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28 year old woman | Toronto, ON, CAN | Seeking men 25-30 within 30 miles
Make the most of life and don't take it too seriously. Everything happens for a reason so enjoy!
I am a passionate, fun-loving girl who wants to try everything at least once! I am very easygoing and I am looking for a guy who doesn't take life too seriously. I love to have fun and laugh as much as possible and I want a guy who can laugh at everything with me. I love to get out and explore the city, trying new things and discovering new places. Passion, confidence, humour and drive are important and sexy! I grew up in a small town not far from the city so I'm still a small town girl! I moved to the city a year ago and have no desire to move any time soon. I love how much the city has to offer, it's impossible to be bored! Some of my favourite things: -Outdoors -Seinfeld -Football -Cottaging -Golfing -Spending time with my family and friends -Swimming -Dexter -Fishing -Jays games -Movies -Travelling -Live Music -Going out for drinks
Music and concerts , Travel/Sightseeing , Watching sports
Aerobics, Basketball, Billiards / Pool, Dancing, Football, Golf, Running, Swimming, Walking / Hiking, Weights / Machines
I exercise 3-4 times per week.
I like Dogs
I don't have a dog right now but I grew up with dogs and can't wait to get one of my own! I love big breeds so that's a must!
Middle of the Road
The Underground, The Maddy, Gracie's, Insomnia, Kensington Market, Mill Street, Bellwoods.
I love really in-depth/dramatic TV shows that keep me watching because they're so exciting! For movies I love comedy! For food I love trying new things and will give almost anything a try! I love going to concerts and will go see any band live!
I love going to sports games - football, baseball, basketball, lacrosse, soccer - they're all fun! I like exploring new places whether they're shops, restaurants, galleries, or hiking trails, anything can be fun!
Sex On The Moon By Ben Mezrich
Z{{Cta}} {{#if HasMoreInfo}} ?{{CtaSecondary}} {{/if}}
|What she is looking for
|5'8" (172cms)
|5'9" (175cms) to 6'5" (195cms)
|Body type:
|Athletic and toned
|Slender, About average, Athletic and toned
|Black, Blue, Brown, Grey, Green, Hazel
|Dark brown
|Black, Light brown, Dark brown, Blonde, Dark blonde
|No Way
|No Way
|Social Drinker
|Social Drinker
|Other profession
|No preference
Project Management
|$35,001 to $50,000
|No preference
|Never Married
|Never Married
|Have kids:
|Wants kids:
|White / Caucasian
|Latino / Hispanic, White / Caucasian
|Spiritual but not religious
|No preference
|Bachelors degree
|Some college, Associates degree, Bachelors degree, Graduate degree, PhD / Post Doctoral


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News: Motor Row Gallery Opens its Doors with a Group ExhibitionCraft Work, Galleries & Museums, News etc., Painting, Sculpture, South Loop No Comments »
The new Motor Row Gallery (MRG) has emerged on Chicago's historic Near South Side in the heart of what is known as the Motor Row District. The fact that the gallery is sheltered in the unsuspecting venue of a U-Haul rental facility, well, that's just the kind of inimitable type of beauty you'd expect to find in Chicago.
The gallery is cozily embedded inside of a Motor Row Lofts building owned by Suzanne Weaver, who has also been running a U-Haul business with her husband from there for the past two and a half years. Motor Row Gallery is an alternative gallery space curated by Weaver's friend of thirteen years Pamela Staker with a special focus on pop-up art exhibitions and special events. For instance, Staker and Weaver have future plans to hold art expositions outdoors in the warmer months, making use of the extra U-Haul vans that aren't rented out. Artists would rent a truck where they could display anything from paintings and sculptures to functional and installation work. Since the space would ultimately belong to the artists, they would have free reign on how they chose to present their work in their creative space.

subset: commoncrawl


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Packing Musts for a Tropical Honeymoon
Sometimes you're given the glorious opportunity to play harder than you work—such is the case as I was invited on a press trip to the British Virgin Islands. Obviously, I squealed, "Yes!" and was beside myself, especially since it had been years since my last vacation and, I've never been to the Caribbean.
I immediately started thinking, "What am I going to pack?" because, yes, I'm a planner, but more so, a shopper, so here are my 10 things I'm bringing to the BVI that are also perfect for a tropical honeymoon!
The Luggage
As tough as it can be to par down on my packing list, it's all about traveling light (at least that's what they say…). I want to guarantee my luggage doesn't get lost or crushed, so I'm bringing it on board and this carry-on is the chicest route since it looks like a tiny steamer trunk from the days when flying was actually stylish.
SHOP NOW: Bellagio 21" Carry-On Spinner Trunk, $525, Bricstore.com
Read the rest on TheNest.com…
More Must-Clicks:
9 Dreamy Resorts With Hammocks Worthy of Your Next Nap


text: The Return of "Wizard of Gore" — Drugged-Out Scifi the Way It Was Meant… Annalee Newitz and 30 othersEditDelete Invite manuallyPromoteXDismissXUndismissBlock for io9Hide Meet today's most excellent mad scientist. He's calling himself a wizard, he's played by Crispin Glover (yes!) and he specializes in wowing audiences of young hipsters with his gore-soaked sideshow. In his act, the Wizard makes it appear that he's eviscerated lovely volunteers from the crowd, though in the end they… » 8/20/08 11:20am 8/20/08 11:20am


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Rugby Union
Select a team
Six Nations
- England
- France
- Ireland
- Italy
- Scotland
- Wales
Aviva Premiership
- Bath
- Exeter
- Gloucester
- Harlequins
- Leicester
- London Irish
- London Welsh
- Newcastle
- Northampton
- Sale
- Saracens
- Wasps
The Rugby Championship
- Australia
- New Zealand
- South Africa
Guinness PRO12
- Cardiff Blues
- Connacht
- Dragons
- Edinburgh
- Glasgow
- Leinster
- Munster
- Ospreys
- Scarlets
- Treviso
- Ulster
- Zebre
Top 14
- Bayonne
- Bordeaux-Begles
- Brive
- Castres
- Clermont Auvergne
- Lyon
- Montpellier
- Racing Metro
- Stade Francais
- Toulon
- Toulouse
- Grenoble
- La Rochelle
- Oyonnax
|Home team
|Away Team
|26 - 10
Match Links
- European Rugby Champions Cup
- Pool 6
- 19th Oct 2013
- Ground: Thomond Park, Limerick
Munster v Gloucester - Live
80 Keith Earls races clear and crosses the line with the final act of the game but he is called back for a forward pass. It matters little as the result is already Munster's. Thanks for joining us, cheers.
79 A long break in play as Ben Morgan gets some attention from the medics. He finally rejoins for the last scrum.
77 Another Gloucester attack fizzles out under a pile of Munster bodies. The home side will feed a scrum just outside their 22. The red jerseys are in no hurry.
75 HA! Mike Tindall hands off Keith Earls like he was a little boy but the Irishman keeps coming and gets him at the second attempt. They'll have a laugh about that at full-time.
73 Gloucester are getting very loose - too loose - as they chase a try. They're actually losing ground in the face of a determined Munster defence.
71 Gloucester push hard for a score that will get them within range of a losing bonus point but Ian Keatley kills their spirits with a long touch-finder after a turnover in midfield.
69 CONVERSION, KEATLEY. 26-10. You can't see Gloucester coming back from here.
67 TRY, MUNSTER, MURPHY. 24-10. That should be good enough for the win! Johne Murphy collects Keatley's perfectly-weighted cross-field kick in acres of space and he can race home untouched.
66 PENALTY, MUNSTER, KEATLEY. 19-10. We finally have the first points of the second half as Munster's pressure is finally given some reward.
64 A big scrum from Munster puts Ben Morgan under pressure as he tries to break off the back. The Munster forwards pile in and win the next scrum feed, 20m out.
63 Scintillating attacking from Keith Earls, who steps his way into some space and bursts away. Johne Murphy is up in support but his offload to Conor Murray goes to ground. The scrum-half was clear if he could just have held on!
61 Impressive scrambling defence from Munster as the Gloucester backs cut loose with some impressive offloading and clever lines. The visitors are getting some momentum at last.
57 It's been all-Munster since the interval but the hosts can't find a way to break down this Gloucester defence. Donncha O'Callaghan is penalised for not releasing, meaning Munster must start from scratch at a line-out in their own half.
55 Casey Laulala looking very dangerous once again. He steps between Mills and May but his fancy offload goes into touch.
