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Nice to meet you here at ringtones service Audiko.net, where you can get the best ringtones for your cell. Today we have a ringtone of "The Wizard" by Uriah Heep as well as 315 other ringtones by Uriah Heep. "The Wizard" is the best track from the "Demons and Wizards" album that was issued by Uriah Heep at their label together with other 7 tracks. You can listen to all 9 Uriah Heep – "The Wizard" ringtone variations available here and download it for free in a click. You can also look at other 23 albums produced by Uriah Heep for all time. "The Wizard" is referred to Classic Rock, and we bring you a huge amount of Classic Rock artists absolutely for free. And finally, we have collected lyrics for this track and you can view it here as well. So feel free to browse, listen, download ringtones at Audiko.net. Make your phone sound cool!
Uriah Heep The Wizard Ringtone
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KALISPELL - Flathead Valley Community College will dedicate the new Rebecca Chaney Broussard Center for Nursing and Health Science this week.
The Flathead Valley Community College invites the community to attend a very special dedication of the new Rebecca Chaney Broussard center for Nursing and Health science.
"We are very excited because it's a privately funded building thanks to a very generous donation from Drew Broussard and his family," FVCC President Jane Karas said.
Rebecca Broussard was a well-known nurse and philanthropist in the Flathead Valley. She was known as a humble person who had a passion for education. Her life came to an end in 2011 when she died of breast cancer.
"Nursing programs were near and dear to her heart and helping the community. Helping students get into their education is a focus of the Broussard family and so thanks to their great generosity we were able to move forward with this building," Karas said.
This 4,000,032-square foot building will house FVCC's nursing and health related programs as well as the college's first student health clinic. Paramedic Program Director Kris Long said the extra classroom space will accommodate both students and instructors.
"We were in a classroom for a lab before where it was about 500 square feet so all the equipment that we have in our area with two labs and two simulations was in one room," Long said.
The building will also include a wall dedicated to Rebecca, new equipment, two labs and two simulation rooms for students to practice their skills.
"We spent a lot of time of actually moving students in the hallway to practice skills and now we can actually be in our labs and working in our labs and not have to take down and put up equipment all the time," Long said.
As the college prepares to welcome students into the new building Karas said she is eternally grateful to the Broussard family and the community for supporting the college.
"Just the generosity of the Broussard family and the generosity of the community and Flathead County and Northwest Montana to help us build this building," Karas said.
The dedication will take place Tuesday at 4 p.m. on the college campus. Building tours and light refreshments will follow.
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subset: commoncrawl


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My son is learning his times tables so to help him out I decided to write a simple game in python. I call it Maths Ball. It's a 2 player game where the idea is to score 3 goals to win.
The game starts with a ball heading towards player 1′s goal. A maths question appears at the top of the screen along with 4 possible answers. Player 1 needs to answer the question before the ball reaches their goal. If they choose an incorrect answer, they concede a goal. If they choose the correct answer, the ball starts heading towards player 2′s goal. The quicker they answer the question the less time the other player will have to respond. It's very effective in helping kids learn their times tables because they get to answer a lot of questions in a small amount of time.
I've put the code for Maths Ball up on github. If you are a python programmer and want to help make it better I'd love you to help. I have the following ideas that would make the game better:
- Add an option to play against the computer
- Allow players to enter their names
- Allow players to choose a level which controls the speed of the ball
- Allow players to choose multiplication, division, subtraction or addition
- Store more stats for each player like questions answered correctly and incorrectly
- Improve the graphics for the splash screen (function splashScreen)
- Improve the graphics for the winning screen (function winningScreen)
- Improve the graphics for the final screen displaying statistics (function showStats)
- Improve the graphics for the goal scored screen (function showGoal)


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Casio G-5600E Tough Solar - World Time, Alarm, Stop Watch, Timer, Oct. 2011
The DW-5600E on the left shows the time 11:49
when in the stop watch mode. The G-5600E measures up to 24 hours and therefore displays the hours in the upper right window. I have actually always liked chronographs that display the time in the stop watch mode. In this picture it also looks as if the LCD displays have been developed further.


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A Penn State internal investigation into the Jerry Sandusky sex abuse scandal has found the university's most powerful men failed to protect children victimized by the former assistant football coach for 14 years.
Former FBI director and federal judge Louis Freeh said Thursday that Penn State president Graham Spanier, head football coach Joe Paterno, vice president Gary Schultz, and athletic director Tim Curley "never demonstrated, through actions or words, any concern for the safety and well-being of Sandusky's victims until after Sandusky's arrest."
Freeh said Penn State officials planned to go to authorities after a former graduate assistant, Mike McQueary, said he witnessed Sandusky with a young boy in a locker room shower, but after Curley consulted with Paterno they changed the plan and decided not to make a report.
"Their failure to protect the February 9, 2001 child victim, or make attempts to identify him, created a dangerous situation for other unknown, unsuspecting young boys who were lured to the Penn State campus and football games by Sandusky and victimized repeatedly by him," Freeh said.
Freeh said Spanier, Paterno, Schultz and Curley further exposed the child to additional harm by alerting Sandusky, who was the only one who knew the child's identity, about what McQueary saw in the shower.
"In order to avoid the consequences of bad publicity, the most powerful leaders at Penn State University - Spanier, Schultz, Paterno and Curley – repeatedly concealed critical facts relating to Sandusky's child abuse from the authorities, the Board of Trustees, Penn State community, and the public at large," Freeh said.
The investigation also found that the four men also knew about a 1998 criminal investigation of Sandusky after an incident with a young boy in a Penn State football locker room shower.
"The evidence shows that Mr. Paterno was made aware of the 1998 investigation of Sandusky, followed it closely, but failed to take any action, even though Sandusky had been a key member of his coaching staff for almost 30 years," Freeh said.
Freeh said even though the four all knew about the 1998 incident, the best they could do to protect Sandusky's victims was to ask Sandusky not to bring his "guests" into Penn State facilities.
You can read the full report here: http://www.abc27.com/link/573149/the-freeh-report
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On Pacers.com the team has released a calendar of "big things" that will happen between now and the season opener. One of the "big things" was a "special announcement coming December 22 for Pacers fans."
I can't find anything online about what this could possibly be, but the first thing that came to my mind was the possibility of Reggie returning as a front office guy. I think this would be great for the franchise and would certainly get the community excited, but I'm curious if anyone agrees with me about this possibly being the "big announcement." Any thoughts?


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St. Augustine murder from 1974 remains unsolved | News
ST. AUGUSTINE, Fla. -- Police are still working to solve the death of a woman who's body was found in an empty field in November 1974.
On November 3, 1974, neighbors told police they saw Frances Bemis take her nightly walk, according to a news release from Mark Samson, Public Information Officer for the St. Augustine Police Department.
Samson said the next morning, Bemis' body was found in an empty field just south of 6 Marine St.
An autopsy performed by the medical examiner determined Bemis died of blunt force trauma, Samson said.
When police first interviewed neighbors, they reported hearing screaming around 7 p.m. November 3 and hearing what sounded like someone being choked.
Police searched the area at the time, Samson said, but did not find anything unusual.
Samson said Bemis owned the Maria Sanchez Apartments at 144 Marine St. She was also active in the community, involved with the Humane Society, Art Association and was outspoken about the treatment of carriage horses.
If you have any information that could help police, you are asked to contact St. Augustine Police Detective Corporal Charles Simpson at 904-209-3484 or the St. Augustine Police Tip Lines at 904-209-3482.
You may also contact Crime Stoppers at 1-888-277-TIPS. You could be eligible to receive a reward up to $5,000 from Crime Stoppers of Northeast Florida.
Top St. Augustine Stories
St. Augustine Deals
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Green Cove Springs News
- More firefighters on the job in Clay County
- Dogs at Clay County Animal Care and Control in need of homes
- Harvey's Supermarket closing in Green Cove Springs as is a distribution center, lost jobs
- Teen spends 35 days in jail after mistaken arrest
- Former IT specialist accused of stealing $19K in computer equipment
Julington Creek News
- If women can run nations and corporations, they can certainly learn to run a boat
- Advocacy group believes aquarium would provide much-needed lift for downtown
- Lisa Almeida of Freedom Boat Club named Up and Coming Entrepreneur of the Year at the Women in Busin
- Freedom Boat Club promotes Dock Manager to Director of Dock Operations
- Freedom Boat Club to hold Open House


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Pitches on the water! by Potsdam_Pitches - over 4 years ago
"We are an a cappella group from SUNY Potsdam. This picture shows us hanging out by a lake during some of our downtime from our Spring Tour in Bennington, VT. CustomInk helped us to get our shirts in time for Tour and overall we're very pleased with the company!"


