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.