53 Mike Tindall is penalised for pushing Felix Jones into touch without the ball as he chases Laulala's kick ahead... but Ian Keatley's shot at goal sails just wide. 16-10 it remains.
51 Gloucester clear their lines from the scrum. A few phases later Akapusi Qera finds some space after a turnover but gets thumped in centre feed and Laulala can pinch it back.
49 Brilliant work from Gloucester's Dan Robson to pounce on the ball as it pops out the side of the scrum. Ryan Mills tries to clear a few phases later but is charged down. Murray knocks on as he tries to regather.
48 Munster hammer away a few metres short of the line but the Gloucester defence holds. The ball is trapped in a ruck and Munster are lucky to be awarded the scrum feed. Great opportunity to score here for the home side as they pack down 5 metres out.
46 Ian Keatley uses the tail wind to his advantage to set up a line-out on the Gloucester 22m line.
44 Referee Jerome Garces loses patience with the Munster front row after series of collapsed scrum. Gloucester go for touch from the penalty but Donnacha Ryan steals the line-out!
42 It's Munster who have made the brighter start to the second period, Paul O'Connell in particular carrying the ball with real venom.
41 Here we go for the second half! Can Gloucester get an early score and put pressure on the hosts?
Munster 16-10 Gloucester
39 PENALTY, GLOUCESTER, BENTLEY! 16-10! Three welcome points for the Cherry and Whites as the referee spots a Munster hand in the ruck. Up steps Bentley from almost bang in front to cut the gap to six.
36 CONVERSION, MUNSTER, KEATLEY! 16-7! And that's an excellent kick from the touchline from the fly-half.
34 TRY, MUNSTER, VARLEY! 14-7! The try is awarded after approval from the TMO. It was a pushover score.
33 Interesting call from Munster captain O'Mahony as he chooses to turn down three points and instead opts for the corner. They are going for the jugular here then with a five-metre line-out coming up.
31 Munster win a penalty here as the visitors are penalised at scrum time. So this will be healthy territory for the hosts, who go wide off first-phase ball to full-back Jones. This looks ominous.
28 Gloucester survive and in fact reclaim possession as Earls spills the ball after being met well by Lokotui. No doubt about it, this has been high on tempo with both teams showing willingness to run.
25 No further scores to mention but it's noticeable that Gloucester have grown in confidence since that Sharples score. However, Tindall's poor clearance off the boot is set to put pressure back on them.
18 CONVERSION, GLOUCESTER, BENTLEY! 9-7! A great kick from the touchline - a nerve settler for Bentley.
16 TRY, GLOUCESTER, SHARPLES! 9-5! A slip ball from Robson finds Sharples who dives in from close range.
15 It's been an entertaining opening and none more so than then as Munster's offloading game down the left wing was followed by a Gloucester breakout that ended with Murphy running the ball into touch.
13 PENALTY, MUNSTER, KEATLEY! 9-0! Another breakdown offence from Gloucester and from a similar position to his last effort, Keatley knocks over his third penalty of the game so far. Simple points for him.
9 PENALTY, MUNSTER, KEATLEY! 6-0! The lead is doubled at Thomond Park after Mills is offside at a ruck. Keatley slots this one easily from 30 metres out in centre-field. Bentley's miss is compounded then.
7 Munster are penalised for holding on at ruck time so it is a first chance for fly-half Bentley to level matters. But it's wide from the Heineken Cup debutant as Thomas exits, replaced by Rob Cook.
4 PENALTY, MUNSTER, KEATLEY! 3-0! Good work from the pack and Gloucester eventually offend at the ruck. Munster don't get any joy out of the passage so back we come for the offence, with Keatley on target.
2 It's a superb start from Munster. O'Mahony ran well from the kick-off before breakdown work when the visitors were in possession saw the Irishmen win a kickable penalty. Ian Keatley goes for the corner.
1 The teams are out and we are good to go here. Munster - and their sixteenth man - look fired up today.
0 For Munster, Johne Murphy takes the place of the injured Simon Zebo on the wing and in the pack boss Rob Penney calls in Damien Varley, BJ Botha, Donncha O'Callaghan and Peter O'Mahony. Meanwhile, Will James captains a Gloucester side which retains just three players from last week's selection - Jonny May, Ryan Mills and Mike Tindall - as boss Nigel Davies lives up to his promise to rotate the squad.
0 Hello and welcome to another live offering of this Heineken Cup Saturday. We are in Limerick for this game as Munster host Gloucester, with the latter hoping to make it two wins from two in the pool. The Irish meanwhile will surely be reeling after losing at Edinburgh so it should be a cracking contest.
Updates to appear here at 17:45 BST.
Sky Bet Odds:Click Here for Your Free Bet
|Aviva Premiership
|Saturday 30th May
|Aviva Premiership
|Bath 16 - 28 Saracens
|Saturday 23rd May
|Bath 47 - 10 Leicester Tigers
|Northampton 24 - 29 Saracens
|Saturday 16th May
|Exeter 44 - 16 Sale Sharks
|Leicester Tigers 22 - 14 Northampton
|Bath 50 - 30 Gloucester
|Newcastle Falcons 37 - 21 Harlequins
|London Irish 40 - 40 Wasps
|London Welsh 17 - 68 Saracens
Rupert Cox's Off Load: Clermont, England Sevens and Jean Deysel come under scrutiny
Rupert Cox shares his highlights from the week's rugby action in his round-up blog.
Hits and tries
The biggest and toughest hits and tackles plus the best tries from the world of rugby union.
Rupert Cox's Off Load: Hurricanes, Toulouse and Nick Williams all come under scrutiny
Rupert Cox shares his highlights from the week's rugby action in his round-up blog.
Experts & Blogs
Phil Edwards
Read the thoughts and opinions of Phil Edwards with skysports.com


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African Regional Organization for Standardization (ARSO)
Overview: ARSO is an African inter-governmental organization established in 1977 mandated to promote standardization activities in Africa. To achieve this goal, ARSO has developed a program on standardization and related activities for its member States. This program is based on the blueprint for Africa's economic development as outlined in the Lagos Plan of Action for the Economic Commission for Africa which envisages the establishment of an African common market through integration of the various sub-regional economic groupings on the continent. The program is designed towards removal of technical barriers that hinder intra-Africa trade and integration. The technical work is concerned with the operation of activities in the following fields: preparation and issuance of African regional standards; quality assurance activities; African regional accreditation and certification marking schemes (ARAS); metrology; laboratory testing; technical information and consultancy services in standardization; and international liaison and participation.
IPR Policy:
Current Status: Active


text:
Coldwell Banker Previews International welcome discerning buyers to view West House, a luxury double fronted detached home offering stunning sea views over Dublin Bay. This Granite faced home was built in 2005 to the highest standards. The meticulous attention to detail is evident throughout the 4000 sq. ft. of contemporary light filled living space. Situated just off the prestigious Colimore Road and a short stroll from the delightful Dalkey Village, the location is most definitely second to none.
If you are looking for the perfect family home, look no further! West House spans over three floors and along with an abundance of space, it offers every convenience for modern day living.
The spacious entrance hallway comes with an atrium providing an abundance of natural light. Just off the hallway to the right there is a generous formal reception / dining room with balcony and spectacular sea views. On the opposite side of the hallway double doors lead to the open plan kitchen / living room. This has its own balcony offering the same sea views. On this ground floor level there is also a fully fitted utility room, guest WC and storage/ cloak cupboard. A double bedroom with shower en suite completes the ground floor layout.
A solid oak staircase with glass side panels and stainless steel handrail lead upstairs to the landing . There are five bedrooms on this level, two of which have private balconies, again with breathtaking views to the sea. The master bedroom is en suite and comes with both walk in and built in wardrobes. There is also a luxurious family bathroom with a large corner Jacuzzi bath.
On the lower ground floor there is yet more living space...a huge family/ entertainment room with French Doors to the patio. Also at this level there is an office/ study, Guest WC, storage room and an inviting Turkish Steam Room complete with seating and attractive mosaic tiling.
The terraced garden to the rear is in lawn with mature shrubs, trees and several split level decked patios.
West House is tucked away in Dalkey Sound behind remote controlled electric gates on a generous site allowing parking for several cars. Dalkey Sound is on Green Road and literally minutes from all local amenities.
