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New testing methods

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Twotimer

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Slow Evolution for Protein Detection, Characterization

Old tools will likely remain a mainstay for the foreseeable future, but new approaches aim to get data faster, reproducibly, and with smaller samples

Lori Valigra
Valigra is a freelance writer
based in Cambridge, Mass.

A Texas cow whose tissue tested positive for mad cow disease in June turned some time-honored protein tests into household words. Consumers and the cattle industry alike debated whether the US Department of Agriculture (USDA) had performed rigorous enough tests on the animal, which initially was deemed negative for bovine spongiform encephalopathy (BSE).

But it wasn't biosensors or other cutting-edge technologies that confirmed the deposits of abnormal prions in a postmortem tissue sample. Instead, it was a positive result

About 12 proteins comprise up to 96% of the protein mass in plasma. (Source: Plasma Proteome Institute and Beckman Coulter)
from an old standby, the Western blot, that prompted the USDA to dig beyond earlier inconclusive and negative results from enzyme-linked immunosorbent assays (ELISAs) and immunohistochemistry (IHC) tests (see sidebar on "Anatomy of BSE Detection"). The USDA and The Veterinary Laboratories Agency, a world reference lab for BSE in Weybridge, UK, conducted additional tests and concurred the sample was positive. The Texas case led the USDA to change its BSE protocol. All inconclusive ELISA tests now will be followed by both an IHC and Western blot rather than just the IHC alone, says Jim Rogers, a spokesman for the USDA's Animal and Plant Health Inspection Service (APHIS). If either confirmatory test is positive, the sample will be considered positive for BSE.

The BSE case underscores the continuing importance of conventional tests, as well as the frequent need to use more than one tool to study proteins, which by nature are more complex than nucleic acids. Many advanced genomics tools already are available as a result of the race to sequence and study the human genome. But proteomics lacks an amplification technology similar to polymerase chain reaction (PCR) used for DNA and protein arrays are harder to develop because of the comparatively broad range of binding conditions for target proteins. Those are some of the reasons why ELISA, IHC, Western blot, one-dimensional (1D) and two-dimensional (2D) gel electrophoresis and mass spectrometry (MS) persist in the market as protein detection and characterization techniques for diagnostics and drug discovery and development. These technologies are being improved by using capillaries to speed testing, and they are also being used with complementary technologies such as liquid chromatography (LC) and MS. "Proteins are so important that you're going to see everybody developing new techniques constantly," says Linda Cahill, president and CEO of Cell Biosciences Inc., Palo Alto, Calif.

Simplifying Western blots
The mere mention of a Western blot can elicit moans from graduate students who serve as free labor for a tedious process that has changed little since it was first published

Western blotting requires about 500,000 cells for analysis and highly specific antibodies for phosphoprotein differentiation. By contrast, the Blotless Nano Western requires fewer than 4,000 cells, and it separates and detects phospho and non-phospho species in one run with a single antibody. (Source: Cell Biosciences Inc.)
in 1979. The paper explained how to transfer proteins from a polyacrylamide gel to a sheet of nitrocellulose to obtain a faithful replica of the original gel pattern. No longer trapped inside the gel, the immobilized protein could be detected by antibodies and studied with a variety of analytical procedures. At the time, it was a breakthrough technology and is still a critical confirmatory assay. Cahill conducted an informal study of the test's prevalence. She looked at life science journals for four months and tracked each time a Western blot was used as the confirmatory assay: it was 85% of the time.

"With all the new protein techniques coming along, you have to ask yourself why Westerns are the dominant method," says Cahill. "The reason is they give you the physical separation of the protein so you can get more specificity out of your antibodies." But a Western blot protocol can seem like something from the 17th century, she says: one to two days of manual labor with as many as 100 to 200 different pipetting steps, each of which could lead to mistakes. "But it works."

Cell Biosciences, which develops instruments and reagents, will start beta testing a capillary-based system inspired by the Western blot later this summer. It will run smaller samples automatically and quickly, with reproducible results, Cahill says. "A Western blot might take you two days, but this takes 30 minutes."

The new system uses what the company calls Blotless Nano Western technology. The technique is based on a capillary immunoassay format that automates the basic steps of Western blotting using nanoliter volumes and physically separating the protein in the capillary. The initial system will require 500 to 1,000 cells in a sample, and it will be able to accommodate multiple capillaries running simultaneously. "We're going to look for whether the protein is there, if it is a good druggable target, and even if it isn't, how the protein is interacting in cells." It is scheduled for introduction in early 2006.

