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CAT-221 - CA Clarity PPM v13.x Professional Certification - Dump Information
Vendor | : | CA-Technologies |
Exam Code | : | CAT-221 |
Exam Name | : | CA Clarity PPM v13.x Professional Certification |
Questions and Answers | : | 50 Q & A |
Updated On | : | December 8, 2017 |
PDF Download Mirror | : | CAT-221 Brain Dump |
Get Full Version | : | Pass4sure CAT-221 Full Version |
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QUESTION: 45
Which CA Clarity PPM Connector helps you to integrate with BMC Remedy?
- Service Connect
- Schedule Connect
- Agile Vision Connect
- Product Vision Connect
Answer: A
QUESTION: 46
In which circumstance should you use a dynamic lookup?
- When the user might want to reorder values in a list
- When you want to display changing values from tables
- When you want to perform multi-tiered lookups with a drill-down tree
- When you want to set up a display mapping to associate a value with a description and a color or icon
Answer: B
QUESTION: 47
A drilldown portlet can be (Choose two):
- A Grid portlet
- A Filter portlet
- A Graph portlet
- An HTML portlet
Answer: A, C
QUESTION: 48
Which technology is Generic Execution Language (GEL) based on?
- Oracle Java
- CA Clarity PPM
- Apache Jakarta Jelly
- SAP Business Objects
Answer: C
QUESTION: 49
What are the three operations in the Audit Trail? (Choose three)
- Move
- Insert
- Delete
- Update
- Archive
Answer: B, C, D
QUESTION: 50
Before saving a Generic Execution Language (GEL) script in a process, it is recommended that you:
- Validate the GEL script
- Remove all FTP Library tags
- Delete all namespace declarations
- Test the GEL script in a duplicate process
Answer: D
CA-Technologies CAT-221 Exam (CA Clarity PPM v13.x Professional Certification) Detailed Information
See how CA certification helps you and your company get ahead.Getting certified isn’t just about increasing your skill set. It’s also about reducing how much you rely on outside support, so you can speed issue resolution time and position yourself as a critical member of your team.
35%
Trained customers make 35% fewer calls to CA Support.
Get certified and increase customer confidence in your expertise.
Choose the right CA Technologies certification level based on your position and responsibilities within the CA solution lifecycle.
Proven Professional
The Proven Professional credential validates knowledge of the application infrastructure and typical use cases that influence how CA Technologies solutions are installed, configured and optimized for use by administrators, business analysts and end users.
Certified Expert
The Certified Expert credentials are designed for CA Partners and employees and are awarded by a panel of CA subject matter experts who validate the time spent completing actual work and your ability to perform work in the future. These credentials are product-specific and must be renewed annually.
Visit the CA Partner Portal for details >
Certified Business Analyst
Unique to CA Project & Portfolio Management (CA PPM), the Business Analyst credential validates the functional analyst’s and user’s abilities to demonstrate application management best practices—including resource and scheduling management as well as any related user-level troubleshooting.
Ready to get certified or accredited? Start here.
Get Certified.
To become certified as a Proven Professional or Business Analyst, you must pass a certification exam. Review our study materials and resources to help you master these topics.
Register for certification exam >
View Certification FAQs >
Learn about our enhanced testing experience >
Become CA Accredited.
CA wants to recognize those of you who have demonstrated superior levels of business, sales and technical expertise. Customers can register through the Customer Catalog. Partners can find out their accreditation options in the program’s Rules and Regulations, then register through the Partner Catalog. You can take your accreditation exams at a CA learning center or your home/office.
Where can I take a certification exam?
You can register to take an online proctored certification exam from your home or office, or schedule your exam at a Kryterion testing center location near you. Kryterion has testing centers in over 750 locations across the globe.
What is the format of a certification exam?
The certification exam is timed and comprises multiple choice questions. It is a proctored exam offered at a third party facility. Typically you will be presented with four answer choices, and you will select one correct answer. In the event there are two or more correct answers, you will be prompted to select the number of correct answers.
For how long is the certification valid, and what is the recertification policy?
Certification credentials are based on the major product release (such as r14), and the credential is issued as r14.x to account for subsequent minor point releases. There is no annual requirement to recertify for the same major release of a product (r14) or a minor point release (r14.1). An updated certification exam will be available for each major product release (r15, r16, and so forth). CA Employees and Partners are expected to get recertified for each major product release for which they require a credential.
Periodic updates to the exams ensure compatibility with minor point releases (such as r14.1).
On rare occasions CA Technologies may deem a minor point-release (such as r14.5) significant enough to warrant certification, in which case a new certification exam will be made available with the credential that bears that specific point release (for example, r14.5).
Which products have certifications?
Check the CA Technologies Certification page for a current table of products and related credentials. Click on the credential of interest to access resources that will help you prepare for the exam.
What is the duration of the exam?
The exams are generally 90 minutes in length.
How do I create an account and register for an exam?
Create your user account at webassessor.com/catechnologies. You may use your account to take any number of certification exams.
How can I receive help and support with account creation and registration on the Webassessor.com/CATechnologies site?
Contact the Kryterion Testing Network Support team at 800-403-6199 (US and Canada) or +1 602-281-3591 Monday 7:00 a.m. through Saturday 7:00 a.m. (Pacific Time) or send an email to ktnsupport@kryteriononline.com.
How can I receive help and support with Online Proctored (OLP) Services? (Installing software; Biometric enrollment; Test launch process)
Login at webassessor.com/catechnologies and follow the instructions to prepare for your online proctored exam. You will need to install the Sentinel software and create your biometric profile, after which you can register for your exam and schedule a date and time. You must create your biometric profile at least 24 hours prior to your online proctored exam. Refer to the guide below to review the technical requirements for your external webcam, microphone, and computer:
Preparing for Your Exam
If you have system or setup related questions the instructions are not able to answer, contact the Kryterion Online Proctored (OLP) Services Support team at olpsupport@kryteriononline.com.
How can I receive help and support finding a testing center or finding available dates and times to travel to a testing center?
Contact the Kryterion Testing Network Support team at 800-403-6199 (US and Canada) or +1 602-281-3591 Monday through Friday from 7:00 a.m. to 4:00 p.m. (Pacific Time) or send an email to ktnsupport@kryteriononline.com.
Is there a cost associated with a Certification exam?
Yes, the price for a Certification exam is USD 150-200 depending on your country, and pricing is posted at webassessor.com/catechnologies in the online registration page.
How do I pay for an exam?
The exam fees are collected and administered directly through the KryterionOnline Webassessor website.
What is the cancellation policy and can I reschedule my exam?
If you registered to take your exam at a Kryterion testing center, you can cancel or reschedule your exam up to 72 hours before your scheduled exam time. To reschedule, log into your account at webassessor.com/catechnologies, click on the details of your registered exam, and select 'Reschedule'.
