right-handed reliever Jonathan Albaladejo. Bauer, Arizona's highly touted first-round draft pick in 2011, has struggled with his control in four starts since being recalled from Reno in late June. He has a 1-2 record with a 6.06 ERA after suffering a 4-0 loss in Cincinnati on Tuesday night. In three innings of work, Bauer walked five, threw two wild pitches, made an errant pickoff throw and gave up all four of Cincinnati's runs. Albaladejo, 29, ranks second in the Pacific Coast League with 17 saves and is 3-1 with a 3.35 ERA. He has 39 strikeouts in 35 relief appearances with the Aces in 2012. He's made two appearances for the D-backs this year, giving up two earned runs in two innings. He has previous major league experience with the Nationals and Yankees, posting a 6-3 recor...
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Narrow your search
The analytics company is intent on doing away with the age of the e-mail survey to make polling users for feedback more enjoyable all around.
The latest data shows growing adoption for Apple's current mobile OS as iOS 9 is expected to be unveiled in June.
The rate of adoption picked up steam with the release of iOS 8.1.3, which fixed some bugs and eased the software's demand for storage space on devices.
The number of people jumping to the latest version of Apple's mobile OS has slowed down dramatically, according to Apple's latest stats.
Apple's latest version of its mobile OS has picked up a bit more steam following the holidays.
The newest version of Apple's mobile OS didn't get off to as fast a start as its predecessor, but it's picking up steam.
The new figure shows steady but slow growth from the 46 percent reported by Apple more than a month ago.
Following its official launch Wednesday, Apple's new mobile OS has already shot up the ranks, according to mobile analytics firm Mixpanel.
A mobile analytics firm pegs iOS 8's adoption rate at 44.5 percent since launch 20 days ago. By contrast, iOS 7 had reached nearly 70 percent adoption over the same period last fall.
The percentage of users updating to Apple's latest mobile OS has remained virtually flat since September 21, which was four days after its release.


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It's one thing to create a team, but quite another to create teamwork in the workplace. Just as it's one thing to join a team, but quite another to perform as a team member. To put it simply, teams don't work without teamwork.
MyStyle believe that to succeed at the task in hand everyone involved needs to combine their efforts. If everyone does their job well, then it increases what the team can accomplish. This teamwork has to be recognised by everyone and know that great things can happen if individuals master the fundamentals and work together as one unit. Everyone has their own unique role, but each person's individual role must be recognised and appreciated.
Teamwork is something that must be a high priority and given constant attention. Every player needs to understand how important it is for them to work smoothly together if they want to be successful. Each player must be dedicated to the whole team and be willing to act unselfishly. When challenges arise (as they always do), the team needs to have the resources, accountability and commitment to deal with them in a constructive and positive manner. A sense of teamwork will play an integral part in this.
MyStyle Marketing take this in to high regard when recruiting. An interview must understand the strong team unit - qualifications and experience are a fantastic attribute - but the consultancy team need to make sure that they would fit in with the group with no conflict.
T.E.A.M. - Together Everyone Achieves More.


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Book Search
Excellence in University Assessment
Learning from award-winning practice
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What is life really like for the elite athlete? How does the experience of being a professional sports person differ from the popular perceptions of fans, journalists or academics? Why might elite sports people experience mental health difficulties away from the public gaze? In the first...
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Reconceptualising Feedback in Higher Education
Developing dialogue with students
Feedback is a crucial element of teaching, learning and assessment. There is, however, substantial evidence that staff and students are dissatisfied with it, and there is growing impetus for change. Student Surveys have indicated that feedback is one of the most problematic aspects of the student...
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text:
Faith Under Fire: Exploring Christianity's Ten Toughest Questions, A DVD Study
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Product Description
Product Information
|Format: DVD
Vendor: Zondervan
Publication Date: 2012
Availability: In Stock
Series: Faith Under Fire
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$10.99Save 18% ($2.00)Availability: In StockCBD Stock No: WW687863
Publisher's Description
In this ten-session small group Bible study DVD, Faith Under Fire: Exploring Christianitys Toughest Questions, Lee Strobel hosts interviews and debates between knowledgeable and passionate experts discussing what they believe and why they believe it.
This dynamic study answers tough questions about the existence of God, Christianity and Islam, the reliability of the Bible, faith and science, and many others issues.
In it, best-selling author Lee Strobel hosts spirited discussions between well-respected Christians, people of other faiths, and people with no faith at all, on the some of the most important spiritual and social issues of our time.
Each DVD session includes video in which leading Christian and non-Christian voices engage in forceful but fair debate. You and your group will consider what youve heard and discuss. Strobel provides additional comments to guide small group discussion. Designed for use with the Faith Under Fire Participant Guide (sold separately).
Sessions include:
1. Is the Supernatural Real?
2. Is Jesus a Prophet or the Son of God?
3. Did Jesus Rise from the Dead?
4. Do All Roads Lead to God?
5. Is the Bible Bogus?
6. Does Science Point to a Creator?
7. Is Anything Beyond Forgiveness?
8. Why Does God Allow Pain and Suffering?
9. The Mystery of the Trinity?
10. Do Christians and Muslims Worship the Same God?
Author Bio
Lee Strobel was the award-winning legal editor of The Chicago Tribune and is the best-selling author of The Case for Faith, The Case for Christ, and The Case for a Creator, all of which have been made into documentaries by Lionsgate. With a journalism degree from the University of Missouri and a Master of Studies in Law degree from Yale, Lee wrote 3 Gold Medallion winners and the 2005 Book of the Year with Gary Poole. He and his wife live in Texas. Visit Lee's website at: www.leestrobel.com.
As Willow Creek Community Church's key evangelism leader for over sixteen years, Garry Poole is the innovator of seeker small groups and a strategist of creative outreach initiatives. Passionate about reaching people for Christ, Garry and his team have trained thousands of leaders to launch seeker small groups in their own settings. His award-winning book, Seeker Small Groups, provides a detailed blueprint for facilitating small group discussions that assist spiritual seekers with investigating Christianity. He also wrote The Complete Book of Questions, a collection of 1001 conversation starters and numerous group study guides including The Three Habits of Highly Contagious Christians; The Tough Questions Series, and Experiencing the Passion of Jesus (with Lee Strobel) to accompany Mel Gibsons film, The Passion of the Christ. In 2005, it became the first discussion guide ever to receive the prestigious Charles Kip Jordon Christian Book of the Year award. Garry lives in suburban Chicago.
Product Reviews
Ask Christianbook
Q: Does Lee Strobel's DVD series have a workbook with it? Lee has a new DVD out with his buddy Mark Mittleberg... 10 questions ... series - does this come with a workbook & study guide? I'd like to use this for discussions.BibleGateway and Amazon don't affiliate them together if they exist.Thank youTodd
This video-based small group study is designed to be used with the Faith Under Fire Participant's Guide, CBD stock # WW687863.
Q: How long is each DVD segment?
The DVD segments range from about 6 to 15 minutes in length:
Session 1 - 9:02
Session 2 - 10:02
Session 3 - 6:26
Session 4 - 13:07
Session 5 - 13:55
Session 6 - 12:32
Session 7 - 10:22
Session 8 - 12:22
Session 9 - 12:18
Session 10 - 14:32
Bonus Session - 8:23


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About This Listing
Space where guests can cook their own meals
Towels, bed sheets, soap, and toilet paper
Space where guests can cook their own meals
Towels, bed sheets, soap, and toilet paper
Central heating or a heater in the listing
In the building, free or for a fee
In the building, free or for a fee
Continuous access in the listing
Breakfast is provided.
The listing can accommodate a gathering of 25 or more attendees
Easy access to the building and listing for guests in wheelchairs
Private or Shared
Free, in the building or nearby
Posted emergency information and resources
The Space
Friendly welcoming home in peaceful surroundings with lounge,free wifi, car parking, access to kitchen Central to Wexford town, Rosslare Harvour, Kilmore Quay. Johnstown Castle
Kilmore Quay is 20 minutes away by car. It is a quaint little fishing village with thatched cottages and lovely pubs and restaurants. The Saltee Islands are a 20 minute boat trip away. It is a bird sanctuary and trips go every day from the Quay
.The Ferry to Dunmore East and Waterford is 30 minutes away by car.