Faster, more sensitive
According to a Cell Biosystems comparison, Western blotting requires about 500,000 cells for analysis and highly specific antibodies for phosphoprotein differentiation. It can
Anatomy of BSE Detection
In June, the tissue of a cow in Texas tested positive for mad cow disease following a battery of time-honored protein tests The bovine spongiform encephalopathy (BSE)-positive cow was selected for testing at a Texas pet-food plant because it was non-ambulatory; its remains were incinerated, so it never entered the human or animal food chains.

According to the Animal and Plant Health Inspection Service, a part of the US Department of Agriculture (USDA), the first postmortem test on the cow's tissues in November 2004 was a Bio-Rad Laboratories Inc. ELISA rapid test that was inconclusive. The sample was then sent to the USDA's National Veterinary Services Laboratories in Iowa, where two internationally accepted confirmatory immunohistochemistry (IHC) tests were conducted in November, 2004. Both were negative for BSE.

The USDA's Office of the Inspector General recommended further testing in early June using another internationally recognized confirmatory test, the Western blot. It came back positive. The following week the USDA conducted another IHC test using different antibodies than the November, 2004 test. It was weakly positive for BSE. In addition, tissue samples were sent to a world reference laboratory for BSE, the Veterinary Laboratories Agency in Weybridge, UK, which conducted rapid enzyme-linked immunosorbent assays, IHC and Western blot tests, and concluded the sample was positive. Those IHC tests used different antibodies than those used by the USDA in November, 2004.

However, Weybridge also confirmed the accuracy of the results of the USDA's November confirmatory IHC tests, and concurred that the case could not have been confirmed on the basis of the sample. USDA and Weybridge scientists are trying to determine the cause of the conflicting IHC results. Several factors could have contributed, one being that the cow had a very low level of infectivity and the USDA's routine IHC test might not have been sensitive enough to detect the disease. In addition, Weybridge experts indicated that the deposits of the abnormal prion were not uniformly distributed in the brain tissue and were present at a low concentration. That means adjacent samples of brain tissue might have yielded different results.
take two days to complete, and it has limited quantitative capability. It is a manual procedure that has a difficult blotting step that works differently for every protein. By contrast, the Blotless Nano Western requires fewer than 4,000 cells, and single-cell sensitivity is anticipated in the future. It separates and detects phospho and non-phospho species in one run with a single antibody.

"We get better separation because we're doing it on a capillary electrophoresis device, which gives us much better control," Cahill says. Presorting is done by isoelectric focusing, so it is done by the charge of the protein and not just its size. "The ability to do this repeatedly with standard measurements against it, so that what you do on Monday morning is the same as what you do on Thursday afternoon, is critically important to biology." And with Western blot technology being used more often to confirm BSE cases, the market potential is large.

Roche Applied Science, Indianapolis, has taken an incremental approach to improving technology. Their LumiLight and LumiLight Plus products have improved the sensitivity and length of the chemiluminescence signal possible when developing a Western blot. "But we're putting a lot of our attention on the production of the specific proteins that are detected using Western blot technology," says Jeffrey Emch, a product manager at Roche. "Customers are struggling to produce the proteins to detect, to isolate. That's one of the significant bottlenecks we hear from academia, pharma, and biotech."

Roche developed a technology for in vitro protein expression called Rapid Translation System. It uses Escherichia coli or a wheat germ lysate for protein production. "When you're producing a protein in vitro it can be the dominant protein, but when you're isolating it from a cellular system you have additional proteins that need to be stripped away from your protein of interest," Emch says. "It's essentially reducing the size of the haystack to find your needle."

Improving gels
Like Western blots, gels have also made grad students cringe: pouring gels, staining, and doing quantitation to get protein characterization is time-consuming. Bio-Rad Laboratories Inc., Hercules, Calif., is working on improving a variety of products including Western blots and 1D and 2D gels. The 1D gels detect proteins by size and are often used with Western blots, and the 2D gels detect proteins by size and charge. The 2D gels can look at thousands of proteins in one experiment.

"The 2D gels today still represent the highest resolution technique available for the greatest number of proteins in one assay that you can do," says Gustavo Salem, division manager of Bio-Rad's protein separations division. "We don't see any specific technology that will displace 2D gels, but there are opportunities to continue to improve how they are used, especially as it relates to sample preparation to try to provide greater resolution of specific proteins." When looking for low-abundance proteins, for example, the sample preparation step is vital to how clean a gel is and how reproducible it will be, especially when trying to make comparisons.