Viewing of this well presented home is recommended and strictly by appointment
About Dalkey Village
Dalkey nestles on the coast in south east Dublin. The locality provides all the amenities of a large town but still retains that village feel and friendly atmosphere. The area oozes charm with its quaint stores, cafes, restaurants and pubs, not to mention its heritage attractions like Dalkey Castle. It is indeed the perfect place to call home.
Links that may be of interest: www.ilovedalkey.com , www.dalkeycastle.com
Basement/Lower Ground floor
Office / Study (3.6 x 3.8)
With solid oak wide plank flooring
Guest WC
With WC, WHB, tiling to floor, part tiling to walls
Turkish Steam Room (2.1 x 1.37)
With mosaic tiling to floor and walls, large shower head and glass door
Family Room / Entertainment Room (9.7 x 4.9)
With solid wide plank oak flooring, open fireplace, floor to ceiling French doors to front garden, Side door to patio area.
Storage Room
Tiling to floor, water tank, cabling
Ground Floor
Hall (6.76 x 3.26)
Light filled entrance hallway with atrium, solid oak flooring, oak staircase with glass paneling and stainless steel handrail.
Formal Lounge / Dining room (5.1 x 3.4) (5.1 x 4.9)
Large formal reception room with fireplace, floor to ceiling French doors to balcony with views to the sea. Dining area with French doors opening to a split level decked patio, with steps leading up to the mature garden.
Open Plan Kitchen / Living room (5


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Holman, NM - United States
Member Since:
July 10th, 2011
About Scott Haley
Formerly I have been a working cowboy, a high school and college science instructor, an independent environmental regulatory compliance consultant, and a hazardous materials specialist for Sacramento County. I started making art, specifically pen & ink drawings, in 1979; about eight years ago, in 2006, I switched to abstract and impressionistic painting. I started photography in the mid-1980s, stopped after a few years, and then took it up again in 2008. I've been a full-time artist since 2009.
The original inspiration of my passion for art came with an elective course in Aesthetics at Colorado State University in the early '60s. The professor who taught that course stressed over and over: beauty is in the eye of the beholder...100%. Also--- perceptions change with time and events. A good example would be: when Jackson Pollock first started painting his 'drip' or 'pour' pieces, the so-called art experts said that those works looked like 'matted spaghetti'. They deemed them of little or no value. [Google 'Lavender Mist'.] Much later, one of his 'matted spaghetti' pieces sold for millions of dollars. Pollock is recognized as a giant in abstract expressionism. It's all in the eye of the beholder.
My favorite painters are (in no particular order): Charles Russell, Claude Monet, Jackson Pollock, Lee Krasner, Jean-Michel Basquiat, Willem de Kooning, Perle Fine, Mark Rothko, Robert Motherwell, Joan Miro, Franz Kline, Allen Lehman, Paul Klee, Erik von Ploennies, Susan Sadoury, and Maxim Grunin.
Scott Haley is hosting all of the following contests. To view contests that Scott Haley is participating in, click here
Accepting Entries
Scott Haley is not hosting any contests which are currently accepting submissions.
Voting in Progress
Scott Haley is not hosting any contests which are currently voting.
Recently Finished
Scott Haley is not hosting any contests which have finished within the past 90 days.


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Bob Staake is the author and/or illustrator of over 50 books and is a contributor to the New Yorker. He entertains and challenges his readers, but with this latest project, he's created something that is kind of messed up. In the best possible way. Staake has taken the design and ideas from books like The Little Golden Book series (which began way, way back) and has made them into visual parodies that are kind of demented. The 1940s-50s imagery and lettering is totally thrown off by Staake's dark and strange titles. We especially like the one of the little kids playing with the dollhouse – it's title, though? A Good Place To Hide The Bodies. And the children looking out to sea, called Dead Whales Can't Wave Back. Evil genius. See them all HERE.
Amsterdam on a Shoestring
No money, no problem
Follow Contiki
- Follow @contikiUSA
3rd May 2013


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Account Handler jobs
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A well-known insurance Broker seeks an Account Handler in their West London office to specialise in construction business.
Construction is a key area for this Broker and they are looking to compliment their team with an experienced Account Handler. The role will involve dealing with a range of SME/mid-market Construction related risks including EL, PL, CAR and JCT. The Account Handler will support the Account Executives with both renewals and new business broking and providing an excellent level of service to clients.
The ideal candidate for this role…
Hales have an oppurtunity for an experienced Account Handler. Our client based in Peterborough are looking for an experienced Account Handler to join their growing company.
As an Account Handler you will have knowledge in:
* Have an understanding of the Insurance Market,products and Services. An awareness of how an Account Handler role supports the business.
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• Taking briefs from clients, handling day to day requirements for smaller clients
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Job Title: Account Handler
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- Resolve various customer service queries and requests
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Adecco are delighted to be working closely with our hugely successful client based in Colchester on the position for an Account Handler
This is a full time, permanent position based in Colchester. The company are rapidly expanding and looking for individuals who ideally have a background in Marketing services.
My client is seeking ambitious, hardworking, efficient and highly organised individuals with exceptional relationship building skills.
Main duties include:
Being involved in day to day contact with clients - generating business, taking briefs, making marketing suggestions, actioning and managing each project to its completion…
Our client is a leading insurance broker and is looking for an immediately available Account Handler to work on a 12 week temporary contract which could potentially become permanent for the right person.
You will work with a friendly team providing support and advice to their commercial clients.
• Providing clients with day-to-day technical and administrative support with regards their products and services
• To understand the clients insurance needs and identify the correct insurer market
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Insurance Account Handler
Would you like to work in fabulous offices in the centre of Sevenoaks, in a dynamic and professional team? Do you have commercial broking experience? (preferably from a broker rather than an insurer? Do you have general insurance experience?
The role will entail giving instructions to insurance providers, checking policy documents, handling invoices of premiums due, processing correspondence, managing own files and other ad hoc duties
If you have general knowledge of insurance principles, product knowledge, customer care, claims, market knowledge and excellent communications skills, then please…
We have an amazing permanent job opportunity for an Account Handler that offers a guaranteed basic salary of £16'000 to £20'000, plus holiday, pension, ample free car parking and a friendly work environment.
They are looking for someone who has previously managed the order process from start to finish. Ideally you would have received orders over the telephone or by email, processed the orders onto a database. You will also have knowledge of transport, liaising with different companies to ensure the stock arrives on time.
Your experience has ideally be gained from a Logistics company, or a 3PL company (3rd party logistics company…
AJ Chambers are recruiting for a Renewal Account Handler for a specialist Insurance brokers based in Rochford.
Key Responsibilities
You will responsible to renew a book of commercial insurance clients covering mainly: Combined Liability
Cross sell and Up-sell where possible: Contractors All Risks, Office Combined, Commercial Combined, Property Owners, Motor Fleet, Commercial Vehicles, Professional Indemnity, D&O, Personal Accident, Legal Expenses, XOL, Excess Protection & JCT cover.
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to £30,000
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The Role
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Insurance Account Handler
Mon-Fri 9am-5pm
Job Profile:
A key customer servicing role within a small team, working in tandem with and supporting our customer facing Commercial Insurance Account Executives, to provide quick accurate Commercial Insurance responses to our valuable EXISTING customers.
Core duties, reporting to a Team Leader and the Commercial Director, include:-
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An exciting new job has arisen in Insurance for a sales focused Account Handler in the Croydon area of Surrey. The salary is up to £25k however the bonuses expected for KPI hitting Account Handlers will mean the successful applicant should earn up to £33,000.
The job is suitable for:
A skilled SME Commercial Account Handler who has over a years' experience in the industry and has delivered new business results in the past
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My client is a 100% client focused, market-leading independent insurance brokerage with branches all over the UK, with an emphasis on offering a truly personal and local service to both its Personal Lines and Commercial customers.
They are now looking to grow further their office in central Camberley by recruiting an Account Handler who will be responsible for dealing with incoming enquiries and renewals from Commercial-type customers.
As a Commercial Account Handler you will be looking after all new business enquiries, the majority of which will be from small to medium sized companies. You will also be inviting renewal quotes…
Working for a leading award winning broker, our client is looking to recruit an Account Handler within their Corporate team.
The Account handler will be responsible for the date to day servicing and growth of nominated corporate insurance accounts, including visits to clients when necessary
Offer support to the account directors account executives and relationship managers and directors who deal with the clients
Help identify cross selling opportunities and potential gaps in client cover. Working on a number of binders carrying a good underwriting authority.