We have a friendly pub in the same field serving the best of seafood from Kilmore Quay. Beaches all around within 15 minutes. Rosslare Strand being the closest boasting one of Irelands leading Hotels, Kellys. Restaurants and food facilitys very good in the area.
Local Gaelic Club with huge activities every evening.
There is a bus stop at the top of our road aproximately 1 kilometer away.
We are a family living in a detached house. Our nearest neighbour is across the road. It is a very quiet area but so central.
Our house is so central to everywhere and so welcoming to all our friends and family. We have a big garden front and back.
Guest Access
Double Bedroom. Bathroom. Lounge. Dining Room. Kitchen. Garden. Parking beside bedroom window.
Interaction with Guests
As much as they wish and we can do to make them feel at home.
The Neighborhood
We live on a country road off main route to busy Rosslare Harbour. We have a lovely Castle just 2 miles away open to public for walks and coffee in Summer months. A pub with food ( good local fish)!in walking distance. Cinema, McDonalds and lovely drop in shopping and cafe 2 miles away on N25. Kilmore Quay fishing village with thatched cottages (very pretty) 12 kilometres away. Locally caught fish in pubs and restaurants there. Boat trips to Saltee Islands bird sanctuary daily. Lots of other amenities.
Getting Around
You need a car.
Other Things to Note
We are a very friendly Irish family home.
No indoor smoking.
Respect our home and feel as at home in it as you would in your own home.
5 Reviews
Peg sent us an adequate description how to find the house. The welcome was warm and the room small but nice. No toilet at the room, but a few steps away.
Breakfest was great.
We had a warm welcome from Peg. Tea and cheesecake, yum! She accommodated our late hours because of wedding with a key to the house. Very sensible! Grand Irish breakfast with 2kinds of homemade bread. Would recommend highly!
Myself and my two children had a most enjoyable stay with Peg and her family. We were made to feel so welcome in her beautiful home that I felt like I was saying goodbye to family when we were leaving. I would highly recommend this accommodation and wish Peg and her family all the best.
We had a great night at Peg's very big and clean house. The atmosphere was very familiar as we also met her family. We arrived late in the evening and Peg even treated us to some dinner. The Irish breakfast was very delicious.
Peg was the most wonderful host togetether with her family they made us most welcome and helped us out in many ways also took us & our luggage to ferry terminal we would recommend her as the hostess with the mostest in everyway
About the Host, Peg
A wonderful "Mammy" of three fine grown lads, Peg is a warm & welcoming person; you will receive a wonderful irish welcome in her lovely home; she has a great amount of local knowledge for all tastes - history enthusiasts, fitness & activity buffs or just those looking for a little "craic agus ceol" and a good pint of Guinness.


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USF DT Todd Chandler Squats 700 Pounds
By Sports Talk Florida
News Desk
Follow @ SportsTalkFLA
News Desk
Follow @ SportsTalkFLA
Not many people can say that they can lift 700 pounds at once.
According to the Bulls Facebook page, USF defensive tackle Todd Chandler squatted 700 pounds and it's pretty amazing to watch.
Post by USF Football.
Tags: Todd Chandler, USF Football


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I have my Master's in Public Health and (what I used to feel was) an impressive resume of international experience. We moved to Tampa... Read Now
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What's most important to you with regards to GTX 460?
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Join Date: Jul 2004
Location: Michigan
Posts: 974
Re: What's most important to you with regards to GTX 460?
Originally Posted by
At certain points the $200 market was HUGE and VERY well represented here at nvnews. GF3 ti200 and GF4 ti4200 attracted swarms of people in this price segment. Things cooled off during Geforce FX days UNTIL the 5900XT showed up. That card took off like a rocket in these parts. That momentum mostly carried over to the 6800NU, and I think that's when the $200 segment has gone through a steady and consistent slump as far as market impact.
It appears as though NVIDIA wants this market demographic to become more pronounced and impactful again. Amen to that, I'm down with it.
Oh, I agree, that is a huge market.
It's just that its been heavily populated recently by older GTX 285's, 280's, etc... to the point where I doubt the 460 will look attractive because it won't perform as well as the "old" higher end cards.
For example. The GTX 465 was recently released, you'd know, you own one. I compared the benchmarks and honestly it's a great card, but the performance is almost identical in most games compared to a GTX 285. Having snatched that card used half a year ago for less than the $280 they were asking (at the time) for a 465 I largely saw it as a wasted price-bracket.
The point I'm trying to make is that these new cards "suck" at these price-points because of the fact that the old generation's high-end has been flooded with a new generation, displacing them. If you really want DX11 or something from that generation then I suppose its great..... but I don't even know if that's true.
For example.... I just got a 470 today. I've been playing with it and yeh, it's a very powerful card... so was my 285. I'm having a hard time understanding why I really made the purchase other than really really wanting to get some time in with CUDA 3.0. I can say right now that the upgrade from a 285 to a 470 is less than appreciable in most games unless dx11 is a huge motivating factor.
To answer your first question. What's the most important about a 460? PRICE!!! This card needs to be around the performance of a 465, or at the least be even to a 285 / 280 / 4890 for me to consider it in builds for friends.... otherwise I'd really have to recommend they pick up 2, $100, used GTX 260's and SLI them....
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I ordered a product from the store and I get two files to download. One ends with trx.exe and the other is dpc.exe
I use studio 188.8.131.52
Thank you for any help
johnnv - 26 November 2012 10:18 PMI ordered a product from the store and I get two files to download. One ends with trx.exe and the other is dpc.exe
I use studio 188.8.131.52
Thank you for any help
dpc.exe is the one you want.
Stuff in Clouds Photos, lots of 'em
What product? Can you post a link just to make sure it can be used in DS3 as some new content can only be used in DS4/4.5 but the chances are that all is well.
Somethings to Consider when starting to learn CG
My Gallery
My Render Thread and Tutorials
Proud member of the Bald Wizards Club and Co-operative
The product is a new release called "Curator Aquarum" for $1.99
The answer to your question is "it depends…"
* Some items with both Poser and DAZ|Studio installers have only D|S materials presets in the D|S installer, which will usually have a much smaller file size than the Poser installer. You will need to run both installers.
* Some items have full Poser and D|S installers, you will only need to run one or the other, and the file sizes should be not too different. (They won't be the same, because of the different way Poser and D|S do things.)
* Many items released in the last year or so include D|S files in the Poser installer. In this case there might not be a D|S installer.
* Many newer items will only work in D|S 4 or 4.5 — these should have "trx" in the file name, although some have the "trx" when they shouldn't (work OK in D|S3) or don't have it when they should (usually texture sets for Genesis stuff, which can't be used in D|S3 anyway).
It should tell you on the store page if any of these apply, but sometimes the store page info is… less than completely accurate. If this happens, the DAZ 30-day return policy is your friend.
"Despite rumor, Death isn't cruel — merely terribly, terribly good at his job."
Terry Pratchett, Sourcery
I am a bit confused myself to be honest now. I see DAZ format listed so in theroy it should work.
My advice is to pop over to this thread [url=http://www.daz3d.com/forums/viewthread/184/]JACK TOMALIN APPRECIATION SOCIETY [JAS III][/ul] and you can either ask the man who man the product or another might see it and advise. I dom't have it myself otherwise I would take a look having DS3A installed and been used too.
well put SpottedKitty
Just noticed you mentioned which item it is — most of Jack Tomalin's stuff seems to be made for D|S3 (and also works in 4.0 and 4.5). The few recent things he's done with 4.5 files have, I think, every time also included files to let it work in D|S3 as well. In this case, the Poser installer has D|S3 files included in the MATs and the D|S installer has D|S3 scene files. You should be OK, then.


text:
Denison Brand Awareness Survey
Posted: 05/19/2014 - DENISON, TX -- The city of Denison is conducting a survey on the city's branding efforts.
Posted: 05/19/2014 - DENISON, TX -- The city of Denison is conducting a survey on the city's branding efforts.
Posted: 05/16/2014 - DURANT, Okla. – The Regional University System of Oklahoma Board of Regents has named Sean Burrage as the 20th president of Southeastern Oklahoma State University.
Posted: 05/14/2014 - ADA – Linzi Thompson is not the kind of person who kicks back with rest and relaxation during her breaks from pursuing an East Central University degree.
