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ODT Online
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Lucy Summers was named this week as the new marketing and
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Mrs Summers (40) was born in Newfoundland and emigrated to
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English husband David and children Will (6) and Charlotte
''I wanted to come back to Dunedin, because it's home and
because of the lifestyle here,'' she said yesterday.
''I'm really enjoying it here. It's so creative. It's a
fantastic place to work.''
The next play at the Fortune is Lungs, which opens on August
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text:
Vegetarian Cassoulet (Cassoulet De Legumes)
Total Time
Prep 1 hr
Cook 3 hrs 30 mins
Though cassoulet is not usually a vegetarian dish, this variation on the classic is very good. For the white beans one can use flageolet, or cannellini or great northern beans. You can also cook the beans a day ahead of when you plan to make the cassoulet. The formation of the crust twice, with the first one mixed into the cassoulet, is well worth the extra 20 minutes it takes. As Julia Child would state, cassoulet is not a fast dish but a delicious one. This recipe was adapted from: "The Vegetarian Bistro", by Marlena Spieler.
Ingredients Nutrition
- 2 cups dried white beans
- 1 bay leaf
- 1 red bell pepper, diced
- 1 carrot, diced
- 12 garlic cloves, peeled
- 1 potato, peeled and diced
- 3 tablespoons olive oil
- 1 teaspoon herbes de provence, crushed
- 1⁄4 teaspoon dried thyme leaves, crushed
- 1 1⁄2 cups dry red wine
- 2 cups diced tomatoes
- 1 1⁄2 cups vegetable stock
- salt and pepper
- 1 cup fresh breadcrumb
- 3 tablespoons minced fresh parsley
- Cook beans according to package directions; can be cooked in a pressure cooker or crockpot. Cook until tender but don't overcook.
- Preheat oven to 325 degrees F.
- Reserve 5 of the garlic cloves.
- In a large heavy saute pan, over medium-high heat, saute the pepper, carrots, potato and remaining garlic cloves, in 2 tablespoons of olive oil until lightly browned, about 5 to 8 minutes.
- In an earthenware (or corningware) casserole, layer the cooked drained beans, sauteed vegetables, diced tomatoes, herbes de Provence, thyme, red wine, and stock, sprinkling each layer with salt and pepper.
- Cover casserole with a tight fitting lid and bake for about one hour.
- Add more liquid if needed to keep the beans from burning.
- Mince the reserved garlic cloves.
- Combine minced garlic with the bread crumbs, minced parsley and 1 tablespoon olive oil.
- Increase oven heat to 400 degrees F.
- Remove casserole from the oven, remove lid and spread half of the crumb mixture over the top.
- Return uncovered casserole to the oven and bake about 15 minutes, or until a golden crust has formed.
- Break the crust and stir it into the cassoulet.
- Repeat by spreading the remaining half of the crumb mixture over the top of the cassoulet.
- Return uncovered dish to the oven and bake until the second and final crust has formed.
- Then serve.
Most Helpful
I'm not giving this a star rating because I changed several things so I don't think that would be fair. (I used canned beans and canned tomatoes and subbed more broth for the wine.) However, I wanted to make a couple comments.

I thought there was not nearly enough crumb topping for the amount of beans. When I saw that, I doubled the amount, but it still didn't really make a crust that covered the top. Maybe you need a smaller, deeper casserole dish than I used.

Also, I though it was too bland, Used the full amount of spices and even added an onion to the vegetables, but still it lacked something. Maybe it was the wine, but maybe not.
A tasty, wintry bean dish - and lethally garlicky! The method is a bit fiddly, in terms of preparing things separately then combining them, but the end result is worth it, and it was simpler than it looked in the recipe. Right up until serving time, I was worried that the cassoulet was too liquid, but stirring in the first crust seems to have absorbed all the excess wine-flavoured juices, and in the end I wondered where all the liquid had disappeared to! The crust topping was so delicious I would be tempted to make double next time. I made it by whizzing bread, garlic and parsley in the food processor, and it was gorgeous (useful for other dishes, as well). Reviewed for Pick A Chef, Fall 2007.

subset: commoncrawl


text:
Java Authentication and Authorization Service (JAAS)
The Java Authentication and Authorization Service (JAAS) is a set of application program interfaces (APIs) that can determine the identity of a user or computer attempting to run Java code and ensure that the entity has the right to execute the functions requested. In this context, authentication is the process of determining whether or not an entity is who or what it declares itself to be; authorization is the process of giving an entity permission to do, use, or obtain something. Logically, authentication precedes authorization.
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text:
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Yesterday's Halloween episode of Ellen was Hallow-rious! First off, let's talk about her costume. She came out as Snooki's POOF! Brilliant! Her entire costume was the hair-do, with a small cabbage patch doll at her feet as the body. Amazing. Ellen's Halloween episodes are like a tradition for me now. Some of my favorite things she does on her show are the pranks, so when she does pranks and scares people left right and center on her Halloween episode, I am on the floor laughing my head off.
In yesterday's episode, she sent one of her writers (Amy), who is notorious for getting scare easily, to a haunted house at Universal Studios, and of course filmed the entire thing. It's hilarious. If I was in Ellen's shoes, I would have done the exact same thing.


text:
Type: Posts; User: tishyrae; Keyword(s):
I remember you from GOSH I can't remember the site...the big one from 2009ish!! LOL Anyways...hello again!
Hi! I have some friends in MN and now another! HI!!!!!!!!!!!!
I love this one. I always use a ton of photos on a page! PERFECT! Thanks!
I just tried really downgrading the image quality and it still gave me this error.
f52028936.jpg (exceeds filesize limit)
OK. I was trying to give this uploading deal a try. (It has been a while)
I made sure my file was 300x300 (even tried 250x250).
Also checked the size-112KB.
Any suggestions?
Hi Joyce. I am a Wisconsinite too!! Where abouts are you from? I am near Appleton!
My name is Trisha and I live in Wisconsin, where we are TRYING to have Spring arrive. Mother Nature is a bit confused here lately! :) My husband, Andy, and I will be married for 15 years in...
Sent my app in! :)