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Aaron Maybin to Get More Playing Time
Aaron Maybin is set for more playing time with Bryan Thomas (Achilles) done for the year. In his first regular season game as a Jet last Sunday night, Maybin came off the edge for a sack and forced a fumble. Now he's left as one of the only pure pass-rushers on the roster. The former first-round pick will be used plenty as a situational player against the Patriots Sunday. Via New York Post


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Every person who seeks cancer treatment has a unique body, mind and life history. At Good Samaritan Hospital's Cancer Center, we develop a sophisticated treatment plan tailored specifically to your condition, diagnosis and circumstance.
Our dedicated and experienced physicians combine clinical expertise, trials and research with advanced diagnostics and treatment options.
Why Choose Advocate Good Samaritan Hospital
Learn more about our services and treatments.View a virtual tour of our cancer center.Quality Measures in Oncology Practice.View more about cancer genetics.
Cancers We Treat
Services and Treatments
Learn more about our services and treatments.
Meet Our Care Team
Our board-certified medical and radiation oncologists and surgeons work as a team to develop your diagnosis and treatment program. Our specialists collaborate with a team of nurses dedicated to cancer care, included nationally-certified oncology nurses. Social workers, chaplains and genetic counselors are also available as resources for you.
We know cancer treatment is a stressful journey, That's why we provide a personal Nurse Navigator, to help you understand treatment, answer your questions and coordinate appointments. We want your process to be personal and comfortable so you can focus on your healing.
Read more about our team. Find a Good Samaritan Hospital cancer specialist near you.
Additional Information
View a virtual tour of our cancer center.
Quality measures in oncology practice
Contact UsCancer Care Center630.275.23003815 Highland AvenueDowners Grove, IL 60515Map and Directions
Cancer EducationPrevention, Detection, andPatient Resources


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Girl Scouting offers girls the opportunity to explore their skills and interests which will inspire them to reach their personal best through a variety of fun and enriching leadership experiences. But more importantly, no matter where or how they participate, girls get the necessary guidance they need from adult volunteers and council staff to develop their leadership skills and understand how those skills can be used to make a difference in the world.
Rather than just meeting in a Troop setting, Girl Scouts in Nassau County have the opportunity to participate in events and programs.
Before registering for a GSNC Program, please review important registration information.
Click on the name of the event that interests you to learn more and to request placement. Completion of the online request is NOT a guarantee of placement. Cookie Credits can be used to pay for any of these programs.
ADDITIONAL program offerings (last-minute opportunities that are not in Possibilities)
Programs for Girl Scouts (In order by date)
Here's an easy way to teach your Daisy Girl Scouts the basics of money and have them earn one or both of the financial literacy badges.
WHO: Volunteers
DATE & TIME: Tuesday, August 12, 2014 at 7:00pm–8:30pm Or Thursday, August 14, 2014 10:00am–11:30am
PLACE: GSNC Service Center, 110 Ring Road West, Garden City, NY 11530
COST: $10 per Adult (Includes program materials) Badges not included
QUESTIONS: Contact Fran McAllister at email@example.com or 516.741.2550 ext. 249
TAKE ACTION FOR THE GOLD AWARD - At Capacity
If you are planning to earn the Gold Award, this workshop is for you! Learn the guidelines for the award, what it means to do a Take Action project, important deadlines, the approval process, and what your interview will be like. You will also hear how to make a lasting impact on your community as you live the Girl Scout Law: To make the world a better place!
WHO: Senior & Ambassador
DATES: Tuesday, August 26, 2014
PLACE: GSNC Service Center, 110 Ring Road West, Garden City, NY 11530
TIME: 7:00pm–8:30pm (all dates)
QUESTIONS: Contact Patte Conway by email at email@example.com or by phone at 516.741.2550 ext. 219
Take Action for the Gold Award - Tuesday, August 26, 2014 has reached capacity. No more registrations can be accepted.
If you are planning to earn the Silver Award, this workshop is for you. Learn the award guidelines, including hands-on activities that show what it means to do a Take Action project, how to map your community, and how you can make the world a better place.
WHO: Cadette
DATE: Thursday, August 28, 2014
PLACE: GSNC Service Center 110 Ring Road West Garden City, NY 11530
TIME: 7:00pm-8:30pm (all dates)
COST: $10 per registered Girl Scout or Troops (includes certificate for Journey book), $5 per adult
IMPORTANT: No Tag-Alongs
QUESTIONS: Contact Patte Conway by email at email@example.com or by phone at 516.741.2550 ext. 219
REGISTER: Register for Take Action for the Silver Award on Thursday, August 28, 2014>>
DATE: October 16-19, 2014
PLACE: Calvin L. Rampton Salt Palace Convention Center, 100 South West Temple, Salt Lake City, Utah
FOR MORE INFORMATION: www.girlscouts.org/convention/default.asp
Please do not call about program availability! This site is regularly updated to indicate those sessions that are full.
Completion of the online request is NOT a guarantee of placement. Once you are placed in the event, you will receive a final confirmation from "firstname.lastname@example.com". Please add this email address to your "safe sender" list. IF YOU HAVE NOT RECEIVED A CONFIRMATION, YOU ARE NOT REGISTERED FOR THE EVENT. Your credit card will not be charged unless you are placed in the program.
Registration Fees are NOT REFUNDABLE unless Girl Scouts of Nassau County is unable to place the Girl Scout in the program/event requested. Refunds are not available for no-shows or early departures.
If you choose to have your confirmation sent via email, it must be to the email address on your membership record. If your information (address, phone, e-mail) has changed since you filed your membership registration, please let us know.
Non-Council events are not available for on-line registration. You must register directly with the program provider listed with the activity.
All events are for registered Girl Scouts. Girls who are not Girl Scouts may register as a Girl Scout member, and then request placement in any of our programs.
Want to pay using Cookie Credits? Learn how.
Any programs that feature the Healthy Living Initiative Symbol are programs that are considered to be in line with Girl Scouts of Nassau County's Healthy Living Initiative.
The Healthy Living Initiative brings a unique perspective to keeping girls healthy. Girls define health in their own terms, placing as much emphasis on emotional and social well-being as physical health, nutrition and physical fitness. In addition to healthy eating and physical fitness, the Healthy Living Initiative focuses on increasing girls' self-esteem, media image awareness and how to develop healthy relationships.


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> View this now
Published on: November 08, 2012
Type of content: VIDEO
Price: FREE
Cloud computing allows IT pros to more efficiently and cost effectively manage and support SAP applications. However moving SAP applications to the cloud affects the entire organization, so you need a cloud provider that has the knowledge and resources in order to ensure the transition to the cloud is a success.
Access this exclusive video to learn about a leading cloud provider that helps you support your most critical SAP applications by reducing capital expenditures and increasing service levels.


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Harrisburg police have identified a suspect in the abduction of a Harrisburg Area Community College student.
Clarence Shaffer, 52, no fixed address, may be armed and should be considered dangerous, police said Tuesday.
An arrest warrant charging Shaffer with a Megan's Law violation has been issued so far. It lists a Kennett Square address.
Police said that Shaffer has family in the general area, but is said to be estranged from all, and he might have access to a vehicle.
Anyone with information regarding Shaffer's whereabouts is urged to call their local police department immediately.
The victim, an 18-year-old suburban Harrisburg woman, told police she was abducted Monday morning from the West parking lot of HACC's Main Campus.
She said a stranger opened her car door and forced her into the passenger seat of her car, then drove her at gunpoint to the Enola area, where he pulled into a parking lot where the Conodoguinet Creek meets the Susquehanna River, forced her to walk to a wooded area and tried to sexually assault her, police said.
She said she was able to run when the attacker put down his gun, police said. She went to a nearby home for help and was not harmed, police said.
The abductor took the woman's car back to the parking lot at HACC and left it there, police said.
Cumberland County District Attorney Dave Freed said Shaffer was sentenced to 4-14 years in prison in 1999 after pleading no contest to unlawful restraint and aggravated indecent assault for a similar incident in East Pennsboro Township in 1995.
Shaffer was released from state prison on November 28, five days before the abduction at HACC, Freed said.
Shaffer is registered as a lifetime offender under Pennsylvania's Megan's Law. He has also used the names William Murray and Clarence Shaffer.
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main index
Queenadreena are a British alternative rock band (although the exact genre is up for dispute- some people refer to them as 'goth'n'roll') which was formed in 1999 in London by vocalist Katie-Jane Garside and guitarist Crispin Gray, both formerly of the punk band Daisy Chainsaw. They initially wrote their name Queen Adreena, but made it a single word soon after the release of their first album, Taxidermy. Since then the band have had a steady and stable career despite frequent lineup and label changes, but no breakthrough success.The band's music is dark and often quite heady, eerie and desperate sounding, with the lyrics, when not open to multiple interpretations as they so often are, usually tackling difficult subjects such as anorexia ("My Silent Undoing") and the murder of Jon Benet Ramsey ("FM Doll"). The videos match: strange and grainy, and occasional doses of horror.Many members of the band have side projects, which they develop alongside Queenadreena. Katie Jane sings for Ruby Throat, and has a released a collection of her own songs under the name 'Lalleshwari', and in 2006 Crispin and Nomi founded a band of their own, the Dogbones. The band have also composed a live soundtrack to David Cronenberg's film Crash.Members:
This band provides examples of the following:


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You're John Dorsey. You are the general manager of the Kansas City Chiefs, by all accounts a bright guy, and you are searching for any competitive advantage you can get.
