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Questions

mcagrawal mcanfvet at sancharnet.in
Fri Dec 21 01:14:41 EST 2001


With this message I forward two questions from Dr. Agrawal to you. If
anybody can comment on these questions, please forward the reply to
schisto at net.bio.net
(The moderator)


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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