text:
WATCH: Southern Mississippi DB Kalan Reed Shows Off His 41.5-Inch Vertical Jump as His Teammates Go Nuts
Kalan Reed is just 5-foot-11, but we found out today that the defensive back from Southern Mississippi can jump about half his height in the air. Kalan Reed vertical: 41.5 (!) pic.twitter.com/H7gJnlo4e4 — Jason Munz (@munzly) March 24, 2016 Today is the Golden Eagles' Pro Day, and Reed showed off his incredible leaping ability by hitting 41.5 inches on the Standing Vertical...
Southern Miss snaps 7-game skid, beats North Texas 81-70
HATTIESBURG, Miss. (AP) Kourtlin Jackson made his final home game count, hitting five 3-pointers and pouring in 27 points to help Southern Mississippi snap a seven-game losing streak by beating North Texas, 81-70 Saturday night. The graduate transfer from Iowa State entered the game after scoring a career-high 31 points against Rice. Jackson leads Southern Miss in scoring average...
Via Associated Press
February 27, 2016 | Discuss
Jackson leads Southern Miss past Texas-San Antonio, 86-70
HATTIESBURG, Miss. (AP) Kourtlin Jackson scored 20 points and Southern Mississippi cruised past Texas-San Antonio, 86-70 on Saturday night. The Golden Eagles shot an efficient 22 of 42 from the field (52.4 percent), including 14 of 28 from beyond the arc. Southern Miss also collected 17 assists on its 22 made baskets. Southern Miss led by six at intermission and extended its...
Via Associated Press
January 31, 2016 | Discuss
WATCH: Southern Miss WR make absurd TD catch
In what may be the coolest catch you will see from Saturday, Southern Mississippi wide receiver Mike Thomas caught a seven-yard touchdown pass Odell Beckham style. Thomas reeled the pass in with one hand, used his body to control the football and maintained possession all the way to the ground. The catch gave his team a 21-3 lead over Louisiana Tech and is as good as any you'll...
Via Larry Brown Sports
November 28, 2015 | Discuss
Blackout? Nah ... Southern Miss, UTEP win Halloween uni contest
A Halloween "blackout?" That's no big deal; it's become a pretty regular thing over the last couple seasons in college football, and there are numerous schools -- including Texas A&M, Utah, Washington and Kentucky -- planning to roll out alternate black threads Saturday night.But Southern Miss and UTEP are taking things to another level with a perfectly Halloween-themed uniform...
Via FOX Sports
October 31, 2015 | Discuss
Mullens leads Southern Mississippi over Austin Peay
HATTIESBURG, Miss. (AP) -- Nick Mullens threw three touchdown passes and ran for another score to help Southern Mississippi beat Austin Peay 52-6 on Saturday. Mullens was 22 of 40 for 275 yards passing. Ito Smith finished with 135 yards rushing on 10 carries and a touchdown for Southern Mississippi (1-1). A blocked punt set up Mullens' 2-yard touchdown run in the first quarter. Mullens...
Via Fox Sports Southwest
September 12, 2015 | Discuss
Knee injury jeopardizes George Payne's season
Southern Mississippi Golden Eagles rising junior running back George Payne suffered a non-contact knee injury and could miss most if not all of the upcoming season. Head coach Todd Monken broke the news to Jason Munz of the Hattiesburg American. Payne rushed for 271 yards and six touchdowns on 86 carries last season, and also caught eight passes for 109 yards and a touchdown. Were...
Via Campus Sports Net
June 17, 2015 | Discuss
Southern Miss WRs Arrested for Armed Robbery and more
A pair of Southern Mississippi football players could be in some very hot water. Per the Hattiesburg American, wide receivers James Cox and Kyle Foster were arrested yesterday morning on felony charges of armed robbery, kidnapping and "burglary of an occupied dwelling." Police offered no further details, but university spokesman Jack Duggan offered a brief statement. "We have...
Via Campus Sports Net
May 21, 2015 | Discuss
Southern Miss beats Missouri 71-63 in NIT
COLUMBIA, Mo. (AP) Neil Watson scored 18 points and Aaron Brown added 16 to help Southern Mississippi beat Missouri 71-63 in the second round of the National Invitation Tournament on Sunday.
Via FOXSports.com
March 23, 2014 | Discuss
Southern Miss tops Toledo 66-59 in NIT
HATTIESBURG, Miss. (AP) Michael Craig scored 16 points, Neil Watson added 12 and Southern Mississippi rallied to beat Toledo 66-59 on Wednesday night in the first round of the National Invitation Tournament.
Via FOXSports.com
March 19, 2014 | Discuss
Southern Miss tied for first place in C-USA
Southern Miss Golden Eagles opened the game against the Marshall Thundering Herd with a 3-2 lead, that would be their only lead until senior guard Neil Watson's three-pointer with 2 seconds remaining, to take a 58-57 lead. After a failed possession and an intentional foul, the Golden Eagles added two more free throws to end the game. Southern Miss defeated the Thundering Herd in...
Via isportsweb.com
February 10, 2014 | Discuss
Southern Miss overwhelms St Catharine 96-60
Daveon Boardingham scored 23 points on 9-of-11 shooting and Matt Bingaya added 17 as Southern Mississippi rolled past Division-III St. Catherine 96-60 Saturday night. The Golden Eagles (9-1) held a 20-18 lead midway through the first half, but surged ahead with an 11-0 run capped by a layup and dunk from Boardingham. Two more Boardingham dunks put Southern Miss up 43-29 at halftime...
Via Associated Press
December 14, 2013 | Discuss
Southern Miss puts it all together against UAB
The Southern Miss Golden Eagles finally put it all together in the final game of head coach Todd Monken's first year. The Golden Eagles erupted with 28 points in the 3rd quarter against the UAB Blazers, for a final score of 62-27. The Golden Eagles snapped the nation's longest losing streak of 23. The Eagles dominated in all facets of the game Saturday versus the Blazers. Accumulating...
Via isportsweb.com
December 02, 2013 | Discuss
Southern Miss remain optimistic in season finale
The Southern Miss Golden Eagles will be traveling to Legion Field, in Birmingham to face the Alabama-Birmingham. This game versus UAB is the final opportunity this season for the Golden Eagles to record that elusive first victory. The Eagles head into the game 0-11, while the UAB Blazers are 2-9 this season with one victory within the conference. Southern Miss Golden Eagles quarterback...
Via isportsweb.com
November 29, 2013 | Discuss
Southern Miss shows fight in loss to Middle Tennessee
The Southern Miss Golden Eagles (0-11) season, faced the Middle Tennessee State Blue Raiders (7-4), the Blue Raiders outgained the Golden Eagles 418 to 317 in the total yards category. The Blue Raiders outscored the Eagles, 28-7 in the first half. Middle Tennessee finished the game with 42 points to win, 42-21. The Golden Eagles offense started the game off well, the offense moved...
Via isportsweb.com
November 24, 2013 | Discuss
Southern Miss routs William Carey 99-54
Chip Armelin scored 16 points as Southern Miss had six players in double figures, scored 61 first-half points and routed William Carey 99-54 on Saturday and remained undefeated. Neil Watson made 4 of 5 from beyond the 3-point arc and scored 14 points for the Golden Eagles (6-0). Matt Bingaya also had 14 points, Michael Craig had 12, Daveon Boardingham had 11 and Aaron Brown had...
Via Associated Press
November 24, 2013 | Discuss
Southern Miss holds off Houston Baptist 67-62
Daveon Boardingham and Neil Watson scored 15 points each to lead Southern Miss to a 67-62 win over Houston Baptist in the CBE Hall of Fame Classic on Saturday. The Golden Eagles (5-0) trailed by as much as 10 points in the first half but took the lead midway through the second half on a Jerrold Brooks 3-pointer and didn't let it slip away. Michael Craig and Deonte Houston were able...
Via Associated Press
November 23, 2013 | Discuss
Golden Eagles looking to finish strong
December 24, 2011 was the last time the Golden Eagles won a football game, 24-17. Southern Miss ended the season ranked 21st in BCS. Since that game, Larry Fedora has left the program and a replacement for arguably the second best quarterback statistically, Austin Davis. Last season was a historic one for the Southern Miss program, a streak of 18 winning seasons in a row was snapped...
Via isportsweb.com
November 22, 2013 | Discuss
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text:
City leaders say unity necessary in Newburgh
There are 10 comments on the Your News Now story from Mar 10, 2012, titled City leaders say unity necessary in Newburgh. In it, Your News Now reports that:Regina Lewis is one of several Newburgh residents who angrily lash out at city officials.
Join the discussion below, or Read more at Your News Now.
#1 Mar 11, 2012
I watched the new conference. By saying that this so called angry resident lashed out is putting it mildly. She spewed racist comments that is all she did and so did the other black woman. They accomplished nothing but to show thier true racist personality and it was disgusting to watch. No matter the out come of this investigation these people will call it a murder and white cops killing a black man. Pull the cops off the streets and let these animals police themselves. I bet in about 48 hours these people would be begging for the police to respond after the thugs and gangs start ripping them off and hurting thier families. Racist people live in City of Newburgh and they are mostly black. Live in the garbage that you people created. You always do anyway.
#2 Mar 11, 2012
if you wanna improve newburgh-bulldoze it.
#3 Mar 11, 2012
I agree. Bulldoze that garbage city but only certain portions of it. Some of the city is nice. Only the area around liberty and landers street. Nothing by shit lives in that area. They created the shit hole they live in so leave it to them to police themselves. Only problem is they will move somewhere else and create another shit hole to live in. People like them always expect something for nothing and they create the garbage that they live. They do nothing to pick themselves up to make a better life for themselves. I bet if you go there and walk into any of those apt. that the animals live in everyone will be disgusting and they think its ok to live like that. So let them. Put up some barbed wire and let them fend for themselves. Interesting isnt it??/
#4 Mar 11, 2012
VIA-section 8.
#5 Mar 12, 2012
This is what happens when NY state has the larest black population in the USA. Most are only 1 or 2 generations removed from being from some Southern state- send em back.
#6 Mar 12, 2012
Why wasnt that lady arrested for trying to instigate a riot at the meeting? She repeatedly threw out "white" in her rants. And threatened the Police Chief saying she'd take him out herself.
#7 Mar 13, 2012
Well,if anything, the comments so far posted on this site certainly reflect racist opinions. Newburgh certainly does not need that, from either the blacks or whites in the city. Racism is a disease of the mind and soul: Racist, heal thyself.
Now, to the matter at hand. All of this ranting and raving over this shooting will do nothing to heal the city or unite it. Let the investigation run its course. My question is this: Where is the same outrage, the same demonstrations in the streets, the same call for meetings from the clergy, when black on black violence, children killing children runs rampant? Where are these same "united" voices in helping the police in confronting the drug plague that infects Newburgh a plague that has such a negative influence on the improvement and stability of our city neighborhoods? This kid was no innocent and to make him into a martyr, an innocent victim is not helpful at all. If he had used that knife to kill a cop, where would the ranting crowds be? We have to move beyond all this racial crap and recognize that we are all in this together, united to stop the drug dealing, the violence and killing and to make our streets an neighbourhoods safe and attractive, not trash strewn embarrassments.
#8 Dec 12, 2013
If this isn't a smoke screen and reverse psychology my name isn't Regina Lewis and for you racists to create a web site to promote your ignorance and lack of insight is typical of lack of knowledge.
#9 Dec 26, 2013
Since: Mar 14
Location hidden
#10 Mar 23, 2014
Fark newburgh, you created that cess pool now live with the consequences.
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I'd like to create some aesthetic structures in Minecraft and am particularly interested in making some floating spheres. Are there templates, tips, tricks, or other methods anyone has found on the best ways to construct them? I'm thinking spheres with a diameter of 10 blocks for starters.
merged by badp♦ Apr 21 '11 at 7:18
This question was merged with What tools do you use to help you build structures? because it is an exact duplicate of that question.


text:
300 W Pershing Blvd Street View
US Post Office 300 W Pershing Blvd, North Little Rock Arkansas 72114 (AR)
US Post Office in North Little Rock
U.S. Postal Service
300 W Pershing Blvd
North Little Rock Arkansas 72114 AR
4.5/5 Based on 1 Review(s)
US Post Office Reviews: Take Our Quick 5 Question Survey And Leave A Comment
About US Post Office
US Post Office is primarily involved in U.S. Postal Service. US Post Office operates in North Little Rock Arkansas. This establishment is involved in U.S. Postal Service as well as other possible related aspects and functions of U.S. Postal Service. In Arkansas US Post Office maintains its local business operations and might carry out other local business operations outside of Arkansas in additional functions related to U.S. Postal Service.
300 W Pershing Blvd Street View
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This section is for posting general comments and disussion about this business. If you would like to leave a full review, Click Here to write a full review.