You decide on a smokescreen. Here is how you deploy this noxious mix of wistful gazes at Geno Smith and the sweet, hickory scent of a potential draft day trade.
- You trade multiple picks to make Alex Smith your starting quarterback
- You lure Chase Daniel away from the Saints to be your backup/potential heir apparent
- You actively shop Branden Albert, potentially opening up a gaping hole at tackle
- You then rave about Geno Smith
Now, no offense to Mr. Dorsey, but you'd have to be a complete idiot to fall for this. The Chiefs could sink the #1 overall pick into Geno Smith, but if they let Albert walk and have Smith and Daniel on the roster, long-suffering Chiefs fans might throw him onto I-70. They would be justified in doing so.
Remember: what the Chiefs or others say doesn't matter. What they DO does matter. Draft picks = Alex Smith's the man— Arrowhead Pride (@ArrowheadPride) March 27, 2013
Rotoworld saw through this. I saw through this, and my knowledge of the Chiefs is admittedly limited. Yet teams persist in trying out these smokescreens, which would seem to suggest that they're successful sometimes. That would seem to further suggest that NFL front offices are dotted with extremely gullible people, which is a depressing thought.
Do they work, though? I'm going the small sample size route because I'm an inveterate lazybones, but I decided to take a look at the 2012 NFL Draft and see if this held up. After all, the Buccaneers and Rams were rumored to be considering moving down, the Vikings were going to pass on Matt Kalil in favor of Morris Claiborne and golly, who knew who was going to end up with Robert Griffin?
As it turns out, last year was a perfect example of the smokescreen having more than a little effect on teams. The Redskins jumped up to take Robert Griffin, knowing he'd be off the board after the second pick, but they never really bought that the Colts weren't zeroed in on Andrew Luck at the top of the draft. The Vikings moved down one pick, behind the Browns, and took Kalil as expected. The Browns got Trent Richardson, the Jaguars got Justin Blackmon and the Cowboys jumped up ahead of the Buccaneers to take Claiborne. The Buccaneers used Jacksonville's pick to grab Mark Barron, though it's entirely possible they wanted in on Claiborne. Fletcher Cox, widely rumored to be a possible top five pick, went 12th overall to the Eagles.
That's a dizzying array of trades, and I'm just cherry-picking here to make a point. The point is that with player salaries clocking in lower than ever, the smokescreen game is actually getting more effective for teams that play it right. All you need to do is plant a seed of doubt that you intend to snap up a player another team desperately wants and they may be willing to cut a deal with you. We'll see if 2013 mirrors 2012, but I wouldn't be surprised if it does.
Still, Dorsey's extremely clumsy attempt to link the Chiefs to Geno Smith probably isn't going to work. Teams are and should be much more worried about the Jacksonville Jaguars, who have been linked to Smith all along. David Caldwell isn't one to play those games.
What do you think of the smokescreen game?


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Do your homework
GOG #17 vs. New York - because you want to be reminded of that series again
The Questions
1. Series outcome (3 games) [3 points]
2. Travis Blackley made the short trip to the right side of the bay a couple weeks ago, and now he's starting on Memorial Day in Minnesota. In four games with the Grizzlies, he compiled a 0.39 ERA, then was promptly lit up when brought up to the Giants. Facing him is Scott Diamond, who oddly enough has the Twins' highest batting average. For each pitcher, predict IP/H/K/ER [2 points each; 16 total]
3. Mannywatch may finally be coming to an end. Ramirez is eligible to join the A's on Wednesday, his 40th birthday. How many AB/H/BB/K for Manny on Wednesday? If he does not join the A's on Wednesday, then we'll use same stats for Ramirez in the Rivercats' game vs. Fresno on Wednesday. [2 points each; 8 total]
4. After a long wait, Mr. Change of Scenery is back with the A's. After going 6 for 7 (!) with two home runs in a rehab stint with the Cats, Brandon Inge is set to rejoin the A's in Minneapolis. Guess Brandon's AB/H/RBI/K for the series [2 points each; 8 total]
5. Starz and Goatz: High(er) roller edition. Name as many players as you want (either team) who will have a positive cumulative WPA for the series. You can name as few or as many players as you like, but here's the catch: for every correct guess, you get 2 points. For every incorrect player, you lose 2 points. If a player doesn't play in the series, or ends up with a WPA of zero, it doesn't count for or against you. [anywhere between -100 and +100]
6. I think I found the problem with the Twins. Their pitching staff has the highest ERA in the majors (5.47), has given up the most earned runs (by a long shot at 249), given up the most home runs (also by a long shot at 67), and has recorded the fewest strikeouts so far (261 - the next lowest team is your lovable muppets at 281). For the Twins' three starters, guess over, under, or equal for the following: 18 H, 14 ER, 10 BB, 13 K's, 4 HRs, 2 wild pitches, 18 IP. [2 points each; 14 total]
7. Ex A's outfielder Compliant Pork is having a respectable year so far for the Twins with a .270 AVG, 41 hits, and 28 RBIs. Nearly equaling Josh is new A's right fielder Josh Reddick with .271/48/26. For each category (for the series), guess Willingham, Reddick, or tie: More hits, more Ks, more walks, more XBHs. [1 point each; 4 total]


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Are you planning to build or remodel your home? Maybe you're looking at house plans, working with an architect or designing your own home. Or maybe you're looking for a home to buy. Whatever your project...
The House Plans Helper website is here to help you with home design
There are lots of sites out there with beautiful pictures of home decor. This site is a little different. HPH concentrates on the bones of a home - layout and architectural features - both of which are essential to a successful home design.
Some homes are a joy to live in. Others – well, not so great. The flow of rooms doesn't quite work or you just don't feel at home for some reason.
House Plans Helper provides information and resources so that everyone can learn about home design.
Taking the time to explore this site will put you in a much better position to think about your house plans. You'll be able to brief your architect or designer effectively and analyze floor plans to see how they measure up to what you need.
For most of us buying, building or making changes to our homes means big decisions (and lots of decisions) with large amounts of money at stake. It's something we're anxious to get right. Using the information and exercises on this site will arm you with better knowledge to help you make good choices. It will take a bit of time and thinking on your part and it's well worth the effort!
Thank you for visiting House Plans Helper. I wish you all the best for your project.
House Plans Helper Alerts
Free Floor Plan Symbols
Free Blueprint Symbols
Search this site
Like if you just love home design


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A device for collecting and transporting aqueous fluid from the oral cavity to a lateral chromatographic strip for test is disclosed. The lateral chromatographic strip is placed within and extend along a cavity defined in a housing. At least one inspection site to the lateral chromatographic strip is provided to enable inspection of selected sites on the lateral chromatographic strip for test results. A porous wick material protrudes from the housing to a collection site exterior of the housing at one end and communicates to the lateral chromatographic strip at the other end. The porous wick material has particulate construction, the particles adsorbing aqueous oral fluid to transport the fluid from the mouth to the lateral chromatographic strip without substantial absorption. The particles of the porous wick material are bound together to define a continuous interstitial volume for the flow of oral fluid to be transported and are treated to be hydrophilic to the adsorbed oral fluids. The porous wick material readily releases oral fluid to the lateral chromatographic strip. Prevention of reverse flow to the oral cavity from the lateral chromatographic strip naturally occurs due to the circuitous flow path of the porous wick material. A bite plate is coupled to the housing and insertable between the teeth of the patient to position the porous wick in the oral cavity for collecting the oral fluid. The bite plate is typically held in place by the occlusal force of the teeth, preferably the molars and/or the bicuspids, to position the porous wick in the buccal space. By observing the lateral chromatographic strip while the test device is in the mouth immediate test results are obtained.
1. A device for assay of oral fluid, comprising:
an assay portion housing a lateral flow assay strip, the assay strip containing at least one reagent that is used to detect one of the presence and absence of at least one analyte in a fluid; and
a neck portion extending from the assay portion, the neck portion forming a channel for delivery of fluid to the assay strip, the channel being defined by a first, narrow part proximal to the assay portion and a second part including an opening for receiving the oral fluid, wherein the second part includes a channel width that is substantially wider than the channel width at the narrow end;
a collection strip in fluid communication with the lateral assay strip, the collection strip having a first portion disposed within the channel and a second portion protruding outwardly from the neck portion opening; and
a blocking strip coupled between and in flow communication with the lateral flow assay strip and the collection strip.