Posted: 05/13/2014 - Sherman, TX –The Sherman Chamber of Commerce will now be hosting Hot Summer Nights, along with Season Sponsors Knight Furniture and Landmark Bank. Hot Summer Nights is held every Thursday night in June and July, with the exception of July 3rd.
Posted: 05/12/2014 - DURANT, Okla. – Thirty-three honor graduates were recognized during Spring Commencement exercises Saturday at Southeastern Oklahoma State University.
Posted: 05/12/2014 - Gabriella Ogletree of Dodd City won the top prize in the children's art contest sponsored by the Fannin County Children's Center.
Posted: 05/07/2014 - DURANT, Okla. – The Board of Regents of the Regional University System of Oklahoma has scheduled a meeting for May 15 to interview five candidates for the position of President of Southeastern Oklahoma State University.
Posted: 05/07/2014 - DSP&R is excited to announce that the inaugural season of Shakespeare in the Grove will feature a FREE production of "A Midsummer Night's Dream" and four workshops FREE to the public.
Posted: 05/07/2014 - ADA – Thanks to a valued partnership between two institutions and the Texoma Health Foundation, the East Central University at Southeastern Nursing Department in Durant is receiving a $55,000 grant to update its facility. The update will help expand opportunities for nursing candidates.
Posted: 05/07/2014 - ARDMORE, Okla. — Robert Wells has a history of farm accidents. On his 8th birthday, Wells had a horrific mishap with a lawn mower that permanently damaged his foot and forever changed his life.
Posted: 05/05/2014 - Norman, Okla., – From the Chickasaw Nation Ambassador who helped hundreds of Native Americans gain acceptance into law school to a former attorney turned tutor, education was a common thread connecting five Chickasaws inducted into the Chickasaw Nation Hall of Fame.
Posted: 05/01/2014 - Susan Phau Yeager will perform her one-woman show, Broadway & Me on May 8, 2014 at the Little Dixie Home Ownership Center, 500 E. Rosewood, Hugo, OK at 7:00 PM.
Posted: 05/01/2014 - TISHOMINGO, Okla. – Chickasaw Nation Governor Bill Anoatubby reached back in time to move toward the future here April 28, dedicating a statue honoring arguably one of the most pivotally important and influential Chickasaws in modern history.
Posted: 04/25/2014 - Serving as Chickasaw Nation Governor for one term, from 1878-80, Benjamin Crooks Burney was a proponent of education.
Posted: 04/25/2014 - ADA – East Central University congratulates Sister Rosemary Nyirumbe, who was named to the Time Magazine List of 100 Most Influential People in the World, it was announced on Thursday.
Posted: 04/25/2014 - SHERMAN, TEXAS—Austin College's Hispanic student group, Los Amigos, will host a Cinco de Mayo celebration on campus Sunday, May 4, from 5 to 9 p.m. in Sid Richardson Center of the Robert T. Mason Athletic-Recreation Complex. The event is free and open to the public.
Posted: 04/23/2014 - ARDMORE, Okla. — A pond can be a significant asset to any farming or ranching operation, but maintaining optimal fisheries production and water quality often requires appropriate knowledge and effort.
Updated: 04/22/2014 - BELLS, TX -- Bells residents got the chance to gril their city council candidates with questions at a public forum Tuesday night.
Posted: 04/21/2014 - ADA – As many as 50 companies and graduate schools will be on hand as East Central University hosts the Spring 2014 Career Expo on Tuesday, April 22, from 9 a.m. to 1 p.m. in the ECU Foundation Hall of Chickasaw Business and Conference Center.
Updated: 04/23/2014 - DURANT, OK -- Staff at the Bryan County Crisis Control Center are busy preparing for their 4th annual Kick Up Your Heels event.
Posted: 04/18/2014 - SULPHUR, OK– Wild blue indigo, Ragworts, and Fleabane daisies – just some of wildflowers carpeting the forest and prairie at Chickasaw National Recreation Area.
Posted: 04/18/2014 - SULPHUR, OK–Saturday, April 26th has been designated National Junior Ranger Day in conjunction with National Park Week (April 19-27), an annual presidentially proclaimed week for celebration and recognition of the country's nearly 400 national park areas.
Posted: 04/18/2014 - Personal beauty care and hygiene will be the focus of a brand new youth summer camp offered at North Central Texas College in June. The Young Ladies Cosmetology Summer Camp will be held on two days for girls ages 8-13. The first camp will be Tuesday, June 17 and the second camp will be Thursday, June 19. Both camps will run from 1 to 4:30 p.m. each day. Cost of the camp is $20 and each clinic is limited to 10 participants.
Posted: 04/17/2014 - SHERMAN, TEXAS—Austin College's Center for Environmental Studies will host a public lecture by Julia Marton-Lefèvre, director general of the International Union for Conservation of Nature (IUCN), on Monday, April 21, at 12:30 p.m. in Wright Campus Center, Room 231. The lecture is free and open to the public.
Posted: 04/16/2014 - ARDMORE, Okla. — Noble Foundation Human Resources Administrator Emily Bynum was recently named one of 39 Oklahoma young leaders in The Journal Record's 2014 Achievers Under 40. Bynum will be honored at an awards luncheon on Thursday, May 22, 2014, and profiled in a special publication.


text:
- Albania
- Bosnia and Herzegovina
- Bulgaria
- Croatia
- Kosovo
- Macedonia
- Montenegro
- Romania
- Serbia
- All Balkan Countries
The mystery of who ordered the tapping of the Deputy PM and President's phone records highlights broken police chain of command.
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Starting with no more than rumours of a colossal atrocity, investigators and prosecutors recall how they put together a convincing case that the Bosniaks of Srebrenica were the victims of genocide 20 years ago.


text:
This list will also likely include petit fours, mini éclairs, trifle, and chocolate and lemon mousse.
It should be soft and light enough to spread easily over the top of the trifle in a not-too-thick layer.
early 13c., trufle "false or idle tale," later "matter of little importance" (late 13c.), from Old French trufle "mockery," diminutive of truffe "deception," of uncertain origin.
"treat lightly," 1520s, from trifle (n.). Related: Trifled; trifling.


text:
Showing 881 - 900 of 998
Whipped cream and a whole lot more
There's a mess of whipped cream cans perched on the front desk of Nordewier-Vanderwerp Hall and no pumpkin pies in sight. This is a dorm service auction, and the whipped cream is meant for faces, not pies.
November 26, 2008 | Allison Graff
Mourning the loss of Ricky Mosher
Calvin mourns the loss of Ricky Mosher.
November 24, 2008 | Phil de Haan
Modern-day slavery
When Adel Abadeer saw an ABC News special on a young boy named Iqbal Masih back in 1994, he couldn't get it out of his head or his heart.
November 21, 2008 | Phil de Haan
Wikki winners selected
The fourth-annual Wikki Stix (sticks of wax-covered yarn) contest wrapped up at noon, Friday, Nov. 14.
November 20, 2008 | Katie Landan
Middle schoolers hone writing skills
More than 800 middle school students flocked to Calvin's campus on Thursday, Nov. 13, to attend the sixth-annual Middle School Writer's Workshop.
November 19, 2008 | Katie Landan
Faculty Profile: Daniel Garcia
Calvin CAS and filmmaker Daniel Garcia has made films about child soldiers in Uganda and philanthropists in Grand Rapids.
November 18, 2008 | Katie Landan
Election 2008: Calvin reacts
Today we queried Calvin students, faculty and staff about the election. How do they feel about the results of yesterday's historic election?
November 14, 2008 | Myrna Anderson , Allison Graff
Peter Fish swims again
At LOFT, Peter Fish were distributed as a campus-wide effort to raise money to combat world hunger.
November 14, 2008 | Katie Landan
Examining sprawl, reimagining our cities
"A Field Guide to Sprawl," on display at the gallery from November 14 through December 20, includes 50 aerial images, each cataloging a different ungainly aspect of urban development.
November 14, 2008 | Myrna Anderson
Inspiring a new generation of givers
"The Gift of All: A Community of Givers" is a documentary about local philanthropy directed by Calvin film professor Daniel Garcia.
November 14, 2008 | Myrna Anderson
Halloween events bring campus to life
Hundreds of costumed children from the community meandered through the apartment buildings in Knollcrest East on Halloween for the annual Light in the Night celebration. Meanwhile, students gathered at dusk on Friday, October 31 for a candlelit vigil to raise awareness about domestic violence.