If you registered to take an online proctored (OLP) exam, you can reschedule your exam up to 24 hours before your scheduled exam time. To reschedule, log into your account at webassessor.com/catechnologies, click on the details of your registered exam, and select 'Reschedule'.
You will not be refunded any portion of the exam fee, or any portion of the value of a coupon or voucher, if you do not reschedule in time or if you miss your exam appointment. Exceptions will be made for bereavement or illness supported by appropriate medical documentation.
What happens if I miss my exam appointment?
If you miss your exam appointment, you will be charged the full amount and will not be eligible for reimbursement. Terms and details will be provided to you at the time you schedule your exam. Note for CA Technologies employees: If you use a voucher to register for an exam and fail to cancel or reschedule, CA Technologies is still charged a fee, and these charges are reported to CA Technologies management.
Do customers or partners receive discounts on exams?
No, there are no standing discounts available to customers and partners for the CA Technologies certification exams. The CA Partner Program may leverage the Cost Relief program during certain periods in order to distribute vouchers for nominal discounts. However, the exams are priced reasonably. Check webassessor.com/catechnologies for pricing by country.
Will customers or partners be reimbursed for exams?
No, there are no reimbursements available for the CA Technologies certification exams.
Can partners leverage the Cost Relief program for Certification?
Yes, the Cost Relief program applies to both training and certification. The Channel Account Manager must make a request online using the Partner Education Training approval form.
Do college students receive discounts on exams?
Yes. If you are a full-time college student in good academic standing, please contact us at certification@ca.com and attach a JPEG image of your current student identification card. We will email you a coupon which will enable you to receive up to a 60% discount. You are limited to two coupons per 12-month period.
Are there any resources available to help me prepare for an exam?
Recommended Learning Paths and other resources are available to help you prepare for the certification exams.
Please visit the CA Technologies Certification page to download the Exam Study Guide that outlines the recommended training for each product certification and the topic areas tested.
Where can I register for product training?
Customers: You can register for product training through the CA Education Portal. To quickly access the recommended product training in the CA Education Portal, click on each course in the Learning Path.
Partners: You can register for product training through the Partner Catalog of the CA Education Portal. To access the Partner Catalog, log into Communities at CA using your partner credentials, go to CA Education, and choose “Find a Course.” It is important to note that unless you are logged into Communities at CA with your partner credentials, you will not be able to view the Partner Catalog. For questions, contact CA Customer Care.
What if I would like to take a course to prepare for certification, but the course is not available in the current course calendar?
You can study the information at your own pace. Alternatively, you can put yourself on the course request list, and with enough interest, a course will be made publically available. Go to the Course Catalog, find the course on the CA Education Portal, and click on Request Information to make your request.
Can I take an exam without taking preparatory courses?
Yes, if you feel you possess the required knowledge, you may register and take an exam. However, CA Technologies recommends that you take the associated courses, perform all labs, and gain hands-on experience prior to attempting an exam. Be sure and review the Exam Study Guide that shows the topic areas being tested.
When will I find out if I passed my exam?
After you have completed your exam, you will immediately receive a message by email with your score and a topic-level report.
What if I do not pass the exam on my first attempt? Can I retake the exam?
You may register for your first retake immediately. For subsequent retakes you are required to wait at least thirty (30) calendar days from the date of your last attempt.
Is there a limit to the number of times I can take an exam?
You are allowed two retakes for an exam, for a total of three attempts. Be aware that there is a waiting period between exams after the first two attempts.
If I did not pass an exam on my first attempt, will I receive a discount on future attempts?
The published fee associated with the first exam will be required for all future attempts. There are no discounts offered for multiple attempts.
Will I receive a Certification distinction?
Yes, upon successful exam and certification activity completion, you will receive an email from CA Technologies within five business days that contains the Certification from CA Technologies diploma and logo usage guidelines.
How can a third party verify my certification status?
Your email report and CA certification diploma can be used to verify your certification status. If a third party requires verification, they may contact certification@ca.com.
Do the exams change, and do the topics covered by an exam change?
CA Technologies may modify an exam, test objective/topic, and the requirements for obtaining or maintaining certification at any time in its sole discretion. It is your responsibility to keep informed of such modifications. The posted Exam Study Guides identify exam objectives and serve as a basis for exam preparation.
What is the exam security policy at CA Technologies?
Only qualified candidates who demonstrate competency by successfully passing the CA Technologies certification exam will be awarded certification credentials. Prospective certification candidates are encouraged to prepare for the exam using the Exam Study Guide and other materials available on the CA Certification page.
To safeguard the integrity of the certification program, CA Technologies requires all candidates to behave in an ethical manner and to comply with the rules of the third-party test center for on-site proctoring and on-line proctoring. The validity of the certification program depends on the exam content remaining secure and undisclosed to other potential certification candidates. CA Technologies will not tolerate cheating, fraud, or misconduct, which includes but is not limited to the following:
Obtaining, whether through purchase or otherwise acquiring, unauthorized exam preparation materials from any non-CA website
Obtaining, whether through purchase or otherwise acquiring, unauthorized exam questions from any non-CA website, including brain dump websites
Acquiring or attempting to acquire exam questions through online communities, chat rooms, forums, and social networking sites
Acquiring or attempting to acquire exam content through CA Subject Matter Experts, CA Support Staff, or CA Certified Professionals
Using or attempting to use a proxy to sit for the exam in your place
Taking or attempting to take an exam for someone else
Receiving or giving assistance during an exam
Sharing exam questions or answers with a potential certification candidate
Violating testing center rules
Falsifying a diploma or score report
Using a CA Technologies certification logo or credential to which you are not entitled
CA Technologies reserves the right to take any actions it deems reasonably appropriate in the case of suspected misconduct or violation of the terms of the Non-Disclosure Agreement, including, but not limited to, cancelling an exam result, revoking exam or certification status, terminating use of the Designation, requiring a candidate to retake an exam, banning a candidate from the certification program, and reporting misconduct to the candidate’s employer. CA Technologies considers cheating a serious offense that warrants disciplinary action, up to and including termination of employment.
For distinguished individuals who have shown superior levels of expertise
Accreditation is recognition and validation for those who have demonstrated superior levels of business, sales and technical expertise that address customer solution needs in areas of operation, administration, design, implementation and troubleshooting.
Accreditation involves successfully completing a timed exam in an open-book environment. The exam may be taken at a CA learning center or from the comfort of your living room or office.
Customer Registration
Customers can search the Customer Catalog, entering the product name followed by “exam.” You can also search by course code. Then select the Add to Cart button to register.
Search Catalog >
Partner Registration
Our Partners should consult their Partner Program Rules and Regulations to find out what Accreditation options are available to you, then access the Partner Catalog.
View Partner Education Portal >
FAQs
What is CA Accreditation?