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81 Reviews
WOW! I really love these cinnamon oranges. It's important to powder the cinnamon very fine in a mixer for this recipe. We(dad and me) had this at lunch this afternoon for dessert. I used Navel oranges, came out very sweet and "addictive" to be honest. The combo of cinnamon with these oranges went very well. Easy to fix, eggless, healthy and tasty - it's got everything you need and look for in a recipe - EXCELLENT DISCOVERY, Friedel;)!
It doesn't get much simpler than this and the cinnamon-orange combo is wonderful, with the cinnamon bringing out the orange's natural sweetness. Pays to have a light touch with the cinnamon.
I tried this with Valencia oranges, which are very sweet & juicy. It wasn't bad, but personally I just like the taste of oranges without the cinnamon.
What a beautiful combo! The flavors complimented eachother perfectly I would have never thought of this. Considering the health benefits of cinnamon this is a great way to get it in your diet!
Beautiful, next time I would serve them with mint springs for the look and taste. I just used oranges and a fresh ground cinnamon. Remember to wash the oranges before slicing. We had this as a late night snack along with other fruit. I will make this again for breakfast too.
I saw this awhile back and was like no way would I eat that. Was about to eat an orange and remembered. So I tried it and was really suprised! It tastes great!!!!
Love this, been making it for years!!
I can't believe how good this is! I never would have thought about sprinkling cinnamon on oranges and I will be doing that a lot now. Keep it simple, right? Made for February Sun and Spice. Thanks Sackville! :)
What a treat. I made this to go along with some Turkish kabobs and it was a refreshing taste. DD, however wasn't a fan (hence the 4 star rating), but DS and I loved it. I will continue to add this to our meals when making certain foods that could use the palate cleanser. Thank youf or sharing with us .


text:
Life Notes From The Prayer Warrior
posted by April 17 at 10:16 AMon
The Prayer Warrior, in D.C. today on a lobbying trip, pauses to reflect on the Virginia Tech tragedy but then pushes forward with his efforts to prevent hate crimes legislation…
Dear Prayer Warriors,
Due to the terrible tragedy at Virginia Tech yesterday, the decision was made to cancel the press conference regarding the hate crime bill. As God is so gracious and sovereign in all His ways, He has allowed us to give individual interviews to various TV and Radio shows here in the DC area. Please continue to pray for the families of those who lost their lives in the shootings at Virginia Tech and for the family of the young man who did the shooting. God will make something good out of this.
At 11:00 AM PST, 2:00 PM EST, I will be addressing the Conservative Group of Congress regarding the hate crime bill and all that it will do to negatively impact churches, if it is passed. Please pray that I will be Gods voice to speak the truth and that they will hear the truth and act accordingly.
Thank you again for your prayers, they are fervent and righteous!
Pastor Hutch


text:
Example application to demonstrate using Guice for dependency injection when using Apache Droids
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Terms of Participation.
Copyright © 2014, Oracle Corporation and/or its affiliates. All rights reserved.
Oracle and Java are registered trademarks of Oracle and/or its affiliates.
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text:
Top Definition
A dookie or a dump taken while playing sodoku on the toilet.
Guy 1: Man, I just took a huge Sodoki in your bathroom. You ran out of toilet paper so I wiped with your Sodoku book.
Guy 2: I hate you.
Guy 2: I hate you.
от M00ps 17 декември 2010
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Cognitive Therapy and Research
, Volume 17, Issue 1, pp 83-98
First online:
The specificity of attributional style and expectations to positive and negative affectivity, depression, and anxiety
- Anthony H. AhrensAffiliated withDepartment of Psychology, The American University
- , David A. F. HaagaAffiliated withDepartment of Psychology, The American University
Rent the article at a discountRent now
* Final gross prices may vary according to local VAT.Get Access
Ninety-four undergraduate subjects completed measures of trait positive and negative affectivity, anxiety, depression, optimism, hopelessness, and attributional style. After writing about negative events or hearing a tape describing a positive academic experience, they completed measures of state positive and negative affect and of self-efficacy expectancies. Positive affectivity was associated with attributional style for positive, but not negative, events. Negative affectivity was associated with attributional style for negative, but not positive, events. Negative event attributional style was specifically associated with anxiety; expectancies and positive event attributional style with depression. Attributional style predicted state positive affect following completion of negative essays, but not negative affect, nor either affect following the positive tape. Effects of attributional style on affect were partially independent of expectations. Results are discussed in terms of the importance of distinguishing between processes related to positive and negative affect in order to distinguish anxiety from depression.
Key wordsattributional style expectations depression anxiety affect cognitive specificity
Reference tools
- Title
- The specificity of attributional style and expectations to positive and negative affectivity, depression, and anxiety
- Journal
Cognitive Therapy and Research
Volume 17, Issue 1 , pp 83-98
- Cover Date
- 1993-02
- Print ISSN
- 0147-5916
- Online ISSN
- 1573-2819
- Publisher
- Kluwer Academic Publishers-Plenum Publishers
- Additional Links
- Topics
- Keywords
- attributional style
- expectations
- depression
- anxiety
- affect
- cognitive specificity
- Industry Sectors
- Authors
- Anthony H. Ahrens (1)
- David A. F. Haaga (1)
- Author Affiliations
- 1. Department of Psychology, The American University, 4400 Massachusetts Avenue, NW, 20016-8062, Washington, DC, USA


text: 08.01.2014 – Day two of menswear presentations in London heralded a celebratory debut. Fantastic Man's fabulous former assistant (not to mention first ever menswear designer to win "Collection of the Year" at the London College of Fashion's BA Graduate Show) Mr. SEBASTIAAN PIETER GROENEN presented his first post-college collection, a wonderful combination of tailoring and knitwear. The label has been baptised "PIETER", and the first presentation stems from its founder's dislike of the telephone app "Grindr", another example of a medium whereby real-life interaction has been traded for yet more staring at smartphones. With next winter's collection, PIETER offers a seductive sartorial alternative via this knitted jumper screaming 'FUN NOW'. Other designs, featuring funny half-length sleeves and cropped waists (see above, right) are sure to facilitate all sorts of touching and stroking.


