Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
2000-02-07
2002-09-24
Scheiner, Laurie (Department: 1648)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S005000, C435S007900, C435S007920, C435S339000, C435S810000, C530S388100, C530S388300
Reexamination Certificate
active
06455264
ABSTRACT:
BACKGROUND OF THE INVENTION
The genera Pestivirus , Flavivirus and “hepatitis C virus group” constitute the family Flaviviridae. The genus Pestivirus currently comprises three members, bovine viral diarrhoea virus (BVDV), classical swine fever virus (CSFV), and border disease virus (BDV). The presence of a fourth separate group of Pestiviruses comprising isolates from cattle and sheep has been recently described, and it is now generally accepted to refer to this additional species as BVDV-2; consequently, classical BVDV strains are named BVDV-1. BVDV-1 and BVDV-2 are considered world-wide as important pathogens of cattle. Both can cause acute infections (diarrhoea, fever, hemorrhagic syndrome) as well as abortion, malformation, and persistent infection. Persistently infected animals represent the major reservoir of the virus; such animals may come down with fatal mucosal disease (MD). With regard to economical impact the reproductive losses are most significant. The assay outlined below aims at the identification of infected and especially persistently infected animals. In control programs established in some countries persistently infected animals are removed in order to obtain BVDV free herds.
Persistently infected animals are generated by the infection of a foetus with a non-cytopathogenic (noncp) BVDV strain during the first trimester of gestation. Such animals acquire immunological tolerance to the particular noncp strain (no antibodies against the persisting virus detectable). The persistently infected animals shed virus in considerable amounts during their life and represent the main reservoir for BVDV. Persistently infected animals may be born without clinical symptoms. Therefore, the identification and eradication of persistently infected animals is an important goal in the control of the disease.
Boulanger et al. (J. Gen. Virol. 72, 1195-1198, 1991) and Corapi et al. (Am. J. Vet. Res. 51, 1388-1394, 1990) disclose the characterisation of a large number of anti-BVDV monoclonal antibodies (Moabs). Most of these Moabs do not recognise an antigen determinant present on all BVDV strains under examination. Corapi et al. identified one Moab which is reactive with a linear antigen determinant expressed by all tested BVDV strains. No diagnostic application of these Moabs is suggested in these documents.
For the diagnosis of BVDV infection in cattle, several methods are available in the art. Until now, the detection of BVDV is sometimes performed by means of the “tissue culture method” in which leukocytes are prepared from blood samples and inoculated on susceptible cells. Infection of the cells by BVDV is detected by immunostaining techniques using specific serological reagents. This diagnostic method is considered as the “golden standard” with regard to specificity and sensitivity. However, this method is quite laborious, time consuming, cannot be automated and is thus not applicable to very large numbers of samples.
Antigen-capture ELISAs have become commercially available for BVDV detection and these assays overcome some of the disadvantages associated with the tissue culture method (Brinkhof et al., Veterinary Microbiology 50, 1-6, 1996; Fritzmeier et al., AFT-Tagung BVD-Bekämpfung, October 1998, Hannover, Germany).
The set up of all these commercially available assays is: (a) coating of microtiter plates with monoclonal antibodies (Moabs) or polyclonal monospecific antiserum against the non-structural protein NS2/3 for capturing of antigen, (b) detection of the viral antigen after incubation with a sample (obtained from leukocytes or organ samples) by either a Moab or polyclonal monospecific antiserum. The sensitivity of the available assays turned out to be less than 100%. Moreover, the presently available assays require the use of leukocyte preparations of anti-coagulated blood, anti-coagulated blood or tissue samples from organs of the test animals.
BVDV antigen detection by means of FACS (fluorescence activated cell sorter) analysis has also been described. This technique also uses leukocytes and BVDV infected cells are identified with antibodies against the BVDV NS2/3 antigen. This diagnostic method is generally considered comparable to infection of culture cells. This method also requires the preparation of blood and the availability of highly specialised laboratory equipment is required.
Thus, there exists a need for a sensitive, rapid and practical diagnostic method for determining Pestivirus infection in animals, in particular for determining BVDV infection in cattle, especially in the framework of eradication programs where large numbers of cattle are to be monitored.
Moreover, there is a need for a diagnostic reagent for use in such diagnostic methods which is highly specific (i.e. no false positive samples) and highly sensitive (no false negative samples).
DETAIL DESCRIPTION OF THE INVENTION
To that aim the present invention provides in a first aspect a monoclonal antibody which is capable of specifically binding to an E
RNS
antigen determinant of a Pestivirus which either is or cross-competes with a monoclonal antibody expressed by hybridoma cell line 50F4-10-INT, deposited at the ECACC, Salisbury, UK, on Feb. 5, 1999 under accession no. 99020505.
It has been found that a monoclonal antibody according to the invention recognises a very conserved epitope on the E
RNS
(RNS=ribonuclease soluble) protein of all tested Pestiviruses, including many different BVDV, CSFV and BDV strains. Detection of the Pestivirus encoded glycoprotein E
RNS
(or antibodies against this glycoprotein) in a test sample indicates that the animal from which the test sample is derived is infected with the virus. Moreover, it has been found that a monoclonal antibody according to the invention is highly specific, i.e. the monoclonal antibody is able to discriminate between negative and positive samples from animals, such as cattle, in contrast to a polyclonal monospecific E
RNS
antiserum.
The E
RNS
protein represents a structural glycoprotein and as such forms part of the Pestivirus. In addition it is secreted from infected cells (for example leukocytes) leading to a soluble form of E
RNS
. Accordingly, both forms of E
RNS
-soluble as well as the virus-associated form—are present in cells as well as serum/plasma of infected animals. This is in contrast to the NS2/3 protein which is only detectable in infected cells (Colett et al., Virology 165, 191-199 and 200-208, 1988; Thiel et al., J. Virology 65, 4705-4712, 1991; Weiland et al., J. Virology 66, 3677-3682, 1992; Meyers et al., Advances in Virus Research 47, 53-118, 1996; Rümenapf et al., J. Virology 67, 3288-3294, 1993).
A monoclonal antibody that cross-competes with the specifically deposited monoclonal antibody is an antibody that binds to the same conserved (conformational) antigen determinant as the deposited monoclonal antibody. Cross-competition experiments are relatively easy to carry out (Waters et al., Virus Res., 22, 1-12, 1991) and so it is a straightforward matter to determine whether a given antibody cross-competes with the monoclonal antibody specifically referred to above.
Briefly, such cross-competing monoclonal antibodies reactive with the same conserved antigen determinant can be obtained by using a spectrum of defined pestiviruses for screening after immunisation of mice with pestivirus infected cells, purified virus or, preferably, purified E
RNS
protein (Wensvoort et al., J. Gen. Virol. 71, 531-540, 1990; Hulst et al., Virology 200, 558-565, 1994), followed by fusion. First, monoclonal antibodies are selected which react with an antigen determinant present on all pestivirus strains under examination. Subsequently, the selected monoclonal antibodies can be used in a standard competition ELISA with the deposited monoclonal antibody ECACC no. 99020505 to identify monoclonal antibodies which bind to the same conserved antigen determinant as the deposited Moab.
The monoclonal antibody technology has become well established since the original work by Kohler and Milstein (Nature, 256, 495, 1975) and there are
Baumeister Judith
König Matthias
Stark Robert
Thiel Heinz-Jürgen
Akzo Nobel N.V.
Blackstone William M.
Milstead Mark W.
Scheiner Laurie
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