Accreditation is recognition and validation which distinguishes individuals who have demonstrated superior levels of business, sales and technical expertise that address customer Enterprise solution needs in areas of operation, administration, design, implementation and troubleshooting.
CA Accreditation involves successfully completing a timed exam in an open-book environment. The exam may be taken at a CA learning center or in your living room or office. CA Accreditation testing allows you to use your real-world problem-solving experiences to solve real-world IT problems.
Do I have to pay for Accreditation exams?
There is no cost to take an Accreditation exam at this time.
How do I become CA Accredited?
You must successfully complete the Accreditation exam with a passing score. You may take the exam at a CA learning center or online in your home or office.
How do I prepare for my exam?
The CA Accreditation exam assesses your knowledge of a CA product. Students who complete the associated CA Education courses achieve higher scores on CA Accreditation Exams.
What do I need to have ready when I take the exam?
You can take an Accreditation exam at a CA Training Center, in your home, or at your office. For your convenience, all Accreditation exams are available through the CA Education Portal. Exams are delivered online and are open book. You should test your Internet connection ahead of time, and you may refer to course materials during the exam.
What is the format of an Accreditation exam?
CA Accreditation exams are timed exams consisting of multiple choice questions.
What does a typical exam question look like?
Example Question: Which protocols does Single Logout support?
1. SAML 1.0
2. SAML 2.0
3. SAML 1.1
4. Liberty V1.x
5. Web Services Federation (WS-Federation)
A. 1, 2, and 3
B. 2, 3, and 4
C. 2, 4, and 5
D. 3, 4, and 5
Example Question: Which component of CA Single Sign-On installs SAML authentication schemes?
A. Affiliate Agent
B. Application Agent
C. Web Agent Option Pack
D. Policy Server Option Pack
Example Question: What is the MOST appropriate step to perform when initialization fails following installation of eTrust Federation components?
A. Check the configuration environment.
B. Uninstall and reconfigure all components.
C. Compare all library files installed to identify the failure reason.
D. Ensure installed products are version compatible with one another.
How do I register for an exam?
Customer, partner and employee exams are hosted online in the CA Education Portal. To take an Accreditation exam, follow these steps:
Customer and Partner Registration
Go to the CA Education Course Catalog:
Customer Catalog
Partner Catalog Login
In the Keywords field, enter the product name followed by “exam.” You can also search for your exam by entering the course code.
To register for an exam, click the Add to Cart button. At that point, you will be prompted to log in, or register for the first time, before you can continue.
After you complete your registration and enrollment, review your selections and click the Finish button. You can then view your selection in the Training to Do List under the My Learning tab.
When you are ready to take the exam, select Go to Content to launch the exam. Please do not select Go to Content until you are ready to complete the exam; otherwise, it counts as an attempt.
Additional Information for Partners
Partners are eligible* to receive:
Free web-based training (including all 100-400 level sales, technical and support courses)
Discounted instructor-led training
For assistance accessing these benefits, please contact CA Customer Care.
*Partner must be an active CA partner.
Please note that if you do not see the course or exam that you are looking for, it is necessary to first log in to the CA Education Portal, and a complete list will be available.
Employee Registration
Go to the CA Employee Education Portal Course Search page.
In the Keywords field, enter the product name followed by “exam.” You can also search for your exam by entering the course code.
When you are ready to take the exam, select Go to Content to launch the exam. Please do not select Go to Content until you are ready to complete the exam; otherwise, it counts as an attempt.
Difficulty accessing the exam?
We recommend that you use a reliable connection to sit for the exam. If you lose network connectivity while writing the exam or if you prematurely exit the exam and can no longer access it, take the appropriate corrective action:
For Customers and Partners:
Please contact CA Customer Care. Note the exam code, and let the Support personnel know that you require an exam reset due to network connectivity causing a lock-out.
For CA Employees:
Open a Service Desk issue to request Exam User Reset:
Select Create a New Request.
If prompted to search knowledge base, select Cancel.
In the Applications section, look under E for Education Portal, and then Education Portal Support.
Be sure and provide the exam code.
What is the exam passing score? Does it change?
CA makes best efforts to maintain passing scores of 80%. The difficulty level of each Accreditation exam is adjusted by a team of internal and third-party experts to allow specific passing and fail rate thresholds. Exceptions to the 80% score occur during Beta testing of new exams, when the passing score may be lowered temporarily (for Beta test takers and early-adopter test takers) while the optimal difficulty level is being established. All of these efforts are aimed at establishing and maintaining the fairness, validity and reliability of CA exams.
Can I retake the exam if I fail?
If you fail an exam you may take the exam again immediately or at your convenience. You have two attempts at exams. If you are not successful, then you should take available training.
How do I print my Accreditation certificate?
After you have passed the exam, you may print your CA Accreditation certificate. In the CA Education Portal, go to My Learning, then Training Completed.
CA Education Portal Login:
Customers and Partners
Employees
For learning path completion diplomas, please contact accreditations@ca.com indicating the specific role-based path, and we will email the diploma to you in about three business days. If you do not receive your diploma within two weeks of successful learning path completion, please email us and we will be glad to email you a new one. Be sure and add accreditations@ca.com to your allowed sender list.
How do I obtain a copy of my score report?
Log in to your student account in the CA Education Portal, select My Learning, then Training Completed to see a list of your completed exams and scores.
CA Education Portal Login:
Customers and Partners
Employees
Why can’t I see all of my exam records in the CA Education Portal?
Exams taken prior to January 19th, 2010, will be updated in your Training Completed transcript.
How long is my accreditation valid? Does it expire?
CA Accreditations do not expire. Your Accreditation will always be valid for the product release for which you were accredited. Additional exams will be required for Accreditation on future product releases.
Personalized learning. Powerful doing.
Business is pass or fail. Make sure your people get the education and training to take your business to the next level. CA Education offers a library of over 400 courses to help your teams get the very most from every CA solution. It’s learning designed with doing in mind—helping you to improve user adoption and productivity, reduce your reliance on external support and maximize your investment to create a competitive edge.
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Pandemic influenza vaccine: characterization of A/California/07/2009 (H1N1) recombinant hemagglutinin protein and insights into H1N1 antigen steadiness abstract background
The contemporary H1N1 influenza
pandemic illustrated the shortcomings of the vaccine manufacturing
process. The A/California/07/2009 H1N1 pandemic influenza vaccine or
A(H1N1)pdm09 become accessible late and briefly provide as a result of
delays in construction caused with the aid of low yields and terrible
antigen balance. Recombinant expertise offers the opportunity to shorten
manufacturing time. A trivalent recombinant hemagglutinin (rHA) vaccine
candidate for seasonal influenza produced the usage of the baculovirus
expression vector device (BEVS) changed into proven to be as helpful and
safe as egg-derived trivalent inactivated vaccine (TIV) in human
medical studies. in this study CAT-221, we describe the characterization
of the A/California/07/2009 rHA protein and examine the H1N1 pandemic
rHA to different seasonal rHA proteins.
outcomes
Our statistics show that, like other
rHA proteins, purified A/California/07/2009 rHA forms multimeric
rosette-like particles of 20–forty nm that are biologically energetic
and immunogenic in mice as assayed via hemagglutination inhibition (HAI)
antibody titers. however, proteolytic digest analysis published that
A/California/07/2009 rHA is more liable to proteolytic degradation than
rHA proteins derived from different seasonal influenza viruses. We
identified a specific proteolytic website conserved throughout assorted
hemagglutinin (HA) proteins that is likely greater purchasable in
A/California/07/2009 HA, perhaps because of differences in its protein
constitution, and can contribute to lessen antigen steadiness.
