Development and evaluation of a novel antigen capture assay for the detection of classical swine fever virus antigens

An antigen-capture enzyme immunoassay (EIA) was developed to detect classical swine fever virus (CSFV) antigen directly from 10% w/v tissue suspension. The assay, based on the sandwich principle, uses a biotinylated monoclonal antibody bound to streptavidin-coated microplates as the capture system and a swine anti-CSFV antibody and rabbit anti-swine HRPO-conjugate as the detector system. The antigen-capture EIA was compared with conventional virus isolation and polymerase chain reaction (PCR) for detection of CSFV in tissues. The ability of the antigen-capture EIA to discriminate classical swine fever (CSF) from bovine viral diarrhea and African swine fever viruses was also tested. The assay was shown to detect 21 different strains of CSFV and was unreactive with tissues from uninfected animals. Signal to noise (S/N) ratios were calculated from the EIA absorbance values. Readings from samples positive by virus isolation (n=47) averaged a S/N ratio of 5.34. In contrast, samples negative by virus isolation (n=96) demonstrated a mean S/N ratio of 0.16. At S/N cut-off value of 1.0, all samples that yield virus isolation and PCR negative result were negative in the antigen-capture EIA. Compared with virus propagation in tissue culture using PK15 cells (followed by indirect peroxidase assay detection) and PCR, the EIA had a specificity of 98.7% and a sensitivity of 91.4%. The EIA is simple, can be performed in 4 h and lends itself to automation for screening of tissues sample from pigs suspected of CSFV infection.     

Detection of bovine respiratory syncytial virus using a heterologous antigen-capture enzyme immunoassay

Based on the marked antigenic similarities that exist between antigens of the human and bovine strains of respiratory syncytial virus (RSV), an enzyme immunoassay (EIA) designed to detect human RSV was used to detect bovine RSV. The commercial test kit (RSV EIA) consists of a solid phase (beads) coated with a capture antiserum prepared against the Long strain of human RSV. The RSV EIA test was compared with the method of inoculation of cell cultures and fluorescent antibody (FA) staining of lung tissue for the detection of bovine RSV. Using a cell culture-propagated stock of strain 375 of bovine RSV, the threshold of sensitivity of the EIA test for the cattle strain of RSV was determined to be less than or equal to 10(2.3) CCID50/ml. In addition, RSV EIA detected the bovine RSV in nasal samples obtained from 3 experimentally inoculated cattle. The RSV EIA exhibited a sensitivity of greater than or equal to 80% during the period that shedding of infectious virus took place. All of the bovine RSV FA-positive lung samples (n = 37) were positive by the RSV EIA. Twenty-six of the remaining 214 bovine RSV FA-negative lung samples were positive by the RSV EIA. The RSV EIA was also used to test 137 nasal swabs obtained from cases of bovine respiratory disease. Of these, 38 tested positive by RSV EIA. All samples that tested positive by EIA were confirmed by blocking assays using hyperimmune serum anti-bovine RSV and a pool of monoclonal antibodies specific for that virus.     

Detection of bovine herpesvirus-1-specific IgM using a capture enzyme immune assay with isotype-specific monoclonal antibodies

The detection of virus-specific immunoglobulin M (IgM) antibodies in acute-phase serum samples offers the possibility of making an accurate and rapid serologic diagnosis. We have developed a solid-phase capture assay that uses murine monoclonal antibodies specific for bovine IgM to separate the whole IgM fraction of a bovine serum sample. The IgM specific for bovine herpesvirus-1 (BHV-1) is then detected by the addition of viral antigen, which in turn is detected by BHV-1-specific monoclonal antibodies conjugated to horseradish peroxidase. A BHV-1 IgM antibody response was detected during the early postinfection period (7-40 days PI). Bovine herpesvirus-1 IgM antibody was not detected in sera taken from 3 animals following dexamethasone-induced viral reactivation. This method compares favorably with viral isolation, antigen detection in the clinical samples, and paired serology in the diagnosis of BHV-1 infection at a herd level.     