While many in the industry believe Western blots are here to stay, some question the longevity of 2D gels. "At thousands of different proteins in a sample, it hits a ceiling," says Gavin MacBeath, PhD, assistant professor of chemistry and chemical biology at Harvard University, Cambridge, Mass. He believes LC and capillary multidimensional column chromatography (MDCC) coupled with MS will give researchers as many dimensions as they want. "You have smaller sample sizes with MDCC and MS, so you can drill deeper into the proteome," he says.

"I see a lot of interest to move away from 2D gels and replace it with chromatographic techniques," says Jeffrey Jensen, president of Eksigent Technologies LLC, Livermore, Calif. His company has multidimensional LC separation products, but he doesn't see them as direct replacements for 2D gels. The products are focused at detecting low-abundance proteins when used with mass spectrometry. To get at the low concentration proteins requires an extremely low flow rate, which Eksigent says it gets with its Microfluidic Flow Control (MFC) technology. MFC uses meters to continuously monitor the flow rate of each mobile phase with microprocessor feedback to a pressure source, so nanoscale flow rates as low as 20 nL/min can be obtained without flow splitting. Flow splitting involves trying to control the flow rate by leaking off 99% or more of the sample in an effort to get the tiny amount they want into the mass spectrometer.

Complementary technologies
Another trend is the combination of different separation technologies to try to get fractionation of a particular class of proteins. For example, an LC system and a gel

When studying proteins, researchers begin by expressing their protein of interest or isolating the protein from biological tissue. Then they prepare and simplify their sample, then characterize it using multiple techniques. (Source: Invitrogen Corp.)
could be used to get the purification capability of an ion-exchange resin on a column and then a size-based separation on a gel, and then it can be taken to a mass spectrometer, Salem says. There is more and more demand lately for isoelectric focusing separations, and Bio-Rad has a system called the Rotofor that can be used with gels and LC.

Bio-Rad is not alone in bringing together complementary technologies. Invitrogen Corp., Carlsbad, Calif., recently launched a SILAC (stable isotope labeling using amino acids in cell culture) kit for protein identification and quantitation. Previously, most SILAC technology was home brewed. SILAC can be used, for example, to purify proteins going through a 1D gel and then to a mass spectrometer. SILAC is a metabolic labeling method that can measure different protein levels between a normal and diseased state. "One isotope is heavy and one is light, so you can run it through a mass spectrometer and see small-fold changes that you couldn't see with a 2D gel," says Cheri Walker, PhD, vice president of proteomics at Invitrogen.

"With consumables our plan is to put together matched reagents that work together. We have some protein expression systems that help with hard-to-express proteins like membrane proteins." Invitrogen also has a system called Zoom Benchtop Proteomics that includes a mini-gel format to profile proteins and 2D gel electrophoresis. It performs sample fractionation, first and second dimension gel electrophoresis, and staining with MS-compatible stains. It uses wide and narrow Zoom Strips, with the narrow range strips running from pH 4 to pH 5.5. Walker says companies with big genomics groups 10 years ago are rapidly changing to more proteomics-focused groups. "They're looking for these types of tools because the workflows aren't standardized today."

High-performance chromatography is being adopted along with MS and moving from experts' laboratories into biologists' labs. "There is a revolution going on, being driven by what you can do with MS today," says Patrick Carberry, LC-MS marketing manager, proteomics, Agilent Technologies Inc., Santa Clara, Calif. Carberry predicts that within the next year, there will be systems on the market priced around $125,000 that incorporate a chromatographic front and a high-performance MS back end with easy-to-use software.

Agilent has a variety of technologies, including a high-performance LC (HPLC) system line whose most recent member is an HPLC chip that can produce narrower peaks that it says are better resolved than a standard nano-column. But the company is taking a systems approach to proteins. "We'll evolve separation equipment and technologies around proteins and peptides along with MS and MS analyzers, informatics and data handling tools that will be integrated with common control, application, and informatics software. And you'll find all these things will become miniaturized. The current HPLC may be able to fit in the palm of your hand and be made of microfluidics components."