Conclusion
We conclude that, comparable to the
recombinant seasonal influenza vaccine, recombinant A(H1N1)pdm09 vaccine
is probably going to function comparably to licensed A(H1N1)pdm09
vaccines and could present manufacturing merits.
key phrases: Recombinant hemagglutinin, Influenza pandemic vaccine,
H1N1, Baculovirus expression vector system (BEVS), Flublok, A(H1N1)pdm09historical past
A novel influenza a virus (H1N1) of swine
origin emerged in Mexico and the USA in March and early April 2009. The
virus right now unfold international via human-to-human transmission
ensuing on the earth fitness organization elevating the influenza
pandemic alert to the maximum degree (part 6) on June 11, 2009 [1-3].
The outbreak and spread CAT-221 of the primary influenza pandemic of the
twenty first century challenged licensed vaccine manufacturers to
unexpectedly mobilize and generate a prophylactic vaccine. start of
initial doses of vaccine to the U.S. public coincided with the 2d height
of the pandemic, too late to give timely insurance policy and
highlighting the need for choice production platforms [2,4].
Two types of licensed influenza vaccines can be found in
the U.S.: trivalent inactivated vaccine and are living attenuated
influenza vaccine [5,6], both produced in embryonated bird eggs. The
method of preparing a brand new vaccine seed stress suitable for boom in
eggs can be fairly lengthy, because it involves re-assortment between
the genes of a excessive yielding donor virus, akin to A/Puerto
Rico/8/34, and the hemagglutinin (HA) and neuraminidase (NA) genes of
the new influenza pressure [7]. The candidate seed virus strains are
then extra chosen for prime growth ability in eggs earlier than they can
also be used for the creation of vaccines. This manufacturing procedure
isn't simplest lengthy however also limited in scalability because of
its dependence on the availability of embryonated fowl eggs.
The production of purified recombinant hemagglutinin (rHA)
subunit vaccines by means of the baculovirus expression vector system
(BEVS) is a leading option platform for influenza vaccine manufacture.
probably the most superior influenza vaccine candidate produced the use
of this technology (below the alternate name Flublok®) is a trivalent
composition of three rHA proteins similar to the complete size HA
proteins of the seasonally circulating influenza traces [8-11]. clinical
trials of Flublok have established that the vaccine is well-tolerated,
immunogenic (as assessed by way of the induction of hemagglutination
inhibiting [HAI] antibodies), and provides protection against drifted
influenza viruses [8-10]. The rHA proteins in Flublok are produced the
use of genetically modified baculoviruses in lepidopteran insect cells.
The proteins are extracted and purified from mobilephone pellet the use
of a mix of filtration and column chromatography methods. Bulk vaccine
will also be produced within seven weeks of receipt of the HA gene
sequence [9], making it a stunning platform for pandemic vaccine
manufacturing as proven all over the preliminary outbreaks of H5N1 [12].
The original influenza seed viruses used for the
egg-primarily based production of the A(H1N1)pdm09 vaccine grew slowly,
produced particularly low portions of HA antigen and showed poor
steadiness [7,13]. The rHA derived from A/California/07/2009 also
printed alterations compared to different rHA proteins, because the
pandemic rHA protein changed into greater sensitive to proteolytic
degradation and reacted uniquely within the single radial
immunodiffusion (SRID) potency assay.
The objectives of this look at had been
(1) to study CAT-221 the houses of the A/California/07/2009 rHA protein
and compare it to different rHA proteins derived from seasonal influenza
lines and (2) to enhance an knowing of the cause of the instability
accompanied with this antigen.
effects
Biochemical and biophysical characterization
The electrophoretic mobility of purified
A/California/07/2009 rHA protein was in comparison to purified rHA
derived from A/New Caledonia/20/1999, A/Solomon Islands/03/2006, and
A/Brisbane/59/2007 H1N1 seasonal influenza lines the use of reducing and
non-reducing SDS-page (figure ). Purified rHA proteins usually migrate
as monomers and disulfide-linked oligomers beneath non-reducing
situations. The simple full-length HA0 band migrates at approximately
62 kDa, and dimer and trimer bands are approximately a hundred and
twenty and one hundred eighty kDa, respectively. As proven in figure ,
the A/California/07/2009 rHA protein displayed electrophoretic mobility
comparable to that of the seasonal rHA H1 proteins. the shortcoming of
protein bands with molecular weight greater than that of HA0 beneath
reducing situations indicates that the oligomeric kinds observed beneath
non-cutting back situations have been disulfide-linked. The
A/California/07/2009 HA0 band migrated a little faster than the HA0
comparators, possibly as a result of A/California/07/2009 HA protein has
most effective a single glycosylation web site within the globular head
(total 5 glycosylation sites) in distinction to the 9 websites
identified within the HA from A/Brisbane/fifty nine/2007 H1N1 virus
[14,15]. moreover, much less cleavage of A/California/07/2009 HA0 into
HA1 and HA2 changed into followed.
reducing and non-reducing SDS-page of H1N1 rHA proteins. For each
pattern, the respective purified rHA protein become diluted to a
awareness of one hundred μg/mL in decreasing or non-decreasing SDS-web
page pattern buffer, and 1 μg turned into ...
A/California/07/2009 rHA protein eluted as a single height
prior to a thyroglobulin molecular weight commonplace (~670 kDa) akin
to other rHA proteins when analyzed by way of HPLC-SEC (determine ),
demonstrating that rHA proteins kind high molecular weight complexes. No
peaks indicative of rHA monomers, dimers, or trimers have been
detected. The retention time of A/California/07/2009 rHA became longer
(ordinary of 36.0 minutes) than that of B/Brisbane/60/2008 rHA
(31.4 minutes) or A/Perth/sixteen/2009 rHA (31.1 minutes). This outcomes
indicates that A/California/07/2009 rHA kinds a a little bit smaller
complicated than A/Perth/sixteen/2009 or B/Brisbane/60/2008 rHA.
HPLC-SEC chromatograms of rHA proteins. Volumes comparable to 17.5 μg
of the indicated rHA proteins had been injected onto a Biosuite 450
measurement exclusion column as described in materials and methods.