November 03, 2008 | Allison Graff
How will Calvin College students vote?
Media relations manager Matt Kucinski catches up with Calvin students on the Commons Lawn and asks them questions about what will influence them when they step into the voting booth on Tuesday.
October 31, 2008 | Matt Kucinski
Quechuan film enjoys Hollywood moment
Hope of the Quechua filmed by Calvin CAS professor Brian Fuller was shown at the Arpa International Film Festival, held October 24–26, at Grauman's Egyptian Theater.
October 31, 2008 | Myrna Anderson
Learning to cook with Marie Catrib
Among the many things that Calvin students learned at a cooking demonstration with local restauranteur Marie Catrib is that there is no such thing as too much garlic.
October 24, 2008 | Allison Graff
Hamersma gets toes tapping with first album
"It was after my retirement that I was inspired to attempt this project," said Hamersma, 79, of his first recording. "Since I now have time to practice regularly, I play better."
October 24, 2008 | Lynn Rosendale
Former soldier brings war awareness to campus
The War Awareness Lecture Series will run for two weeks at Calvin, from Monday, Oct. 13 through Monday, Oct. 24.
October 17, 2008 | Myrna Anderson
Taking the hassle out of the passport process
For three years now, Calvin has been making the process of getting a passport less of a fuss.
October 13, 2008 | Katie Landan
Turning tightropes into bridges
Two geology professors collaborate on a book about Christianity and the age of the earth.
October 10, 2008 | Phil de Haan
Making sense of the financial crisis
Calvin business professor Leonard Van Drunen and economics professor Scott Vander Linde sat down with communication and marketing's senior writer Myrna Anderson for a conversation about the crisis.
October 08, 2008 | Myrna Anderson
Unlearn Week begins
"The goal of Unlearn Week is to start a campus-wide, yearlong dialogue about racism," said Rhodes, Calvin's assistant dean of multicultural student development.
October 06, 2008 | Myrna Anderson


text:
That may have been true last month for students from Warroad High School in Warroad, Minn., who reported violent Internet threats against a female student. Some believe it may have prevented a deadly shooting.
Chat-room transcripts obtained by ABC affiliate WDAZ in Grand Forks, N.D., read: "I will bring a gun to school. … My grandpa does have a 44 mag and I will bring it to school." The student asked others whether they wanted to see another school shooting. Another student said, "I'm going to beat the hell out of you before I kill myself."
Other students in the chat then asked the threatening students to stop the intimidation. One even threatened to call police. Two students have since been charged in the incident.
Back in Mashpee, Brown said he hoped the new T-shirts would continue to encourage students to "start snitching" so he could stop violence.
"Now it's about getting information," he said.


text:
13-13.5mm Cultured Baroque Pearl and Rose Quartz Drop Earrings in 14kt Gold Over Sterling
Item#: 830553
Ultra-classy and feminine, these baroque pearl drop earrings stand out from the crowd. Freshwater pearls hang delicately from rose quartz medallions, surrounded by a halo of cool, sparkling CZs, .30 ct. t.w. Post/clutch backing. 14kt gold over sterling silver earrings. CZ weights are diamond equivalents.
Product Details (for 13-13.5mm Cultured Baroque Pearl and Rose Quartz Drop Earrings in 14kt Gold Over Sterling)
1/2 inch
14kt close 14KT: Karat (abbreviated kt or K) is a unit that indicates the purity of gold in the metal. 14kt is 58.33% gold. In the United States, 14K yellow gold is most favored and used in jewelry. Gold Over Sterling close STERLING SILVER: Also known as "Standard Silver," sterling silver is silver alloyed with other metals to increase the hardness and durability. Generally, sterling is 92.5% silver and 7.5% other metals. Some sterling silver jewelry is plated with 14kt-18kt gold and referred to as "vermeil," or "gold over sterling." close GOLD OVER STERLING: Sometimes referred to as "vermeil," this metal is gold-plated sterling silver. It is one of the most desirable gold-plated metals and can come in 14kt-24kt yellow, white, or rose. Silver
Polished close POLISHED: A buffed-out finish used to produce a mirror-like shine.
Post/Clutch close POST/CLUTCH: An earring with a post insert and a slide-on clutch backing to hold it in place.
Hanging Length:
1 3/8 inches
Gemstone 1:
Cultured Freshwater Pearl close CULTURED PEARLS: Likened to the moon, pearls have been valued for their luminous glow and natural beauty for thousands of years. Today, high-quality cultured freshwater pearls make pearl jewelry affordable to all. Pearl is the traditional birthstone for the month of June. (Color: White, Quantity: 2, Shape & Cutting Style: Baroque close BAROQUE: is the term used to describe oversized and irregularly-shaped pearls. Baroque pearls add interest and intrigue to classic jewelry styles. , Size: 13-13.5 mm)
Gemstone 2:
Quartz close QUARTZ: From the icy shimmer of rock crystal quartz to the delicate pink of rose quartz to the moody hues of smoky quartz, this gemstone offers something for everyone. Quartz, found in endless colors and varieties, is a popular choice for bracelets, necklaces, rings and earrings. (Color: Rose, Quantity: 2, Shape & Cutting Style: Oval close OVAL: An elliptical shape that resembles an egg. , Weight: 4.00 ct. t.w., Size: 9x7 mm)
Gemstone 3:
Cubic Zirconia close CUBIC ZIRCONIA: once called the poor cousin to the diamond, CZs have since become a fashion statement in their own right. Perfect for travel and a little extra bling, CZ Jewelry is one of man's flawless creations. (Color: Colorless, Quantity: 44, Shape & Cutting Style: Round Brilliant close BRILLIANT: A gemstone cut to enhance brilliance without sacrificing much carat weight. Typically has 58 facets. , Weight: 0.30 ct. t.w., Size: 1 mm)
All weights and measures are approximate.
Ross-Simons Presentation Box Your purchase includes an elegant Ross-Simons presentation box.
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text:
Competition Law and Policy in the EC and UK
4th Edition
By Barry Rodger, Angus MacCulloch
Routledge-Cavendish – 2009
Routledge-Cavendish – 2009
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text: Sierra Club Utah, BLM Sign Land Swap Deal By Dan Bammes • May 8, 2014 Utah Institutional Trust Lands Administration Utah's governor and the state director of the Bureau of Land Management have formalized a land exchange involving 60-thousand acres in southern and eastern Utah. The land exchange has been in the works since 2009, when then-Senator Bob Bennett and Congressman Jim Matheson worked to pass a bill authorizing the deal. But it's taken years to finalize the details. Alton Coal Mine Expansion Still Facing Opposition By Dan Bammes • Sep 10, 2013 SUWA Environmentalists are reminding the Bureau of Land Management that public opposition to expanding a coal strip mine in Kane County hasn't gone away. The Sierra Club and other groups went to the BLM office in Salt Lake City to deliver more than 45-thousand public comments opposing the expansion of the coal mine. The mine currently operates on private land near the town of Alton. The agency is about to issue a supplemental environmental impact statement on the plan that could allow it to expand onto public land in the same area. Groups Ask UDOT to Stop West Davis Corridor By Dan Bammes • May 7, 2013 Dan Bammes A coalition of community and environmental groups is asking the Utah Department of Transportation to reconsider building a new freeway along the west side of Davis County. Their so-called "Shared Solution" asks U-DOT to study improving east-west roads and walkable communities as an alternative. Community activist Lori Kalt wants to avoid a new freeway cutting through her neighborhood on the west side of Farmington.


text:
Tiered Cake POPs
|Cake balls
|20 Ounce (See Recipe -Http://Www.Ifood.Tv/Recipe/Pop-Basics-Making-The-Cake-Balls 20 Pieces, 1 Ounce Each)
|Blue candy melts
|14 Ounce (1 Package, 400 Gram)
|White candy melts
|7 Ounce (200 Gram)
|Nonpareils/Sprinklers of your choice
|1⁄2 Cup (8 tbs)
1. To make the tiers for the cake, flatten the cake ball
and cut out the largest shape using the larger cookie cutter. Gather the remaining pieces
of cake ball and push them into the smaller cookie cutter to create the second tier.
2. Prepare the candy melts (see page 25). Dip the base of the top half of the cake into the blue candy melt and attach to the bottom half of the cake.