Use of a monoclonal antibody against envelope protein gp51 of bovine leukemia virus in a test for the diagnosis of enzootic bovine leukosis

A highly specific monoclonal antibody (mAb) directed against envelope protein gp51 and effectively bonding the antigen (Ag) on account of its high affinity from an unpurified Ag preparation was chosen for use in a double-sandwich enzyme immuno-assay (EIA) for diagnosis of bovine leukaemia virus (BLV). The epitopes recognised in bovine sera by the gp51-specific antibodies were at the same time properly exposed. Some parameters of major importance to testing were optimised (Ab and Ag quantities, dilution of bovine sera for testing). Preliminary testing of the double-sandwich EIA on selected bovine sera and comparison with both the immunodiffusion test and anti-BLV EIA confirmed its good diagnostic specificity and sensitivity. Hence, this double-sandwich EIA, developed by means of an mAB against gp51, on account of the possibility to use as Ag culture supernatant of the FLC cell line, is a sensitive, low-cost alternative to the anti-BLV EIA Dessau MTP which had so far been used. The double-sandwich EIA is recommended for use in final sanitation for its high analytical and diagnostic sensitivity.     

Rotavirus antibody assays on monkey sera: a comparison of enzyme immunoassay with neutralization and complement-fixation tests.

An enzyme immunoassay (EIA) for the detection of rotaviral antibodies was developed, using a purified, cell culture-grown SA 11 viral antigen and alkaline phosphatase as an enzyme label. This technique was evaluated by comparative testing with tube neutralization and complement-fixation assays on a collection of simian sera. There was close correlation between positive and negative results obtained by EIA and by neutralization. The EIA was as easy to perform as complement fixation testing, but showed greater sensitivity and fewer nonspecific reactions. Thus, EIA was shown to be a very suitable test for routine detection of rotaviral antibodies in serum. Results of neutralization tests suggested that the monkeys (mostly rhesus macaques) in the present study were infected with viruses varying in their antigenic relatedness to SA 11 virus and to a British isolate of calf rotavirus.     

New ELISA test for detection of bovine leukemia virus infections in cattle, using bacterially synthesized p24

A new ELISA test is described for the detection of antibodies to bovine leukemia virus protein p24. This test employs a bacterially synthesized p24 antigen which represents a hybrid protein consisting of beta-galactosidase and about 70% of the mature viral p24. The antigen preparation was enriched from Escherichia coli cells to 95% purity and was used for the detection of antibodies in cattle. In a selected set of 100 positive field sera, 97 could be verified by the new test.     

Development and evaluation of a rapid immunomagnetic bead assay for the detection of classical swine fever virus antigen

Classical swine fever (CSF) is a highly contagious and severe viral disease of swine resulting in substantial production losses in different farming systems in many regions of the world. The accurate and rapid detection of CSF outbreaks is reliant on sensitive and specific laboratory testing and is a key component of disease control. Specific detection of CSF virus can be achieved by virus isolation in tissue culture, antigen capture or the detection of viral RNA using molecular techniques. In order to reduce the time taken to achieve a diagnostic result and simplify testing methods, an antigen capture ELISA using immunomagnetic beads (IMB) as the solid phase was developed and compared to a microplate-based antigen capture (AC)-ELISA. The IMB-ELISA has up to 64-fold greater analytical sensitivity than the AC-ELISA and initial estimates of diagnostic sensitivity and specificity are 100%. The IMB-ELISA has a highly robust, rapid and stable test format and is simpler to perform than the AC-ELISA. The IMB-ELISA has the added advantage that a result can be sensitively and specifically determined by eye, lending it to the possibility of adaptation to a near-to-field test with minimal equipment or expertise needed.     