Retention times for the respective rHA ...
Dynamic mild Scattering (DLS) information showed that for
all rHAs evaluated, the measured dimension corresponded to multimeric
protein particles, with the bulk falling between 20 and 40 nm in
diameter (figure ). A more suitable percent of the rHA proteins from
A/California/07/2009 and B/Brisbane/60/2008 had particle diameters
beneath 20 nm in comparison to A/Brisbane/59/2007 and
A/Perth/sixteen/2000 rHA. despite these minor modifications, the DLS
effects reveal that the A/California/07/2009 rHA protein types particles
with dimension distribution akin to that of different seasonal rHA
proteins.
dimension choice of rHA complexes through Dynamic easy Scattering.
The results show the percent of rHA protein within the size increments
indicated. The results plotted are capability of quantity-ordinary
diameters in the unique size latitude got from assorted ...
Electron microscopy images of the purified
A/California/07/2009 (H1), A/Brisbane/fifty nine/2007 (H1),
A/Perth/16/2009 (H3) and B/Brisbane/60/2008 rHA proteins are shown in
figure . A/California/07/2009 rHA formed multimeric rosette-like
structures in step with the different purified rHA proteins however have
been less distinct compared to the H3 rHA protein, which formed clearer
rosette-like structures than both the H1 or B rHA proteins. the entire
rHA rosette-like buildings measured approximately 30 – 40 nm in
measurement, in step with the measurements decided by using HPLC-SEC and
DLS.
Transmission Electron Microscopy photographs of purified rHA
proteins. All photographs had been got at nominal 52,000× magnification.
The white scale bar represents 200 nm.
Purified A/California/07/2009 rHA protein confirmed
elevated sensitivity to trypsin in comparison to other rHA proteins.
typically, HA0 is cleaved into HA1 and HA2 subunits when handled with
trypsin, and we found that A/California/07/2009 rHA became digested into
HA1 and HA2 peptides in an identical manner (determine ). besides the
fact that children, we additionally found that A/California/07/2009 HA2
turned into moreover digested into two admired peptide fragments of
about 18 and 6 kDa (special HA2a and HA2b, respectively), suggesting the
presence of an extra trypsin proteolytic site.
Trypsin digestion of rHA proteins. rHA proteins had been digested
with 50 μg/mL trypsin for 30 minutes at 2-eight°C. about 1 μg of rHA
protein become loaded per lane below decreasing situations. the two
extra ...
so as to better represent HA2a and HA2b and identify their
cleavage web page(s), the protein bands have been isolated and
subjected to N-terminal (Edman) sequencing. The amino acid sequencing
effects from the Edman analyses are proven in desk . The results confirm
that purified A/California/07/2009 rHA is produced in its mature kind
(N-terminus sequence for HA0 and HA1) with a conserved trypsin cleavage
web page at Arginine position 324 [16]. (notice that amino acid
numbering is in accordance with full length sequence.) youngsters, the
HA2 polypeptide was found to own an extra cleavage web page at Lysine
position 419 (and potential cleavage websites at Arginine-420,
Lysine-426 and Lysine-427).
Edman sequencing consequences for H1 A/California/07/2009 trypsin digested fragments
Alignment of the amino acid sequences of
A/California/07/2009 with A/New Caledonia/20/ninety nine,
A/Brisbane/59/2007 and A/Solomon Islands/03/2006 H1 HA proteins was
generated to determine even if sequences and secondary constitution
predictions might clarify the trypsin digest results (determine ). The
alignment consequences exhibit that the three capabilities additional
trypsin cleavage websites (i.e., Arginine-420, Lysine-426 and
Lysine-427) are conserved amongst all 4 HA proteins; however, Lysine-419
is exciting to A/California/07/2009. for this reason, Lysine–419 is the
without doubt fundamental protease digestion site that generates HA2a
and HA2b. interestingly, according to ExPASy proteomics tools, amino
acids 377 – 427 are estimated to form a coiled coil domain (indicated by
way of a eco-friendly over-line in determine ). A/California/07/2009
has six amino acid changes during this area, including Lysine-419, in
comparison to the different HA proteins. These sequence modifications
might affect protein conformation and make this place extra attainable
to trypsin digestion.
CLUSTAL multiple sequence alignment and secondary structure
prediction for H1 A/California/07/2009, H1 A/New Caledonia/20/ninety
nine, H1 A/Brisbane/59/2007 and H1 A/Solomon Islands/03/2006. A
constitution prediction changed into established the use of ExPASy
Proteomics equipment ...organic recreation
A/California/07/2009 rHA protein turned
into evaluated for purposeful exercise by means of picking out its
hemagglutination activity the usage of red blood cells (RBCs) and
evaluating it to the actions of the 2000–2010 seasonal H1 influenza
strain rHAs, A/New Caledonia/20/ninety nine, A/Solomon Islands/03/2006
and A/Brisbane/fifty nine/2007. A prerequisite for hemagglutination
pastime is the formation of trimers and the organization of these
trimers into greater order structures that may crosslink corresponding
sialic acid receptors on cells. The A/California/07/2009 rHA validated
hemagglutination recreation with guinea pig and turkey RBCs however not
chicken RBCs (table ). This changed into most akin to the
hemagglutination endeavor followed for A/Solomon Islands/03/2006 rHA
with guinea pig and turkey RBCs.
Hemagglutination exercise of purified H1 A/California/07/2009, H1
A/Brisbane/59/2007, H1 A/New Caledonia/20/99 and H1 A/Solomon
Islands/03/2006 rHA
A/California/07/2009 rHA protein undertaking become also
analyzed using the SRID assay. This assay measures the potency of
influenza vaccines by means of quantification of purposeful HA protein
[17]. four loads of A/California/07/2009 reference antigens comparable
to re-assortants X-181 and X-179A acquired from both the middle for
Biologics evaluation and research (CBER) and the country wide Institute
for biological requisites and handle (NIBSC), and two loads of the
A/California/07/2009 rHA (matching Genbank accession
#"type":"entrez-protein","attrs":"text":"ACP41953","term_id":"227831808","term_text":"ACP41953"ACP41953)
were demonstrated towards three different antisera generated against
hemagglutinin from A/California/07/2009 (figure ). The antisera have
been acquired from NIBSC, CBER and Protein Sciences employer, and were
generated using HA from egg, E. coli and BEVS-insect cellphone sources,
respectively. each the CBER and Protein Sciences antisera had been
experimental and produced in opposition t recombinant HA proteins (the
CBER antiserum in opposition t the HA1 fragment [18] and the Protein
Sciences antiserum against full length rHA).