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 device of
15. The device of
16. A kit for the detection of antibodies in oral fluids comprising:
an device of
a buffer, reagent, or detection reagent for collection and lateral flow chromatography of an oral fluid.
17. The device of
18. The device of
19. The device of
20. The device of
21. A kit for the detection of antibodies in oral fluids comprising:
an device of
a buffer, reagent, or detection reagent for collection and lateral flow chromatography of an oral fluid.
This application claims benefit under 35 U.S.C. �6 120 as continuation-in-part of U.S. application Ser. No. 09/280,269, filed Mar. 29, 1999, and as a divisional of U.S. application Ser. No. 09/292,293, filed Apr. 15, 1999, now U.S. Pat No. 6,303,081, U.S. application Ser. No. 9/280,269 claims benefit under 35 U.S.C. �119 from U.S. Provisional Application 60/079,958, filed March 30, 1998.
This invention relates to the assay of oral fluids typically in lateral flow chromatography. A single unit, continuous in-line, one step rapid assay format suitable for oral specimen collection and testing is disclosed. More particularly, a hydrophilic capillary matrix is disclosed as a transport for oral fluids to a lateral chromatographic strip. This enables rapid assay of oral fluids while a disposable testing device is held in a patient's mouth.
Numerous analytical methods have been developed for determining the presence or absence and/or quantifying the amount of various analytes in tissues and fluids of organisms. Currently most diagnostic testing is done with blood, urine, fecal material, or tissue biopsy. Testing based on these materials, however, entails substantial invasion of privacy and poses a significant safety hazard (particularly with the testing of blood). In contrast, the collection of oral fluid including saliva and/or mucosal transudate for testing entails relatively little invasion of privacy, is relatively safe, and can be accomplished rapidly with relative ease.
The use of oral fluid for detection purposes has been discussed in scientific and clinical research for some time. A multitude of researchers have investigated using oral fluid as a possible clinical specimen for diagnosis of specific disease states or altered metabolic activity (see, e.g., Annl. New York Acad. Sci., Vol. 694: Saliva as a Diagnostic Fluid, Malamud and Tabak, eds., N.Y. Acad. Sci. Pub. (1993)). There is a preponderance of evidence that suggests that oral fluids might be extremely useful samples for the detection of certain analytes. The basic technological premise is that analytes present in blood will pass through the oral mucosa and/or salivary glands into the oral cavity where they can be detected. Further it is assumed that the concentration of analyte in oral fluid will be indicative of the blood concentration. There is thus considerable interest in the development of devices for the collection, transport, and sample handling of oral fluids and in the development of oral fluid-based assays; in particular assays for various antibodies and metabolites.
Typically in tests performed with samples such as blood, urine, or fecal material, there is an ample supply of test material and high volumes of analytes are available for assay. In addition, since assays of such materials are performed outside the body, there is no issue of contamination of the body with assay reagents.
This is illustrated, for example, in the assay device described by de Zoeten et al., U.S. Pat. No. 5,611,995, issued Mar. 18, 1997. In this device an absorbing body is supplied with a handle and held in the stream of urine being expelled from the body. When the absorbing body is saturated, it is then inserted into a holding device having a test strip. The saturated pad comes into contact with a test strip, is compressed, and deposits urine to be tested on the test strip. A gap at the side of the hold device holding the test strip assures evaporation of excess fluid to prevent backflow along the test strip.
Previously described assays of biological samples, in particular assays for analytes in oral fluid, have typically required at least two different actions. First the sample, e.g., blood, or urine is collected. Then the collected sample is either stored, e.g., for later assay in a laboratory, or is assayed in or by an assay device which is typically a device other than the collection device. Such assays, requiring multiple components, are often expensive to manufacture and cumbersome for home use.
Moreover, particularly with respect to assaying oral fluid samples, oral fluid is often in short supply, particularly under circumstances where the test subject is stressed (e.g., when testing for drugs of abuse or life-threatening illnesses, which may make it difficult to use such multi-component assays).
When typical absorbent pads are used to recover oral fluid, the pads must typically be compressed to release the trapped oral fluid. The manipulations associated with the compression step can result in sample contamination. In addition, such Atraditional pads have a significant void volume requiring that the sample often be collected in a volume significant greater than is actually required for analyte assay itself.
This invention provides improved devices and methods for one-step collection of oral fluid for detection and/or quantification of analytes in the oral fluid. The devices and methods require extremely low volumes of oral fluid, and require no subsequent sample manipulation after collection. Adequate sample collection is immediately verified and the risk of sample contamination is minimized. The assays are direct, rapid, and require no complicated steps. The devices and methods are therefore ideally suited for use in homes, in work or office settings, and generally do not require the presence of trained medical personnel.
Unlike prior art oral fluid collection devices that typically utilize an absorbent pad made of paper, cellulose, cotton or sponge and which require compression or centrifugation of the collection pad to release the oral fluid sample, the devices of this invention utilize a relatively rigid capillary matrix also referred to as a capillary matrix. The capillary matrix, when inserted into the oral cavity of a mammal (e.g., a human) rapidly wicks up oral fluid (e.g., via capillary action) and delivers it to the receiving area of a lateral flow chromatography strip. The oral fluid is rapidly released from the capillary matrix to the lateral flow chromatography strip without any manipulation (e.g., compression) of the matrix.
In one embodiment this invention provides an apparatus for lateral flow chromatography of an oral fluid. The apparatus comprises a capillary matrix having exposed a surface for insertion into an oral cavity; and a lateral flow chromatography strip where the lateral flow chromatography strip is attached to the capillary matrix such that when the capillary matrix is contacted to an oral mucosa in an oral cavity, the capillary matrix wicks up oral fluid and delivers the oral fluid to a receiving area of a lateral flow chromatography strip. In another embodiment, the apparatus comprises a capillary matrix having exposed a surface for receiving oral fluid; and a lateral flow chromatography strip where the lateral flow chromatography strip is in communication with the capillary matrix such that when the capillary matrix receives oral fluid, the capillary matrix wicks up the oral fluid and delivers the oral fluid to a receiving area of the lateral flow chromatography strip.
In a preferred embodiment, the capillary matrix is composed of a material different from the material comprising the lateral flow chromatography strip or the receiving area or sample pad of such a strip. The capillary matrix is composed of a material such that saturation of the capillary matrix with an oral fluid does not substantially alter the morphology of the capillary matrix. Thus, neither the average pore size nor the void volume of the capillary matrix is substantially altered. In addition, the volume of the capillary matrix is substantially constant. Saturation of the capillary matrix typically effects less than 30%, preferably less than 25%, more preferably less than 20% and most preferably less than about 15%, 10%, 5% or less than about even 1%. The capillary matrix preferably has an average pore size ranging from about 40 μm to about 250 μm, more preferably from about 60 μm to about 200 μm, and most preferably from about 80 μm to about 120 μm and a void volume of less than about 60 μL/cm3. Particularly preferred porous matrix materials have pore sizes that range from about 45 μm to about 90 μm, from about 90 μm to about 130 μm, or from about 80 μm to about 120 μm. Preferred capillary matrix materials are plastics (e.g., porous matrices of a high density polyethylene (HDPE), an ultra-high molecular weight polyethylene (UHMW), a polypropylene (PP), a polyester, a polyvinylidene fluoride (PVDF), a polytetrafluoroethylene (PTFE), a nylon 6 (N6), or a polyethersulfone (PES)). The plastics may be hydrophilic or treated (e.g., with a surfactant such as sodium N-methyl cocoyl taurate) to be hydrophilic.
In a preferred embodiment, the capillary matrix, when contacted to an oral mucosa takes up oral fluid from the oral cavity and readily releases the oral fluid to the receiving area of the lateral flow chromatography strip in under about 5 minutes (preferably under about 2 minutes, more preferably under about 1 minute) without compression, altered air or fluid pressure, or other manipulation of the matrix material. The capillary matrix, when contacted to an oral mucosa takes up oral fluid from the oral cavity and releases the oral fluid to the receiving area of the lateral flow chromatography strip most preferably in under about 30 seconds. Under these conditions, the capillary matrix, is preferably saturated with oral fluid in under about 1 minute, and saturation typically utilizes less than about 300 μL of oral fluid. Generally speaking, the capillary matrix will released sufficient oral fluid to saturate the receiving area of the chromatographic strip.
The apparatus can optionally further include a blocking strip placed between the capillary matrix and the lateral flow chromatographic strip. The blocking strip can contain a blocking reagent (e.g., BSA, deoxycholate, sodium -n-lauroylsarcosine, etc.) and/or one or more buffers. In certain embodiments, the blocking strip can also prevent backflow of reagents from the lateral flow chromatography strip to the capillary matrix.