Deeper into the proteome
Beckman Coulter Inc., Fullerton, Calif., is working on improving its technology offerings. Its hallmark system is the ProteomeLab PF 2D, a protein fractionation system that allows for 2D chromatography to fractionate the target sample and obtain fractions in a liquid format. It can detect low-abundance proteins and characterize them. The company's ProteomeLab PA 800 protein characterization system resolves and quantifies proteins by their isoelectric point and molecular weight. It can serve as a front-end separation to MS. The ProteomeLab IgY, partitions away the 12 most abundant proteins that make up to 96% of the protein mass in plasma or serum.

Beckman Coulter's aim, like that of many companies in proteomics, is to make it easier for scientists to conduct extensive protein research. And when instruments give scientists an extra inch, they want an extra mile. "To me, proteins are like real estate. It's location, location, location," says Kenneth Bloom, MD, chief medical officer of Clarient Inc., a San Juan Capistrano, Calif., company that provides products and services to characterize, assess and treat cancer. "It's really not just a question of whether a protein is or isn't there. It's a question of how much protein is there and more importantly, where it is in the cell."
 

flounder

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USDA 2003

We have to be careful that we don't get so set in the way we do things that
we forget to look for different emerging variations of disease. We've gotten
away from collecting the whole brain in our systems. We're using the brain
stem and we're looking in only one area. In Norway, they were doing a
project and looking at cases of Scrapie, and they found this where they did
not find lesions or PRP in the area of the obex. They found it in the
cerebellum and the cerebrum. It's a good lesson for us. Ames had to go
back and change the procedure for looking at Scrapie samples. In the USDA,
we had routinely looked at all the sections of the brain, and then we got
away from it. They've recently gone back.
Dr. Keller: Tissues are routinely tested, based on which tissue provides an
'official' test result as recognized by APHIS
.

Dr. Detwiler: That's on the slaughter. But on the clinical cases, aren't
they still asking for the brain? But even on the slaughter, they're looking
only at the brainstem. We may be missing certain things if we confine
ourselves to one area.


snip.............


Dr. Detwiler: It seems a good idea, but I'm not aware of it.
Another important thing to get across to the public is that the negatives
do not guarantee absence of infectivity. The animal could be early in the
disease and the incubation period. Even sample collection is so important.
If you're not collecting the right area of the brain in sheep, or if
collecting lymphoreticular tissue, and you don't get a good biopsy, you
could miss the area with the PRP in it and come up with a negative test.
There's a new, unusual form of Scrapie that's been detected in Norway. We
have to be careful that we don't get so set in the way we do things that we
forget to look for different emerging variations of disease. We've gotten
away from collecting the whole brain in our systems. We're using the brain
stem and we're looking in only one area. In Norway, they were doing a
project and looking at cases of Scrapie, and they found this where they did
not find lesions or PRP in the area of the obex. They found it in the
cerebellum and the cerebrum. It's a good lesson for us. Ames had to go
back and change the procedure for looking at Scrapie samples. In the USDA,
we had routinely looked at all the sections of the brain, and then we got
away from it. They've recently gone back.

Dr. Keller: Tissues are routinely tested, based on which tissue provides an
'official' test result as recognized by APHIS
.

Dr. Detwiler: That's on the slaughter. But on the clinical cases, aren't
they still asking for the brain? But even on the slaughter, they're looking
only at the brainstem. We may be missing certain things if we confine
ourselves to one area.


snip...


FULL TEXT;


Completely Edited Version
PRION ROUNDTABLE


Accomplished this day, Wednesday, December 11, 2003, Denver, Colorado



-------- Original Message --------
Subject: INCONCLUSIVE USDA
Date: Thu, 24 Feb 2005 15:55:45 -0600
From: "Terry S. Singeltary Sr." <[email protected]>


United Press International: USDA vet: Texas mad cow breach not unique...
Published 5/4/2004 5:01 PM WASHINGTON

http://www.upi.com/view.cfm?StoryID=20040504-012834-2365r


United Press International: USDA orders silence on mad cow in ...
Published 5/11/2004 10:16 PM WASHINGTON

http://www.upi.com/view.cfm?StoryID=20040511-015527-4917r


Feds reviewing Texas mad cow breach - (United Press International)
By Steve Mitchell United Press International.
Washington, DC, May. 5 (UPI) -- The US Department ...

http://washingtontimes.com/upi-breaking/20040505-064316-1509r.htm


No mad cow tests at Texas firm in 2004
By Steve Mitchell
United Press International
Published 5/14/2004 11:06 AM

WASHINGTON, May 14 (UPI) -- The U.S. Department of Agriculture did not test any cows for mad cow disease in the past seven months at the same Texas facility where federal testing policies for the deadly disorder were violated last month, United Press International has learned.
http://www.upi.com/view.cfm?StoryID=20040513-065401-5285r

USDA Ordered that Suspected Mad Cow in Texas Not Be Tested.
USDA's San Angelo vets and techs ordered not to test suspect cow.
by Daniel ...