SRID evaluation of recombinant and egg-derived HA proteins and
anti-serum against H1 A/California/07/2009. Reference antigens are
recognized by using re-assortant (X-181 or X-179A) and source (CBER or
NIBSC), and had been diluted to 30 μg/mL based ...
the entire antisera produced immunoprecipitin rings with
all reference antigens and the A/California/07/2009 rHA protein
(figure ), indicating that the rHA protein turned into antigenically
related. youngsters, the NIBSC antiserum generated in opposition t
egg-derived antigen produced bigger, greater diffuse rings for the rHA
(figure ) that corresponded to calculated efficiency values that were 2-
to five-fold better than the amount of purified rHA protein inoculated
into sample wells (information now not shown). In distinction to the
NIBSC antiserum, extra well described rings for rHA have been achieved
the use of antiserum generated against recombinant antigens (determine
and ). curiously, each the reference antigens and two separate licensed
A(H1N1)pdm09 monovalent vaccines (from Novartis and Sanofi Pasteur)
reacted differently with the three antisera, suggesting a different
interaction of each antiserum with every hemagglutinin created from the
A/California/07/2009 pandemic H1N1 virus. These ameliorations had a
major affect on the calculated potency of the commercial vaccines
(desk ) and reveal the should have well-matched reagents for the
pandemic H1 vaccine antigens in manufactured products.
efficiency by using SRID for licensed monovalent H1N1 vaccines calculated using different antisera and reference antigens
eventually, trivalent formulations of purified rHA vaccine
corresponding to the 2008–2009 and 2010–2011 seasonal influenza traces,
the latter of which contained A/California/07/2009 rHA, had been
organized to compare the immune responses of the diverse vaccine
add-ons. A business egg-primarily based 2009–2010 vaccine (FluLaval,
GSK, Lot # AFLLA599BA, multi-dose components) became protected as a
handle. CD-1 mice had been administered two doses of the respective
formulations at 21 day intervals, and hemagglutination inhibition (HAI)
antibody titers have been determined three weeks after each dose. The
immunogenicity effects are supplied in table .
Immunogenicity of trivalent vaccine formulations
There changed into a transparent dose
dependence of the HAI response to all vaccine accessories throughout the
look at various formulations, and the magnitude of the response
extended from Day 21 to Day 42 after the 2d immunization. The immune
response generated in opposition t A/California/07/2009 (H1) rHA antigen
changed into equal to or slightly enhanced than that of
A/Brisbane/59/2007 (H1) rHA antigen and the business egg-based
A/California/07/2009 (H1) handle by means of Day 42. The immune
responses were also constant for the H3 and B vaccine antigen
accessories. These effects exhibit that rHA antigens, including
A/California/07/2009 rHA, produce a strong immune response.
discussion
The A(H1N1)pdm09 influenza vaccine
turned into purchasable late and in brief deliver as a result of delays
in production caused by using low yields, bad antigen balance and
absence of virus stockpile. Recombinant hemagglutinin-based vaccines are
inherently less at risk of production challenges and are a leading
choice for influenza vaccine manufacture. essentially the most advanced
recombinant influenza vaccine candidate is a trivalent method of
seasonal rHA proteins that can be produced drastically faster than usual
egg-based influenza vaccines and has been shown to be as positive and
protected as egg-derived trivalent inactivated vaccine (TIV) in human
clinical trials. Pandemic rHA vaccines dangle an identical promise.
This in-depth characterization of A/California/07/2009 rHA
showed that it is biochemically, biophysically, and antigenically akin
to seasonal rHA antigens. A/California/07/2009 rHA had an
electrophoretic mobility corresponding to that of seasonal rHA proteins
and shaped better order, multimeric rosette-like particles of
approximately 20–40 nm. this is in distinction to the findings of
Khurana et al. who expressed full size A/California/07/2009 HA in a
bacterial expression device and located that the purified protein
migrated predominantly as a monomer [18]. The cause of this difference
is unknown however could relate to the diverse expression platforms.
A/California/07/2009 rHA confirmed significant organic exercise and
elicited a powerful immune response in mice according to that generated
by way of commercial egg-derived A/California/07/2009 vaccine both
during this examine and up to now [19,20]. together, these data guide
the suitability of A/California/07/2009 rHA as a plague influenza
vaccine alternative. An initial scientific study CAT-221 has established
the defense and immunogenicity of this rHA [21].
The antigenic balance of the A(H1N1)pdm09 vaccine became
discovered to be at the beginning poor [13]. Trypsin digestion of
purified A/California/07/2009 rHA uncovered a special susceptibility of
the protein to proteolytic cleavage no longer found in the seasonal rHA
comparators. N-terminal (Edman) sequencing published that this cleavage
occurs in a subdomain of the HA2 vicinity of the protein that for most
HA proteins are predicted to be structured as a coiled-coil. We
postulate that the six amino acid adjustments in this domain in
A/California/07/2009 HA may also disrupt this constitution, leading to
decreased antigenic steadiness. additional experiences are necessary to
check no matter if the virus re-assortants in the end used for egg-based
mostly A(H1N1)pdm09 vaccine manufacture possessed adjustments that
impacted protein constitution in this location, enhancing antigen
steadiness. Preliminary evaluation suggests that a purified recombinant
rHA derived from re-assortant virus NIBRG-121xp [7] really remained
unstable although its interplay with sialic acid receptors turned into
superior (records no longer proven).
eventually, the antigenic potency of
A/California/07/2009 rHA, as determined by using the SRID assay,
confirmed dramatic heterogeneity (≥ 2-fold) reckoning on the assay
reagents used. an identical effect changed into followed for licensed
egg-derived monovalent vaccines. this modification suggests that
training of reagents for efficiency testing (antisera and reference
antigens) with novel pandemic influenza viruses requires further
assessment to accommodate recombinant manufacturing ideas purchasable
for quick pandemic response. in addition, the construction of option
potency assays which are less stylish upon selected antigen-antibody
interactions that can be suffering from the manufacturing platform is
warranted.
Conclusions
These outcomes reveal that the
production of purified recombinant hemagglutinin (rHA) subunit vaccines
by means of the baculovirus expression system is a leading alternative
platform for influenza vaccine manufacture. The biochemical, biophysical
and immunological characterization of a purified recombinant
A/California/07/2009 (H1N1) hemagglutinin has been compared to
distinctive seasonal rHA proteins and egg-produced A/California
reagents. The records display that purified A/California/07/2009 rHA
molecules exist in high molecular weight complexes and form rosette-like
particles of 20 – forty nm in size. Biochemically, the protein displays
hemagglutination activity and a superior sensitivity to tryptic
digestion with additional cleavage in the HA2 subunit. The pleasing
constitution of this particular HA antigen might also account for bad
steadiness. The A/California/07/2009 rHA protein became found to be
antigenically akin to egg-derived virus and confirmed immunogenicity and
construction of neutralizing antibody titers in mice.
strategies
rHA cloning and baculovirus technology
The H1 A/California/07/2009 cDNA
was generated by using influenza viral RNA as a template in a reverse
transcriptase PCR reaction (RT-PCR). The cDNA changed into cloned into
baculovirus transfer vector pPSC12, and advantageous clones had been
established by means of DNA sequencing to be just like the H1
A/California/07/2009 reference sequence (GenBank accession #
"classification":"entrez-protein","attrs":"text":"ACP41953","term_id":"227831808","term_text":"ACP41953"ACP41953).
