A Direct Neutralizing Peroxidase‐Linked Antibody Assay for Detection and Titration of Antibodies to Bovine Viral Diarrhoea Virus

Abstract

Summary A neutralization peroxidase‐linked antibody (NPLA) assay in which the peroxidase‐linked antibody is a BVDV specific gammaglobulin (direct NPLA assay) is described. Multiplication of BVDV isolate 0712/80 in MDBK cells, cultivated in nutrient medium with different serum supplements (5% V/V), yielded varying degrees of inhibitory effect on the replication of the virus. Infectivity titres of the virus were considerably higher in medium supplemented with horse serum than with ox or two different batches of commercial foetal calf sera. The relationship between virus dose (TCID50) and neutralizing dose (ND50) was assayed by direct NPLA. Using the 0712/80 isolate of BVDV, a neutralization slope of 2.54 was obtained for sequential sera of a calf inoculated intranasally with the same virus isolate. Consequently, an increase or decrease of 1 log10 unit in the TCID50 would result in an inverse change of approximately 0.4 log10 in the ND50. A comparison was made between the NPLA assay and the microtitre neutralization test (MNT) for the detection of BVDV neutralizing antibodies in field cattle sera. Out of 425 field cattle sera tested, 48% were positive by the NPLA assay while 45% were positive by the MNT. In pigs inoculated intranasally with different strains of BVDV (NADL or A 1138/69), antibodies against these strains were detectable much earlier by the NPLA assay than by the MNT. The antibody titres were also remarkably higher in NPLA assays than in MNT with the difference being more prominent in sera of pigs inoculated with the NADL strain than with strain A1138/69 of BVDV. Zusammenfassung Es wird ein Neutralisationstest ‘NPLA’ (Neutralization peroxidase‐linked antibody) beschrieben, bei dem der Peroxidase‐gekoppelte Antikörper ein BVD‐virusspezifisches Gammaglobulin darstellt (direkter NPLA‐Test). Bei der Vermehrung des BVD‐Virusisolates 0712/80 in MDBK‐Zellen, die in mit unterschiedlichen Seren (5% v/v) supplementierten Nährmedien kultiviert worden waren, zeigten sich inhibitorische Effekte unterschiedlichen Ausmaßes auf die Virusreplikation. Die Infektiositätstiter des Virus waren deutlich höher bei Medium, welches mit Pferdeserum supplementiert worden war, als bei Verwendung von Medium, dem Rinderserum oder zwei verschiedene Chargen von kommerziellem fötalen Kälberserum zugesetzt worden war. Es wurde der Zusammenhang zwischen der Virusdosis (KID50) und der Neutralisationsdosis (ND50) im direkten NPLA ermittelt. Unter Verwendung des BVD‐Virusisolates 0712/80 konnte bei sequenziell gewonnenen Serumproben von einem intranasal mit demselben Virusisolat infizierten Kalb für die Neutralisation ein Regressionskoeffizient von 2,54 ermittelt werden. Daraus folgte, daß ein Anstieg oder Abfall der KID50 um eine log10‐Stufe eine entsprechende umgekehrte Änderung der ND50 um 0,4 log10 bewirkte. Für das Auffinden von neutralisierenden Antikörpern gegen BVD‐Virus in Feldseren von Rindern wurde vergleichsweise der NPLA und der Mikrotiter‐Neutralisationstest (MNT) eingesetzt. Mit Hilfe des NPLA wurden unter 425 Serumproben 48...

Isolation from an Ant Myrmecia gulosa of Two Inducible O-Glycosylated Proline-rich Antibacterial Peptides

“…Antiviral and Cell Culture Assays-A modified virus neutralization peroxidase-linked assay was carried out in 96-well cell culture plates (25). The virus was a non-cytopathogenic Australian bovine viral diarrhea virus (pestivirus) isolate (Trangie) (26).…”

Comparative Investigation of Border Disease Virus Infection in Sheep Flocks with Abortion Problems in Konya Province

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Identification of conserved epitopes on a hog cholera virus protein

“…For identification and titration of hybridoma cell cultures that produced neutralizing antibodies, a modified neutralization IM-EIA (NIM-EIA) was used [21,23]. Fifty gl of serial twofold dilutions of purified antibodies (prediluted 1 : 5) were incubated with 50 gl of virus suspension (see Table 1) containing 50 TCIDso for 1 h at 37 °C in microtitre plates.…”