text:
Top Definition
A phrased used to irritate corporate customers in an Indian outsourced technical support environment, suggesting lunacy, moronic tendencies and possible idiocy. A worded used in a similar language not unlike Pooty Tag but not completely definable in the English language.
Customer, "my internet connection is not working. Is there anything wrong with your connection today?". Indian Tech: "fisedup sir, fisedup!.
by KennyD October 28, 2004
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I like to engage in "Technology Serving Community" via free software.
Consultant, Data engineer, Ubuntu member, Wikipedia contributor since 2002, ElectionAudits developer, Boulder Community Network co-founder, Internet2 consultant for IDtrust, author of pgpstat, Android enthusiast, ex-Bell Labs web/security/standards maven....
See my web site for way more: http://neal.mcburnett.org/
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text:
|Publication number
|Publication type
|Application number
|Publication date
|Mar 11, 1997
|Filing date
|Mar 31, 1995
|Priority date
|Mar 31, 1995
|Fee status
|Also published as
|Publication number
|John W. Deutsch
|Original Assignee
|Lee Laboratories, Inc.
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The invention relates generally to the production and purification of Clostridium difficile Toxin A. More particularly, the invention relates to purified Toxin A and its use in preparing antibodies for use in diagnosis of pseudomembranous colitis.
Clostridium difficile produces an assortment of gastrointestinal diseases in humans and animals ranging from mild diarrhea to life threatening pseudomembranous colitis. It is widely accepted that C. difficile causes pseudomembranous colitis in humans as a result of the elimination of the normal flora of the colon by antibiotic usage and subsequent growth of this toxin producing bacterium. The disease usually occurs in hospitalized patients where it causes a massive diarrhea with extensive inflammation of the colon. Mortality rates as high as 44% have been reported. Treatment of the disease is possible but relies on a proper diagnosis which may be accomplished by establishing the presence of the causation organism and demonstrating the characteristic lesions in the colon.
C. difficile produces two toxins, designated Toxin A and Toxin B, that are cytotoxic for tissue cultured mammalian cells. In addition to its cytotoxicity, Toxin A also possesses enterotoxin activity. Toxin A causes an accumulation of fluid in the intestine loops and causes extensive damage to the gut mucosa. Although both toxins are typically produced during disease, the principle laboratory diagnostic methods for detection of C. difficile have been directed towards detection of Toxin A. For example, a commercial latex test for the presence of Toxin A was marketed by Marion Scientific, a division of Marion Laboratories, Inc. of Kansas City, Mo. This commercial test was, unfortunately, found by subsequent researchers to be non-specific for Toxin A. See Lyerly et al., J. Clin. Micro, 23: 622-623 (1986).
One method for detecting pathogenic C. difficile involves the culture of human feces, which requires specialized facilities for long periods of incubation and which has the disadvantage of interference by non-pathogenic C. difficile strains. Another method for detecting Toxin B, but which does not work well for detecting Toxin A, is cytotoxicity assays using tissue culture cells. Because Toxin A is a much less potent cytotoxin it is more difficult to detect in tissue culture assays.
Other methods developed for Toxin A detection have been based on the use of specific antibodies to Toxin A. These methods include the enzyme-linked immunosorbent assay (ELISA) as taught by Lyerly et al., J. Clin. Micro., 17: 72-78 (1983) and Laughton et al., J. Infect. Dis., 149: 781-788 (1984). Lyerly et al. reported detection of Toxin A at 1 ng (5 ng/ml) quantities while Laughton et al. reported detection at 0.1 ng (1.0 ng/ml) levels. An ELISA assay using a monoclonal antibody to Toxin A was reported by Lyerly et al., J. Clin. Micro., 21: 12-14 (1985), which could detect 4 ng (0.02 μg/ml) of Toxin A. This detection level is usually adequate to detect the toxin in stool samples from patients with C. difficile-related diarrhea. Another antibody dependent test is the Latex Agglutination Test (LAT) wherein antibody is immobilized on latex beads and agglutination of said beads by soluble Toxin A is visualized.
U.S. Pat. No. 4,530,833 to Wilkins et al. teaches a method of purifying Toxin A from cell culture supernatant which comprises an ion-exchange chromatography step followed by isoelectric precipitation. The patent teaches that purified Toxin A is achieved by adjusting the pH and molarity of a solution of impure Toxin A to, preferably, 5.5 and 0.01, respectively. One disadvantage of using isoelectric precipitation for protein purification is that other proteins with similar properties as the desired protein aggregate and coprecipitate with the desired protein to form the isoelectric precipitate. Thus, the precipitate must be washed by resuspension and recentrifugation. This results in loss of the desired protein. In addition, the process prior to and including the precipitation step must be very carefully controlled to control the precipitation step. If the fractions from the ion-exchange column are not carefully and consistently pooled, more or less proteins will co-precipitate with the Toxin A.
Another disadvantage of isoelectric precipitation with respect to purification of Toxin A is that denaturation of Toxin A may result if high speeds are used to pellet the precipitated protein. Thus, the protein will not be active in immunoassays or when used as an immunogen for antibody production. Isoelectric precipitation of proteins may also cause changes in the tertiary structure of the protein. In particular, Toxin A is an extremely hydrophobic protein and its exposure to a low ionic strength buffer for several wash steps can result in its partial denaturation.
Another method of Toxin A purification is taught in U.S. Pat. No. 5,098,826 to Wilkins et al. This method comprises contacting an impure solution of Toxin A with an immobilized reagent containing one or more of the terminal non-reducing structures characteristic of any of the human antigens X, Y, or I. These structures are known to be polysaccharides, and they can be immobilized by binding them to an insoluble matrix, e.g., silica gel, agarose, latex beads, or the like. The Toxin A binds to the immobilized reagent and is then eluted from the reagent in a more pure form. This method requires one of the antigens X, Y, or I, which are relatively expensive to obtain, and thus is an impractical method for purification of useful quantities of Toxin A.
Production of polyclonal antibodies to Toxin A is known in the art. For example, Ehrich et al., Inf. Immun. 28: 1041-43 (1980) teaches a procedure of culturing C. difficile and inoculating rabbits to produce polyclonal antibodies to an impure C. difficile preparation. Such polyclonal antibodies are not mono-specific for toxin because of the large number of proteins present in culture supernatant. Thus, the performance of toxin specific polyclonal based EIA have been disappointing because of the relatively low toxin specific titer and the high level of nonspecific reactivity of the polyclonal antisera.
Therefore there is a need for a method for the purification of Toxin A as well as a rapid and accurate assay for diagnosis and detection of C. difficile Toxin A.
It can be seen by the above that a method for purifying Toxin A which results in a consistently pure and non-denatured protein and which can be used to purify large quantities of protein is needed. Antibodies produced by such a highly purified, active protein will themselves be of higher activity.
The present invention is drawn to a method for the purification of Toxin A from Clostridium difficile. Substantially purified Toxin A is provided which is useful for the production of antibodies to Toxin A. Thus, the invention is also drawn to antibodies, particularly polyclonal antibodies with a high sensitivity for Toxin A. Kits for the diagnosis and detection of C. difficile Toxin A are also disclosed.
Additional objects, advantages and novel features of the invention will be set forth in part in the description which follows, and in part will become apparent to those skilled in the art upon examination of the following or may be learned by practice of the invention. The objects and advantages of the invention may be realized and obtained by means of the instrumentalities and combinations particularly pointed out in the appended claims.
Compositions and methods for the detection of pseudomembranous colitis caused by C. difficile are provided. Specifically, a new method for purification and production of Toxin A is disclosed. Further, polyclonal antibodies are provided which are specific for Toxin A of C. difficile. The antibodies find use in assays for the detection and diagnosis of C. difficile.
The term "substantially pure" or "purified Toxin A" as used herein indicates that the Toxin A preparation is substantially free of contaminating substances when examined by resolving techniques known in the art.
The term "partially pure" or "partially purified Toxin A" refers to Toxin A preparations having some, but not all, contaminants removed.
The term "active Toxin A" refers to Toxin A which gives a positive result on Toxin A assays currently used in the field including ELISA's and the Latex Agglutination Test which uses purified rabbit anti-C. difficile antibody immobilized onto Latex beads.
The term "denatured" refers to protein or more specifically, Toxin A, which has lost its native or natural structure or properties.
The term "mono-specific antibody for Toxin A" refers to an antibody which does not have any determinant sites for antigens of C. difficile other than Toxin A.
The present invention comprises a highly reproducible method for purifying Toxin A having high activity. The reproducibility of the present process is a very desirable feature of protein purification processes in general and especially for the purification of proteins to be used as antigens. The more pure a preparation of antigen is, the more specific the antibodies produced will be towards that antigen. In addition, reproducibility is of high priority for processes involved in the preparation of products submitted for FDA approval.
Toxin A is produced by C. difficile cells which may be cultured as is known in the art. The following references describe the culturing of C. difficile and are incorporated herein by reference: Sterne and Wentzel, J. Immun. 65: 175-183 (1950); Ehrich et al., Infect. Immun. 28: 1041-43 (1980); Sullivan et al., Infect. Immun. 35: 1032-40. The present invention comprises reversibly binding the crude or partially purified Toxin A to immobilized polyclonal antibody. Polyclonal antibodies are oxidized and are then bound or immobilized to a hydrazide group containing resin, such as hydrazide activated agarose gel. The resulting immobilized reagent is highly selective for Toxin A and its use as a purification reagent results in a highly purified Toxin A. The purification process is much more reproducible than processes using precipitation because the immuno-affinity step is very selective for Toxin A and the protein product is of consistent composition, namely highly pure and highly active Toxin A. Also, multiple potentially denattiring washing and resuspension steps are not necessary. In addition, the process of selectively binding and eluting Toxin A to the resin does not permanently denature or inactivate the Toxin A. Therefore, more active mono-specific, polyclonal antibodies may be produced by using the purified, highly active, Toxin A of the present invention.
The process of the present invention is more economical than prior art immuno-affinity processes because the immuno-affinity resin is stable and reusable for many uses. The resin materials are fairly inexpensive and the immuno-affinity resin has a high binding capacity because it presents to the Toxin A containing solution two Toxin A binding sites per bound antibody molecule.
The purification method of the present invention involves an immuno-affinity purification. An advantage of the immuno-affinity purification is that the protein is not subjected to centrifugation/rehydration steps during the procedure. The process is thus controlled and very reproducible.
Culture filtrate of Clostridium difficile can be applied directly to an immuno-affinity column as described herein. Alternatively, additional steps for purification of the Toxin A prior to application to the immuno-affinity column may be performed. Such steps include high speed centrifugation followed by filtration, precipitation such as ammonium sulfate precipitation, and chromatography such as ion exchange, affinity, or size exclusion chromatography.
The immuno-affinity columns of the present invention utilize hydrazide activated agarose. That is, the antibodies are immobilized onto hydrazide activated agarose through oligosaccharides. Such methods are known in the art. See, e.g., O'Shannessy and Wilchek, Anal. Biochem. 191: 1-8 (1990); Domen et al., J. Chromat., 510: 293-302 (1990); Palmer et al., J. Biol. Chem. 238: 2393 (1963); Schneider et al., J. Bio. Chem. 257: 10766 (1982); all of which references are incorporated by reference herein.
The use of hydrazide group containing resins for biomolecule purification is a known technique. See O'Shannessy and Wilchek supra. Commercial preparations are available wherein agarose beads are provided with spacer arms having terminal hydrazide groups. These hydrazide groups will react with aldehyde groups such as are produced by the oxidation of carbohydrate groups.
Carbohydrate groups are located primarily in the Fc region on antibody molecules. The antigen binding sites are, on the other hand, located on the Fab region of the antibody. Thus, antibodies bound to a hydrazide group containing resin are oriented so that both of their antigen binding sites are presented to antigens such that these immuno-affinity resins may function as better affinity materials. In addition, the long spacer arms of such hydrazide-agarose gels appear to allow for higher binding capacity for antigen, as compared to other types of antibody-resins.
When antibodies elicited to acetate precipitated Toxin A are compared with antibodies elicited to immuno-affinity purified Toxin A, using the purified antibodies immobilized onto latex in the LAT, an average endpoint of approximately 25-35 ng/ml is found with the antibodies from acetate precipitated Toxin A while an average endpoint in the range of about 12.5-25 ng/ml is found for the antibodies from the immuno-affinity purified Toxin A.
The LAT is a more tightly controlled assay than the ELISA and gives more consistent results. The LAT can be carried out as described in U.S. Pat. Nos. 4,879,218 and 5,098,826 to Wilkins et al., and also as described in Lyerly et al., J. Clin. Micro., 21: 12-14 (1985), the disclosures of which are incorporated herein by reference.
Purified Toxin A is useful in studying diseases caused by C. difficile and in designing detection tests and treatment plans. In particular, purified Toxin A is necessary for preparing antibodies to Toxin A, which antibodies are used in ELISAs, LATs, and other detection tests such as those described in the above cited references for detecting the presence of Toxin A in biological samples. In the development of any immunoassay, the first consideration is the quality of the antiserum that will be used in the assay. If the antiserum is not sensitive or has cross reactivities with other undesired or related proteins, the assay will not have the desired sensitivity. Thirty (30) or more proteins expressed by C. difficile are present in the culture supernatant. Any of these proteins which are present in the final Toxin A product will diminish the efficiency of the product used as an antigen. Therefore, an improved method for purifying large amounts of Toxin A is needed which provides highly pure, nondenatured, Toxin A. An economical production method is needed which is highly specific for Toxin A and which is highly reproducible.
Mono-specific antibodies can be produced in accordance to the present invention by immunizing appropriate animals with Toxin A purified as disclosed herein. It is also anticipated that monoclonal antibodies can be prepared using the Toxin A of the present invention. Mono-specific polyclonal antibodies show high activity in ELISA tests and in the LAT. Antibodies raised against Toxin A of the present invention were up to twice as active in the LAT as antibodies raised against acetate precipitated Toxin A.
In a second embodiment of the invention, polyclonal antibodies can be produced with the purified, highly active Toxin A through methods known in the art. For example, the methods taught by Ehrich et al., Infect. Immun., 28: 1041-43 (1980) and in E. Harlow & D. Lane, Antibodies-A Laboratory Manual, Cold Spring Harbor Laboratory (1988), may be used. The initial step is the preparation of a toxoid and immunization of rabbits with the toxoid. Toxin A-adjuvant is then prepared and used for immunization. The collected sera is then purified over protein A.
The above described production and purification of C. difficile Toxin A, and its use in preparing antibodies, are illustrated by the following example, which is not intended to be limiting.
Protein Purification
C. difficile was cultured in 0.85% sodium chloride in dialysis bags suspended in brain-heart infusion (BHI) broth media as generally described by Sterne et al., J. Immun., 65: 175-183 (1950) and Lyerly et al., J. Clin. Micro. 17: 72-78 (1983) (incorporated herein). Five dialysis bags holding 300-500 ml each of 0.85% NaCl were placed in 15L of BHI broth media in a 20L Bellco Spinner flask. Each tube was inoculated with 3 ml of a 18-24 hr culture diluted 1:10 with 0.85% chloride. The cells were cultured anaerobically for about four days at 39� C. �1� C. After sedimenting the cells by centrifugation at 10,000 RCF for 30 minutes at 2�-8� C., the supernatant was decanted off and clarified by filtration through a 0.45 micron filter.
The culture supernatant was concentrated and washed using an apparatus such as the Minitan Ultrafiltration System with PTHK 100,000 MW packet type ultrafiltration membranes. The supernatant was washed with 50 mM Tris buffer, pH 7.5 containing 0.02% sodium azide using twice the starting volume of the filtered culture supematant. After washing, the washed culture supernatant was concentrated to approximately 250-300 ml.