Serological assessment of chlamydial infection in the koala by a slide EIA technique

A rapid and simplified slide enzyme immunosorbent assay (EIA) was developed for the diagnosis of chlamydial infection in the koala. HeLa 229 cells infected with koala strain Chlamydia psittaci were fixed on the surface of multiwell slides and used as the antigen. The assay consisted of first reacting koala antiserum with the fixed C psittaci antigen, followed by reaction with biotinylated rabbit anti-koala IgG, ABC reagent and substrate. The chlamydial EIA antibody titres obtained were compared with those of a complement fixation (CF) test using koala strain C psittaci as antigen. Of 35 koala sera tested, 16 CF positive sera (greater than or equal to 1:8) also had a positive titre (greater than or equal to 1:200) in the slide EIA test (sensitivity 93.8%, 15/16). Nineteen CF negative sera were also negative in the slide EIA (specificity 100%, 19/19). Sixty-eight samples of koala blood were collected by ear-prick using a sampling paper method and were assayed by both tests. Sensitivity of the slide EIA was 100% (15/15) and specificity of the test was 96.2% (51/53). To simplify the slide EIA for use as a practical screening test, a 3-point serum dilution series (1:100, 1:200, 1:400) was used. This 3-point slide EIA was compared with the CF test using sheep strain chlamydial antigen. Thirty-nine sera were assayed by both tests. The sensitivity of the 3-point method was 85.7% (6/7) and the specificity was 71.9% (23/32) as compared with the sheep antigen CF test.(ABSTRACT TRUNCATED AT 250 WORDS)     

Prevalence of bovine viral diarrhea virus infection in bulls in artificial insemination centers in Poland

This study was directed at the evaluation of the prevalence of bovine viral diarrhea virus (BVDV) infection in bulls in artificial insemination centers. Both serological and virological examinations were performed. Blood samples were tested in micro-seroneutralization test for BVDV antibodies. Virus isolation was performed in cell culture and BVDV antigen was detected in an indirect immunofluorescence assay with monoclonal antibodies. One hundred and seventy-five serum samples and 219 whole blood samples for virus isolation were tested. Neutralizing antibodies were found in 86% of the bulls. Persistent infection (PI) was detected in 0.9% of the analyzed blood samples.     

Enzyme-linked immunosorbent assay for detection of equine infectious anemia antibody to purified P26 viral protein

An indirect enzyme-linked immunosorbent assay (ELISA) was developed for the detection of equine infectious anemia (EIA) antibody in horse sera. Purified P26 viral protein was the antigen; alkaline phosphatase linked to rabbit anti-horse immunoglobulin G was the conjugate. The ELISA detected EIA antibodies in horse sera as early as 11 to 14 days after experimental inoculations. There was full agreement between the results of ELISA and the agar-gel immunodiffusion tests on EIA proficiency test sera. The ELISA readily detected EIA antibody in horse sera that had weak positive reactions on agar-gel immunodiffusion.     

Use of monoclonal antibody against major internal protein p24 of bovine leukemia virus in capture ELISA

A monoclonal antibody (4H4) against the major internal protein, p24, of bovine leukemia virus (BLV) is described. It recognizes a sequence determinant on the p24-molecule and displays high affinity to its antigen. The monoclonal antibody 4H4 was applied in capture ELISA for diagnosis of enzootic bovine leukosis, using crude BLV-preparation as antigen. This test is more sensitive than the immunodiffusion test and at least as sensitive as direct ELISA.     

An immunodiffusion test for detection of bovine viral diarrhea virus antibodies in bovine serum.

An immunodiffusion test (IDT) was developed for detecting bovine viral diarrhea virus antibodies in bovine serum. The antigen utilized in the IDT was prepared from bovine viral diarrhea virus-infected monolayer cultures. Results of the IDT were obtained within 48 hours and correlated with the virus-neutralization test.     

