Production of a Monoclonal Antibody Reacted Broadly with Feline Calicivirus Field Isolates.

Abstract

ABSTRACT. A monoclonal antibody (MAb) reactive with 36 field isolates and 2 laboratory strains of feline calicivirus (FCV) was produced by immunizing mice with the mixture of FCVs. The MAb (4D7) reacted with FCVs in an enzyme-linked immunosorbent assay (ELISA), but had no neutralizing activity against the F4 strain of FCV. MAb 8G1, previously produced against the FCV F4 strain, also reacted in ELISA with all FCVs used in the present study. However, the epitopes recognized by 4D7 and 8G1 were different. Using these two MAbs and a polyclonal rabbit antibody, we attempted to develop a sandwich ELISA for detection of FCV antigen. The combination of 4D7 and the polyclonal rabbit IgG was most sensitive. Using this system, all the field isolates of FCV cultured in vitro were detected. However, among the 36 swab samples, from which FCV was isolated, 4 were negative.-KEY WORDS: feline calicivirus, monoclonal antibody, sandwich ELISA.J. Vet. Med. Sci. 60(2): 155-160, 1998 Serial passages of 9 field isolates, F4 and F9 strains were carried out and the high-passaged viruses, passage number of 100, were also used.The 90-8A strain of FHV1, isolated in 1990 from a cat with respiratory disease, was also used in the present study.Preparation of virus antigen: Virus antigen for immunization and ELISA was prepared from culture fluid of CRFK cells inoculated with each strain of FCV. The culture fluid of FCV was collected and centrifuged at 1,000G for 10 min. For precipitation of the virus, 22.8 g of ammonium sulfate was added to 100 ml of the culture fluid. After centrifugation at 10,000G for 20 min, the virus was suspended in Dulbecco's phosphate buffered saline (PBS) and dialyzed overnight against PBS at 4°C. Following centrifugation at 700G, the protein concentration was measured by using the Bio Rad protein assay (Bio Rad Laboratories, Inc., CA, U.S.A.). For immunization, four types of antigen were prepared. Antigen A was a mixture of all strains of FCV. Antigen B and antigen C were each a mixture of 13 strains of FCV field isolates. Antigen D was a mixture of 10 field isolates, together with the F4 and F9 strains of FCV.Anti-FCV monoclonal antibodies: Six week-old BALB/ cCrSlc mice were immunized with antigen A intraperitoneally followed by a second immunization with antigen A subcutaneously 1 week later. The mice were then immunized subcutaneously with antigen B, C and D at 5 day intervals. The last immunization was performed subcutaneously using antigen A. Three days after the last immunization, the mice were killed and the spleen cells fused with the myeloma cells, P3X63Ag8.653 using polyethylene glycol 1,500 (Boehringer Mannheim GmbH, Germany). Hybridomas were selected using HAT medium Feline calicivirus (FCV) is one of the major pathogens for cat upper respiratory diseases. Although vaccination for FCV has been available for almost twenty years, a high proportion of vaccinated cats present with clinical signs of FCV infection [1]. A number of different strains of FCV have been isolated and the serological relationships...

Prevalence of antibodies to canine respiratory coronavirus in some dog populations in Japan

“…Antibodies to crc o v were tested by a modification of a previously reported elisa (Erles and others 2003, Decaro and others 2007). The culture fluid of Mardin‐Darby bovine kidney ( mdbk ) cells (American Type Culture Collection) that were infected with the Mebus strain of bc o v (Stair and others 1972) was concentrated by the addition of ammonium sulphate (Sigma) (Tajima and others 1998). The precipitate was dialysed to phosphate‐buffered saline ( pbs ) and used as the positive antigen.…”

Reactivities of Feline Calicivirus Field Isolates with Monoclonal Antibodies Detected by Enzyme-Linked Immunosorbent Assay

“…Previously Tohya et al reported the existence of subunit particle of FCV F4 strain in culture fluid. These particles were non-infectious but had a neutralizing epitope recognized by MAb 13B4 [9]. In the present study, noninfectious F4 and 91-6 reacted with 10F9 and/or 16E8 but not with the rest MAbs.…”

“…ELISA antigen was prepared from culture fluid of CRFK cells inoculated with each FCV as described previously [9]. The antigen was diluted with 0.05 M carbonate buffer, pH 9.6, to a protein concentration of 10 µg/ml.…”

“…ELISA using MAbs and peroxidase-conjugated antimouse IgG goat serum (Biosource International, U.S.A.) was carried out essentially as described [9]. A twofold serial dilution, starting from 1:10,000 to 1,280,000, of the MAb was reacted with each FCV antigen and then a neutralizing epitope 4.…”

Eligibility of Feline Calicivirus for a Surrogate of Human Norovirus in Comparison with Murine Norovirus, Poliovirus and Coxsackievirus

“…The samples were processed by sodium dodecyl sulfate SDS -polyacrylamide gel electrophoresis PAGE , and proteins were transferred onto a nylon membrane. The membrane was probed with anti-FCV VP1 monoclonal antibody 8G1 Tajima et al, 1998 and horseradish peroxidase-conjugated anti-mouse IgG antibody Santa Cruz Biotechnology, Santa Cruz, CA . Proteins were detected by using a chemiluminescence substrate for HRP, Luminata Forte Western HRP Substrate Millipore , followed by analysis using a LumiCube imaging analyzer Liponics, Tokyo, Japan .…”