USDA Ordered that Suspected Mad Cow in Texas Not Be Tested

USDA's San Angelo vets and techs ordered not to test suspect cow

by Daniel Yovich on 5/5/04 for Meatingplace.com

It was a trio of Agriculture Department staff < two veterinarians and one
technician < who were supposed to follow agency protocol by testing what
they determined was an older cow that likely had a central nervous system
disorder when it arrived April 27 at the Lone Star Beef plant in San Angelo,
Texas.

One government source and another within the industry, both of whom say they
have firsthand knowledge of events that day, said the final call on not to
test the animal was made by an APHIS supervisor in Austin, Texas, after an
APHIS technician at the plant advised her supervisor she was preparing to
take a tissue sample from the culled animal for BSE testing. Both sources
spoke to Meatingplace.com on condition of anonymity, and USDA officials did
not return telephone calls Tuesday seeking comment and confirmation of the
allegations.

What USDA has confirmed is that the agency's standard operating procedures
call for animals condemned due to a possible CNS disorder be kept until
APHIS officials can collect samples for testing. That clearly was done in
this case. The animal sat for more than 90 minutes and less than two hours
after it was condemned, stunned and killed before the APHIS tech told Lone
Star Beef management to dispose of the animal "in a routine manner."

As a condemned cow, there was never any chance that the meat from the animal
would enter the food chain. What is less clear is what went wrong at USDA
and why.

USDA spokesman Ed Loyd said the agency was conducting an investigation into
the issue < attempting to establish a timeline and chronology of who was
involved and who made the decisions last week in San Angelo.

What is clear, in the mind of the two sources who spoke to Meatingplace.com
, is that all three of USDA's key decision makers on the ground at the San
Angelo plant were overruled by a staffer with more authority in Austin.

"Everybody expected a test, and then the word came that there wasn't going
to be any test," one source said. "I'm not sure why that decision was made,
and I'm not going to speculate about the reasons for it. But I think what
USDA is going to find is that the final decision was made up the food chain,
and I think a lot of people will be interested in why that decision was
made."

http://www.organicconsumers.org/madcow/notest050704.cfm


Forums - Congressman Waxman's Letter and Texas Mad Cow http://www.prwatch.org/forum/showthread.php?t=4292&goto=nextnewest
June 12th, 2004, 01:59 PM, #1. Terry. Registered User. Join Date: Oct 2002. Location:
Bacliff, Texas. Posts: 408. Congressman Waxman's Letter and Texas Mad Cow. ...


Letter and Texas Mad Cow
http://www.prwatch.org/forum/archive/index.php/t-4359.html
Forums > Books by PR Watch Staff > Mad Cow USA > Congressman Waxman's Letter
and Texas Mad Cow. View Full Version : Congressman Waxman's ...

-------- Original Message --------
Subject: MAD COW CONFIRMED TEXAS COW (rumor)
Date: Mon, 22 Nov 2004 17:16:23 -0600
From: "Terry S. Singeltary Sr."
Reply-To: BSE-L



http://www.vegsource.com/talk/madcow/messages/93583.html


Re: MAD COW CONFIRMED TEXAS COW (rumor)

http://www.vegsource.com/talk/madcow/messages/93584.html


BSE ( test results ;-) Texas Animal Health Commission News Release ...

Counting that one and the other positive, positive, inconclusive, and finally
declared and documented as negative cows (NO WB), the USA in my ...

http://www.vegsource.com/talk/madcow/messages/93884.html

Re: ''INCONCLUSIVE'' IS NEGATIVE or so they claim...OFFICIAL ...

would not be telling us of any 'inconclusive', but > they ... because the likelihood >
of it > being positive was very ... tell you why, they wanted a negative so bad ...

http://www.vegsource.com/talk/madcow/messages/93990.html


Forums - Mad Cow USA
... 2nd Positive Inconclusive Negative For Bse Usa; 1st Positive Inconclusive Is Negative; ...
PUBLIC COMMENTS ON USDA’S DOWNED ANIMAL BAN: [Docket No. ...

http://www.prwatch.org/forum/archive/index.php/f-11.html


Forums - Dr. Ron DeHaven DOING THE MAD COW TEXAS TWO-STEP AGAIN
... Dec 2, that IHC- DOES NOT MEAN IT IS NEGATIVE. ... so many
errors (i am assuming X meant
inconclusive), why are ... at the sheep that tested IHC- but were
positive''. ...

http://www.prwatch.org/forum/showthread.php?t=5264


CJD WATCH... positive and histopathology and immunohistochemistry negative) with the ... 2005 positive
The case was confirmed on ... Origin of infection: unknown or inconclusive. ...

http://disc.server.com/discussion.cgi?disc=167318;article=1927;title=CJD%20WATCH

Re: BSE 'INCONCLUSIVE' IN USA, FROM TEXAS ???