Maintain good working relationships with clients, colleagues, insurers…
Commercial Account Handler
My Client
My Client are an independent insurance brokers which provide commercial & personal insurance/ My client is urgently recruiting for a Commercial Account Handler to be based in Nottinghamshire - ideally with experience in Vintage / Classic Car Insurance & Personal Lines Insurance.
The Role
To provide a complete Commercial Lines Insurance service for exisiting & potential new customers and to support the Directors & Account Executives in their provision of this.
Key Responsibilities
Provide new business quotations and advice for Commercial products
Draw clients' attention to all information pertinent…
A new opportunity for an experienced commercial account handler to join a rapidly expanding insurance group within their Orpington office.
Due to continual growth this professional organisation requires a commercial account handler with 3 years plus experience in a commercial broking environment. You will have a good knowledge of commercial general insurance products and be well versed in writing detailed insurance renewal reports for Account Executives. You will also be dealing with adjustments and general policy queries, ensuring a first class service at all times.
Preference for Cert II or study towards, knowledge of the Acturis system…
Commercial Account Handler (Insurance)
Location – Leamington Spa, Warwickshire
Salary – £ 20,000 - £ 25,000 + OTE and Excellent Benefits
Award winning and forward thinking Insurance Group principally known for its niche Motor and Commercial Insurance Products are seeking to attract a Commercial Insurance Professionalto join the group based in the Leamington Spa.
The Commercial Account Handler will be responsible for developing and maintaining a portfolio of clients across the region, providing expert advice to clients on their Commercial Insurance requirements and levels of risk exposure.
This is a unique opportunity to be part…
Our client is a market leading Insurance Organisation that offers a broad range of Commercial Insurance products and prides themselves on their outstanding customer service.
A permanent role has arisen for an experienced Commercial Account Handler to join their team.
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Insurance Account Handler
I am currently recruiting for an Insurance Account Handler with a professional Broker in Ipswich. I am looking for candidates with at least 2 years insurance experience, in either the Personal Lines (Home and Motor) or the Commercial sector - ideally from a Broking environment.
The position will entail handling small Commercial insurance from New Business/Quote stage through to Renewal, including Mid Term Adjustments and general enquiries. You will therefore need to have strong set of communication and organisation skills as well as well as a good eye for detail.
The role will suit someone that is currently…
My client a leading insurance broker requires an account handler with previous experience specialising in Professional Indemnity, you will be dealing with clients aiding in the overall service proposition to clients ensuring that the business is retained and developed.
The account handler will be responsible for the book of business and will manage the renewals from start to finish, negotiating terms with Insurers, preparing documents and presenting terms to the client. You will be targeted on the retention and growth of the book.
The right individual will have strong communication skills and have the ability to build and maintain good…
Commercial Insurance Account Handler
to £25,000 + benefits
Bromley area, Kent
Busy friendly Commercial Insurance Broking office, with long standing client relationships.
Their client portfolio covers a broad range of SME to larger mid market and corporate clients, covering the full range of Property Risks, Commercial Combined, PL, EL, Commercial Motor, Professional Indemnity, D & O etc.
Their Account Handlers service all aspects of new business, renewals and adjustments. While this is an office based role, there are opportunities to meet with clients from time to time.
They quote and place business with a wide panel of Insurers…
A new opportunity has arisen for an experienced commercial/corporate account handler to join a leading Insurance Brokerage within their specialist UK Corporate business area.
You will be supporting the Account Executive dealing with the insurance requirements of a broad range of Corporate UK businesses. This will include renewal preparation, adjustments, client liaison and general policy advice. Premiums dealt with will be in the £20k- £300k bracket.
Ideally you will have previous experience dealing with corporate clients, but my client will also consider people from a general commercial background. It is essential…
An exciting opportunity has arisen to join an expanding Insurance Broker based in Newport as they are looking for a bright Commercial Account Handler to join their growing team.
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text:
Headphone Amplifiers
V-MODA VAMP Headphone Amplifier
" So my Impressions: Introduction I have said before, I am against selling what you win or are given, and I am not an apple fan, and have no interest in getting an iPhone. So what was I to do with the VAMP I just won? Well the original idea I had was to give it to my mother to troll around with with her K550's. But it seems fate had other ideas, my friend, helped me win..." --WiR3D
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text:
Member since February 18, 2008
Right now, I'm reading:
Between the Assassinations by Aravind Adiga
My favorite story of all time is:
The Hobbit.
In bed I like to read:
a chapter before falling asleep.
What six words would I tell my teen self if I could go back in time?
Hang on: SSRI's soon become available.
©2005 - 2014 Six Words, LLC


text:
A chemical sensor includes a patterned layer having discrete sections, a -dimensional detector array, and a two-dimensional lens array that focuses an optical signal from the patterned layer onto the two dimensional detector array. Typically, the two-dimensional detector array is a charge-coupled device array and the lens array is a graded index of refraction lens array. The chemical sensor maintains good resolution throughout its field of view.
1. A chemical sensor for detecting a plurality of analytes in a sample, said sensor comprising:
a patterned signal layer comprising a two-dimensional array of discrete, optically transparent test sections at known locations thereon, each of said test sections comprising an immobilized specific binding partner for one of said plurality of analytes, such that specific binding of said analyte to said immobilized specific binding partner generates an optical signal in said test section indicative of the presence of said analyte;
a two-dimensional light sensor array which detects said optical signal in a corresponding discrete detection section; and
a two-dimensional array of graded index of refraction lenses which focuses each said optical signal from each said test section into each said corresponding detection section.
2. The chemical sensor of claim 1 wherein said patterned signal layer is a waveguide comprising said discrete test sections of said immobilized specific binding partners.
3. The chemical sensor of claim 2 wherein said waveguide is a glass coverslip or a microscope slide.
4. The chemical sensor of claim 1 wherein each of said specific binding partners are antibodies covalently attached to said signal layer, said analytes are antigens, and at least one of said test sections further comprises a conjugate of a detectable label and said antigen or an analog thereof specifically bound to said immobilized antibody.
5. The chemical sensor of claim 4 wherein said detectable label is a fluorescent label and said sensor further comprises:
a light source which irradiates said fluorescent label at a known excitation wavelength such that the fluorescent label emits light at a known emission wavelength to provide said optical signal and
a filter positioned between patterned signal layer and said two-dimensional light sensor array, wherein said filter removes transmitted light of the known excitation wavelength before said optical signal is detected by said two-dimensional light sensor array.
6. The chemical sensor of claim 5 wherein said filter is positioned between said two-dimensional lens array and said two-dimensional light sensor array.
7. The chemical sensor of claim 6 wherein said filter is sandwiched directly between said two-dimensional lens array and said two-dimensional light sensor array.
8. The chemical sensor of claim 1 wherein said two-dimensional light sensor array is a charge-coupled device array.
9. The chemical sensor of claim 1 wherein said two-dimensional array of graded index of refraction lenses is positioned between said patterned signal layer and said two-dimensional light sensor array.
10. A method of simultaneously detecting a plurality of analytes in a sample, comprising:
(a) providing a chemical sensor, said sensor comprising:
a patterned signal layer comprising a two-dimensional array of discrete, optically transparent test sections at known locations thereon, each of said test sections comprising an immobilized specific binding partner for one of said plurality of analytes, such that specific binding of said analyte to said immobilized specific binding partner generates an optical signal indicative of the presence of said analyte in said test section;
a two-dimensional light sensor array which detects said optical signal in a corresponding discrete detection section; and
a two-dimensional array of graded index of refraction lenses which focuses each said optical signal from each said test section into each said corresponding detection section;
(b) contacting said sample to said patterned signal layer; and
(c) detecting the presence or absence of said focused optical signal in each of said detection sections, whereby the presence of said focused optical signal in said detection section indicates the specific binding of the analyte in the test section and thereby indicates the presence of the analyte in the sample.
11. The method of claim 10 wherein said contacting step further comprises contacting said sample and said patterned signal layer with a labeled reagent comprising a fluorescent label conjugated to (i) either said analyte or an analog thereof which is specifically bound by said immobilized specific binding partner or (ii) a second specific binding partner which specifically binds to said analyte at a site which does not intefere with the specific binding between said analyte and said immobilized specific binding partner.
12. The method of claim 11, wherein said sensor further comprises a light source for irradiating said fluorescent label at a known excitation wavelength such that the fluorescent label emits light at a known emission wavelength to provide said optical signal and said method further comprises a step of irradiating said patterned signal layer with said excitation wavelength after said contacting step, thereby producing the focused optical signal detected in the detecting step.