The apparatus can optionally further include a conjugate strip that contains one or more chromatography reagents (e.g., labeled microparticles). In addition, a single strip can double as a blocking strip and a conjugate strip.
The apparatus can further comprise a housing having a cavity, wherein the lateral flow chromatography strip extends into the cavity along the housing to an inspection site on the housing; and at least one inspection site from an exterior of the housing to the lateral chromatographic strip to enable visual inspection of reagents at selected sites on the lateral chromatographic strip. The housing can act as a handle for inserting the capillary matrix into the oral cavity. The housing can be fabricated out of any convenient material (e.g. HDPE, LDPE, PP, polystyrene, acrylic, polycarbonate, etc.).
In accordance with another embodiment, an apparatus for collection and lateral flow chromatography of an oral fluid comprises a lateral flow chromatography strip including a receiving area; and a capillary matrix having a surface. A bite portion is coupled to the capillary matrix and insertable between the teeth of a subject to position the surface of the capillary matrix for receiving an oral fluid of the subject. The capillary matrix is in communication with the lateral flow chromatography strip to wick up and deliver the received oral fluid to the receiving area of the lateral flow chromatography strip. In a specific embodiment, the bite portion comprises textured or non-textured surfaces for contacting the teeth, and is insertable between the teeth of the subject to optionally contact the tongue of the subject. The bite portion may include a saliva-stimulating substance selected from the group consisting of citric or other acids (e.g., tartaric, fumaric, ascorbic, malic, etc.) acid, salt, and natural sweetener (e.g. fructose, glucose, sucrose etc.) or artificial sweetener (e.g. Nutrasweet�). The bite portion can optionally include other materials such as artificial or natural flavors and/or aromatic substances, and/or polysaccharides, gelatin, shellac, or other coating materials. The bite portion is connected to a housing having a cavity in which the lateral flow chromatography strip is at least partially disposed.
In accordance with yet another embodiment, an apparatus for collection and lateral flow chromatography of an oral fluid comprises a lateral flow chromatography strip including a receiving area; and a bite portion insertable between the teeth of a subject. The apparatus further comprises collection means coupled to the bite portion to contact an oral mucosa of the subject for receiving oral fluid of the subject, and in communication with the lateral flow chromatography strip for wicking up and delivering the received oral fluid to the receiving area of the lateral flow chromatography strip.
In another embodiment, this invention provides methods of detecting an analyte in the oral fluid of a subject. In preferred embodiments, the methods involve inserting the apparatus (collection devices) described herein into the oral cavity of said subject such that the bite portion of the apparatus is held between the teeth of the subject. retaining the apparatus in the oral cavity until an oral fluid sample is collected; and reading the presence or absence of the analyte from an indicator region on the apparatus.
This invention also provides kits for the detection of an analyte in an oral fluid. The kits include an apparatus for collection and lateral flow chromatography of an oral fluid as described herein and instructional materials describing the use of the apparatus.
As used herein, the term �analyte� is used to refer to a moiety that is to be detected in a particular assay. Analytes can be atoms (elements), molecules, or groups of molecules. Analytes commonly detected in the assays of this invention include, but are not limited to, antibodies, antigens, growth factors, enzymes, therapeutic drugs, drugs of abuse, other ligands (e.g. protein A, avidin or biotin, conconavalin A, etc.), and the like. Particularly preferred analytes include antibodies and antigens relevant to infectious and non-infectious diseases, therapeutic drugs or drugs of abuse, and metabolites of the same.
As used herein, an �antibody� refers to a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes or fragments of immunoglobulin genes. The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as the myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.
The basic immunoglobulin (antibody) structural unit is known to comprise a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one �light� (about 25 kD) and one �heavy� chain (about 50�70 kD). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms variable light chain (VL) and variable heavy chain (VH) refer to these light and heavy chains respectively.
Antibodies may exist as intact immunoglobulins or as a number of well characterized fragments produced by digestion with various peptidases. Thus, for example, pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab)′2, a dimer of Fab which itself is a light chain joined to VH�CHl by a disulfide bond. The F(ab)′2 may be reduced under mild conditions to break the disulfide linkage in the hinge region thereby converting the F(ab)′2 dimer into an Fab′ monomer. The Fab′ monomer is essentially an Fab with part of the hinge region (see Fundamental Immunology, W. E. Paul, ed., Raven Press, N.Y. (1993) for a more detailed description of other antibody fragments). While various antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that such Fab′ fragments may be synthesized de novo either chemically or by utilizing recombinant DNA methodology. Thus, the term antibody, as used herein, also includes antibody fragments either produced by the modification of whole antibodies or synthesized de novo using recombinant DNA methodologies.
The term �oral fluid�, as used herein, refers to one or more fluids found in the oral cavity individually or in combination. These include, but are not limited to, saliva and mucosal transudate. It is recognized that oral fluid (e.g., saliva) can comprise a combination of fluids from a number of sources (e.g., parotid, submandibular, sublingual, accessory glands, gingival mucosa and buccal mucosa) and the term oral fluid includes the fluids from each of these sources individually, or in combination. The term saliva refers to a combination of oral fluids such as is typically found in the mouth, in particular after chewing. The term �mucosal transudate�, as used herein, refers to fluid produced by the passive diffusion of serum components from oral mucosal interstitia into the oral cavity. Mucosal transudate often forms one component of saliva.
The terms �capillary matrix� or �porous matrix� are used herein to refer to a highly porous material characterized by a pore size sufficiently small that the material rapidly takes up aqueous solution (e.g., of oral fluid) predominantly by capillary action or �wicking�.
The term �wick up� is used to refer to the uptake of a fluid predominantly by adsorption and capillary action.
A �lateral flow chromatography strip� refers to a test strip utilized for lateral flow chromatography. Lateral flow (chromatography) assays typically involve the application of a liquid test sample suspected of containing an analyte to be detected to an application zone of a lateral flow (immunochromatographic) test strip. The strip is comprised of a matrix material (e.g., paper, nitrocellulose, etc., see, e.g., U.S. Pat. No. 5,569,608) through which the test fluid and analyte suspended or dissolved therein can flow by capillary action from the application zone to a detection zone where a visible signal, or absence of such, reveals the presence or absence of the analyte. Where the detection of the analyte utilizes an antibody or antibody fragment, the assay may be referred to as a lateral flow immunochromatography assay and the strip a lateral flow immunochromatography strip.
A �receiving area or sample pad� of the lateral flow chromatography strip refers to the area of the lateral flow chromatography strip to which a sample is first applied.
A �signal� on the lateral flow chromatography strip refers to an indication, typically in a particular predefined region of the chromatography strip that indicates the presence or absence or quantity of analyte or the sufficiency of sample within the chromatography strip. The signal can be calorimetric, fluorescent, enzymatic, electroluminescent, radioactive, etc.
As used herein, �housing� refers to any member which encases or supports but does not react with the lateral flow chromatography strip.
As used herein, �cavity� refers to any receiving volume on or within the housing for holding the lateral chromatographic strip.
The �lateral flow chromatographic strip� is any absorbing member capable of transporting analyte and reagents to the visual inspection site. The strip can be nitro cellulose, cellulose acetate, paper, nylon, cellulose, glass fiber, polyester, or any other suitable bibulous material.
As used herein, an �immunoassay� is an assay that utilizes an antibody or antigen to specifically bind to the analyte. The immunoassay is characterized by the use of specific binding to a particular antibody as opposed to other physical or chemical properties to isolate, target, and quantify the analyte.
The phrase �specifically binds to an analyte� or �specifically immunoreactive with�, when referring to an antibody refers to a binding reaction which is determinative of the presence of the analyte in the presence of a heterogeneous population of molecules such as proteins and other biologics (i.e., such as may be found in oral fluid). Thus, under designated immunoassay conditions, the specified antibodies bind to a particular analyte and do not bind in a significant amount to other analities present in the sample. A variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular analyte. For example, solid-phase ELISA immunoassays are routinely used to select monoclonal antibodies specifically immunoreactive with a protein. See Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, N.Y., for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity.
A �label� is a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Useful labels in the present invention include magnetic beads (e.g., Dynabeads�), fluorescent dyes (e.g., fluorescein isothiocyanate, texas red, rhodamine, green fluorescent protein, and the like), radiolabels (e.g., 3H, 125I, 35S, 14C, or 32P), enzymes (e.g., horse radish peroxidase, alkaline phosphatase and others commonly used in an ELISA), and colorimetric labels such as colloidal gold, silver, selenium, or other metals, or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads. Patents teaching the use of such labels include U.S. Pat. Nos. 3,817,837, 3,850,752, 3,939,350, 3,996,345, 4,277,437, 4,275,149, and 4,366,241. Means of detecting such labels are well known to those of skill in the art. Thus, for example, radiolabels may be detected using photographic film or scintillation counters, fluorescent markers may be detected using a photodetector to detect emitted illumination. Enzymatic labels are typically detected by providing the enzyme with a substrate and detecting the reaction product produced by the action of the enzyme on the substrate, and colorimetric labels are detected by simply visualizing the colored label.