... Date: February 1, 2005 at 3:08 pm PST. In Reply to: Re: BSE 'INCONCLUSIVE' IN USA,
FROM TEXAS ??? posted by TSS on November 19, 2004 at 9:41 am: ...

http://www.vegsource.com/talk/madcow/messages/93989.html


BSE 'INCONCLUSIVE' IN USA, FROM TEXAS ???

http://www.vegsource.com/talk/madcow/messages/93563.html


Forums - View Single Post - Re: MAD COW CONFIRMED TEXAS COW (rumor ......
Harrison >> November 22, 2004 >> >> Test results for the BSE inconclusive are not ...
was just this...damn, >> i will not sleep tonight/// >> TSS >> >>

http://www.prwatch.org/forum/showpost.php?p=12049&postcount=3


Forums - Bse Usa 'inconclusive' Test Reported Nov. 18, 2004
... Because this test is only an inconclusive test result, we ...
animal presented for slaughter is sampled for BSE, holding the ...
tss USDA News [email protected]

http://www.prwatch.org/forum/showthread.php?t=5160


Nebraska Outdoor Forum: Study of Atypical Bse Project Number: 3625
.. ars.usda.gov/is/AR/archive/dec04/ TSS Terry S ... mice from the experimental cow brain
had been inconclusive. ... clinical signs of brain lesions characteristic of BSE. ...

http://www.ngpc.state.ne.us/cgi-bin/ultimatebb.cgi?ubb=get_topic;f=12;t=000385


CJD WATCH

... that the USA is now facing, an epidemic of > ''INCONCLUSIVE'' TSEs...TSS > > Terry
S ... 5:15 this evening, we were notified that an >> inconclusive BSE test result ...


http://disc.server.com/discussion.cgi?disc=167318;article=1490;title=CJD%20WATCH


-------- Original Message --------
Subject: US CHOICE OF MAD COW TEST QUESTIONED
Date: Wed, 24 Mar 2004 16:12:06 -0600
From: "Terry S. Singeltary Sr."
Reply-To: BSE-L


US CHOICE OF MAD COW TEST QUESTIONED

The US plans to measure the incidence
of mad cow disease in its cattle with a
test that its own officials have said gives
too many false positives. Some experts
fear the choice reflects an official desire
to downplay the impact of the first
positive BSE tests that emerge, when
they turn out not to be confirmed.

Last week the US Department of
Agriculture (USDA) approved two tests,
including one made by the Californian
firm BioRad, for screening up to 300,000
cattle for BSE, starting in July. No more
tests will be licensed for months.
Announcing the testing plan, chief
veterinary officer Ron DeHaven cautioned
that "there will be positive results",
many of them false.

BioRad's antibody-based test for the
prion protein that causes BSE has given
numerous false positives in Belgium and
Germany. And in Japan only 8 of 113 cattle
that repeatedly tested positive with
BioRad were confirmed by slower tests
that do not give false positives.

The USDA even wrote last May that
"it is well known" that tests like
BioRad's give false positives. It states
that other kinds of quick tests are more
suitable for testing for very low levels of
BSE, which are expected in the US.

The second quick test approved by
the USDA, made by Maine-based IDEXX,
could also in theory give false positives.
It remains unclear how reliable it is,
because there has been little practical
experience with the test so far. It is not
yet approved for use in Europe, where
the vast majority of BSE tests are done.