13. The method of claim 12, wherein said sensor further comprises a filter positioned between patterned signal layer and said two-dimensional light sensor array, wherein said filter removes transmitted light of the known excitation wavelength before said optical signal is detected by said two-dimensional light sensor array and said detecting step further comprises removing said transmitted light of the known excitation wavelength before detecting said focused optical signal.
14. The method of claim 10 wherein said two-dimensional light sensor array is a charge-coupled device array.
15. A method of simultaneously detecting a plurality of analytes in a sample, comprising:
(a) providing a chemical sensor, said sensor comprising:
a patterned signal layer comprising a two-dimensional array of discrete, optically transparent test sections at known locations thereon, each of said test sections comprising an immobilized antibody which specifically binds to one of said plurality of analytes such that the specific binding generates an optical signal indicative of the presence of said analyte in said test section;
a two-dimensional light sensor array which detects said optical signal in a corresponding discrete detection section;
a two-dimensional array of graded index of refraction lenses which focuses each said optical signal from each said test section into each said corresponding detection section; and,
a light source for irradiating a fluorescent label at a known excitation wavelength such that the fluorescent label emits light a known emission wavelength to provide said optical signal;
(b) contacting said sample, said patterned sign layer and a labeled reagent comprising said fluorescent label conjugated to (i) either said analyte or an analog thereof which is specifically bound by said immobilized antibody or (ii) a second antibody which specifically binds to said analyte at a site which does not interfere with the specific binding between said analyte and said immobilized antibody;
(c) irradiating said patterned signal layer with said excitation wavelength after said contacting step, thereby producing said focused optical signal; and
(d) detecting the presence or absence of said focused optical signal in each of said detection sections, whereby the presence of said focused optical signal indicates the specific binding of the analyte in the test section and thereby indicates the presence of the analyte in the sample.
1. Field of the Invention
The present invention relates generally to chemical sensors and more specifically to multi-analyte chemical sensors employing arrayed charge-coupled imaging devices.
2. Background of the Invention
Multiple-analyte-detecting chemical sensors, particularly biochemical sensors, have been previously described. In some embodiments, these sensors include a patterned layer including discrete sections, and a light sensor array (e.g., a charge-coupled device (CCD) array or a photodiode array). Each discrete section, in response to the presence in a sample of an analyte of interest, alters light transmitted through, absorbed by, or emitted from that section. In some cases (e.g., sandwich assays), this response appears only after the reaction with a third, labelled molecule). Each section is specific for a single analyte. Light from the patterned layer is then transmitted, typically through an appropriate filter, to a sensor array. The light sensor array produces an image or electrical signal that can then be correlated with the patterned layer to simultaneously determine the presence of a variety of analytes.
Typically, it is desirable to perform multiple-analyte chemical and biochemical detection in the field. Field use requires a compact, light-weight device that may be easily transported and used. Thus, a high density of sections in the patterning layer is desirable. The need for good resolution between optical signal from nearby sections (to prevent optical signals from nearby sections from overlapping at the light sensor array layer), however, limits the usable density of sections. A lens improves resolution, and thus usable density, but only for a small portion of the field of view.
Accordingly, it is an object of this invention to chemically detect multiple analytes using a small, compact sensor.
It is another object of the present invention to enhance the usable density of discrete sections throughout the field of view in a patterned layer of a multiple analyte chemical sensor.
It is a further object of the present invention to enhance the optical resolution between signals from nearby sections throughout the field of view in the patterned layer of a multiple-analyte chemical sensor that detects those signals using a two-dimensional light sensor array.
These and other objects are accomplished by using a two-dimensional lens array to focus optical signals from discrete sections in the patterned layer of a multiple-analyte sensor onto a two-dimensional light sensor array.
A more complete appreciation of the invention will be readily obtained by reference to the following Description of the Preferred Embodiments and the accompanying drawings in which like numerals in different figures represent the same structures or elements, wherein:
FIG. 1 illustrates the construction of antibody pattern on a coverslip for use with the present invention.
FIG. 2 is an exploded view showing a typical apparatus according to the present invention.
FIG. 3 is a graph showing the response of a biosensor according to the present invention to Cy5-labeled antibody. The responses were determined from the subtracted images for each concentration obtained as described in the "EXAMPLES" section below. The average fluorescence intensities for each of the three circular regions was averaged and normalized to the average intensities from the rectangular regions for the row of interest. Data shown are for (∘) rabbit anti-goat IgG, (□) rabbit anti-chicken IgG, and (Δ) rabbit anti-mouse IgG.
The sensor of the present invention is a multilayered device. The outer, exposed layer of the device has a plurality of discrete sensing sections formed in a two-dimensional pattern on the surface of the layer. Each of these sensing sections, in the presence of a specific analyte, provides a detectable optical signal. Different sensing sections within the patterned surface are specific for different analytes. Thus, a plurality of different analytes within a sample contacted with the sensing layer may be simultaneously detected.
The selection of material for use as the sensing layer is not critical to the advance achieved according to the present invention. Any materials useful as the sensing surface in conventional multiple-analyte sensors may be used according to the present invention. For example, sensing surfaces useful in the method of the present invention may be flat glass or plastic surfaces. Typically, and conveniently, the sensing surface is the upper surface of a waveguide substrate, such as a glass coverslip. Throughout the present specification and the claims that follow, a waveguide substrate is defined as a material that acts both as a substrate for the pattern and as a medium for the excitation light and/or light emitted from or transmitted through the pattern. The low cost, light weight, and disposability of glass coverslips make them particularly advantageous as waveguide substrates for use in portable chemical and biochemical sensors.
A pattern of sensing sections may be formed in the sensing surface by any method, including conventional methods. Typically, and as shown in FIG. 1, layer 100 of photopolymerizable optical adhesive is applied between glass coverslip 102 and wax film layer 104 (for example, PARAFILM) coated unto a suitable material, such as glass. TEFLON (tetrafluoroethylene) or other non-adherent spacers 106 space coverslip 102 from the PARAFILM coating. This assembly is exposed, through photomask 108, to photopolymerizing uv radiation from light source 110. The PARAFILM and unexposed portions of the adhesive layer are then removed, leaving behind wells formed within a layer of moderately polymerized adhesive, with the glass coverslip forming the bottom of the wells. The bottom of these wells is then silanized to allow crosslinking to antibodies or other molecules or biomolecules that specifically react with an analyte of interest.
Biospecific, or chemically-specific, molecules are then added to each well, for example with a pipette, micropipette or similar dispenser. Each well may be filled with a molecule specific to a different analyte. The moderately polymerized polymer layer is then removed, by conventional methods, to expose the remainder of the coverslip. The newly exposed areas of the coverslip are then blocked to prevent non-specific binding thereto.
The optical signal obtained in the presence of the analyte may be either an increase or a decrease in the light emitted by a section. Typically, the optical signal is either an increase or decrease in the fluorescence emitted from the section to which the analyte of interest has bound. Phosphorescence and other luminescent phenomena may also be used as the optical signal. Whether optical signal obtained is directly or inversely proportional to the concentration of an analyte of interest will depend largely, and in a known manner, upon the type of assay performed, e.g., competitive assay, sandwich assay, direct assay, or displacement assay.
The type of assay performed will also determine, in a known manner, which, if any, molecules are labelled. The present invention may be used in any type of immunoassay using an optical signal to indicate the presence of an analyte of interest. These assays include direct assays, competitive assays, displacement assays, and sandwich assays. In most competitive assays, an antibody to the analyte is attached (typically covalently) to the supporting surface. During the assay, free, unlabelled analyte and a labelled analog of the analyte having essentially the same immunoreactivity as the analyte compete for free analyte-binding sites on the attached antibody. In a typical displacement assay, the analyte-binding sites on the support-attached antibody are saturated with a labelled analog of the antigen. The support is then briefly contacted with the analyte. Some of the unlabelled analyte displaces some of the bound, labelled analyte. In a typical sandwich assay, one immunoreactive molecule (typically, an unlabelled antibody), is attached (typically covalently) to a support surface. The support surface is then allowed to react with and bind unlabelled analyte from the sample. Then, a labelled antibody is reacted with the bound, unlabelled analyte. In the specification and the claims the follow, a molecule and the labelled analog of that molecule have essentially the same immunoreactivity toward their binding partner (i.e., the labelled analog and the unlabelled molecule are immunoequivalent) if, for the purposes of the assay being performed, the differences in reactivity are insignificant. However, labelling may not always be required. For example, if specific interaction of the attached molecule and the analyte of interest forms a fluorescent molecule, or quenches fluorescence emitted from a molecule attached to a section of the pattern, labelling may not be required.