Let harden in the freezer for 10 minutes.
3. Dip the tip of a lollipop stick into the blue candy melts and insert it three-quarters of the way through the cake, so that both tiers are attached. Dip the entire POP into the candy melts. Let set.
4. Fill a small piping bag with the white candy melts. Pipe around the edge of the bottom tier and along the sides. While the candy melt piping is still wet, sprinkle with nonpareils. Let dry, cover, and store in the refrigerator.
This recipe was contributed by Clare O'Connell, author of Pop Party.
To purchase POP Party visit www.amazon.com.


text:
An assay pad for measuring the concentration of HDL-associated cholesterol in a sample, a method for using the pad, and a diagnostic assay device for carrying out the method are described. The assay pad includes a polymer adhesive or heat laminate bond. The assay design prevents interference by reagents used for such removal with the HDL quantification reaction or with other assays carried out on the same sample. If desired, removal of non-HDL lipoproteins and assay of HDL cholesterol can be carried out without interruption of the assay.
1. An assay device for measuring serum cholesterol associated with high-density lipoproteins (HDL) in a blood fluid sample containing lipoproteins other than HDLs of the type including a sample distribution array effective to distribute a blood fluid sample from a sample application region to one or more sample collection regions, the device comprising:
a HDL test pad in which HDL concentration can be assayed being spaced apart from said array, said HDL test pad being affixed to a mounting means; and
a reagent pad containing a binding reagent effective to selectively bind and remove non-HDLs from the fluid sample;
wherein said HDL test pad and said reagent pad are joined by a bond selected from the group consisting of a heat formed bond and heat activated adhesive bond;
wherein said mounting means are effective (i) to maintain the device in a sample-distribution position, wherein said joined HDL test pad and reagent pad are spaced apart from said array, and (ii) to transfer said device to a test position, whereby the joined HDL test pad and reagent pad are in contact with said array.
2. The device of
3. The device of
4. The device of
5. The device of
6. The device of
7. The device of
8. The device of
9. The device of
10. The device of
11. The device of
12. The device of
13. The device of
14. The assay device of
15. The device of
16. A method of preparing a device suitable for measuring serum cholesterol comprising:
providing a reagent pad and an HDL test pad, wherein at least the reagent pad is formed of an asymmetric polysulfone membrane having a small pore side and an open pore side;
heating to adhere said HDL test pad and said reagent pad; and
applying (i) HDL test reagents to said HDL test pad, and (ii) a reagent effective to selectively bind and remove non-HDLs from a fluid sample to said reagent pad to form said device.
17. The method of
18. The method of
before said heating, orienting the reagent pad with the HDL test pad such that the small pore side of the reagent pad contacts the open pore side of the HDL test pad.
19. The method of
20. The method of
21. A method of preparing a device suitable for measuring serum cholesterol comprising:
coating a reagent pad with heat activated adhesive;
applying (i) HDL test reagents to an HDL test pad, and (ii) a reagent effective to selectively bind and remove non-HDLs from a fluid sample to said reagent pad;
heating to adhere said HDL test pad and said reagent pad to form said device.
22. The method of
23. The method of
24. The method of
25. The method of
26. The method of
27. The method of
28. The method of
29. The method of
30. A method of measuring serum cholesterol associated with high-density lipoproteins (HDL) in a blood fluid sample containing lipoproteins other than HDLs, comprising:
contacting the sample with a sample reservoir through which the sample is distributed to one or more sample collection sites;
bringing into contact with such a sample collection site a laminate comprising (i) a reagent pad containing a reagent effective to selectively bind and remove non-HDLs from the fluid sample, and (ii) an HDL test pad having a detectable indicator of HDL cholesterol;
wherein said blood fluid sample passes through said laminate by capillary action and/or gravity through said laminate to permit measurement of HDL concentration; and
measuring the level of HDL cholesterol in said sample by detection at said HDL test pad.
31. The method of
32. The method of
33. The method of
34. The method of
35. The method of
36. The method of
37. The method of
This application claims benefit of priority to U.S. Provisional Application No. 60/460,079, filed Apr. 2, 2003.
The present invention relates to a high density lipoprotein (HDL)-associated cholesterol assay pad, a method for using said pad, and a diagnostic assay device for carrying out the method.
The amount of cholesterol present in the blood is known to be related to the risk of coronary artery disease. Cholesterol circulates in the blood predominantly in protein-bound form. The proteins which transport cholesterol are the lipoproteins, which are subdivided into three classes based on their density. The very-low density lipoproteins (VLDL) are triglyceride-rich lipoproteins which are synthesized in the liver and ultimately converted to low-density lipoproteins (LDL), which transport most of the plasma cholesterol in humans. The high-density lipoproteins (HDL) are lipoproteins which are involved in the catabolism of triglyceride-rich lipoproteins, and in the removal of cholesterol from peripheral tissues and transport to the liver. An inverse relationship between serum HDL levels and risk of coronary disease has been established. In particular, if the proportion of serum cholesterol associated with HDL is low, the risk of coronary disease is increased.
In view of the importance of relative serum cholesterol levels in risk assessment and management of atherogenic disease, considerable effort has been spent screening large populations of both normal and high-risk individuals for serum levels of HDL, LDL, as well as total cholesterol and triglycerides. The effectiveness of treatments of high-risk individuals has been monitored by regular testing of serum levels of cholesterol in the various lipoprotein compartments.
One method for specific HDL cholesterol testing is based on the selective precipitation of non-HDL lipoproteins in serum by a polyanionic compound, such as dextran sulfate, heparin, or phosphotungstate, typically in the presence of a group II cation, such as Mg2+, Mn2+, or Ca2+. The specificity and degree of precipitation are dependent on a variety of factors, including the type and concentration of the precipitating reagent. In general, the order of precipitation of serum cholesterol particles, with increasing concentration of polyanion, is VLDL, LDL, and HDL. HDL usually remains soluble at concentrations of heparin or dextran sulfate which completely precipitate lower density particles, although minor apoE species of HDL may be co-precipitated with lower density particles. By selective precipitation of lower density particles, HDL serum cholesterol levels can be determined.
In a typical lipid assay procedure, a small volume of blood is drawn and centrifuged to produce a clear plasma or serum sample fluid. The sample fluid is then aliquoted into several assay tubes, for determination of (a) total serum cholesterol, (b) triglycerides, and (c) HDL cholesterol. The HDL sample is precipitated, as above, and the lower density particles are removed by filtration or centrifugation prior to cholesterol detection. The samples are then reacted with an enzyme mix containing cholesterol esterase, cholesterol oxidase, peroxidase, and a dye which can be oxidized to a distinctly colored product in the presence of H2O2. The tubes may be read spectrophotometrically, and the desired total, HDL and LDL cholesterol values determined.
Despite the accuracy and reliability which can be achieved with the liquid-phase cholesterol assay just described, the assay has a number of limitations for use in widespread screening. First, the method uses a venous blood sample, requiring a trained technician to draw and fractionate the blood sample, and aliquot the treated blood to individual assay tubes. At least one of the sample tubes (for HDL determination) must be treated with a precipitating agent and further processed to remove precipitated material. Although some of these procedures can be automated, analytical machines designed for this purpose are expensive and not widely available outside of large hospitals.
Co-owned U.S. Pat. Nos. 5,213,964, 5,213,965, 5,316,196 and 5,451,370, each of which is incorporated herein by reference, disclose methods and assay devices which substantially overcome many of the above-mentioned problems associated with liquid-assay procedures for measuring serum cholesterol levels. In one embodiment, the device is designed for measuring the concentration of HDL-associated cholesterol in a blood sample also containing LDL and VLDL particles. The device includes a sieving matrix capable of separating soluble and precipitated lipoproteins as a fluid sample migrates through the matrix. A reservoir associated with the matrix is designed to release a soluble precipitating agent, for selectively precipitating LDL and VLDL, as fluid sample is drawn into and through the matrix. This allows HDL separation from the precipitated lipoproteins, based on faster HDL migration through the sieving matrix. The fluid sample, thus depleted of non-HDL lipoproteins, then is transferred to a test surface where it is assayed for cholesterol.
The above-referenced devices, while representing an advance over liquid-phase assays, present the possibility of contamination of the flow transport path with the precipitating reagents. Such reagents could interfere with HDL quantification, or with other assay chemistry taking place on other regions of a multi-assay device. The present invention addresses and overcomes these problems.