Debora MacKenzie,
Brussels correspondent,
New Scientist.
tel +32-2-245-0412
fax +32-2-245-0552
mobile +32-49-754-0444

http://www.newscientist.com/
=======================

Greetings,

odd that the USDA et al approves two US-OWNED tests that are
_known_ to give false positives, when they know other rapid
TSE test are much more reliable. IT's like they purposely do
not want to find any TSE in the USA bovine, so they pick the
worst test available. The USDA own experts think BioRad is
not suitable for supposedly BSE/TSE free and low incidence
areas, so why did they choose this test and or the IDEXX,
which i dont think has even been submitted to the EU for evaluation
and has no commercial experiance to my knowledge. You could
almost get the feeling they are deliberately skipping over
Prionics for the least supperior TSE rapid test. I believe
the Canadians finally did choose prionics. maybe paul or marcus
might comment? seems if North America is going to be a
consolidated BEEF trading market amongst themselves and expect
to export there tainted products everywhere, they could at least
come up with the same TSE rapid Test. how can one use a less
reliable test and the other use a more reliable test, and it
all be the same? i know there is a word Dehaven used, but it
slips my mind now, (consolidated markets) that's not it,
but you get the just of my thoughts, i think;-)...TSS

----- Original Message -----
From: "Terry S. Singeltary Sr."
To: BSE-L
Sent: Wednesday, June 30, 2004 6:57 PM
Subject: Re: [BSE-L] FIRE UP THE PIT, THE FIRST BSE POSITIVE INCONCLUSIVE IS NEGATIVE


> greetings list members,
>
> > -------- Original Message --------
> > Subject: Re: ANOTHER POSSIBLE MAD COW CASE IN THE USA
> > Date: Sat, 26 Jun 2004 13:55:42 -0500
> > From: "Terry S. Singeltary Sr."
> > Reply-To: BSE-L
> > To: BSE-L
> > References:
> >
> >
> snip...
>
> > on the other, i wonder if this is another faked incident like the feed
> > bag
> > event in texas a couple of years back ("the system worked!"). surprise
> > surprise this one won't be confirmed. in essence, a drill to train
> > trading
> > partners not to respond to a positive test kit result. dull the
> > response of
> > media and public. a steady drumbeat of "inconclusive" positives and
> > anticlimatic followups 4-7 days later (say friday pm before 4th of july
> > weekend) of which occasionally one will be positive as "expected" so
> > as not
> > to be newsworthy.
> >
> > the lack of detail makes it impossible for the press to follow up,
> > "refused
> > to identify if the
> > suspect animal was a cow or a steer, its age, location or any other
> > information. not going to be any tv crews swarming around a
> > slaughterhouse
> > or interviewing another dave lothan. total control. just a statistic.
> >
> > problem solved...
> >
> > REFERENCE PURINA MILL INCIDENCE RIGHT AFTER THE INFAMOUS
> > 50 STATE USA BSE EMERGENCY CONFERENCE CALL OF JAN. 9, 2001
>
>
> snip...
>
>
> TOM's TAKE TODAY;
>
> >i don't share your view (patty hearst syndrome?) that usda has been
> >transparent or honest. how could they be unaware, during the long selection
> >process leading to BioRad, of the very low false positive rates observed in
> >Europe, yet the chief guy at usda has repeatedly turned the rates
> >completely upside down, from 1 in 1000 to 999 in a 1000 for a biorad
> >positive being confirmed positive.
> >
> >while i don't know how many false positive or total tests japan has done,
> >the rates you cite from japan are not consistent with europe or usda. even
> >at face value, you are quoting a 1 in 5 chance of confirmation. with 2
> >cows, that is 16/25 of both being negative or 9/25 of one or more true
> >positives, that's 36%, making a liar out of the usda guy (who is not a pr
> >person but way up in the professional staff).
> >
> >for a $20 rapid test kit it makes sense to run a presumptive positive
> >another couple of times the same day. this lowers the rate to 1 in 100,000
> >without the ridiculous 4-7 day delay which in my opinion is solely intended
> >to make yet another Friday pm announcement on the biggest meat buying
> >weekend of the year (since they can't stall until christmas eve this time)
> >plus give them 3-6 days to ramp up their pr engine plus tip off friends in
> >the commodities pit again.
> >
> >i think it is a little manipulative not to disclose the ages of the cow and
> >whether they are from the same test lab. like the market is not making
> >speculation now?
> >
> >it is very very clear to me that they do not want to test large numbers of
> >cows in the manner of japan and europe. this is not because of kit
> >economics but because every last country that has done so, has found higher
> >numbers than their ag agencies had ever indicated possible.
> >
> >while we can wait for their next announcement, the truth is we have no idea
> >whether a non-confirmation will be the truth because testing is a totally
> >closed agency shop, eg Creekstone.
> >
> > they would never never never allow a university lab like prusiner's to get
> >their hands on this sample. why don't you throw your weight behind getting
> >some sample retested in europe with biorad and prionics and by prusiner,
> >just to restore confidence in usda?
> >
> >i do feel it is possible for there to be glitches in start-up with so new
> >many labs getting going, though i am not aware of anything technically
> >groudnbreaking, quite the contrary, about the biorad tests
> >
> >
> >
> >
> >
>
> NOW, why are we using the BIO-RAD _if_ PRIONICS is better?
>
> OR maybe PRIONICS is not as complicated as BIO-RAD?
>
> either way, we have some 8,585 (BSE-expanded) test so far and the
> 1st of 2 positive ''inconclusives'' in the 1st month is negative. OH, don't
> forget about the mad cow in TEXAS, that don't count though?
> something seems terribly wrong here.
>
> TSS
>
>
> Terry S. Singeltary Sr. wrote:
>
> >
> >
> > Release No. 0272.04
> >
> > Contact:
> > USDA Press Office (202) 720-4623
> >
> >
> >
> > Statement By Deputy Administrator Dr. John Clifford For The Animal And
> > Plant Health Inspection Service
> >
> > June 30, 2004
> >
> > At approximately, 3:45 p.m. today, we were notified by the USDA
> > National Veterinary Services Laboratories (NVSL) in Ames, Iowa that the
> > inconclusive screening test sample reported on June 25, tested negative
> > for BSE upon confirmatory testing.
> >
> > NVSL used the world-recognized gold-standard test for BSE, the
> > immunohistochemistry test to confirm this finding.
> >
> >
> > http://www.usda.gov/Newsroom/0272.04.html
> >
> >