As shown in FIG. 2, this coverslip 10, with pattern 11 thereon, is then placed on transparent support 12 (typically glass or clear plastic window) of an imaging device and exposed to a sample (not shown). Typically, gravity maintains coverslip 10 in place on support 12. If necessary for light emission from pattern 11, pattern 11 is exposed to a source of excitation light 14. Transparent support 12 transmits light from the coverslip 11 to two-dimensional lens array 16. Generally, this transmitted light will be a combined light that includes both excitation light and the optical signal from the signal patterning layer.
Two-dimensional lens array 16 is sandwiched between transparent support 12 and optical emission filter 18, and focuses light from pattern 11 onto imaging array 20, which supports filter 18.
Focusing light emitted from pattern 11 with a lens array, rather than a single lens, greatly reduces aberration. Consequently, lens array 16 greatly enhances the resolution in the imaging array. Because of this enhanced imaging resolution, the present invention permits a higher useful patterning density, and a more compact design, than available in analogous prior art sensors.
Any two-dimensional lens array may be used in the present invention. Because of its flat surface and profile, a two-dimensional graded index of refraction (GRIN) lens array is particularly advantageous for use according to the present invention. Also, GRIN lens arrays with small focal lengths may be inexpensively produced. Nevertheless, other types of lens arrays, including concave lens arrays, may be used in place of GRIN lens arrays. Also, a two-dimensional lens array may be molded in to the substrate support.
The use of a patterned coverslip 10 is optional. If desired, pattern 11 of sections that specifically recognize various analytes of interest may be placed directly on the glass support 12. The disposability of patterned coverslip 10, however, provides convenience and cost benefits. Alternatively, slides, or capillary tubes having a flattened longitudinal surfaces upon which the desired pattern is formed, may be used in place of coverslips. Where the waveguide support is a capillary tube(s) or other hollow structure(s), the excitation light may be launched into the interior of the hollow structure (i.e., an evanescent excitation wave).
Emission filter 18 removes the excitation light from optical signals transmitted through lens array 16. Preferably, this filter is positioned (as shown in FIG. 2) between the lens array and the imaging array to reduce scattering. Positioning filter 18 between glass support 12 and lens array 16 would increase scattering, in unfocusing the desired image.
Any filter may be used as filter 18. Typically, filter 18 is an absorptive long-pass filter. An alternative is to use an interference filter, which would have a narrow blocking band and potentially allow more of the optical signal through. However, an interference filter usually requires collimated light. This collimation can be achieved by using two 2-d lens arrays, with the first one collimating the light and the second focusing onto the CCD. Placing the filter in between two lens arrays allows the use of interference filters.
The present invention may be incorporated into multianalyte biosensors for many antigens, including biologically hazardous agents, environmental pollutants, explosives, or drugs of abuse (see Ligler et al., Immunomethods 3, 122-127 (1993); Shriver-Lake et al., Sensors and Actuators B 11, 239-243 (1993); and Shriver-Lake et al., Anal. Chem. 34, 2431-2435 (1995), the entireties of which are incorporated herein by reference for all purposes) Additionally, the present invention can easily detect antibody concentrations as low as 50 ng/mL (0.29 nM for ˜170 kD antibody analytes).
Having described the invention, the following examples are given to illustrate specific applications of the invention including the best mode now known to perform the invention. These specific examples are not intended to limit the scope of the invention described in this application.
Materials and Methods
Imaging system
The basic design of the biosensor is shown in FIG. 2, which was discussed above. A scientific grade CCD image sensor (CCD05-30 from EEV, Elmsford, N.Y.) consisting of 1242 (H)�1152 (V) pixels was used as the detection system. This CCD was operated in inverted (MPP) mode to suppress dark current noise. CCD detectors operated in this mode acquire dark current noise up to 20-fold less quickly than non-MPP CCD's, and improve charge transfer efficiency. The combined effect is that for relatively short exposures, these CCDs can be operated at room temperature with significant sensitivity. Half of the vertical pixels were used as a framestore for continuous image acquisition, so each image frame consisted of 1242 (H)�576 (V) pixels. The readout rate (pixels/sec) was maintained at 50 KHz, so that each image took 14.8 sec to acquire.
The glass-covered CCD chip was inserted into an appropriate headboard and mounted underneath a metal stage. The output video signal from the chip and electrical power was controlled by drive circuitry unit (EEV, Elmsford, N.Y.) external to the stage area. The video output signal was collected using a Lab-LC A/D board (National Instruments, Austin, Tex.) mounted in a Macintosh LC475 computer, and a program written in THINK C using the A/D board software libraries. Each acquired frame was saved as a 12-bit digital image.
The stage area above the CCD was constructed from a 0.6 cm thick rectangular aluminum plate with dimensions 21 cm�26 cm. This plate was mounted on four 15.2 cm screws (one at each corner of the plate) and secured to an optical breadboard. A rectangular area 10.1 cm�8.3 cm was removed from the aluminum plate near its center. A 0.2 cm wide and 0.2 cm deep ledge was cut into the aluminum plate around the rectangular cutout, such that a large glass microscope slide (10.2 cm�8.3 cm�0.2 cm (Thomas Scientific)) just fit into the cutout and was flush with the top of the aluminum plate. The bottom of the glass plate was masked with black spray paint, except for a region in the center with dimensions 3.5 cm�7.3 cm. The glass coverslips with immobilized antigens (see below) were placed over the unpainted region for interrogation.
A graded index of refraction (GRIN) lens array was placed beneath the glass plate to focus the surface of the cover slips onto the CCD. Each cylindrical GRIN lens was 0.7 cm long, with a diameter of 0.09 cm and a focal length of 0.38 cm from either end of the lens cylinder. The two-dimensional GRIN lens array was constructed from linear rows of GRIN lenses (Nippon Sheet Glass, Summerset, N.J.) so that it covered the unmasked area under the glass plate. The array was mounted 3 mm below the glass plate and 3 mm above the CCD surface, with the heights adjusted to achieve exact focusing.
The coverslips were interrogated by projecting two beams of light from a halogen projector lamp equipped with two fiber-optic bundles (Dolan-Jenner Industries, Lawrence, Mass.) through two 635�10 nm bandpass filters (Omega Optical, Brattleboro, Vt.) and onto the coverslip surface. Light was projected at a 45� angle from the coverslip surface, and was introduced from two sides to improve lighting homogeneity. A 5 cm�5 cm square 665 nm long pass filter (Schott Glass, Duryea, Pa.) was placed between the GRIN lens array and the CCD to reduce detection of excitation light.
Construction of Antibody Arrays
Antibody arrays were formed on glass coverslips (22�40 mm, No. 1.5; VWR Scientific, Media, Pa.) by generating a series of wells using the photomask shown in FIG. 1. The mask was constructed by printing the pattern onto standard overhead projector transparencies. This mask consisted of 6 rectangular regions that served as control wells for lighting variations and 9 circular regions.
Wells were formed by placing a 0.2 mm layer of Norland Optical adhesive no. 63 (Norland Products, Brunswick, N.J.) between the glass coverslip and a layer of laboratory PARAFILM laboratory film. The pattern was then placed over the glass and unfiltered light from a 100 W xenon lamp was directed onto the pattern for two minutes, as per the manufacturer's directions for pre-curing. The pattern and PARAFILM laboratory film were then removed to expose the moderately polymerized adhesive. An acetone soaked cotton swab was used to remove unpolymerized adhesive from the wells. This left polymer-free wells with glass bottoms within the polymerized coating. The cleaned coverslip with polymer were placed under a 360 nm UV lamp (Fisher Scientific) for thirty minutes. The fully cured polymer on glass was stored overnight in the dark.
The polymer coated glass coverslips were then silanized to activate the wells for crosslinking to antibodies, using the method described in Bhatia et al., Anal. Biochem. 178, 408-413 (1989), the entirety of which is incorporated herein for all purposes. The coverslips were coated with a 4% solution of 3-mercaptopropyltrimethoxysilane (Fluka) in toluene. The silanized coverslips were then incubated with 2 mM g-maleimidylbutyryl succinimide (Fluka) in ethanol for 1 hr. prior to the addition of antigen.
