Billie,

there have previously been a number of very helpful responses to this same
question which are available in the histonet archive. 
One in particular by Jim Burchette from Duke Uni was so good I saved it for
my own future reference. I have included it below. It should answer all of
your questions plus some you didn't ask. Some people respond so well to
histonet questions there should be an annual award for these folk. Jim you
are one of the histonet legends.

Regards,

Mark Donovan
Anatomical Pathology
The Alfred Hospital
Melbourne, Australia

From James Burchette, Duke University  posted 22-04-2003 on Histonet

I use the SV40 T Ag (Ab-2) from Oncogene, catalog # DP02. I have found steam
HIER pretreatment with DAKO Target Retrieval, pH 6.0 and Envision Plus is
the best method for detecting this antigen. We use this antibody at a
dilution of 1:800. Most requests we get for this antibody usually come from
the nueropathology group. The anti-SV40 antibody will also react with BK
polyomavirus. One of two human pathogenic polyomaviruses, JC virus causes a
devastating infection to the central nervous system called progressive
multifocal leukoencephalopathy (PML) of immunocompromised patients.
Generally, oligodendrocytes within the white matter of the brain are the
target cells of this virus.

For detecting BK virus large T antigen, I use Chemicon's monoclonal
antibody, catalog # MAB8505. BK virus infects a variety of urinary tract
epithelia of immunocompromised patients and with increasing frequency renal
transplant recipients. This antibody does not react with the JC
polyomavirus. 

IHC for BKV in FFPE sections of human renal transplant biopsies, requires
proteolytic enzyme digestion rather than HIER methods. The use of stable
trypsin solution for pretreatment is my choice. Tissue sections are placed
on charged glass slides. Sections are digested with the stable trypsin
solution (see below) for 13 minutes at 37-40 C. One hour primary with the
BKV diluted 1:16,000 and 30 minutes with DAKO's Envision Plus for mouse
primary antibodies followed by 4 minutes of DAB results in a very nice
immunohistochemical preparation. The use of a biotin free detection system
has allowed us to consistently have high signal to low noise IHC. We have
noticed sections that contain high inflammation, have increased background
or non-specific staining of these inflammatory cells. This is thought to be
from the antigen source used to prepare this clone.

Dave Tacha with BioCare Medical and myself worked up this stable trypsin
reagent a few years ago (12+?). For demonstrating BKV, I find this trypsin
works better than high temperature antigen retrieval (steam or pressure
cooker). There is less tissue disruption, less nonspecific biotin staining,
and the trypsin is faster. This isn't the magic bullet for all antibodies,
but is the ticket for a few. Remember, when working up antibodies, always
compare different enzymes against the many HIER techniques and solutions.
Sometimes no pretreatment is the answer. 

The stable trypsin recipe follows: 

Tacha's Stable Trypsin
Stable trypsin is used for demonstrating Adenovirus and BK Polyomavirus in
formalin-fixed, paraffin embedded tissue sections. Also good for the UEA
lectin and Mac387.

1% Trypsin Stock
1 gram trypsin (Sigma T8138 or T7409)
100 ml distilled water
Dissolve trypsin in distilled H20 and adjust to pH 3.5.
Prepare 3-4 ml aliquots. The shelf life* of stable trypsin is at least
six-month's at 4°C or indefinite when frozen at -70°C.

*Note: I never had problems with bugs growing in T8138. However, since Sigma
has discontinued T8128 and offers the replacement T7409, I have noticed the
stability of 1% stock solution of T7409 to be less than 6 months. A thawed
3-4 ml alloquot of trypsin stock should last at least a month. You can
always freeze smaller alloquots of stock trypsin solution if low use
dictates this need. Keeps forever at -70C or lower.

Buffer Stock Solution
100 ml 1X Automation Buffer (Biomeda M-30)
100 µl of Tween 20
100 mg calcium chloride
10 mg sodium azide
Adjust pH to 7.8. Shelf life is at least six-month's when stored at 4=B0C.
Aliquot's may be frozen and stored indefinitely at -70°C for future use.

Working Trypsin solution 0.25%
Mix:
100 µl Trypsin Stock
300 µl Buffer Stock
Prepare fresh before using.

Use at 37-40°C for 12-15 minutes. Rinse with D H20 to stop enzyme reaction.

* Sigma # T8128 has been replaced with # T7409. Sigma indicated this is same
product and is being produced by Sigma rather than an outside vendor. I now
use #T7409 for all of my stable trypsin pretreatment needs.

Reference:
DIAGNOSIS AND MANAGEMENT OF BK POLYOMAVIRUS INFECTIONS IN RENAL TRANSPLANT
RECIPIENTS. DN Howell, SR Smith, DW Butterly, PS Klassen, HR Krigman, JL 
Burchette, SE Miller. Transplantation, Vol. 68, No. 9, pages 1279-1288,
November 1999.

I hope this information is of help to you.

Sincerely,
Jim Burchette, HT(ASCP)
Duke University Medical Center
Immunopathology Laboratory
Box 3712
Durham, NC 27710
919-681-3973
burch007@mc.duke.edu 

 







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