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Bovine coronavirus is an important cause of scours in calves and winter dysentery in cattle. In the Midwestern United States, about 20% of cases are caused by bovine coronavirus. The virus affects the jejunum, ileum, spiral colon, mesenteric lymph node, and rectum. Kansas State University has developed a monoclonal antibody, 8F2 that specifically detects bovine coronavirus. This antibody also reacts with other ruminant coronaviruses, including elk coronavirus, which are antigenically, biologically, and genetically related. We have characterized this monoclonal antibody for its specificity and have found it to be sensitive and specific for detecting the virus in formalin-fixed tissues. This research has been published and the references appear below. |
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8F2-This monoclonal reacts with antigenic group II
viruses of the family Coronaviridae, thus reacting with bovine coronavirus and elk
coronavirus nucleoproteins. However, this monoclonal does not recognize antigenic group I
virus such as transmissible gastroenteritis virus (TGEV). |
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Monoclonal antibody 8F2 also detects
bovine respiratory coronavirus in tracheal, nasal gland, and lung
tissue. |
Rural Technologies, Inc. holds an exclusive, worldwide license to market and sell the 8F2 monoclonal antibody. To place an order, please contact them directly at
rtilab@ruraltechinc.com or refer to their website
http://www.ruraltechinc.com/mono.html for more information.
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Bovine
Coronavirus Immunohistochemistry Procedure |
Section slides at 4mm and air dry. Heat slides at 56°
for 20 to 25 minutes.
Deparaffinize by placing in three changes of xylene (or "Hemo-De", Fisher), 5
min each.
Rehydrate by passing through graded ETOH 100% (2 changes, 2 min each), 95% (2 changes, 2
min each), 80% (1 change for 2 min), distilled water (2 to 5 min).
Heat retrieval method:
While slides are deparaffinized/rehydrating, place citrate buffer in steamer and heat for
20 minutes (Biogenex "Antigen Retrieval Citra"). Remove slides from distilled
water, place in hot citrate buffer and continue to heat for 20 min. After the steamer
shuts off, leave slides in buffer, in the steamer, for 10 min.
*If not using heat retrieval: Incubate slides with trypsin-EDTA for 10 min at room
temperature. Place in distilled water bath for 2 to 5 min.
For peroxidase stains place slides in 3% H2O2 for 5 min to quench
endogenous peroxidase activity. Place in distilled water bath for 2 to 5 min. (Skip this
step if using an alkaline phosphatase enzyme).
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Place
in PBS/T (phosphate buffered saline with .005% Tween 20) for 10 min.
Block endogenous charged sites by incubating with PBA (Protein Blocking Agent,
Shandon-Lipshaw) for 10 min at room temperature. This product consists mostly of milk
casein.
Drain excess PBA from slide and add primary antibody (8F2 at
1:300). Incubate slides for 60 min at 37°C. Rinse slides and place in PBS/T bath for 10
min.
Add secondary antibody (Vector Labs biotin-labeled equine anti-mouse diluted 1:200 in PBS)
and incubate for 15 min at room temperature. Rinse slides and place in PBS/T bath for 10
min.
Add ABC (Avidin-biotin-enzyme complex, Vector Labs) and incubate for 15 min at room
temperature. Rinse slides and place in PBS/T bath for 10 min. (For alkaline phosphatase
enzymes I use Streptavidin Alk-Phos, from Dako, diluted 1:200 for 30 min at 37°C).
Place slides in dH2O bath for 5 min. Remove excess water and apply chromogen
(DAB, Vector Labs or Fuschin, Dako).
Place slides in dH2O bath for 5 min. Counterstain (Gill's 1 hematoxylin, Fisher
Scientific). Dehydrate (If using Fuschin the slides should be quickly dipped 10-20 times
in 95% ETOH, 100% ETOH, then xylene as the chromogen is slightly soluble in organic
solvents) and mount.
For further information on protocols contact Cindy Chard-Bergstrom at 785-532-4427.
Kapil, S. and Austin, K.M. 2000.
Global impact of bovine coronavirus infections.CABI
Animal Health and Production Compendium (AHPC)-Infectious Disease. In Press
Daginakatte,
G. C., C. Chard-Bergstrom, G. A. Andrews, and S. Kapil. 1999.
Production, characterization, and uses of monoclonal antibodies against recombinant
nucleoprotein of elk coronavirus. Clin Diagn Lab Immunol. 6:341-4.
Gaber, Fathy and Sanjay Kapil.
Development of an antigen spot test for detection of Coronavirus in bovine fecal samples.
Clin Diagn Lab Immunol. 6:542-544.
Kapil, S. and Goyal, S.M. 1995. Bovine
coronavirus-associated respiratory disease. Comp. Cont. Edu. Pract. Vet. 17: 179-181.
Kapil, S., S. M. Goyal, and A. M. Trent.
1994. Cellular immune status of coronavirus-infected neonatal calves. Comp Immunol
Microbiol Infect Dis. 17:133-8.
Kapil, S., K. A. Pomeroy, S. M. Goyal, and A. M. Trent. 1991. Experimental infection with a virulent pneumoenteric isolate of
bovine coronavirus. J Vet Diagn Invest. 3:88-9.
Kapil, S., K. L. Richardson, T. R. Maag, and S. M. Goyal. 1999. Characterization of bovine coronavirus isolates/from eight different
states in the USA. Vet Microbiol. 67:221-30.
Kapil, S., K. L. Richardson, C. Radi, and C. Chard-Bergstrom. 1996. Factors affecting isolation and propagation of bovine coronavirus in
human rectal tumor-18 cell line. J Vet Diagn Invest. 8:96-9.
Kapil, S., A. M. Trent, and S. M. Goyal.
1994. Antibody responses in spiral colon, ileum, and jejunum of bovine
coronavirus-infected neonatal calves. Comp Immunol Microbiol Infect Dis. 17:139-49.
Kapil, S., A. M. Trent, and S. M. Goyal.
1990. Excretion and persistence of bovine coronavirus in neonatal calves. Arch Virol.
115:127-32.
Schoenthaler, S. L., and S. Kapil.
1999. Development and applications of a bovine coronavirus antigen detection enzyme-linked
immunosorbent assay. Clin Diagn Lab Immunol. 6:130-2.
Zhang,
Z., G. A. Andrews, C. Chard-Bergstrom, H. C. Minocha, and S. Kapil. 1997.
Application of immunohistochemistry and in situ hybridization for detection of bovine
coronavirus in paraffin-embedded, formalin-fixed intestines. J Clin Microbiol. 35:2964-5.
Our thanks to the "American Society for Microbiology Journals Department"
for allowing permission to link to their site.
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