The following immunoglobulins were purchased from Jackson ImmunoResearch (West Grove, Pa.): Horse IgG, Cy5-labeled rabbit anti-horse, goat IgG, Cy5-labeled rabbit anti-goat, chicken IgG (IgY), Cy5-labeled rabbit anti-chicken, mouse IgG, and Cy5-labeled rabbit anti-mouse. A 0.25 mg/mL solution of each IgG in phosphate buffered saline (PBS) was pipetted into separate wells. The horse IgG was placed into the rectangular wells to serve as the control sample. The goat, chicken, and mouse IgG's were placed in the three wells in each row, so that each measurement could be made in triplicate. After incubating with these antibody solutions for one hour, the coverslips were rinsed with distilled water and stored in 1.5% bovine serum albumin (BSA) in PBS at 4� C.
Direct immunoassays were performed using Cy5-labeled rabbit anti-species IgG diluted in PBS/0.05% TWEEN-20 surfactant detergent (to reduce non-specific binding of antibodies) to the desired concentrations. The polymer coating that formed the wells during antigen deposition was easily removed from the coverslips after soaking the coverslips overnight in BSA-PBS. These exposed coverslips, with antigens immobilized at specific locations, were immersed in 1.5% BSA for 20 min to block newly exposed, non-specific binding locations on the glass surface.
After BSA blocking, the coverslips were rinsed with distilled water and air dried. A PAP Pen (wax in toluene; The Binding Site Inc., San Diego, Calif.) was used to encircle the assay area to reduce the volume of sample required for the experiment. The coverslips were then secured over the transparent area of the glass plate in the imaging platform.
Images of the coverslips in PBS buffer solutions were taken as background images to be subtracted from the immunoassay images. A 150 μL aliquot of 500 ng/mL Cy5-rabbit anti-horse IgG was initially applied to the coverslip. After 15 min, this solution was rinsed off, the slide illuminated, and an image captured to visualize the rectangular areas on the coverslip. The variation in fluorescence between these rectangular areas accounted for lighting non-uniformity within the image. For increasing concentrations of Cy5-labeled rabbit anti-species IgG, a 150 μL aliquot of each solution was placed on top of the coverslip and allowed to incubate for 15 min. The slide was then rinsed twice with PBS-0.05% TWEEN-20, and the coverslip was illuminated with the halogen lamp. The fluorescence from the bound Cy5-labeled antibody was then visualized by capturing the image. Higher concentrations were added sequentially to generate a titration curve. Species were added in the order: rabbit anti-goat IgG, rabbit anti-chicken IgG, and rabbit anti-mouse IgG.
The amount of antibody captured was quantitated from the CCD images using NIH Image (National Institutes of Health, Bethesda, Md.). Each 12-bit image was linearly scaled to 8-bits for processing, and were well within the dynamic range of the CCD. The areas of interest corresponding to each spot on the coverslip were extracted using a binary mask in the form of the photomask (see FIG. 2). The average fluorescence from each of the three circular areas in each row were computed using the quantitation tools in NIH Image, and the average of these 3 values was used as the initial signal from the region (�standard deviation). The rectangular areas at the end of each row were used as a standardization signal by taking the average intensity of each rectangle and obtaining the norm of the two values. The signals from each of the circular regions were then expressed relative to the norm from the rectangular signals. Since the illumination sources for the images were directed parallel to the rows, and since the rows contained the areas of interest, the greatest need for correction for variation in lighting intensity was in the horizontal direction.
Experimental Results
These experiments show the detection of low concentrations of three Cy5-labeled antibodies, as well as the signal from a fourth labeled antibody used for correcting for uneven illumination. The Cy5-rabbit anti-horse IgG (500 ng/mL) bound to the rectangular regions at the edges of the pattern. The resulting fluorescence (after subtraction of a PBS background) indicated non-uniform lighting across the sample, and in particular, the central horizontal row of antibodies would be expected to be excited to a greater extent than those of either the top or bottom rows. Thus, for quantitation of samples, it is helpful to have such continuous controls in the array.
These experiments also indicate a degree of cross-reactivity between the Cy5-rabbit anti-horse IgG antibody and goat IgG. When examined separately, the other antibodies used did not show detectable cross-reactivity (data not shown). This background reactivity must consequently be subtracted from the specific signals detected.
These experiments also show the sequential addition of 300 ng/mL samples of Cy5-labeled rabbit anti-goat IgG, rabbit anti-chicken IgG, and rabbit anti-mouse IgG. Even at these relatively low concentrations of antigen, excellent sensitivity was observed, and each signal could be easily discerned, with a signal difference of 10-30 standard deviations above background noise levels. Typically, the 50 ng/ml fluorescence intensities were 17-23% higher than the PBS background, and 6-8 standard deviations above background noise following PBS image subtraction. This assay could also detect mixtures of labeled rabbit anti-species solutions at concentrations of 100 ng/mL (data not shown), making it a simple and rapid device for detecting multiple analytes within a single 150 μL sample.
Due to the linear response of CCD sensors, quantitation of analyte concentrations could also be performed. Increasing concentrations of Cy5-labeled antibody were placed over the coverslip array and allowed to stand for 15 min. After washing, the images due to fluorescence of Cy5 were collected, and the initial image captured prior to addition of the antibody was subtracted from the image for each concentration. The resulting subtracted images for 100 ng/mL solutions of the three Cy5-labeled rabbit anti-species; indicated, as in the prior case, that the signals from the top and bottom rows are less intense than those in the middle. This is in part due to uneven illumination, but may also contain contributions from the varying avidities of the antibodies.
The average fluorescence signals from the indicated spots were quantitated with NIH Image for 25, 50, 100, and 300 ng/mL (˜0.2-1.9 nM) samples of antibody solutions. These values were then normalized to the average fluorescence from the rectangular regions of the same row in the image (we assume a smooth gradation of lighting intensities across the rows). The normalized data is plotted in FIG. 3 to show the concentration response by the sensor. For both Cy5-labeled rabbit anti-chicken IgG and rabbit anti-mouse IgG, the signal from the 25 ng/mL solution was below the detection limit of the system, while the 50 ng/mL signal was significantly above background. For the coverslip examined in the two above-discussed cases, even in the presence of background Cy5-rabbit anti-horse IgG signals, Cy5-labeled rabbit anti-goat IgG could be detected at 25 ng/mL. However, in routine use of a series of coverslips (n>5), 50 ng/mL was the minimal concentration that could be consistently detected with reliability in our device. Concentrations of 100 ng/mL and higher were easily detected for all antigens examined, as shown in FIG. 3.
Obviously, many modifications and variations of the present invention are possible in light of the above teachings. It is therefore to be understood that, within the scope of the appended claims, the invention may be practiced otherwise than as specifically described.
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54 year old man | Rancho Cucamonga, CA, USA | Seeking women 38-52 within 60 miles of La Verne, CA
Very Romantic man that's exciting to be with.
I'm a happy go lucky man that loves life and fun experiences. I'm very romantic, mysterious, entertaining, and humorous. Looking for a Lady that loves adventure and traveling to exotic places. Looking for my Best Friend, Soul Mate, and the Love of my Life. Here is a little about me, I enjoy shopping, walks along the beach, romantic getaways, dancing, concerts, movies, all kinds of music, Broadway plays, antiques, rollerblading, volley ball, amusement parks, sporting events, just about anything fun. I'm retired Air Force and working a second career with the Department Of Defense. I'm successful, well educated, very kind, generous, and I know how to show a lady a real good time. I'm very romantic, sensitive to a woman's needs, very affectionate, a good protector, adventuress, a good planner but I can also be very spontaneous, looking for "real true love' and no mind games. Looking for a lady to have a very "dreamy relationship" with, a lady that would ride a sea doo with me in the harbor, zip line with me in the rain forest in Hawaii, run naked down a deserted beach with me, a lady that will Marry me and spend the rest her life with me going on fantastic adventures. I also enjoy just relaxing at home kicking back and doing nothing or just hanging out. Families are Everything to me they are Priceless in my eyes and Heart. I'm good with children of all ages and treat all of them like they are my own. I'm handy around the house, with cars, the yard, and everything else.
Camping , Coffee and conversation , Cooking , Dining out , Gardening/Landscaping , Hobbies and crafts , Movies/Videos , Museums and art , Music and concerts , Exploring new areas , Nightclubs/Dancing , Performing arts , Playing cards , Playing sports , Religion/Spiritual , Shopping/Antiques , Travel/Sightseeing , Volunteering , Watching sports , Wine tasting
Cycling, Dancing, Hockey, Inline skating, Running, Tennis / Racquet sports, VolleyBall, Weights / Machines
I exercise 3-4 times per week.