TSS
 

bse-tester

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We showed the USDA that we had a valid and rapid urine test for PrPsc 18 months ago and the then Secretary (Anne V.) refused to acknowledge that it even existed. We also notified the CFIA in Canada that we had it and they also took a vacant attitude towards it. The EFSA in Brussels encouraged us to get it validated and sent us their Validation Protocols and to that end we are going to proceed with validation and clinical trials. Our test was proven at the United States National Prion Surveillance Center located in Cleveland and yet the USDA still doesn't wish to take it and use it even though their people know it works and can detect PrPsc in as little as 1 ml of urine. The problem is not that there are tests out there that work, as ours does, but rather the complacent attitudes within the halls of power. It boggles the mind that the ones in control of the situation can so readily ignore the greater picture that demands action today - right now in fact. Instead they sit back on their thumbs and form more committees to consider optional directions that lead nowhere.
 

PORKER

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. Our test was proven at the United States National Prion Surveillance Center located in Cleveland and yet the USDA still doesn't wish to take it and use it even though their people know it works and can detect PrPsc in as little as 1 ml of urine.
At least this can be done with a live ANIMAL and also on the KILL FLOOR.
 
A

Anonymous

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bse-tester said:
We showed the USDA that we had a valid and rapid urine test for PrPsc 18 months ago and the then Secretary (Anne V.) refused to acknowledge that it even existed. We also notified the CFIA in Canada that we had it and they also took a vacant attitude towards it. The EFSA in Brussels encouraged us to get it validated and sent us their Validation Protocols and to that end we are going to proceed with validation and clinical trials. Our test was proven at the United States National Prion Surveillance Center located in Cleveland and yet the USDA still doesn't wish to take it and use it even though their people know it works and can detect PrPsc in as little as 1 ml of urine. The problem is not that there are tests out there that work, as ours does, but rather the complacent attitudes within the halls of power. It boggles the mind that the ones in control of the situation can so readily ignore the greater picture that demands action today - right now in fact. Instead they sit back on their thumbs and form more committees to consider optional directions that lead nowhere.

After reading the Public Citizen report on the M-COOL lobbying I can see that your test has a fatal flaw--- You have not bought enough Republican politicians.....If you had been one of those big campaign donors, think where your test would be- Halliburton could be distributing it world wide for you :wink: :lol:
 

bse-tester

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After reading the Public Citizen report on the M-COOL lobbying I can see that your test has a fatal flaw--- You have not bought enough Republican politicians.....If you had been one of those big campaign donors, think where your test would be- Halliburton could be distributing it world wide for you

The one thing that will get you nowhere fast in this world is sucking up to a mess of useless politicians who will always take the position that they own you once you give them what they want. So, we will do our fund raising the old fashion way, through hard work and honesty. By the way, our test has no flaws in it at all. It works each and every time!
 

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