The washed and concentrated culture supernatant was next subjected to ion-exchange chromatography. An ion-exchange resin which works well is DEAE-Sepharose. A 300-400 ml column was equilibrated in 50 mM Tris buffer pH, 7.5, 50 mM NaCl, at 2�-8� C. The Toxin A containing solution was loaded onto the column whereupon the Toxin A was retained. Proteins were then eluted using a gradient from 50-250 mM NaCl while the eluant was monitored at 280 nm. Those fractions having an absorbance greater than 0.05 AUFS were subjected to crossed IEP with the fractions run in one dimension and a reference anti-Toxin A antibody in the second dimension. Crude Toxin A fractions were pooled and collected. These fractions can be stored for one week at 2�-8� C. or longer at -20� C.
Immuno-Affinity Column
The immuno-affinity separation column is prepared generally according to the method taught by Pierce Chemical in its 1989 product information brochure on its CarboLink™ Gel. 20 mg of affinity purified anti-Toxin A antibody was oxidized with an equivalent weight amount of sodium meta-periodate by incubating the one to one combination at room temperature for 60 minutes. The mixture was then applied to a 1.6�20 cm Sephadex G-25 column equilibrated and run with 0.1M NaPi, pH 7.0. The eluant was monitored at 280 nm and the protein-containing fractions were pooled.
20 ml hydrazide group containing agarose beads, sold under the trademark "CarboLink" by Pierce Chemical, were washed with the same buffer and then combined with the oxidized antibody. The fixture was incubated 16-18 hours at room temperature and then formed into a small column (2.5�10 cm) where it was washed with the same buffer. The column was monitored at 280 nm to detect uncoupled protein. The column was washed with 5 bed volumes of deionized water followed by 5 bed volumes of 1M NaCl and again with 5 bed volumes of deionized water. The column can be equilibrated and stored in 50 mM Tris, pH 7.5 containing 50 mM NaCl and 0.01% sodium azide.
For use, the immuno-affinity column was equilibrated with about 160 ml 50 mM Tris, pH 7.5. Partially purified Toxin A off the ion-exchange column can be purified over the immuno-affinity separation column as follows. Based on a Bio-Rad protein assay, 75 mg of the crude Toxin A was applied to the column. The column was washed and run with 50 mM Tris, pH 7.5 while monitoring the column eluant at 280 nm until a peak eluted off the column. The column was washed with 100 mM Glycine, pH 2.7 containing 1% Tween 20, while monitoring the observance of the eluant at 280 nm. The purified Toxin A was eluted from the column as one peak and was collected into a container that contained 50 ml of 0.25M Tris, pH 8.0. The protein concentration of the purified Toxin A was measured using a BCA assay. The protein solution was concentrated using an Amicon concentrator such as the Amicon Single Channel Concentrator Model 8050 with a YM 30 membrane to a concentration of 1.2 mg/ml. The purified Toxin A can be stored at -20� C. for up to one year.
Column Regeneration
The immuno-affinity column can be reused up to at least 20 times. After the Toxin A is eluted off the column, the column is washed with 100-150 ml of 1M NaCl followed by 100 ml of 50 mM Tris, pH 7.5. The column is then equilibrated in 50 mM Tris, pH 7.5 containing 0.01% sodium azide and can be stored at 2�-8� C. until ready for reuse.
Toxin A Antibodies
Preparation of antibodies to Toxin A was carded out generally by the method of Ehrich et al., Infect. & Immun. 28, 1041-1043 (1980) (incorporated herein). Due to the toxicity of the purified Toxin A to rabbits, rabbits cannot be immunized initially with the purified Toxin A but rather must be immunized initially with a toxoid of Toxin A. A toxoid is a toxin treated so as to destroy its toxicity but leave it capable of inducing the formation of antibodies on injection. A toxoid was prepared by mixing the Toxin A as purified off the immuno-affinity column with formaldehyde. One ml of 1.2 mg/ml of Toxin A was diluted with 5 ml of sterile saline. One ml of 37% formaldehyde was diluted with 8.25 ml of sterile saline. 6 ml of the prepared 4% formaldehyde solution was added to the diluted Toxin A and the mixture incubated in a 37� C.�2� C. water bath for 18 to 24 hours. The toxoid-Toxin A solution was mixed with an equal volume of Freund's adjuvant and this solution was used to immunize rabbits. Freund's complete adjuvant was used for the first two injections and Freund's incomplete adjuvant used thereafter. An immunization schedule as shown in Table 1 was used for toxoid immunization. A fresh toxoid should be prepared the day before each immunization. Once the emulsion of Toxoid and Freund's adjuvant is prepared, it should be injected within two hours.
TABLE 1______________________________________Immunization Amount (ml) Route: (0.5 ml Day of theNumber Rabbit per Two Sites) Schedule______________________________________ 1 1.0 POP* 1 2 1.0 POP 3 3 1.0 POP 8 4 1.0 POP 10 5 1.0 POP 15 6 1.0 POP 17 7 1.0 POP 22 8 1.0 POP 24 9 1.0 IM* 2910 1.0 IM 31Test Bleed 3611 (boost) 1.0 IM 40Test Bleed 44______________________________________ *POP = popitiel *IM = intramuscular
On day 36�1,2-3 ml of blood was collected from each rabbit and the anti-Toxin A titer measured using an ELISA assay. An endpoint of greater than or equal to 1:5000 was considered acceptable. If the titer was not acceptable, the rabbits were boosted with another toxoid injection and the titer retested on day 44�1.
Once a protective titer against the toxoid of Toxin A has been achieved, the rabbits can be immunized against the purified Toxin A. The Toxin A was diluted to about 0.4 mg/ml with sterile saline. This solution was then diluted to about 0.2 mg/ml with Freund's incomplete adjuvant. Each rabbit was immunized with 1 ml of this solution. Rabbits were immunized according to the immunization schedule of Table 2.
TABLE 2______________________________________Immunization Amount (ml) Route: (0.5 ml Day of theNumber Rabbit per Two Sites) Schedule______________________________________1 1.0 IM 12 1.0 IM 83 1.0 IM 154 1.0 IM 22Test Bleed5 (boost) 1.0 IM 30Test Bleed 35______________________________________
On approximately the 27th day of the immunization schedule, the animals were tested for antibody titer as described above. If the titer was high enough, at about or higher than 1:40,000, the rabbits were bled and the sera collected.
The sera prepared in the foregoing manner was clarified, when necessary, by high speed centrifugation and/or filtration and then precipitated with 70% ammonium sulfate. The precipitated globulin was resuspended in 20 mM NaPi, pH 7.4 and then purified over a protein A column.
Preparation of Latex-Bound Antibodies
For use in a Latex Agglutination Test the antibodies are bound to latex using the following procedure. Rabbit anti-C. difficile Toxin A antibody was prepared and dialyzed into 20 mM NaPi, pH 6.0 with 0.01% sodium azide. The protein concentration of the solution was adjusted to about 2.0 mg/ml. 50 ml of 2.5% (w/v) solids carboxylated polystyrene latex was placed into each of four 250 ml centrifuge bottles. 70 ml of 20 mM NaPi, pH 6.0 was added to each bottle and the bottles centfifuged at 11,000 RCF for 30-40 minutes. The liquid was poured off and the beads were resuspended in 120 ml of the same buffer. (For all latex suspensions, the latex must be resuspended initially in a minimal volume of buffer.) The bottles were centrifuged as before and the washing step was repeated. The beads were then resuspended in 120 ml of the same buffer and 50 mg of 1-ethyl-3-[3-dimethyl aminopropyl]-carbodiimide hydrochloride and 12.5 mg Sulfo-N-hydroxysuccinimide was added to each bottle. The latex suspensions were allowed to incubate for two hours at room temperature while stirring. The suspension was pelleted by centrifugation at 11,000 RCF for 30-40 minutes, the supernatant poured off, and each pellet resuspended in 120 ml of 20 mM NaPi, pH 6.0. The suspension was pelleted as before and this step was repeated. 15 mg of the buffer exchanged antibody was added to each latex suspension and the resulting latex-antibody suspension incubated for 18-20 hours at room temperature while stirring. 2.5 ml of 1M 2-aminoethanol was added to each suspension and the resulting solution incubated for two hours at room temperature while stirring. The latex-antibody was then pelleted at 10,000 RCF for 30-40 minutes, the supematant decanted and each pellet resuspended in 250 ml of 0.1M glycine-NaOH, pH 8.2 with 0.15M sodium chloride, 0.25% Tween 20, 0.2% sodium azide, and 0.2% BSA. The latex-antibody was pelleted at 10,000 RCF for 30-40 minutes and this step repeated.
A volume of Toxin A containing solution was mixed with 0.5 ml specimen buffer in a centrifuge tube and vortexed for 30 seconds. The tube was then centrifuged for 15 minutes at a mininum of 1500�g. Using a typical LAT test card, 35 μl of the specimen supernatant was placed in the test circle and 35 μl in the negative circle of the test card. 35 μl of a C. difficile culture filtrate was placed in the positive circle. 35 μl of the Toxin A coated latex beads were placed in the test and positive circles. 35 μl of normal rabbit antibody coated latex beads was placed in the negative circle. The contents of each circle were mixed with a separate applicator stick and spread over the surface of each circle. Using a clinical rotator, the test card was rotated for the designated time at 100 rpm. The test was read microscopically under a fluorescent light.
Activity of Antibodies
Table 3 shows the results of a comparison of antibodies raised against acetate precipitated Toxin A with antibodies raised against immuno-affinity purified Toxin A. The Toxin A sample was a sample prepared by acetate precipitation. The antibodies were prepared according to the description recited above. After the rest period, the rabbits were bled every two weeks and the bleeds were purified as described above and coupled to latex beads as described above. The LAT procedure described above was used to compare the antibodies.
As can be seen from the data, antibodies prepared in accordance with the present invention and used in the Latex Agglutination Test gave results of an average endpoint of 12.5-25 ng/ml. Antibodies prepared using acetate precipitation gave results of an average endpoint of 25-35 ng/ml.
TABLE 3__________________________________________________________________________Antibodies raised against acetate precipitated Toxin AToxin ABleed Date Bleed Date Bleed Date Bleed DateConc.10/3 12/7 2/22 3/22(ng/ml)2 min. 4 min. 2 min. 4 min. 2 min. 4 min. 2 min. 4 min.__________________________________________________________________________200 3+ 4+ 4+ 4+ 4+ 4+ 4+ 4+100 2+ 3+ 3+ 4+ 2+ 4+ 3+ 4+ 50 1+ 2+ 2+ 3+ 1+ 3+ 2+ 3+ 35 <1+ 1+ 1+ 2+ Vague 1+ <1+ 1+ 25 Neg. Neg. Vague 1+ Neg. <1+ Neg. Neg. 12.5Neg. Neg. Neg. Vague Neg. Vague Neg. Neg. 6.25Neg. Neg. Neg. Neg. Neg. Neg. Neg. Neg.__________________________________________________________________________Antibodies raised against Immuno-affinity purified Toxin AToxin A Bleed Date Bleed Date Bleed DateConc.Bleed Date 1/4 3/14 4/11 5/24(ng/ml)2 min. 4 min. 2 min. 4 min. 2 min. 4 min. 2 min. 4 min.__________________________________________________________________________200 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+100 4+ 4+ 4+ 4+ 4+ 4+ 3+ 4+ 50 4+ 4+ 3+ 4+ 3+ 4+ Vague 2+ 35 3+ 4- 1+ 3+ Vague 3+ <1+ 1+ 25 1 2+ Vague 1+ Neg. <1+ Neg. Neg. 12.5Vague 1+ Neg. Vague Neg. Neg. Neg. Neg. 6.25Neg. <1+ Neg. Neg. Neg. Neg. Neg. Neg.__________________________________________________________________________
The foregoing description of preferred embodiments of the invention has been presented for purposes of illustration and description. It is not intended to be exhaustive or to limit the invention to the precise forms disclosed, and obviously many modifications and variations are possible in light of the above teaching. It is intended that the scope of the invention be defined by the claims appended hereto.
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|Grace M. Thorne; Diagnostic Tests in Gastrointestinal Infections; 1994 Current Science ISBN 1-85922-606-X ISSN 0267-1379.
|Kamiya et al.; Production of Monoclonal Antibody to Clostridium difficile Toxin A . . . Activity; FEMS Microbiology Letters (1991) 311 316.
|Kamiya et al.; Production of Monoclonal Antibody to Clostridium difficile Toxin A . . . Activity; FEMS Microbiology Letters (1991) 311-316.
|Katoh et al.; Purification and Some Properties of Cytotoxin Produced by Clostridium difficile; Microbiol. Immunol.; vol. 32 (6), 551 564, 1988.
|Katoh et al.; Purification and Some Properties of Cytotoxin Produced by Clostridium difficile; Microbiol. Immunol.; vol. 32 (6), 551-564, 1988.
|Lyerly et al., Enzyme Linked Immunosorbent Assa for C. difficile Toxin A, Jrnl. of Clinical Microbiology, Jan. 1983, pp. 72 78.
|Lyerly et al., Enzyme-Linked Immunosorbent Assa for C. difficile Toxin A, Jrnl. of Clinical Microbiology, Jan. 1983, pp. 72-78.
|Lyerly et al., Monoclonal and Specific Polyclonal Antibodies for Immunoassay of C. difficile Toxin A, Jrnl. of Clinical Microbiology, Jan. 1985, p. 12 14.
|Lyerly et al., Monoclonal and Specific Polyclonal Antibodies for Immunoassay of C. difficile Toxin A, Jrnl. of Clinical Microbiology, Jan. 1985, p. 12-14.
|Muller et al., Monoclonal Antibodies Specific for C. difficile Toxin B and Their Use in Immunoassays, Jrnl. of Clinical Microbiology, Jun. 1992, pp. 1544 1550.
|Muller et al., Monoclonal Antibodies Specific for C. difficile Toxin B and Their Use in Immunoassays, Jrnl. of Clinical Microbiology, Jun. 1992, pp. 1544-1550.
|O Shannessy et al., Immobilization of Glycoconjugates by Oligosaccharides: Use of Hydrazido Derivatized Matrices, Analytical Biochemistry 191, 1 8 (1990).
|O'Shannessy et al., Immobilization of Glycoconjugates by Oligosaccharides: Use of Hydrazido-Derivatized Matrices, Analytical Biochemistry 191, 1-8 (1990).
|Pierce Ordering Catalog, pp. 3 10, (1989).
|Pierce Ordering Catalog, pp. 3-10, (1989).
|Production of monoclonal antibody to Clostridium difficile toxin A which neutralizes enterotoxicity but not hemagglutination activity; (1991), vol. 81, #3 pp. 311-315, Fems. Microbiol. Lett.
|Production of monoclonal antibody to Clostridium difficile toxin A which neutralizes enterotoxicity but not hemagglutination activity; (1991), vol. 81, 3 pp. 311 315, Fems. Microbiol. Lett.
|Purification and some properties of cytotoxin produced by Clostridium difficile; Katoh. T, Microbiol. Immunol, 1988 32/6 (551 564).
|Purification and some properties of cytotoxin produced by Clostridium difficile; Katoh. T, Microbiol. Immunol, 1988 32/6 (551-564).
|Robert K. Scopes, Separation by Precipitation, Protein Purification (2d edition) pp. 40 45.
|Robert K. Scopes, Separation by Precipitation, Protein Purification (2d edition) pp. 40-45.
|Ryan et al., Rapid Detection of C. difficile Toxin in Human Feces, Jrnl. of Clinical Microbiology, Dec. 1980, pp. 776 779.
|Ryan et al., Rapid Detection of C. difficile Toxin in Human Feces, Jrnl. of Clinical Microbiology, Dec. 1980, pp. 776-779.
|Tresyl activation of a hydroxyalkyl ligand for coupling a hydrazide gel: stable immobilization of T 2 toxin for affinity purification of T 2 antibody; Allam, K; 1987 Anal. Biochem; vol. 162, 1, pp. 171 177.
|Tresyl activation of a hydroxyalkyl ligand for coupling a hydrazide gel: stable immobilization of T-2 toxin for affinity purification of T-2 antibody; Allam, K; 1987 Anal. Biochem; vol. 162, #1, pp. 171-177.
|Warny et al., Human Antibody Response to C. difficile Toxin A in Relation to Clinical Course of Infection, Infection And Immunity, Feb. 1994, pp. 384 389.
|Warny et al., Human Antibody Response to C. difficile Toxin A in Relation to Clinical Course of Infection, Infection And Immunity, Feb. 1994, pp. 384-389.
|Wolfhagen et al., Toxins A and B of C. difficile, FEMS Microbiology Reviews 13 (1994) 59 64.
|Wolfhagen et al., Toxins A and B of C. difficile, FEMS Microbiology Reviews 13 (1994) 59-64.
|Citing Patent
|Filing date
|Publication date
|Apr 4, 1997
|Oct 12, 1999
|Biosite Diagnostics
|Diagnostic tests and kits for Clostridium difficile
|Apr 3, 2000
|Aug 7, 2001
|Geltex Pharmaceuticals, Inc.
|Anionic polymers as toxin binders
|May 11, 2000
|Sep 18, 2001
|Geltex Pharmaceuticals, Inc.
|Anionic polymers as toxin binders and antibacterial agents
|Jun 11, 2001
|Jul 16, 2002
|Geltex Pharmaceuticals, Inc.
|Anionic polymers as toxin binders
|Nov 28, 2000
|Feb 11, 2003
|Geltex Pharmaceuticals, Inc.
|Anionic polymers as toxin binders and antibacterial agents
|Jul 9, 2001
|Feb 11, 2003
|Geltex Pharmaceuticals, Inc.
|Anionic polymers as toxin binders and antibacterial agent
|May 1, 2001
|May 4, 2004
|Genzyme Corporation
|Anionic polymers as species specific antibacterial agents
|Nov 14, 2002
|May 10, 2005
|Genzyme Corporation
|Anionic polymers as toxin binders and antibacterial agents
|Apr 7, 2005
|Mar 16, 2010
|Genzyme Corporation
|Anionic polymers as toxin binders and antibacterial agents
|Apr 20, 2012
|Jul 9, 2013
|Wyeth Llc
|Compositions relating to a mutant Clostridium difficile toxin and methods thereof
|Mar 22, 2013
|Oct 15, 2013
|Wyeth Llc
|Compositions relating to a mutant Clostridium difficile toxin and methods thereof
|Feb 25, 2009
|Apr 15, 2014
|3M Innovative Properties Company
|Antibodies to Clostridium difficile spores and uses thereof
|Aug 19, 2013
|Dec 2, 2014
|Wyeth Llc
|Compositions relating to a mutant Clostridium difficile toxin and methods thereof
|Jul 10, 2015
|Nov 17, 2015
|Wyeth Llc
|Compositions relating to a mutant Clostridium difficile toxin and methods thereof
|Nov 14, 2002
|Jul 24, 2003
|Geltex Pharmaceuticals, Inc.
|Anionic polymers as toxin binders and antibacterial agents
|Oct 13, 2004
|Apr 13, 2006
|Dominique Charmot
|Toxin binding compositions
|Oct 13, 2005
|May 11, 2006
|Ilypsa, Inc.
|Toxin binding compositions
|Jul 18, 2006
|Mar 8, 2007
|Toni Chancellor-Adams
|Poly(potassium and sodium styrene sulfonate) its manufacture and its uses
|Aug 6, 2008
|Jul 9, 2009
|Caroline Bacon-Kurtz
|Poly(Potassium and Sodium Styrene Sulfonate) Its Manufacture and Its Uses
|Apr 3, 1998
|Jul 19, 2000
|Biosite Diagnostics Inc.
|Diagnostic tests and kits for clostridium difficile
|U.S. Classification
|435/7.32, 435/7.2, 436/533
|International Classification
|Cooperative Classification
|European Classification
|May 22, 1995
Owner name: LEE LABORATORIES, INC., GEORGIA
Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:DEUTSCH, JOHN W.;REEL/FRAME:007663/0145
Effective date: 19950515
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Year of fee payment: 12