I like Birds, Dogs, Fish, Horses
The Beach, PF Changs, Victoria Gardens, Fashion Island, Irvine Spectrum, Scottsdale Fashion Plaza, House of Blues, Yard House, Javier's, Club 3:16
Community College of the Air Force, Maxwell Air Force Base, AL
Antique shoping, Skating with the LA Kings, Singing and Dancing, Shoping, Movies, the Beach, Volley Ball, Nice Resturants, Amusement Parks, Travel, Concerts, Broadway Plays,
Travel, Karokee, Dancing, People Watching, Comedy, Classic Cars
The Note Book
Z{{Cta}} {{#if HasMoreInfo}} ?{{CtaSecondary}} {{/if}}
|What he is looking for
|5'9" (175cms)
|4'0" (121cms) to 5'6" (167cms)
|Body type:
|Athletic and toned
|Blue, Brown, Green, Hazel
|Dark brown
|Auburn / Red, Black, Light brown, Dark brown, Blonde
|No Way
|No Way
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|Political / Govt / Civil Service / Military
|Administrative / Secretarial, Executive / Management, Financial / Accounting / Real Estate, Medical / Dental / Veterinary / Fitness, Political / Govt / Civil Service / Military, Retail / Food services, Sales / Marketing, Self-Employed / Entrepreneur, Student, Education / Teacher / Professor, Technical / Science / Computers / Engineering, Travel / Hospitality / Transportation, Fashion / Model / Beauty
|$75,001 to $100,000
|$25,001 to $35,000, $50,001 to $75,000, $100,001 to $150,000
|Never Married, Widow / Widower, Divorced
|Have kids:
|Yes, and they live away from home (1)
|Yes, and they sometimes live at home, Yes, and they live at home
|Wants kids:
|Definitely, Someday, No, but it's OK if my partner has kids
|White / Caucasian
|White / Caucasian
|Christian / Protestant
|Christian / Catholic, Christian / Protestant, Jewish, Spiritual but not religious, Christian / Other
|Bachelors degree
|High school, Some college
Aeronautical School


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Chicago coach on Hossa: "He seemed OK"
Feb 20, 2013, 12:03 AM EDT
While it's early on in the process, Chicago Blackhawks head coach Joel Quenneville provided a hopeful update regarding Marian Hossa's health following a controversial Jannik Hansen hit from Tuesday's 4-3 shootout win against the Vancouver Canucks.
Here's what he told CSN Chicago's Tracey Myers:
"He seemed OK but we'll know more tomorrow," Quenneville said.
Another bright side: Quenneville said that the 34-year-old forward didn't head to the hospital (at least immediately). Still, Quenneville also admitted that Hossa's concussion history makes the situation a major concern.
As far as whether Hansen delivered a dirty hit, Quenneville avoided passing blame.
"Everybody saw it, everybody can have their own opinion," Quenneville said. "I'll let the people that do that stuff do their job."
(Decide for yourself by watching the hit video here.)
Blackhawks captain Jonathan Toews' take didn't seem too vitriolic, but he did provided a more pointed observation to the Chicago Sun-Time's Mark Lazerus.
"I don't think there's any doubt that there was contact to the head there," Toews said. "We just hope that Hoss is going to be OK."
The concern did put a damper on the team's record-tying start, although said the team "should be proud of the achievement."
- loinstache - Feb 20, 2013 at 1:48 AM
Canucks fan and I really hope Hossa is ok, far too good of a player to see off the ice. Incredibly dumb (malicious or not) play by Hansen and he probably will get punished, but this is completely out of norm for a player who is by no means scummy/dirty.
- mds1990 - Feb 20, 2013 at 7:53 AM
"player who is by no means scummy/dirty"
- loinstache - Feb 20, 2013 at 3:21 PM
LOL you think an interference penalty in the most important game of a player's life makes them dirty? Real hard hittin' journalism, guess every hockey player who's been penalized is one youtube clip away from villainy eh?
- mds1990 - Feb 20, 2013 at 4:09 PM
loinstache laying a guy out who was across the ice not even close to the puck because you can't lose with with some dignity? Yea I'd call that dirty
- canucks18 - Feb 20, 2013 at 2:32 AM
Malicious? Give me a break they both reached up for the puck and he did not hit him hard. It was a unfortunate play yes but it wasn't intent to injur the guy.
- kicksave1980 - Feb 20, 2013 at 3:48 AM
If Hansen was reaching for the puck there, I'd be concerned about his reflexes, because he's a lot slower than a typical NHL player. By the time he got his arm up (and into the back of Hossa's head), Hossa had clearly beaten him to the puck.
Was it an intent to injure? Who knows. I'm not going to go as far as to say that Hansen was trying to injure Hossa, but it didn't look good.
- r4n6er - Feb 20, 2013 at 11:46 AM
People assume that NHLers are so talented that they never miss a puck that they reach for, and missing means it is intentional and dirty. Give me a break, this was full speed action, two guys fighting for the puck.
- rosettast0ned - Feb 20, 2013 at 1:29 PM
Brashear, Bertuzzi, Rome, Torres, Hansen, Burrows… Anyone notice a trend here? Seems to me like Vancouver is notorious for turning out goons, cheap shot artists, and players that have a soft spot for cannibalism (Burrows). And the last time I checked a hand was used to catch things, not an elbow or forearm. We have these amazing things called opposable thumbs as human beings and they work great for catching pucks! And no I am not a hawks fan but I have a lot of respect for Hossa, and it is too bad he has been the victim of two cheap shots from two products of Vancouver.
- indianhead1992 - Feb 20, 2013 at 3:12 AM
that was a dirty hit, last time I checked you dont catch pucks with your forearm/elbow area. you guys were booing duncan keith for his hit on sedin from last year. they are both not hockey plays. at least I can admit when my team plays dirty but canucks fans just seem to always watch a different game from the whole nhl. canucks have pullied keiths hair anf kneed him in the groin, along with some biting in the finals. panzies
- r4n6er - Feb 20, 2013 at 11:49 AM
Funny. You are the only Hawks fan that I could find that admitted Keiths elbow was a cheap-shot. I am sure that you will be 86'd from all Hawks bars now
- kicksave1980 - Feb 20, 2013 at 6:11 PM
I'll admit it was a cheap shot. Most Hawks fans will admit that. The thing that Canucks fans fail to remember is that something led to that. Any team that has guys like Lapierre, Burrows, Kessler and Bieksa on it has absolutely no room to plead innocence when something happens to one of their guys.
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Taking a cue from artists and their studios, Debbie Freeman cultivates her vintage collection with a sharply honed, deeply personal signature style. Independently, each of these tattered home accents, art pieces, and toys is arresting and uncommon. Collectively, they have the mark of a masterful eye—one seeking objects that inspire with raw, bohemian beauty. Enter the kingdom of Duchess D.
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IIrda extended deadline for implementation of new individual product regulations for life insurance industry by three months to Dec 31
SBI Life Insurance plans to relaunch about 10 products by January 1, as part of regulatory compliance to make the policies more customer-friendly.
"We have some approval from the regulator IRDA. I think we would relaunch about 10 products," SBI Life Insurance Managing Director Atanu Sen told PTI.
The Insurance Regulatory and Development Authority (Irda) had extended the deadline for implementation of new individual product regulations for the life insurance sector by three months to December 31, to enable insurers to cope with the system readiness.
The new guidelines are aimed at making insurance policies more customer-friendly.
"All the existing group policies and all the existing individual products not in conformity with the provisions of this regulation shall be withdrawn from August 1, 2013 and January 1, 2014, respectively," Irda had said in a circular.
With regard to group policies, the life insurers have been asked not to enroll these policies after the immediate policy anniversary falling due after July 2013. However, it had said, all group policies at the time of renewal of such policy shall be given an option to switch over to the modified version of the group product, if any, once introduced.
During 6 months ended September, 2013, the new business premium of SBI Life declined by 8.3 per cent to Rs 1,726.77 crore as against Rs 1,884.76 crore in the same period of the previous fiscal.
However, ICICI Prudential, HDFC Standard Life and SBI Life, witnessed a decline in premium collection during the April- Sept period
Govt banks' share in total profit also declined to 48% in FY15 from 52% in FY14
Investors become risk-averse as uncertainties loom over bond market
The RBI deputy governor stresses on strong co-relation between financial inclusion, financial literacy and consumer protection to ensure ...


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