Further methods and devices for measuring HDL cholesterol in blood samples are disclosed in EP 0408223 and EP 0415298 (Rittersdorf et al.), which describe a continuous assay method carried out on a test strip comprising the following steps and corresponding elements. The blood sample is applied to a separation layer for separating cellular blood constituents. Driven by capillary forces or gravity, the sample flows through a further carrier containing soluble precipitating agents, which, after dissolving in the serum sample, precipitate non-HDL lipoproteins contained in the sample. In a further carrier, the precipitated constituents, above, are filtered from the serum sample to prevent their interference with later HDL quantification. In the same carrier, the sample is transported to a position adjacent the HDL-quantification carrier, and is stored until the HDL quantification step is to be started. Finally, the sample is transferred to an HDL quantification layer, where HDL cholesterol in the serum sample is quantified by an enzymatic reaction.
A disadvantage of this assay design is that the carrier functioning as a reservoir allows migration of the precipitated constituents or soluble reagents into the sample, which can interfere with HDL quantification. In addition, during the storage of the serum sample, HDL can be trapped by adhering to the carrier fibers, precipitating reagents can cause further undesired reactions, and the carrier can become clogged by the drying serum sample.
U.S. Pat. No. 5,135,716 (Thakore) discloses additional devices and methods for HDL quantification in a blood fluid sample. In these devices, the fluid sample flows continuously, though an unbroken path, from an inlet well to a carrier for HDL quantification. Accordingly, the ability to control sample volume entering the HDL test carrier, and to control environmental conditions for the HDL assay, is limited. Nor do the devices provide for simultaneous assay of various analytes from a single fluid sample.
It is therefore the object of the present invention to provide a HDL assay device which overcomes the above-noted prior art disadvantages.
In one aspect, the invention includes an assay pad formed by joining a HDL test pad and a reagent pad where the assay pad retains porosity sufficient for the sample to pass through the reagent pad to the HDL test pad. The assay pad further retains the biological activity of the reagents on the reagent pad and HDL test pad.
In another aspect, the invention includes an assay device and method for using the device for measuring serum cholesterol associated with high-density lipoproteins (HDL) in a blood fluid sample containing lipoproteins other than HDLs. The device includes a sample distribution array, a HDL test pad in which HDL concentration can be assayed; and a reagent pad containing a binding reagent effective to selectively bind and remove non-HDLs from the fluid sample. The HDL test pad and reagent pad are joined by an adhesive layer or by heating the pads.
In yet another aspect, the invention provides methods of preparing the joined HDL test pad and reagent pad.
I. Definitions
The terms below have the following meanings unless indicated otherwise.
An element is in "fluid communication" with another element when a fluid is able to travel from one element to the other via capillary action and/or gravity. The elements do not need to be in direct contact; i.e., other elements through which said fluid can pass may be intervening.
A "pad", as used in the context of a "sample distribution pad", "test pad", "HDL test pad", and "reagent pad" intends a thin, flat mat or cushion, or a piece of absorbent material. Pads may be comprised of any material, such as a porous membrane or fibrous strip, which can contain impregnated or immobilized reagents and through which fluid can move via capillary action and/or gravity.
II. Assay Device
The device in accord with the invention is illustrated in
With initial reference to
Sieving pad 22 carried in region 20 functions to partially remove large particulate matter (including blood cells) as the sample migrates through the pad matrix in a bottom-to-top direction as shown in the figure. Pad 22 is preferably formed of a glass fibrous matrix of material designed to draw aqueous fluid by surface wetting, and to retard the movement of blood cells as the blood sample is drawn through the matrix. One exemplary pad is a glass fiber filter, such as a GF/D, PD008, or F145-02 filter available from Whatman, having a packing density of about 0.16 g/cm3, and a thickness of about 1 mm. The pad is dimensioned to absorb a defined volume of sample fluid, preferably between about 15–25 μL. Sieving pad 22 may additionally contain red blood cell capture reagents, such as lectins, antibodies specific for red blood cell surface membrane proteins, thrombin, or ion exchange agents. In one embodiment, the pad may contain reagents for removal of non-HDL lipoproteins, as described further below.
The sample contacts an elongate strip or sample distribution array 26. The sieving pad 22 may be in fluid contact with array 26 between the well 16 and the array 26. In a preferred embodiment, array 26 is formed of three or more separate membranes in fluid communication. In an embodiment where array 26 is formed of three membranes in fluid communication, as seen in
The device further includes four or more test pads 64, 66, 68, and 70, which are wettable, absorbent reaction test pads. Each test pad used in a particular assay contains analyte-dependent reagents effective to produce an analyte-dependent change in the pad which can be detected in a known manner, as described further below. All or any integral subset of the test pads may be employed in a particular assay.
Desirably, the test pads are porous polymer membranes, preferably having a thickness of about 100–150 μm and side dimensions of about 3 mm. The absorption volume of each pad is preferably between about 0.5–1.0 μL. In one embodiment, some or all of the reaction pads are asymmetric membranes; that is, membranes having a porosity gradient across the thickness of the membrane.
In one embodiment, test pad 64 is a HDL test pad containing reagents effective to produce a change in the pad in response to the presence of HDL. In another embodiment, the HDL test pad 64 is also a polymeric membrane, containing reagents for assaying HDL level. One exemplary strip material is a BTS-83 asymmetric polysulfone membrane available from Pall Corporation (East Hills, N.Y.). If desired, HDL assay reagents, such as peroxidase, may be immobilized to the test pad membrane, according to well known methods for enzyme immobilization. (See e.g. U.S. Pat. No. 4,999,287; U.S. Pat. No. 5,419,902; Blum, L. J. et al., Anal. Lett. 20(2):317–26 (1987); Kiang, S. W. et al., Clin. Chem. 22(8):1378–82 (1976); Guilbault, G. G., Ed., Modern Monographs in Analytical Chemistry, Vol. 2: Analytical Uses of Immobilized Enzymes (1984); Torchilin, V. P., Progress in Clinical Biochemistry and Medicine, Vol. 11: Immobilized Enzymes in Medicine (1991)).
A reagent pad 74 contacts the HDL test pad 64 and contains the chemicals for precipitation of non-HDL cholesterol. In one embodiment, the reagent pad is the same material as the HDL test pad, i.e. an asymmetric polysulfone membrane. In a preferred embodiment, reagent pad 74 is composed of a porous polymeric membrane, having pore sizes of about 1 μm or less. In the device shown in
The reagent pad preferably has a thickness of about 100–150 μm, side dimensions of about 3�6 mm, and an absorption volume of about 0.5–1.0 μL. It contains at least one reagent effective to selectively remove LDL and VLDL particles from the fluid sample. The reagent may be, for example, an antibody, or preferably a polyanionic LDL- and VLDL binding reagent. Such reagents, which are known in the art, include sulfonated polysaccharides, heparin, and phosphotungstate, in the presence or absence of a group II cation, such as Mg2+, Mn2+, or Ca2+. A preferred reagent is a sulfonated polysaccharide, such as dextran sulfate, having a typical molecular weight of 50,000 to 500,000 daltons, optionally in combination with magnesium acetate or chloride, optionally buffered to maintain neutral pH. The reagent may be an immobilized reagent effective to bind, and remove from the fluid sample, non-HDL lipoproteins. The reagent pad is effective to entrap bound non-HDL lipoproteins within the reagent pad and prevent them from entering HDL test pad 64. In one embodiment, a reagent, such as catalase, which is effective to decompose any generated hydrogen peroxide that might diffuse downward from test pad 64, may be immobilized in reagent pad 74.
HDL test pad 64 and reagent pad 74 are adhered together using heat alone, as described in Example 6, or an acrylic acid copolymer that can be melted as a heat-activated adhesive, as described in Examples 1–3. The combined HDL test pad and reagent pad is called the assay pad.