The term �subject�, as used herein, refers to the test subject whose oral fluid is to be collected for testing. The subject includes without limitation to a person or a patient such as a mammal (e.g., a human).
The term �teeth�, as used herein, refer to natural or prosthetic teeth of the subject. The teeth include, but are not limited to, the molars and/or the bicuspids.
This invention provides a device for the rapid, one-step, collection and detection of analytes in oral fluid. In a preferred embodiment, the device is inserted into the oral cavity (e.g., preferably juxtaposed to the oral mucosa) where it absorbs oral fluid. After a period of time, the device is removed from the oral cavity and one or more indicators contained in the device are read out (e.g., by visual inspection or by detection in a Areader≅) to provide an indication of the presence or absence, and/or quantity of one or more analytes of interest. The device thereby provides a rapid, one-step, non-invasive assay for the detection of one or more analytes of interest.
The assay devices and methods of this invention can be used for the detection (positive or negative, and/or quantification) of virtually any analyte in oral fluid. Moreover, the devices and methods can be used to detect one or more analytes simultaneously. Such analytes may include, but are not limited to, antibodies to HIV, antibodies to HTLV, antibodies to Helicobacter pylori, antibodies to hepatitis, antibodies to measles, hepatitis antigens, antibodies to terponemes, antibodies to host or infections agents, cellular markers of pathology includign, but not limited to, cardiolipin, lecithin, cholesterol, lipopolysaccharide and sialic acid, antibodies to mumps, antibodies to rubella, cotinine, cocaine, benzoylecgonine, benzodizazpines, tetrahydrocannabinol, nicotine, ethanol theophylline, phenytoin, acetaminophen, lithium, diazepam, nortriptyline, secobarbital, phenobarbital, theophylline, testosterone, estradiol, 17-hydroxyprogesterone, progesterone, thyroxine, thyroid stimulating hormone, follicle stimulating hormone, luteinizing hormone, transforming growth factor alpha, epidermal growth factor, insulin-like growth factor I and II, growth hormone release inhibiting factor, IGA and sex hormone binding globulin; and other analytes including glucose, cholesterol, caffeine, cholesterol, corticosteroid binding globulin, PSA, or DHEA binding glycoprotein with the methods being particularly well suited to the detection of HIV antibodies.
The assay device of this invention relies on the unique cooperation between a capillary matrix (W in
The device can be conveniently assembled such that the capillary matrix comprises a receiving pad for insertion into the oral cavity, while the lateral flow immunochromatography strip comprises the handle of the device. One or more zones on the handle can comprise indicators for readout (e.g., of a calorimetric signal) of the assay results. Of course, other formats of the device are suitable and will be readily recognized by those of skill in the art.
Capillary Matrix
The capillary matrix (porous matrix) material is preferably selected to provide a number of unique properties to the assay device. Such properties include, but are not limited to, a relatively low void volume, a pore size sufficient to provide rapid and effective delivery of the oral fluid to the test strip, low or non-reactivity with the oral fluid or analytes, easy release of the oral fluid to the immunochromatography test strip, and a non-deformable (when wetted) collection pad.
Because oral fluid may be in short supply (patients often suffer a Adry mouth≅during testing) it is desirable to maximize the amount of oral fluid that is transported from the oral cavity (e.g., the oral mucosa) to the lateral flow chromatography strip. This is accomplished by the use of a capillary matrix having a minimum void volume. The capillary matrix should have a void volume less than about 65%/cm3, preferably less than about 57%/cm3, more preferably less than about 48%/cm3, and most preferably less than about 40%/cm3, 35%/cm3, or even 25%/cm3. Capillary matrices having such low void volumes typically deliver a significant amount of the absorbed oral fluid to the lateral flow chromatography strip.
The matrix itself must be of relatively small dimension. Specifically, the interstices is preferably of a dimension where capillary forces cause the fluid to be drawn into the capillary matrix. Thus, the capillary matrix is also selected to have an average pore size small enough to provide rapid uptake of the oral fluid with which it is contacted (e.g., via capillary action). The small pore size also functions to exclude particulate material present in the fluid sample. The pore size, however, is also selected to be large enough that the viscous oral fluid does not clog the capillary matrix and instead rapidly transports through the matrix to the lateral flow chromatography pad. Preferred materials have an average pore size that ranges from about 40 μm to about 250 μm, more preferably from about 60 μm to about 200 μm, and most preferably from about 80 μm to about 120 μm.
In addition to having a pore size (channel size) that results in rapid uptake of the oral fluid, the surfaces of the capillary matrix should be chemically compatible with rapid uptake of the oral fluid. Thus, preferred capillary matrix materials are themselves hydrophilic or treated to be hydrophilic (e.g., by addition of a surfactant also referred to as a detergent or wetting agent). That is to say, water must flow on and be attracted to the surfaces of these materials.
While a number of suitable materials are naturally hydrophilic (e.g., clean sintered glass or fused glass beads), other suitable materials (e.g., plastics) are typically hydrophobic (e.g., do not easily wet). However, such hydrophobic materials can be routinely treated with a wetting agent (i.e., surfactant/detergent) and thereby rendered hydrophilic (wettable). However, since the capillary matrix is used in the oral cavity, it is required that the treating detergent be known not to be harmful to the subject mammal (e.g., human body) and preferably be approved for such use by the relevant regulatory authority (e.g., Food and Drug Administration). In one preferred embodiment, a porous plastic material (e.g., polyethylene or polypropylene foam) can be rendered hydrophilic by taking the untreated matrix material and placing it in a dilute aqueous solution under vacuum, or a dilute alcoholic solution under ambient pressure, of an approved detergent such as sodium N-methyl cocoyl taurate. Thereafter, the treated material is dried, leaving the surfaces of the matrix apparently thinly coated with the detergent.
While N-methyl cocoyl taurate is preferred, it will be appreciated that other detergents can as well be used (e.g., N-methyl oleoyl taurate, sodium lauryl sulfate, Triton X 100). It is only required that the detergent be safe for mammalian oral exposure, not interfere with the test on the lateral chromatographic strip, and produce the required hydrophilic properties on the exterior surfaces of the matrix.
In addition to rapidly taking up and transporting the oral fluid to the lateral flow chromatography strip, the capillary matrix material is selected that preferably readily releases the fluid to the chromatography strip. This should be accomplished rapidly without compression of the matrix material itself. Thus, in a preferred embodiment, the capillary matrix delivers and releases oral fluid to the lateral flow chromatography strip with no manipulation (e.g., no squeezing or compression of the capillary matrix).
From the foregoing, it should be clear that preferred capillary matrix materials have an interstitial spacing that facilitates uptake of oral fluid through capillary attraction in combination with adsorption on the material. This causes the oral fluid gathered from the mouth to be transported to the lateral chromatographic strip in preference to remaining in the mouth. At the same time, when the oral fluid arrives at the lateral chromatographic strip, it is absorbed to the strip in preference to remaining in the capillary matrix.
In a preferred embodiment, the hydrophilic capillary matrix is an essentially nonabsorbing matrix which adsorbs liquid via capillary action. In such adsorption, the volume of the material is not appreciably effected. In addition, the capillary matrix material is relatively rigid such that its morphology remains essentially unchanged during the assay (e.g., when saturated with oral fluid). Thus, saturation of the matrix with an oral fluid does not substantially alter the average pore size or void volume of the porous matrix. In addition, saturation of the capillary matrix with an oral fluid results in a volume change of less than 30%, preferably less than 25%, more preferably less than 20% and most preferably less than about 15%, 10%, 5% or less than about even 1%.
In a particularly preferred embodiment, the capillary matrix can act as a barrier to back flow of reagents from the lateral flow chromatography strip back into the capillary matrix. This can be accomplished, for example, where the chromatography strip has a larger volume for fluid storage than the capillary matrix. In addition or alternatively, where the lateral flow chromatography strip is more hydrophilic than the capillary matrix the capillary matrix can also act as a barrier to backflow.
The capillary matrix materials are selected such that they are not chemically reactive with either the oral fluid or the analytes contained therein. Matrix materials compatible with oral fluid are well known to those of skill in the art and include, but are not limited to, glass, resins, and various plastics.
In one preferred embodiment, the properties described above are achieved by the use of porous plastic materials for the capillary matrix. Suitable porous plastic materials include, but are not limited to, porous matrices of high density polyethylene (HDPE), ultra-high molecular weight polyethylene (UHMW), polypropylene (PP), polyvinylidene fluoride (PVDF), polytetrafluoroethylene (PTFE), polyester, nylon 6 (N6) and polyethersulfone (PES). In a preferred embodiment, the porous matrix materials are either themselves hydrophilic (so as to readily uptake the oral fluid) or are treated.(e.g., with a surfactant/detergent) so as to be hydrophilic.