Spodoptera frugiperda Sf9 cells were co-transfected with linearized
Autographa californica assorted capsid nucleopolyhedrovirus (AcMNPV)
genomic DNA and the pPSC12 transfer vector containing H1
A/California/07/2009 cDNA through calcium phosphate precipitation. This
system generated recombinant baculoviruses harboring the gene encoding
H1 A/California/07/2009 by homologous recombination. Recombinant plaques
had been remoted and used to generate baculovirus shares in expresSF+ ®
(SF+) insect cells. extra particulars on the cloning and expression of
alternative rHAs using this device are described elsewhere [9,22,23].
rHA protein creation
The recombinant baculovirus stock
changed into used to supply the H1 A/California/07/2009 rHA protein.
Virus inoculum from the working virus stock become delivered to 450 L of
SF+ insect mobilephone culture in a 600 L bioreactor at a attention of
2% (v/v) after the insect cells reached a density of 2.0 – 2.5 × 106
cells per mL. The contaminated culture was incubated at 28°C for 40 –
55 hours and harvested at a viability of 70 - eighty%. A cell pellet was
generated by using centrifugation and the recombinant protein become
solubilized the use of a buffer containing non-ionic detergent. Cells
had been removed with the aid of depth filtration, and the clarified
extract became utilized to an ion-alternate column. Recombinant HA
changed into eluted and subsequently certain to a hydrophobic interplay
column. Following elution, the protein was utilized to a Q-membrane to
eliminate any residual DNA. at last, Q filtrate was diafiltered and the
rHA protein formulated in last buffer.
SDS-web page and western blot
Proteins were separated the use of
4-12% NuPAGE Bis-Tris Gels (Cat# NP0323, life applied sciences
organisation, Carlsbad, CA) and 1x MES operating Buffer (50 mM MES,
50 mM Tris, 0.1% sodium dodecyl sulfate, 1 mM EDTA pH 7.three).
Non-reducing SDS-web page gel samples were organized the usage of 2x
non-reducing disruption buffer (a hundred and twenty mM Tris pH 6.8, 20%
glycerol, four% sodium dodecyl sulfate, 0.2% bromophenol blue). cutting
back SDS-page gel samples had been prepared the usage of 2x disruption
buffer (one hundred twenty mM Tris pH 6.8, 20% glycerol, four% sodium
dodecyl sulfate, 0.2% bromophenol blue, 200 mM dithiothreitol). The
SDS-page gels have been fixed in pH 1.1 fixative (25% methanol, 10%
glacial acetic acid, 10% thrichloroacetic acid) for 10 minutes, followed
through staining in Coomassie Blue staining solution (0.1% staggering
Blue R, 7.7 M reagent alcohol, 1.75 M glacial acetic acid) for
60 minutes. The gels were de-stained in 10% acetic acid. For the Western
Blot, the separated proteins were transferred to nitrocellulose
membrane (Cat# IB3010, existence technologies organisation) or to
polyvinylidenefluoride (PVDF) membrane (Cat# IB4010, lifestyles applied
sciences organization) and incubated with a 1:1,000 dilution of
A/California/07/2009 (H1N1) influenza antiserum (Lot #09/152, sheep
506/507, NIBSC, UK) adopted by means of a 1:three,000 dilution of
anti-sheep IgG (total molecule) alkaline phosphate secondary antibody
(Cat# A5187, Sigma, St. Louis, MO). Recombinant H1 A/California/07/2009
proteins had been visualized the usage of chromogenic alkaline
phosphatase substrates, 1x NTB (nitro-blue tetrazolium chloride, Cat#
0329, Amresco, Solon, OH) and 1x BCIP
(5-bromo-four-chloro-three-indolyl-phosphate, p-toluidine salt, Cat#
0885, Amresco) in chromagen buffer (2-amino-2-methyl-1-propanol, Cat#
221, Sigma) per the producers’ guidance.
Trypsin digestion
reaction combinations containing an
rHA attention of 250 μg/mL have been incubated within the absence or
presence of 50 μg/mL trypsin (Cat# T1426, Sigma) for 30 min at 2-8°C.
Digestion was stopped by way of heating the samples in 2x disruption
buffer. Samples had been analyzed by using SDS-page and Western
blotting, as described above.
Hemagglutination assay
The hemagglutination assay become
carried out in U-backside 96 smartly microtiter plates. Recombinant HA
antigen (starting concentration of 1 μg/mL; protein awareness decided by
BCA [Bicinchoninic Acid; Cat# 23225, Thermo Scientific, Rockford, IL])
become diluted by two-fold serial dilution to a final dilution of
4,096-fold. fresh purple blood cells (RBCs) were washed with 1x PBS (pH
7.2) after which brought to the wells. RBCs used consist of 0.5% chook
RBCs from Charles River SPAFAS, Charleston, SC (Lot #A101213), 1% guinea
pig RBCs from ViroMed Laboratories, Minnetonka, MN (Lot#11916), and
0.5% turkey RBCs from ViroMed Laboratories (lot#10984). After one hour
of incubation at room temperature, the plates were scored for
agglutination. The HA pastime is described through the dilution at which
partial agglutination changed into accompanied (i.e., 50% of the RBCs
were agglutinated or the pellet appeared unfastened). If best totally
agglutinated and/or tight pellets were accompanied, the endpoint changed
into described because the common of the dilutions with agglutinated
and tight pellets. If no agglutination became accompanied in any neatly,
then the endeavor of the look at various article was deemed lower than
20 gadgets/μg of protein.
dimension exclusion chromatography (HPLC-SEC)
size exclusion chromatography
(HPLC-SEC) turned into carried out through the use of an Agilent HPLC
equipment (Agilent technologies, Santa Clara, CA) with UV or diode array
detectors and a Biosuite 450, 8 μm HR SEC column (7.eight × 300 mm)
(Waters; Cat#186002166). Samples were run in a mobile phase containing
1x PBS with 300 mM NaCl pH 7.2 at a stream fee of 0.25 mL/min. For each
and every monovalent bulk batch of A/California/07/2009 rHA, 17.5 μg of
protein became used. Sizes of rHA multimers were estimated according to a
common curve generated the usage of reagents from a gel filtration HMW
calibration kit (Cat# 28-4038-42, GE Healthcare Piscataway, NJ).