Device 14 also includes a reaction bar 60, which is an elongate support, which may be transparent or have windows, e.g. window 76 (
The reaction bar is mounted on support 15 by mounting means effective to (a) maintain the device in a sample-distribution position, wherein the test pads and assay pad are spaced apart from the sample distribution array, the sieving pad or the well, and to (b) transfer the device to a test position, where the test pads and assay pad are in fluid communication with the sample distribution array, the sieving pad and the well. The mounting means can also be used to break such fluid communication after a desired amount of sample has entered the assay pad and/or the test pads, and/or after a determined contact time, by transferring the device from the test position to a position in which the assay pad and/or the test pads are not in fluid communication with the sample well (which may be the same as the "sample-distribution" position). Such transferring can be controlled by monitoring the reflectance at the top surface of the test pads, which reflects extent of wetting, as described in co-owned U.S. Pat. No. 5,114,350, the content of which is incorporated by reference herein. Alternatively, when the absorption capacity and rate of sample uptake of the pad materials are known, the quantity of sample can be controlled with sufficient accuracy simply by using a predetermined contact time.
The mounting means can include, for example, a pair of resilient members, such as elastomeric blocks 71, 72, which act to bias the assay pad toward a non-transfer or sample-distribution position, at which the pads are spaced apart from the sample distribution array, the sieving pad, the capillary conduit or the sample well. By compression or release of the resilient members, fluid communication between the assay pad can be selectively established and separated, as shown in
In a further embodiment, the HDL test pad comprises a biosensor, as described, for example, in PCT Publication. No. WO 99/58966 (Dobson et al.), which is incorporated herein by reference. This document discloses a microscale biosensor device, comprising a conducting surface, a layer of dielectric material overlying the conducting surface, and a plurality of pores extending through the dielectric layer. Each of the pores contains a biopolymer in contact with the conducting surface, and can act as a microelectrode, converting a chemical response into an electrical signal. In use, a fluid containing an analyte to be assayed is applied to the pores so as to be in contact with the biopolymer. In the present HDL assay device, this is achieved when the HDL test pad 64 is in fluid communication with the sample, i.e. the test position as shown in
The biopolymer within the microelectrode pores is typically an enzyme, such as, for the measurement of HDL-associated cholesterol, cholesterol oxidase. Cholesterol is oxidized by cholesterol oxidase to the corresponding ketone, liberating hydrogen peroxide, which can then be converted to water and oxygen by the enzyme peroxidase. Either oxygen or hydrogen peroxide can then be measured electrochemically. Electrochemical methods that may be used include amperometric methods, as in the Clark oxygen electrode, which measures current produced by reduction of oxygen or oxidation of hydrogen peroxide, or voltammetric methods. The use of cyclic voltammetry at microelectrodes has been described for measurement of various analytes (see e.g. R. J. Forster, Chem. Soc. Rev. 289–297 (1994)), such as dopamine (Pihel et al., Anal. Chem. 68(13):2084–9 (1996)) and fullerenes (Soucaze-Guillous et al., Anal. Chem. 65(6):669–72 (1993)) as well as hydrogen peroxide (Horrocks et al., Anal. Chem. 65(24):3605–14 (1993); Nowall et al., Electroanalysis 9(2):102–9 (1997); Dequaire et al., J. Am. Chem. Soc. 124(2):240–53 (2002).
III. Preparation of the Assay Pad Using an Adhesive Layer
As seen in
The adhesive layer is preferably formed of an acrylic acid copolymer. The copolymer will typically, but not necessarily, have a melting point below the denaturing temperature of the HDL assay reagents used or below a temperature that is otherwise damaging to the HDL assay reagents. One exemplary copolymer is an Ethylene Acrylic Acid copolymer (EAA). EAA is available as different particle size dispersions from Michelman (Cincinnati Ohio, P/N MP 4990R with a melting point of about 75� C). One preferred particle size is 30–500 nm, with an average particle size of about 90 nm. Other polymers, including polyethylene glycol, polyethylene terephthalate, and polyvinyl alcohol, may also find use as the adhesive layer. These copolymers may be cross-linked and may further be copolymerized with a second, different polymer. In another embodiment, the adhesive layer is formed of a pressure sensitive adhesive or a wax emulsion. In yet another embodiment, the adhesive layer is formed of hot melt adhesives as described in U.S. Pat. No. 6,596,112, which is incorporated herein by reference. It will be appreciated that any substance may be used for the adhesive layer where the assay pad retains a suitable porosity for the sample to flow from the reagent pad to the HDL test pad.
The composite assay pad is typically prepared by dissolving the copolymer in a suitable solvent, often an aqueous-based solution. A layer of the copolymer emulsion or solution is applied to the reagent pad 74. Any suitable method for coating the pad may be utilized, including dipping the pads in the emulsion or solution, spray coating the pad or applying the polymer solution to the pad with a wick. The pad may be coated on one side only or on both sides. The pad is dried for a suitable time, i.e. for about 20 minutes at 50� C. The precipitating reagent is then dispensed onto the large pore (dull) side of the asymmetric membrane.
When the reagent pad and HDL test pad are both formed of an asymmetric membrane, the reagent pad and HDL test pad are oriented such that the small pore (shiny) side of the reagent pad contacts the open pore (dull) side of the HDL test pad.
The copolymer adhesive layer between the HDL test pad 64 and the reagent pad 74 is formed by applying heat and/or pressure to the coated reagent pad 74 and HDL test pad 64, whereby the copolymer forms a bond with the adjacent pad material. The pads may be heated using any suitable device that provides heat at the required temperature and pressure. A preferred temperature for heating the pads is about 80� C. Any suitable lamination machine may be used.
The concentration of copolymer on the reagent pad must be such that the assay pad retains porosity for the sample to flow from the reagent pad to the HDL test pad. Suitable concentrations of copolymer to provide adhesion are about 4.0% to about 10.0% emulsion. Preferred concentrations of the polymer coating are 4%, 5%, 6%, 7%, 8%, 9% and 10%. One preferred emulsion is a 5.5% EAA emulsion.
The HDL reagent may be dispensed onto HDL test pad 64 by any suitable method before or after adhering the test pad to the reagent pad. The HDL reagent is preferably dispensed onto the test pad after adhering the test pad to the reagent pad.
IV. Preparation of the Assay Pad Using Heat
In one embodiment, the assay pad is prepared by adhering the reagent pad 74 containing reagent to the HDL test pad 64 using heat applied at a temperature sufficient to at least partially melt at least one of the reagent pad or the HDL test pad, as described in Example 6. The reagent pad and HDL pad are heated above about 65� C. to about 220� C. as described in U.S. Pat. No. 6,596,112, which is incorporated herein by reference. In a preferred embodiment, when the reagent pad and HDL test pad are heated above about 165� C., the pads become adhered to each other. In other preferred embodiments, the reagent pad and HDL test pads are heated above about 93� C., 121� C., 148� C., 165� C., 200� C., or 205� C. The pads are oriented as described above. The HDL reagent is dispensed onto the HDL test pad before or after adhering the test pad to the reagent pad. In a preferred embodiment, the HDL reagent is dispensed onto the HDL test pad and/or the chemicals for precipitation of non-HDL cholesterol are dispensed onto the reagent pad after adhering the test pad to the reagent pad. In a particularly preferred embodiment, both the HDL reagent and the chemicals for precipitation are dispensed onto the HDL test pad and the reagent pad, respectively, after the test pad and the reagent pad are adhered together.
V. Dispensing Reagents onto Assay Pad Using Printing Process
In another embodiment, one or more reagents are applied to opposite sides of a pre-laminated membrane using a printing process that incorporates an engraved Gravure or Anilox cylinder. In this embodiment, less than a saturation volume of the HDL reagent and the precipitation reagent are used. By using non-saturating volumes, the reagents may be confined to their respective membranes. A volume of about 80 ml/1000 inch2 is a typical saturating volume. Volumes in the range of 25 to 55 ml/1000 inch2 of reagent are suitable, however, in one embodiment, 20 ml/1000 inch2 reagent is used. The precipitation reagent may be applied before or after the HDL reagent. In other embodiments of this printing process, other reagents that contain two components that must be kept separate can be applied in like manner as described for the HDL reagents. Alternatively, a single reagent can be applied to a single unlaminated membrane.
VI. Assay Method
In operation, a blood sample is placed into well 16, and is imbibed through sieving pad 22, where large particulates, including red blood cells, are removed, and thence into the sample distribution array 26. In one embodiment, these steps take place while the device is in a "sample-distribution" stage, such that the assay pad is not in fluid communication with the sample distribution array, the sieving pad, the capillary conduit, or the sample well.