Such porous plastics are commercially available (see, e.g., Porex Technologies, Fairburn, Ga.). Particularly preferred porous plastics are detergent (surfactant) treated polyethylene and/or polypropylene. The treatment typically involves soaking the capillary matrix in a surfactant/detergent and then allowing it to dry naturally or force drying the material.
Particularly preferred porous matrix materials are Porex7 X-4588, 80�120 μm pore size at 0.024 inch of thickness made from polypropylene. Likewise, Porex7 X-4903 at 0.0625 inch, pore size 45�90 μm, and Porex7 X-4913 at 0.0625 inch, pore size 90�130 μm, are suitable. In one preferred embodiment, these materials are treated with sodium N-methyl cocoyl taurate. The capillary matrix materials are soaked in the detergent which is then dried onto the surfaces.
It will be understood that the Porex7 material that are utilized do not retain large volumes of the oral fluid. For example, consider the following data:
At the same time, hydrophilic capillary matrix does not have a high relative retention of the oral fluid. For example, it readily surrenders its fluid to lateral chromatographic strip and absorbent pad (A in
Identification of Suitable Porous Matrix Materials
It will be appreciated that the rate of oral fluid uptake, transport to the lateral flow chromatography pad and release to the pad is a function of both the composition of the capillary matrix and its shape (e.g., area exposed to the oral mucosa, cross-sectional area, and area contacted to the lateral flow chromatography strip. The rates are also effected by the average pore size, hydrophilicity of the capillary matrix material and the relative absorbance characteristics of the capillary matrix and the lateral flow chromatography pad.
These parameters can be optimized according to routine methods well known to those of skill in the art. In one embodiment, this is accomplished by assembling a test device having the desired capillary matrix material and lateral flow immunochromatography strip. The test device can then be contacted with a test oral fluid solution (e.g. natural oral fluid, or synthetic oral fluid, see U.S. Pat. No. 5,695,929 and copending U.S. Ser. No. 08/608,431).
The contacting can be accomplished by actual insertion of the receiving portion (e.g., receiving pad/face) of the capillary matrix into an oral cavity and contact with an oral mucosa (e.g., of a human or non-human test animal). Alternatively, the contacting can be accomplished by touching the capillary matrix with the test fluid disposed on a surface or in a bowl or other receptacle, immersing part or all of the capillary matrix in the test fluid, or contacting the capillary matrix with a test body (e.g., a sponge, cloth, swab, etc.) impregnated with the test fluid.
The test oral fluid will typically be contacted with the capillary matrix for the time period it is desired to run the assay (e.g., less than 5 minutes) and then the lateral flow chromatography strip can be read for the presence, absence or quantity of analyte and/or for the amount of oral fluid taken up. In this manner it will be determined if the assay gives adequate sensitivity and specificity.
In addition, the time period for uptake of oral fluid into the capillary matrix, and lateral flow chromatography strip can be determined. Similarly the total volume of sample required to properly saturate the lateral flow chromatography strip and the amount of fluid retained by the capillary matrix can also be ascertained (e.g., by weighing the various components before and after the assay).
In a preferred embodiment, the capillary matrix, when inserted and held in a mouth is saturated with oral fluid in under about 5 minutes, preferably in under about 3 minutes, more preferably in under about 1 minute, and most preferably in under about 30 seconds. Similarly, the capillary matrix will release sufficient fluid to the chromatography strip for a properly run assay (an assay run to the specifications of the chromatography strip) in under about 10 minutes, preferably in under about 5 minutes, more preferably in under about 3 minutes and most preferably in under about 2 minutes or even in under about 1 without compression of the porous matrix.
A porous matrix will be made that is saturated by less than about 500 μL, more preferably by less than about 500 μL, preferably by less than about 300 μL, more preferably by less than about 200 μL and most preferably by less than about 100 μL.
It will be recognized that where just the oral fluid uptake and release properties are to be assayed, there is no need to utilize a complete chromatographic assay. The base capillary matrix material, lateral flow chromatography strip, and if desired, blocking pad, materials can be assembled. The rate of fluid uptake and delivery to the pad can be quantified by immersing or contacting the capillary matrix pad with a sample fluid (e.g., natural or synthetic oral fluid) and quantifying the rate of fluid uptake and delivery to the pad and/or the amount retained within the capillary matrix (e.g., by weighing the various elements after particular preselected times of exposure to the test fluid). A combination of materials and shapes elements that provides maximal fluid delivery of an oral fluid from an oral mucosa to the lateral flow chromatography strip in the shortest amount of time is preferred.
In one particularly preferred embodiment, the lateral flow chromatography strip is a nitrocellulose strip (e.g., Syntron QuickScan 6, Syntron Bioresearch, Carlsbad, Calif.; Avitar Visualine II, Avitar TEchnologies, Canton, Mass.; Determine HIV-1/2, Abbott Laboratories, Abbott Park, Ill.). A typical chromatographic strip is Millipore SRHF or STHF nitrocellulose membrane (Millipore Corp., Bedford, Mass.) having dimensions 4 mm�50 mm. A porous matrix material measures 7 mm�52 mm (0.0625 inch thick) and overlaps the nitrocellulose membrane by about 64 mm2. In another embodiment, the capillary matrix is paddle-shaped having a total surface area of about 720 mm2 and overlapping the chromatographic strip by about 8 mm2. The small overlap is particularly well-suited to embodiments containing a conjugate pad (G in
Lateral Flow Chromatography Strip
The assay device of this invention can utilize virtually any lateral flow chromatography strip for detection and/or quantification of the analyte or analytes. Lateral flow chromatography assays are well known to those of skill in the art (see, e.g., U.S. Pat. Nos. 5,569,608, 5,120,643, 5,656,503, 4,855,240, and 5,591,645, British Patent GB 2204398A, and European patent EP 0323605 B1) and such assays are commercially available on a retail or OEM basis for numerous analytes.
Lateral flow immunoassays typically involve the application of a liquid test sample suspected of containing an analyte to be detected to an application zone of a lateral flow (immunochromatographic) test strip . The strip is comprised of a matrix material (e.g., paper, nitrocellulose, etc., see, e.g., U.S. Pat. No. 5,569,608) through which the test fluid and analyte suspended or dissolved therein can flow by capillarity from the application zone to a detection zone where a visible signal, or absence of such, reveals the presence or absence of the analyte.
Typically, the strip will include means for immunospecifically binding the analyte to be detected with its specific binding partner (e.g., where the analyte is an antigen, the binding partner is an antibody or antibody fragment, and vice versa) which bears a detectable label. In one such scheme, as disclosed in U.S. Pat. No. 4,446,232, the strip contains an enzyme labeled, mobile binding partner for the analyte which is in a zone downstream from the sample application zone. If analyte is present in the test sample, it will combine with its labeled binding partner to form a complex which will flow along the strip to a detection zone which contains a substrate for the enzyme label capable of providing a signal (e.g., a colored response) in the presence of the enzyme label.
The strip typically contains a zone in which analyte is immobilized, so that the labeled binding partner which does not combine with analyte, due to absence of analyte in the sample, will be captured and thereby inhibited from reaching the detection zone. There have been published various modifications of this technique, many of which involve some competitive specific binding system in which the presence or absence of analyte in the test sample is determined by the detection or lack thereof of labeled binding partner in the detection zone. In U.S. Pat. No. 4,868,108 there is disclosed a similar scheme with the addition of an immobilized capture reagent for the enzyme labeled binding partner in the detection zone to concentrate the enzyme label and enhance its ability to react with the enzyme substrate and thereby render the assay more sensitive.
Not all of the schemes for immunochromatography rely on an enzyme labeled binding partner/enzyme substrate as providing the signal for detection of the analyte. In U.S. Pat. No. 4,806,311 there is disclosed a multizone test device for the specific binding assay determination of an analyte and an immobilized binding partner therefore together with a detection zone for receiving labeled reagent which migrates thereto from the reagent zone. The detection zone contains an immobilized form of a binding substance for the labeled reagent. The labeled reagent bears a detectable chemical group having a detectable physical property which is detectable on the basis of its own physical properties, so that it does not require a chemical reaction with another substance. Exemplary of such groups are colored species fluoresces, phosphorescent molecules, radioisotopes and electroactive moieties. U.S. Pat. No. 4,313,734 describes the use of gold sols as labels for antibodies which are detectable without a chemical change.
Many lateral flow immunochromatography systems utilize particulate (microparticle) markers (e.g., gelatin, dyed latex, or colloidal gold) which are labeled with a binding partner (e.g., antibody or antigen) that binds the analyte of interest.