text:
OKLAHOMA CITY (AP) - Governor Brad Henry is preparing to sign a
bill aimed at helping authorities when they are called to find missing senior citizens.
The bill sets up a "Silver Alert" program.
It is patterned after the Amber Alert program that helps locate missing children.
Like Amber, the Silver Alert will use television, radio and other mediums of communications to alert the public of a missing senior.
Henry planned to sign the bill at a Thursday afternoon news conference.


text:
Book your room at Hotel Europe
Collect 400 points
Offer eligible for stays completed between 1 July 2016 and 30 September 2016. Simply enter your Nectar card number when prompted. Your points will be credited 30 days after you return from your trip. *Offers may apply.*Offers may apply
Exclusive five-star Hotel Europe is situated in the historical heart of Minsk. It is the ideal place for every kind of accommodation and it is endowed of all the modern comforts. The hotel offers together with high class hospitality a relaxing atmosphere. The hotel is ideally located in the very heart of the city next to the City Hall the Palace of the Republic and two main Cathedrals. It is surrounded by deluxe boutiques, art galleries and famous cafes.Would you like to give us feedback? Share it! Opens in a new window
Hotel Europe28 Internatsionalnaya Str Minsk 220030
Hotel Amenities
Hotel Amenities
All of the guest rooms are comfortable and nicely equipped to give a feeling of being home while away from home. The hotel also has a variety of facilities and services that are sure to meet the needs of both business and leisure travelers.
- Total number of rooms - 67
- Number of floors - 6
- Business center
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text:
Eyes on the Road… Hands at 2 and 10
Submitted by chris
Share More
Dante Williams:
July 31, 2012 at 12:46 am
Well the Jews ARE behind everything..
Dan Man:
July 31, 2012 at 4:18 pm
...especially behind other people (if you know what i mean)
Sam Blank:
July 31, 2012 at 8:21 pm
Nice. A couple of anti-Semites.
The E.S.L. Dating Diaries:
July 31, 2012 at 8:57 am
It's the new rear Jew mirror: you have a Jewish person sitting in the backseat holding the mirror.
Alisa Cabral:
July 31, 2012 at 1:12 pm
If your Bubbe was sitting in the backseat you wouldn't have hit that woman. She would have heard your grandmother kvetching even with the windows rolled up.
Christian Kuhl:
July 31, 2012 at 6:36 pm
Jews in the mirror are closer than they appear.
Kamira Jeffreys-coley:
July 31, 2012 at 9:07 pm
lol stop checking out the jews.
Wendy Whitehurst-Dade:
August 1, 2012 at 12:07 am
Lol! :)
Tabby Truxler:
August 1, 2012 at 2:31 am
Lol! Side and rear Jews!
"Best of" Lists


