CLINICAL ANDDIAGNOSTICLABORATORYIMMUNOLOGY, 1071-412X/99/$04.0010 July 1999, p. 542544Vol. 6, No. 4 Copyright 1999, American Society for Microbiology. All Rights Reserved. Development of an Antigen Spot Test for Detection of Coronavirus in Bovine Fecal Samples FATHY GABERANDSANJAY KAPIL* Department of Diagnostic Medicine-Pathobiology, College of Veterinary Medicine, Kansas State University, Manhattan, Kansas 66506 Received 3 March 1999/Returned for modifi cation 21 April 1999/Accepted 26 April 1999 We have developed a rapid and sensitive microimmunodot blot assay, the antigen spot test (AST), for the detection of bovine coronavirus (BCV) antigen from neonatal calf fecal samples. The AST procedure can be completed in 3.5 h, whereas the previously reported immunodot blot assays require 10 to 12 h. Ninety-six samples can be tested per membrane, and 10 membranes (960 samples) may be processed by a single technologist in 1 working day. The effects of detergents, oxidizing chemicals, chaotropic agents, and enzyme substrates in improving the sensitivity and signal-to-noise ratio of the AST were studied. Finally, the sensitivity and specifi city of AST for the detection of BCV antigen were compared to those of a sandwich enzyme-linked immunosorbant assay (ELISA) and a hemagglutination assay (HA). Of 347 fi eld samples tested by all three methods, 94.2% were positive by AST, 91.4% were positive by ELISA, and 86.7% were positive by HA. The sensitivity of the AST was determined to be 100% compared to the results of the ELISA reference method. The specifi city of the AST was 67%, which refl ects a lower limit of detection of 104viral particles per ml in a 10% fecal suspension. Bovine coronavirus (BCV), an enveloped, positive-strand RNA virus, is an important cause of diarrhea in calves of up to 4 weeks of age (13, 22), and it is frequently detected in the feces of adult cattle with winter dysentery (1, 2, 1820, 22). Neonatal diarrhea caused by BCV is responsible for heavy economic losses in dairy and beef cattle (11). BCV is second only to rotavirus as a cause of enteric infection in calves be- tween 1 and 16 weeks of age (8, 9, 12, 14, 16, 17). Virus isolation in cell culture is considered to be the “gold standard” for the detection of most viruses in clinical samples. This technique, however, is rarely used for the diagnosis of BCV infection because no cell culture system which can reli- ably propagate BCV to a high titer from clinical samples has been identifi ed (4, 5, 21). Monoclonal antibody (MAb) tech- nology has facilitated the development of sensitive and specifi c tests for the detection of many microbial and viral antigens in clinical specimens. Several immunological techniques which incorporate the use of MAbs have been described, including enzyme-linked immunosorbent assays (ELISAs) (4, 5, 21, 24), direct- and indirect fl uorescent-antibody tests (9, 20, 23), im- munohistochemical staining of fi xed tissues (6, 26), and immu- noblot assays (10). The advantages of using dot blot immuno- assays for the detection of viral antigens directly from tissues, swabs, washings, and body secretions are (i) culture of virus in cells, eggs, or laboratory animals is not required; (ii) large numbers of specimens can be handled simultaneously; and (iii) immunoassays are sensitive and specifi c and can be performed rapidly. In this study, a rapid blot assay for the detection of BCV spike protein and nucleoprotein developed in this laboratory is described. The assay has increased sensitivity compared to the previously described sandwich ELISA (21). The effects of treatment of the antigen blots with detergent, chaotropic agents (which change the structure of biomolecules, such as protein conformation), and oxidizing solutions and the use of two detection substrates were studied to optimize the sensitiv- ity of the immunoblot technique. MATERIALS AND METHODS Specimens. Fecal samples were obtained from 347 neonatal, diarrheic calves submitted to the Kansas State University Veterinary Diagnostic Laboratory, Manhattan. Fecal samples from naive, BCV-free calves obtained immediately after birth and kept in an isolation facility were used as negative controls. All fecal samples were processed before testing by vortexing 0.5 g of feces in 5 ml of 0.01 M phosphate-buffered saline (PBS; pH 7.0) and then centrifuging at 1,800 3 g for 5 min at room temperature. The supernatants, which were used for testing, were transferred to a sterile vial and were stored at 270C. Stock cultures (108 PFU/ml) of two BCV isolates, California-1 (CA-1) and Wisconsin-1 (WI-1-SK), were used as positive controls for all tests. Antibodies. MAbs Z3A5, to the BCV spike protein (26), and 8F2 (6), to the BCV nucleoprotein, were prepared and tested in this laboratory as described previously (6, 26). Both MAbs were of the immunoglobulin G1 (IgG1) isotype. The secondary antibody used was horse anti-mouse IgGhorseradish peroxidase (HRPO) conjugate (Vector Laboratorie