Variation in E(rns) viral glycoprotein associated with failure of immunohistochemistry and commercial antigen capture ELISA to detect a field strain of bovine viral diarrhea virus

Bovine viral diarrhea virus (BVDV) affects cattle populations causing clinical signs that range from subclinical immunosuppression to severe reproductive and respiratory problems. Detection and removal of persistently infected (PI) calves is the single most important factor for control and eradication of BVDV. Current testing strategies to detect PI calves rely heavily on immunohistochemistry (IHC) and a commercially available antigen capture ELISA (ACE) assay. These viral assays depend on 1 or 2 monoclonal antibodies which target the E(rns) glycoprotein of BVDV. The sensitivity and specificity of these two tests have been reported previously. The purpose of this research was to characterize a strain of BVDV (AU501) that was undetectable using IHC and ACE based on a single monoclonal antibody, but was consistently detected in samples from a Holstein steer using virus isolation and PCR testing. Sequencing of this AU501 viral isolate revealed a unique mutation in the portion of the genome coding for the E(rns) glycoprotein. This unique field strain of BVDV demonstrates the risk of relying on a single monoclonal antibody for detection of BVDV. Multiple testing strategies, including polyclonal or pooled monoclonal antibodies that detect more than one viral glycoprotein may be necessary to detect all PI calves and facilitate eradication of BVDV.     

Prevalence and characterization of bovine viral diarrhea virus in the white-tailed deer population in Indiana.

Bovine viral diarrhea (BVD) is one of the economically important diseases of cattle. For many years, different types of vaccines have been commercially available, yet this disease is hard to control in high-density population areas. Detection and isolation of bovine viral diarrhea virus (BVDV) from any potential reservoir is vital, especially when considering virus eradication from a herd or locale. One potential source is wild ruminants. Ear notches and lymph nodes were collected from the wild population of white-tailed deer (Odocoileus virginianus) during deer hunting season in Indiana and tested for BVDV with a commercial BVD antigen capture enzyme-linked immunosorbent assay. Two samples out of 745 collected samples were positive, and subsequently cp and ncp BVDV was isolated from 1 ear notch and 1 lymph node. These isolates were genotyped as type 1a and 1b based on sequence analysis of the 5' untranslated region (UTR). The results of the present study indicate that the prevalence of BVDV in the white-tailed deer population of Indiana is about 0.3%. Wild ruminants infected with BVDV should be taken into consideration during an eradication program of BVDV from the livestock population.     

Detection of bovine viral diarrhoea virus in specimens from cattle in South Africa and possible association with clinical disease

Studies covering all aspects of bovine viral diarrhoea virus (BVDV) have been conducted in several countries in Europe, Asia and America. In southern Africa, more information is required about the nature of BVDV infection, the prevalence of different strains and the economic importance of the disease. The presence of BVDV in southern Africa has been known since the early 1970s through serological surveys but few reports confirming its presence by virus isolation and correlation with clinical disease are available. Specimens (n = 312) collected in 1998/99, from live and dead cattle from different farming systems, were obtained from private practitioners, feedlot consultants and abattoirs throughout the country. Specimens (n = 37) from African buffaloes (Syncerus caffer) in the Kruger National Park were also included. All specimens were processed for virus isolation in cell culture with confirmation by means of immunofluorescent antibody tests and some also by means of an antigen capture ELISA. BVDV was isolated from 15 (4.7%) cattle and were all noncytopathic biotypes. BVDV was not detected in 37 lymph nodes obtained from buffaloes in the Kruger National Park. Of the clinical signs in cattle from which virus were isolated, respiratory signs was the most frequent (10/15), followed by diarrhoea (5/15). Abortion, congenital malformations, haemorrhagic diarrhoea and poor growth were also included as criteria for selection of animals for specimen collection, but no BVD viruses were isolated from cattle manifesting these clinical signs.     

Serological diagnosis of influenza A infections in the horse by enzyme immunoassay. Comparison with the complement fixation test

An enzyme immunoassay (EIA) using horseradish peroxidase and a type-specific antigen is described for the detection and quantitation of anti-influenza antibodies in the horse. Compared with the complement fixation (CF) test (using the same antigen), EIA proved to be superior with respect to sensitivity and reliability. The internal variation of EIA was low and thus small titres in EIA can be considered of diagnostic significance. However, no strict correlation with CF was observed. The use of an immunoconjugate against equine IgM in parallel with IgG would certainly improve the sensitivity of the test, especially in early stages of infection.