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Nokia E6 smartphone
Nokia E6 review: Another Nokia phone that possesses excellent hardware, but let down by software that just isn't as slick, fast or user friendly as competitors
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text:
Video Industry News Video Company News Technology Industry News Technology Company News Novelty Industry News Novelty Company News Gay Industry News Gay Company News Legal Industry News All Company News
Released: Nov 01st, 2001
Running Time: 80
Company: Heatwave Entertainment
Cast: Platinum (I), Jasper Wade, Devlin Weed, Lola Lane, Julian St. Jox, Wesley Pipes, Sierra (I), Akara Richards, Herschel Savage
Critical Rating: AAA
Genre: All-Sex
The hooker premise is pretty flimsy here, but the sex is workable. Some noteworthy moments: the gonzo-style opener starring adorable Platinum, a true bullshit artist who claims she's surfed and prostituted her way around the world. "So you're the surfin' ho?" laughs the uncredited interviewer. Platinum ends up surfing her way around Jasper Wade's dick, but Wade wipes out early and Julian St. Jox comes in for some very decent butt sex. Also worth a look is Lola Lane and Devlin Weed in a good reverse cow anal.


text:
Yes, white grub worms are the larvae of various scarab beetles, including June bugs, Japanese beetles, dung beetles and northern masked chafers.
June beetle (genus Phyllophaga), also called May beetle or June bug, genus of nearly 300 species of beetles belonging to the widely distributed plant-eating subfamily Melolonthinae (family Scarabaeidae, order Coleoptera). These red-brown beetles commonly appear in the Northern Hemisphere during warm spring evenings and are attracted to lights.
The heavy-bodied June beetles vary from 12 to 25 mm (0.5 to 1 inch) and have shiny wing covers (elytra). They feed on foliage and flowers at night, sometimes causing considerable damage. June beetle larvae, called white grubs, are about 25 mm (1 inch) long and live in the soil. They can destroy crops (e.g., corn [maize], small grains, potatoes, and strawberries), and they can kill lawns and pastures by severing grasses from their roots.
A Grub's Life: Egg to Beetle Life Cycle:
Tip! One of the signs that spring is in full swing is the incessant sound of male June beetles (Phyllophaga spp. and others) crashing into the lit windows of your home at night. More commonly called June bugs, these beetles emerge in late March, April and May to lay their eggs in lawns.
While adult June bugs are a big nuisance, it is their offspring – known as white grubs – that are the real pest. White grubs feed primarily on the roots of warm season grasses in June, July and August, causing massive damage and turf loss. How do you know if your grass is being damaged by white grubs? Here are some signs to look for from Gardenstylesanantonio.com.