Dynamic light scattering
Dynamic light Scattering become
carried out on purified rHA protein the use of a Malvern Zetasizer
Nano-S (Malvern devices, Worcestershire, UK) in accordance with the
manufacturer’s guidelines. Zetasizer application (edition 6.20) turned
into used for information evaluation. Bulk drug substance was analyzed
devoid of dilution through including 60–70 μL of pattern to a
microcuvette and study CAT-221ing (customarily) three units of 12–14
individual scans. The volume-general measurement distributions had been
averaged and binned to <20 nm, 20–40 nm, and >forty nm.
Electron microscopy
Transmission Electron Microscopy
become carried out by means of Nanoimaging services (La Jolla, CA).
Samples have been organized the usage of a continuous carbon grid method
with grids of nitrocellulose supported 400-mesh copper. Three
microliters of 1:100 diluted samples (~6 μg/mL protein) had been applied
to a cleaned grid, blotted with filter paper, and immediately stained
with uranyl formate. Electron microscopy changed into carried out using
an FEI Tecnai T12 electron microscope, working at 120KeV, outfitted with
an FEI Eagle 4 okay × 4 ok CCD digital camera the use of nominal
magnifications of one hundred ten,000x (0.10 nm/pixel), 52,000x
(0.21 nm/pixel), and 21,000x (0.50 nm/pixel) at electron doses of about
10–15 e/Å2. terrible stain grids had been transferred into the electron
microscope using a room temperature stage.
Edman sequencing
approximately 6 μg of trypsin
treated or untreated A/California/07/2009 rHA become loaded on an
SDS-page gel and run beneath cutting back conditions. Protein bands have
been transferred to a PVDF membrane and stained with 0.1% Coomassie
Blue R-250 in forty% methanol and 1% acetic acid, followed with the aid
of de-staining with 50% methanol and water. Protein bands of interest
have been excised from the PVDF membrane and submitted to the Protein
Core Facility at Columbia tuition (new york, big apple) for N-terminal
(Edman) sequencing. The stained bands were sequenced on an utilized
Biosystems 494 protein sequencer based on the company’s guidelines.
Single radial immunodiffusion (SRID) assay
SRID assays have been carried out
as described up to now [17,24]. in brief, an antibody answer at the
greatest working attention became blended with melted 1% agarose (Cat#
50010, SeaKem ME, Lonza, Rockland, ME ) in 1x PBS (pH 7.2) (Cat#
20012–050, life technologies employer) at 54° - fifty six°C. Following
solidification on GelBond movie (Cat# 53734, Lonza, Rockland, ME) at
room temperature, four mm wells have been punched in the gels.
preliminary dilutions of test samples and reference requisites have been
prepared in 1% Zwittergent three–14 (Cat# 693017, Calbiochem,
Darmstadt, Germany), incubated for 30 min at room temperature, and
additional diluted with 1% Zwittergent three–14 in PBS. Twenty
microliter samples had been then utilized to the agarose wells. The gels
were positioned in a sealed moist chamber at room temperature for
18 hours. Following incubation, the gels had been washed first with 1x
PBS (pH 7.2) and then water, dried and stained with Coomassie dazzling
Blue R250 (Cat# BO149, Sigma). After de-staining, the gels have been
dried, and SRID ring diameters had been measured the use of the GT
vision SRID study CAT-221ing program (GT vision LLC, Hagerstown, MD).
The diameters of the precipitin rings were measured in two orthogonal
instructions. Recombinant HA efficiency was calculated in μg/mL by way
of the parallel line bioassay system the usage of reference and test rHA
antigen response curves (log antigen dilution vs. log zone diameter).
Statistical parameters for determining check validity had been in
accordance with correlation coefficients (r ≥ 0.95) and the equality of
slopes (t < 4.604) between test and reference antigens. NIBSC
antiserum lot 09/152, CBER experimental antiserum lot H1-Ab-1004 and
Protein Sciences Protein G purified rabbit antiserum had been used
within the SRID assay. in addition, X-181 CBER antigen lot H1-Ag-1002,
X-181 NIBSC antigen lot 09/294, X-179A CBER antigen lot 69, X-179A NIBSC
antigen lot 09/174, and two diverse purified rHA plenty had been used.
Licensed (egg-derived) monovalent A/California/07/2009 H1N1 pandemic
vaccines from two manufacturers have been additionally evaluated (Sanofi
Pasteur Lot UP088AA and Novartis Lot 110739).
generation of rabbit polyclonal antibody
New Zealand White Rabbit immunization
become performed beneath contract with Harlan Bioproducts for Science,
Inc. (Madison, Wisconsin). An accepted 112 day animal protocol was used.
Recombinant H1 A/California/07/2009 was used as an antigen. Freund’s
adjuvant turned into used to generate the rabbit antiserum.
approximately 2 mg of antigen at a minimum protein concentration of
0.5 mg/mL became used per rabbit. The rabbit antiserum from the closing
bleed was purified over a Protein G Sepharose column (Cat# 17-0618-05,
GE Healthcare) per the brand’s strategies. The eluted antibody fraction
changed into dialyzed in opposition t 1x PBS (pH 7.2) (Cat# 20012–050,
lifestyles applied sciences company). The purified rHA H1
A/California/07/2009 antibody turned into used in the SRID assay. All
work became conducted ethically, with animal welfare as a correct
precedence, in full compliance with countrywide animal welfare
regulations, below PHS Assurance in a completely USDA licensed facility.
[http://www.harlan.com/about_harlan_laboratories/animal_welfare.hl].
Animal immunization and HA inhibition (HAI) assays
The Animal Core Facility at Colorado
State school (CSU) carried out the immunization and resolution of HAI
titers. briefly, 6 – 8 week ancient feminine CD1 mice had been
administered trivalent vaccine formulations containing purified
recombinant H1 A/California/07/2009 rHA, H1 A/Brisbane/59/2007 rHA, or
an A/California/07/2009 egg-based commercial vaccine in trivalent
formulation (FluLaval®, GSK, Lot # AFLLA599BA, multi-dose method) by way
of intramuscular (IM) injections. The formulated doses have been in
response to SRID.
For HAI titer resolution, particular
person serum samples were treated with receptor destroying enzyme (RDE,
from Vibrio cholera Denka-Seiken, Tokyo, Japan) to eliminate
non-specific inhibitors and tested against four hemagglutination
contraptions (HAU) of the respective influenza viruses grown in eggs the
use of 0.5% chook RBCs as prior to now described [25]. All serum
samples had been validated in duplicate at a 1:10 starting dilution. The
HAI titer become defined because the reciprocal of the premiere
dilution that completely inhibited the agglutination of the hen RBCs. A
titer value of 5 changed into assigned to represent responses under the
assay detection restrict. All work changed into performed ethically in
compliance with all federal, state, and local laws, regulations, and
policies, in addition to Colorado State tuition interior policies
http://web.analysis.colostate.edu/ACP/rules.aspx.
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