The microparticles or other detectable moieties attached to an analyte binding moiety (e.g., an antibody or antigen) are dried onto (or otherwise localized in) either a lateral flow chromatographic strip or onto a sample application pad (typically glass fiber) which in turn is affixed to one end of a strip of chromatographic medium such as nitrocellulose. Another material binding to the analyte of interest is affixed to the chromatographic medium at or near the end opposite to the end having the application pad.
The liquid sample to be analyzed is placed on the pad, causing the suspension of the microparticles into the liquid and allowing any analyte in the liquid sample to bind to the analyte-binding material attached to the microparticles. The liquid sample leaves the application pad by diffusion and capillary action and begins to migrate along the nitrocellulose strip carrying the microparticles down the strip along with the liquid. When the liquid containing the suspended microparticles arrives at the region of the chromatographic strip bearing the second binding material, the analyte (if present in the original sample) will form a molecular bridge between the analyte-binding material on the microparticles and the analyte-binding material affixed to the strip, resulting in the immobilization of the microparticles at that point on the strip where the analyte-binding material is affixed. This immobilization of the microparticles results in a visible signal (e.g., a colored band or dot) at this point on the strip. If the analyte is not present in the sample, the microparticles will continue past this location on the chromatographic strip and a visible signal will not be produced.
It will be appreciated that other labels (e.g., fluorescent labels) besides microparticles can be utilized. In addition, a single chromatography strip can contain reagents to detect or quantify a number of different analytes.
It will also be appreciated that the lateral flow strip can use an analyte detection that does not involve an antibody-antigen recognition system. Thus, for example, the strip can be impregnated in a detection zone with a chemical that reacts with the analyte itself to produce a signal.
The devices of this invention can be readily assembled with any of a variety of commercially available lateral flow chromatography assays (e.g., Syntron QuickScan 6, Syntron Bioresearch, Carlsbad, Calif.; Avitar Visualine II, Avitar TEchnologies, Canton, Mass.; Determine HIV-1/2, Abbott Laboratories, Abbott Park, Ill.).
Optional Blocking Strip
The assay devices of this invention can optionally include a blocking strip between the porous matrix and the lateral flow chromatography strip. The blocking strip can be impregnated with buffers to adjust the pH (e.g., to pH 7.5) of the oral fluid for compatibility with the lateral flow chromatography assay. The blocking strip can also include one or more blocking reagents that reduce non-specific binding of the analyte and/or reagents of the assay device and thereby reduce the occurrence of false positives. Suitable blocking reagents include, but are not limited to, bovine serum albumin (BSA), deoxycholate, and n-lauroyl sarcosine. In one particularly preferred embodiment, the blocking solution includes: 4% polyvinyl alcohol (10 kd mw, Aldrich 36,062-2), 4% sodium -n-lauroylsarcosine (Sigma L5777), 2% polyvinyl pyrrolidone (10 kd mw, Sigma P2263) in 60� Tris EDTA (Sigma T9285) or 7.5% sodium -n-lauroylsarcosine (Sigma L5777), 2.5% tectonic 1307 (BASF), 0.00001% polyethyleneglycol compound (Sigma P2263) in 100� Tris EDTA buffer (Sigma T9285).
The blocking pad can be composed of a wide variety of materials as long as they do not impede flow of oral fluid from the capillary matrix to the lateral flow chromatography strip. Such materials include, but are not limited to, paper, cellulose, nitrocellulose, polyester, glass fiber, and the like. Particularly preferred materials will be selected to reduce or eliminate backflow of reagents or oral fluid from the chromatographic test strip to the capillary matrix.
It is noted that the blocking agents can be placed on the capillary matrix instead or, or in addition to, their placement on the optional blocking strip. In some configurations the blocking agents are on the capillary matrix, and in some other configurations, both the blocking agents and the conjugate are on the capillary matrix.
Optional Conjugate Strip
In one embodiment, the lateral flow chromatography reagents (e.g., antibody labeled gold particles, etc.) are disposed in the lateral flow chromatographic strip itself, and/or directly in the capillary matrix. In another embodiment, however, one or more chromatography reagents can be disposed in a conjugate strip (G in
Assembly of the Assay Device
The components (e.g., capillary matrix, lateral flow chromatography strip, optional blocking strip) can be assembled by any of a wide variety of means well known to those of skill in the art. Thus, for example, the components can be welded together or glued together and the like.
In a preferred embodiment, the components are simply pressed together and held in place by a housing (H). Housing (H) can be of any desired construction. For example, in prototype construction, soda straws are utilized which supplied both adequate structure and visibility to gauge accuracy of testing. Acrylic tubing has also been used. In addition, polyethylene, mylar, or other films can be used if they are adequate to provide minimal support to the strip and protect the strip from exposure to thee oral fluid except via the capillary matrix. It will be recognized that other stock tubing materials can be used or specially designed housings can be custom molded or extruded.
The components are assembled such that the lateral flow chromatography strip is disposed lengthwise within the straw and appressed at one end to an end of the capillary matrix (W). The housing (H) thus provides a convenient handle for insertion of the capillary matrix into the oral cavity of a mammal (e.g., a human).
The oral fluid then migrates along the lateral flow chromatography strip (C) where it interacts with various reagents (e.g., antibody or antigen labeled binding partners (e.g., antibodies or antigens)) that are deposited within the chromatography strip (C) and/or within optional conjugate pad (G) which, when present, contacts the chromatography strip (C).
The oral fluid/reagent combination continues to migrate along the chromatography strip (C) until it reaches one or more indicator zones (20). If an analyte is present the indicator zone (20) immobilizes the bound labeled analyte or otherwise produces a detectable signal. One of more of the indicator zones can optionally indicate sample sufficiency (e.g., with a color change) when the necessary sample volume and/or analyte concentration is reached. Sample sufficiency indicators are well known to those of skill in the art and particular advantageous sample sufficiency indicators are described in copending application U.S. Ser. No. 08/456,459 and in U.S. Pat. No. 5,479,937.
The indicator zones (20) can be read (e.g., via visual inspection or other means) through view ports (18). As mentioned above, the device is optionally equipped with blocking pad (B). This pad can be impregnated with a blocking reagent (e.g., n lauryl sarcosine) and/or buffer(s) for adjusting pH of the sample. In addition, the blocking pad (B) can be fashioned out of a semi-permeable material that allows oral fluid flow towards the chromatography strip (C), but prevents backflow of oral fluid and/or reagents into the capillary matrix (W). Further, the far end (away from the capillary matrix) of the chromatography strip (C) can bear affixed thereto an absorbent pad (A) which acts as a reservoir to receive the oral fluid and thereby prevent backflow into the capillary matrix (W).
It will be appreciated that the devices can be assembled in a wide variety of forms. One preferred embodiment is illustrated in
For convenience, the assay device can be optionally equipped with a cover (22) for protection of the capillary matrix surface (3) before and after use. This prevents contamination of the assay device and facilitates sanitary disposal of used devices.
The device (D) of
The reader will understand that with the interaction of hydrophilic capillary matrix (W) and lateral chromatographic strip (C), an extremely simple construction is possible. Further, the end product operates in a manner not unlike a conventional thermometer. The test is fast�with prototypes exhibiting about 2 minutes for complete test results.
Most importantly, the test only requires minimal volumes of fluid from the oral cavity to run a test. For example in the designs shown, the assays utilized only 100 to 200 μL of oral fluid. This is an improvement of at least a factor of 4 over the sample volume requirements of previous assays.
Use of the Assay Device
In use, the assay device of this invention is inserted into the oral cavity of a mammal (e.g., a human, or in veterinary or research applications, a non-human) such that the handle within which is disposed the chromatography strip is outside the mouth. The capillary matrix face (3) is preferably contacted to the oral mucosa and, in a particularly preferred embodiment, is pressed to the oral mucosa at the gingival crest e.g., pressed between the cheek and the gums.
In one preferred method of use, the assay device is kept in place, without mastication, until sufficient sample is collected. This can be for a predetermined time interval or until the capillary matrix achieves a characteristic tactile quality, or appearance of a signal indicator (e.g., a color change in a test window) indicates an adequate sample.
At the recommended time (after completion of the immunochromatography assay), the device is read (e.g., by visual inspection of the indicator zone(s) through the viewport(s)), to determine whether the subject is positive or negative for the analyte(s) of interest or to quantify the analyte(s).
Kits for the Detection of Analytes in Oral Fluids
In another embodiment, this invention provides kits for the detection of one or more analytes in oral fluids. The kits include one or more of the assay devices described herein. In addition, the kits may include instructional materials containing directions (i.e., protocols) for the practice of the assay methods of this invention. While the instructional materials typically comprise written or printed materials they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by this invention. Such media include, but are not limited to, electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD ROM), and the like. Such media may include addresses to Internet sites that provide such instructional materials.
The kits may optionally contain any of the buffers, reagents, detection reagents, and so forth that are useful for the practice of the methods of this invention.
It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated in their entirety by reference for all purposes.
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