text:
How do I get rid of this error... I disabled an re-enabled the incoming mail settings in a document library. When type in the alias i get this error: Unable to assign this e-mail address to the list, because the address is in use.
There is another Document Library with the same email address. Even if you have deleted old Document Library it might still be in the Recycle bin somewhere.


text:
Hello to this forum,
I have a blog made on WordPress Self Hosted Software. Recently my blog was doing perfect and their was no issue with any thing but from last two days I am seeing everything is just removed from Google Search.
My Domain : http://www.GetHow.org
I also checked the problem of my blog for SEO but everything is same as it was, but still I am getting problem.
Help me out from this problem I am loosing my precious visitors.


text:
"The night is far spent, the day is at hand: let us therefore cast off the works of darkness, and let us put on the armour of light" (Romans 13:12).
Monday, September 30, 2013
After 1,000 Rejections, Persistence Pays
This morning I posted an interview on my writing blog about Cheree Alsop, the author of 16 novels, who persisted in spite of 1,000 rejections. A thousand rejections! Cheree proves that persistence pays. If you are in need of some inspiration, I invite you to read the post.
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text:
Spending Mother's Day in Prison
What happens to families when mothers are removed through incarceration?
Released: 6-May-2014 8:00 AM EDTAdd to Favorites
Source Newsroom: University of Illinois at Chicago
Expert Available
Source Newsroom: University of Illinois at Chicago
Expert Available
Newswise — A national study on incarcerated parents in prison found that 62 percent of incarcerated women had at least one child under the age of 18. Among the family issues related to women prisoners are maintaining contact with their children, alternative care for their children and support of the mother's role during incarceration. Patricia O'Brien, UIC associate professor of social work, can comment on the negative effect that the removal of women through incarceration has on families.
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text:
Devils' DeBoer Has Found Success, If Not Job Security
NEWARK—There's a heavy history that comes with being the New Jersey Devils' head coach, and Peter DeBoer was well aware of it when he took the job last July. That history can be distilled to three words: Don't get comfortable.
Over his 24 years as the Devils' general manager, Lou Lamoriello has never hesitated to change coaches. He's fired 10 of them and even assumed the role twice himself. More, DeBoer accepted the position at a rare moment in the franchise's recent past: The Devils had missed the playoffs last season for the first time in 15 years. If Lamoriello is known for keeping his head coach on a short leash, DeBoer's might have seemed shorter than the average shoelace.
"But any coach worth his salt," DeBoer said, "thinks he's going to be the guy who comes in and bucks the trend."
So far, he has. So far. Not only have the Devils returned to the playoffs in DeBoer's first season with them, but their first-round series affords him the chance for a greater measure of personal satisfaction. The Devils' opponent is the Florida Panthers, the only other NHL team DeBoer has coached. Game 1 is Friday night in Sunrise, Fla.
"It's not a bad thing," Devils forward Ilya Kovalchuk said of DeBoer's familiarity with the Panthers, who won the Southeast Division this season. "I think it'll be an advantage for us that he knows some players there and he knows what to expect from the team."
DeBoer arrived in Florida in 2008. Years earlier, the Panthers had emerged as one of the NHL's success stories in the southern U.S., making a surprising run to the Stanley Cup Finals in 1996 and building a cult-like following. For a while, their fans carried on one of the league's more creative traditions: After a player used his stick to kill a rat in the team's locker room, the home crowd began throwing toy rats on the ice after each Panthers goal. But those good times had ended, and DeBoer was charged with trying to revive a franchise that hadn't reached the NHL postseason since 2000.
He had spent the previous seven years as the head coach and manager of the Kitchener (Ontario) Rangers, a major-junior club, and the biggest adjustments to coaching professional hockey in southern Florida, he said, were the obvious ones: the travel, the lifestyle, the weather. "You get up every morning, and the sun's shining," he said. "There's something to be said about that. On the flip side, it also plays a role in that your players, when they leave the rink, aren't heading home to sit on a couch and relax."
The Panthers never finished better than third in their division and never made the playoffs in any of DeBoer's three years there. They fired him on April 10, and the Devils' job became vacant on the same day when that Jacques Lemaire stepped down. Among the prospective replacements for Lemaire, DeBoer's name didn't carry the most cachet. But Lamoriello said he had long been impressed with DeBoer's performance as a major-junior coach (he won 535 games for three teams over 13 seasons) and with the way DeBoer's Florida teams played.
"He had been one of the candidates in a confidential way," Lamoriello said. "I felt comfortable with him in reference to his philosophy, what he felt didn't work for him in Florida, what he learned."
Lamoriello got what he wanted from DeBoer during the regular season: The Devils scored 216 goals—a 45-goal jump from last season—and had the eighth-best goals-against average in the NHL. No one has coached the Devils for two consecutive full seasons since Brent Sutter did it from 2007 to 2009, but with a deep playoff run, DeBoer might last that long and then some. "You really don't think about the end," he said, "even though you know it's there no matter what."
Write to Mike Sielski at email@example.com


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I only needed them for like 3 games. They are mostly useless.
They are just trying to get more money.
Players are never forced to buy kreds, it's purely up to them to decide. The reason tipping was disabled was to prevent money laundering that was done by some developers.
your automatically a loser for buying them
Originally posted by gamerguy75:I only needed them for like 3 games.
I only needed them for like 3 games.
So they are not useless.
I don't understand how money laundering can be achieved. Its only starts when you use dirty money, but the money is coming from gamers, unless the gamers are the dirty mobsters in real life.
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It's grilling at its best! Tasty seasoned burgers and red onion slices both cook on the grill. Rye burger buns add just the right final touch.
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The Little Red Hen: Folk & Fairy Tale Easy Readers Mini-Book & Worksheet
Model your sequencing thought process aloud with your class. Then review the sequence the little red hen used to make bread. See Full Product Description.
Number of pages: 6 Subject: Leveled Reading, Sequencing, Writing, Short Vowels, Independent Reading Grade: Kindergarten - 2 Type: Mini Books NonFiction File Size: 428 KB
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Model your sequencing thought process aloud with your class. Review the sequence the little red hen used to make bread.
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Build fluency by modeling the reading of the dialogue. Draw students' attention to the punctuation used and ask the children to echo your reading, sentence by sentence. This story also lends itself to Reader's Theater.
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There is 8 stones at the total carats of 0.04ct.
The stones are 0.005ct.(half point)
The stones is light yellow.
The price PER stone is R19.00
Any Manufature Defect we will replace it with new pieces
7 Days: Any Manufature Defect we will replace it with new pieces


text:
3 people inherit property so they go to the bank the bank ask for everyones id and ss 2 refuse the bank says to them you do understand by not complying you will essentially have no say in the property ,,, the 2 agree and 3rd is told the morpage lender will be contacting him. what does this mean
correction mortgage lender
Probate Attorney
I suspect what is happening is that due to privacy laws, the bank is unable to deal with anyone until that person agrees to complete paperwork that allows him/her to become their customer. The bank cannot control who has a "say in the property." They can control who they will deal with.
Depending on HOW the property was inherited and how it is now titled, you may need an attorney to help you sort this out.
The mortgage lender cannot foreclose on the property or accelerate the loan as long as payments are kept current, under Federal law.
James Frederick
***Please be sure to mark if you find the answer "helpful" or a "best" answer. Thank you! I hope this helps. ***************************************** LEGAL DISCLAIMER I am licensed to practice law in the State of Michigan and have offices in Wayne and Ingham Counties. My practice is focused in the areas of estate planning and probate administration. I am ethically required to state that the above answer does not create an attorney/client relationship. These responses should be considered general legal education and are intended to provide general information about the question asked. Frequently, the question does not include important facts that, if known, could significantly change the answer. Information provided on this site should not be used as a substitute for competent legal advice from a licensed attorney that practices in your state. The law changes frequently and varies from state to state. If I refer to your state's laws, you should not rely on what I say; I just did a quick Internet search and found something that looked relevant that I hoped you would find helpful. You should verify and confirm any information provided with an attorney licensed in your state. I hope you our answer helpful!
Probate Attorney
It's not clear why they went to the bank in the first place, but hey should contact a probate lawyer to ensure that title is/was transferred properly.
If this information has been helpful, please indicate below. Stephen Pearcy is licensed to practice law in California. The response herein is not legal advice and does not create an attorney-client relationship. The response is for legal education and is intended to provide general information about the matter within the question.
Estate Planning Attorney
Because the term "inherited property" is ambiguous, the first step is to understand how inherited - remainder after daeth of life tenant, joint tenants after the death of one, transfer on death deed, intestacy, will, trust, or other? Dpepdmeomg on the form that may indicate whether passes free and clear or subject to claims.
Second, a mortgage lender does have a mortgage that continues to be a lien regardless of the form referenced above, assuming that the mortgage was erfected properly - and that again requires looking at the title factors first referenced above; could be free and clear, or could be subject to mortgage depending on facts.
The idnetiy could be related to privacy laws. or might be that bank's interpretation of a regulation. Like an attorney you could call and ask for citations to authority. Might be a banking regulation or something else yu are not aware of - look it over to see if disputable or not.
Hope this helps.
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FMCA Commercial Members receive the benefit of one FREE listing in FMCA's biannual RV Marketplace. This directory is printed twice per year in FMC magazine's January and June issues (Deadlines: January issue - November 1, June issue - April 1).
If you would like to be listed under more than one category, please phone the Commercial Membership Services Department at 1-800-543-3622.