The plasma sample travels from sample-application membrane 28 to sample-collection membranes 30 and 32. When the plasma sample reaches sample-collection membranes, the device is adjusted to a test position, e.g. as shown in
Prior to contacting the HDL test pad 64, sample plasma contacts a precipitating or binding reagent, which is contained in a separate reagent pad 74, such that non-HDL lipoproteins are bound to the respective carrier. The device is thus effective to remove non-HDL lipoproteins from the serum, while allowing passage of sample fluid containing liquid-phase HDL to HDL test pad 64 with these elements (e.g.
During operation, in embodiments such as illustrated in
In these embodiments, when the sample fluid contacts reagent pad 74 containing the binding reagents, the latter is in direct contact with HDL test pad 64, thus limiting the temporal contact of the blood sample with the binding reagents prior to the HDL assay reaction. Sample preparation and HDL evaluation are thus carried out in separate steps, where sample preparation includes, for example, filtering of cellular blood components and, optionally, temporary storage of the blood sample and adaptation of the blood sample to such test requirements or conditions as temperature, pressure and environmental atmosphere. Because the temporal contact of the blood sample with the different reagents is reduced, any chemical interference with the HDL evaluation is prevented. If desired, the assay can be interrupted for a desired time after the sample application and removal of cellular components, but prior to contact with binding reagents, e.g. to adjust the surrounding atmosphere or adapt the environmental temperature to support the testing. This is accomplished by maintaining the device in the sample-distribution position. To this end, the sample distribution matrix is designed to additionally serve as a reservoir, if needed.
The HDL test pad contains reagents for quantification of HDL-associated cholesterol. Preferably, these include cholesterol esterase, for releasing free cholesterol from HDL, cholesterol oxidase, for producing H2O2 by reaction with free cholesterol, peroxidase, and a coupled dye system which is converted, in the presence of peroxidase and H2O2, to a distinctively colored signal reaction product. The test pad may also comprise a biosensor effective to electrochemically quantify H2O2 and/or O2, as described above.
The remaining test pads 66, 68, and 70 also contain assay reagents which produce a change in the pad, which can be detected optically, either visually or by a detector, in a known manner. In preferred embodiments of the current device and method, the non-HDL binding reagents are located in reagent pad 74, and not in the sample distribution array or sieving pad. In this embodiment, the possibility of interference from these reagents, in assays of analytes other than HDL, is eliminated.
Preferably, each of the test pads contains reagent components for producing H2O2 via reaction of the analyte with an enzyme; the H2O2 subsequently converts a substrate reagent to a colored signal reaction product, or is measured electrochemically, as described above. Enzymatic color reactions which employ a variety of substrate-specific oxidases, for enzymatic generation of H2O2, and subsequent oxidation of a dye to form a colored reaction product, are well known.
A device having four or more test pads can be used to simultaneously measure HDL cholesterol (HDL), glucose, total cholesterol (TC), triglyceride lipid (TRG), ALT, AST, BUN, and/or creatinine. Each pad contains the above-described common pathway components (peroxidase and a coupled dye system) such that generated H2O2 produces a distinctly colored signal reaction product. The total cholesterol test pad, which is exposed to serum without exposure to a precipitating or binding reagent, and the HDL test pads each include, in addition to the common pathway components, cholesterol esterase, for releasing esterified cholesterol in free-cholesterol form from serum lipoproteins, including HDL, LDL, and VLDL particles, and cholesterol oxidase, for producing H2O2 by reaction with free cholesterol in the sample fluid, as described above. The glucose assay pad includes glucose oxidase, in addition to the common-pathway components. The triglyceride pad includes, in addition to the common-pathway components, lipase, L-glycerol kinase, and L-glycerol-3-phosphate oxidase, for generating H2O2 from triglyceride, via the intermediate L-glycerol-3-phosphate. The serum sample drawn into the TRG pad is not exposed to precipitating or binding reagents, and thus contains all of the serum lipoproteins, so the TRG signal represents total serum triglycerides.
Reference standard pads may also be employed; see, for example, the system described in co-owned U.S. Pat. No. 5,114,350, which is incorporated herein by reference.
The following examples illustrate but are in no way intended to limit the invention.
A BTS-83 polysulfone membrane was loaded with HDL reagent at a loading volume of 77 μl/in2 and dried for 20 minutes at 50� C. in a continuous roll process. Test pads containing the following HDL reagents were prepared: cholesterol oxidase 36.5 Units/ml, cholesterol esterase 215 Units/ml, peroxidase 200 Units/ml, 4-aminoantipyrine 1.88 mg/ml, and TOOS (3-[ethyl(3-methylphenyl)amino]-2-hydroxy propanesulfonic acid) 12.05 mg/ml. Lengths of e.g. 100 feet can be prepared in this manner and cut to fit the assay devices.
An aqueous reagent solution containing EAA dispersion at a concentration of 5.5% was applied to BTS-83 polysulfone membrane in a dip tank and dried for 20 minutes at 50� C. in a continuous roll process. Next, an aqueous reagent solution containing 1–5 mg/ml dextran sulfate (500,000 MW) and 35 mM Mg(OAc)2 was dispensed onto the same membrane using a syringe pump to meter reagent onto one surface of the membrane. Lengths of e.g. 100 feet can be prepared in this manner and cut to fit assay devices.
A HDL test pad and a reagent pad were prepared according to Examples 1 and 2, respectively. The pads were oriented with the layers having the shiny side of the reagent pad (small pore size) contacting the dull or open pore side of the HDL test pad. The two membranes were passed through a lamination machine that heated the membranes to 80� C. for 20 seconds in a continuous roll process.
A typical assay was carried out in an LDX� analyzer, using an assay pad, prepared essentially as described in Example 3. Sections of the adhered pads were hand cut to reaction membrane size and attached to the reaction bar. Alternatively, laminated assay pads can be applied to reaction bars in a continuous roll process using ultrasonic welding. A serum sample was loaded in the sample well. After sample distribution, the reaction bar was contacted with the adhered pads for approximately 4 seconds, a time sufficient to transfer enough serum to fill the HDL test pad, after which the bar was returned to its original position. The color was allowed to develop for 3 minutes and the reflectance was monitored by the LDX� analyzer.
The concentration of EAA on the reagent pad was varied to determine porosity of the membrane. The reagent pad was prepared essentially according to Example 2. Serial dilutions of EAA were applied to the reagent pad starting with a 20% solution. The reagent pads were adhered according to Example 3 to a HDL test pad. The dull side of the reagent pad was tested with a dilute aqueous solution of Evan's blue to determine penetration and/or diffusion of the colored solution through the reagent pad and into the HDL test pad. The highest concentration of EAA that allowed ready penetration and/or diffusion of the solution was about 10% EAA.
Two layers of BTS 83, without any reagents applied, are laminated together in a continues roll process. The orientation of membranes is as described for Example 3. An aqueous solution of Dextran sulfate (50,000 MW) and Mg(OAc)2 is coated onto the open pore side of the laminate using a standard gravure coater. The gravure coater is selected to dispense a non-saturating volume of reagent onto a single layer of the laminate. Following drying, the HDL reagent is applied to the opposite membrane in a similar manner. The reagents are concentrated from the levels shown in Example 1 and 2 to compensate for the reduced volume being applied.
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LAHORE: Players of the Pakistan badminton team cannot take part in the World Badminton Championship, to be held in India, due to their demotion in world ranking.
The World Badminton Championship will be held in Indian city of Hyderabad Deccan from August 10 to 16.
Senior vice president of Pakistan Badminton Federation Syed Naqi Mohsin told Geo News that ranking of Rizwan Azam and Mohammad Ateeq in doubles was 62 while ranking of Wajid Ali in singles was 106 but these players could not participate in any international contest due to non-availability of funds for the last five months due to which their ranking fell to 170 and 250, respectively and now they have become ineligible for taking part in the World Championship.
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'There were four of us and we thought we'd have a change and do some outdoor surfing," Hart told Metro U.K. "Me and a friend put a rope on the car and went for it. We didn't think it would work but it worked like a dream."
In the first section of the video, Hart rides his board upright through the flooded streets of Castleford, West Yorkshire, which, reported, recently suffered the worst storms in more than three decades.
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continued The fact that his father is coming home for the event ices the metaphorical cake. His family wasn't sure if Matt Allen was going to make it home, and they didn't receive confirmation until recently.
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Makoun joins Olympiakos on season loan subject to FIFA approval
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