s1sharma at umassd.edu
Wed Mar 8 11:58:45 EST 1995
In article <Pine.3.89.9503070127.A576888081-0100000 at SKYCAT.USask.CA>, cheol.yun at usask.ca writes:
>I believe you know the book called 'CURRENT PROTOCOLS IN IMMUNOLOGY'.
>It describes all about ELISA and ELISPOT quite well.
>From my personal experience, it seemed very important the numer of
>cells (it's better to have serial dilutions of cells first to
>see the profiles) and the amount of antigen (0.5-1 ug for conA,
>however certain ag can be higher than this) for each system
>not only Igs but also cytokines like IFNg or ILs.
>Nitro-cellulose plate was working lot better than ordinary plate,
>although I was able to got the spot usiing ordinary one.
>Here are few things you should try;
> 1. Plate - mentioned above
> 2. Abs - try to find out the amount of Abs for coating
>and detecting work better (you can use recommended amount from
>protocols, I mentioned above - if you don't have I can find for
> 3. Antigen - make sure your antigen stimulate the system
> 4. Check the spots from your plates again that make sure
>they (esp. IgA) are spots nither background nor interfered
>by remaing debris/cells.
> 5. You can cross check those results from the supernatants
>Cheol H. YUN
>Animal Biotechnology Center
>Dept. Animal and Poultry Science
>University of Saskatchewan
>yun at sask.usask.ca
>On Fri, 3 Feb 1995, Daniela Verthelyi wrote:
>> Hi, I'm developing an ELISPOT system in the lab. So far I've been successful in
>> determinig the number of cells producing IgM spots, but I' run into some probl
>> ems with IgG (too few spots). Has anybody run into the same kind of problems?
>> Any ideas to help me out? Thanks in advance, Daniela.
>> Hi! Daniela, I would like to develop the elispot for Clostridium botulinum
neurotoxin can you teel me the basic procedure and main hinders in this test.
thanks in advance, Shashi
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