Questions

Tam, Milton mtam at path.org
Mon Jan 14 15:48:37 EST 2002


>Dear Dr. Agarwal:
>
>There is not enough information for me to answer the first question.  What
>us the yield of protein in mg expected for 500 flukes and how does this
>compare with the amount found by your Lowry determination?  Has a standard
>been included in your initial protein determinations?  What percentage of
>the worms is protein vs. other substances, i.e. carbohydrate/lipid?
>
>For the IFA, if fixation of schistosomes is similar to other parasites or
>virus-infected cells, then the simplest method could be the best.  Take a
>purified suspension of worms in saline and apply a few microliters to each
>well of a CLEAN 8- or 10-well microscope slide (those with painted wells and
>designed for IFA are commercially available) so that each well has at least
>a few parasites and allow to air dry. After drying, immediately fix in 95%
>ethanol or 100% acetone for 15-30 minutes.  The slides can now be frozen for
>storage at -20 deg or below indefinitely.  Titrations of positive and
>negative test sera and conjugate can then be done, but it is essential to
>use PBS + 1.0% BSA in diluting the test and control sera to inhibit
>background fluorescence.  I would start at 1:10 and then do doubling
>dilutions.
>
>Incubate 15-30 min at anywhere from RT to 37 deg. Aspirate the sample
>carefully from the slide and wash in PBS.  One 5-10 min soak with
>intermittent agitation is usually sufficient.  Similarly, the conjugate
>needs to be diluted in PBS+BSA to reduce background. Some commercial
>conjugates are of high titer, so it may be necessary to dilute them as much
>as 1:500-1:1,000 or more in order to obtain specificity.  Some commercial
>conjugates are better and more specific than others so it might be necessary
>to evaluate 3-4 different ones before a good one is found.
>
>After the last conjugate wash, it may be helpful to use a counterstain such
>as Evan's blue to reduce background, but be careful not to over-stain which
>reduces specific immunofluorescence reactions.  Use tris-glycerol pH 8.0 for
>mounting coverslips. There are substances you can add to the tris-glycerol
>to preserve specific fluorescence and inhibit "bleaching."  Good luck.
>Please contact me and let me know if any or all of the above steps improve
>on the quality of your IFA.
>
>Best regards
>
>Milton Tam
>
>Milton R. Tam, Ph.D
>Technical Director
>PATH
>4  Nickerson Street
>Seattle, WA 98109, USA
>Phone 206-285-3500, fax 206-285-6619
>"All the electrons in this message have been recycled"
>
>
>-----Original Message-----
>From: mcanfvet at sancharnet.in [mailto:mcanfvet at sancharnet.in]
>Sent: Thursday, December 20, 2001 10:15 PM
>To: schisto at net.bio.net
>Subject: Questions

>Subject: Lower  protein concentration in  schistosome homoginate
>
>Dear Colleagues,
>
>At Veterinary College JNKVV, Jabalpur, we are trying  to develop  ELISA
>methods for man  and animals. In the intial stage, we have taken  about  500
>blood flukes of Schistosoma spindale in 4 ml  phosphate buffer saline and
>ultrasonicated with due interruptions.
>The protein estimation by Lorrys method, however, could demonstrate only
>3.03-0.018 mg/ml. Obviously, this  is  a very  low  concentration as we
>expected a
>higher concentration  due to  large numbert of worms.
>We solicite help of  the  scientist , how it is possible to  improve the
>protein yield from
>schistosome homogenate.
>
>Subject: Problem in IFT using schistosome cercariae.
>
>We are also trying to develop some immunodiagnostical methods for checking
>schistosomiasis in animals and man.
>To this end, we have taken cercariae of Schistosoma incoginitum killed by
>air drying
>or by heat. They were processed as follows for  IFT
>1. Fixation was done in 70 % alcohol for 5-10 min., followed by a treatment
>with 1% BSA for 20 min.
>2. Three washes were done with 7.2 pH  phosphate buffer saline (PBS) in 5
>min. intervals.
>3. Treated with  50 ul  test sera with different dilutions: 1: 100, 1:50,
>and
>1:25. Than incubated for 60 min., and in the same way non-infected control
>sera were also tested.
>4. Three washes with PBS
>5. Treated with 50 ul - 1:100 diluted conjugate and incubated for 90 min.
>6. Three washes with  PBS
>7.  Mounted in 50% glycerol for flourescence microscopy.
>
>We hope for suggestion  from experienced scientists, why  our method is
>so non specific. It appears poor compared to CHR. However, in our openion,
>IFT
>with  cercariae will  be more useful as it will illuminate use of alive
>cercariae.
>
>Dr. M.CAgrawal.







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