DE19848937A1 - Diagnosis of bovine viral diarrhea comprises detecting the presence or absence of viral coat protein E(ms) - Google Patents

Abstract

Bovine viral diarrhea can be diagnosed by demonstrating the presence of Erns.

Description

The diagnostic method for detecting infection of cattle with the virus bovine viral diarrhea (BVDV) is based either on the serological Detection of antibodies against antigens of the virus or the direct Pathogen detection.

The pathogen detection is of particular importance in combating this Viral disease too, since this disease causes persistent viremic animals may occur. These animals were called the fetus during a certain phase during pregnancy via the placenta and recognize the proteins of the infection virus as "self". They are lifelong virus eliminators and must be the most important Source of infection from cattle herds can be eliminated. The standard procedure for Evidence of the BVD virus is the cultivation of the virus from leukocyte preparations, Whole blood or serum samples in cell culture. Besides this time consuming There have been several ELISA systems for years with which virus antigens have been used can be detected in leukocyte preparations or whole blood samples.

The principle of the ELISAs currently used for pathogen detection is based on the detection of cell-bound viral proteins. This requires a cell-containing Sample material (whole blood or leukocyte preparations) from which the corresponding viral components are brought into solution with detergents must (Böttcher et al., 1993; Greiser-Wilke et al., 1993). This procedure is time-consuming (leukocyte preparation) or less sensitive (whole blood samples).  

The task was to develop a detection system that works with the same or improved diagnostic specificity and sensitivity without a time consuming Leukocyte preparation or, as a result, unreliable whole blood preparations gets along. In addition, the sample material should be used for different serological examinations of other infectious diseases can be used.

For the detection of pathogens in infections with the classical swine fever virus (CSFV), a virus in pigs closely related to the BVD virus, it could be shown that a viral coat protein, the E0 of the E ^rns , is secreted by infected cells (Rümenapf et al. , 1993) and therefore can also be detected in the plasma or serum of infected animals. In addition to the usual ELISA systems (detection of cell-bound virus proteins from whole blood or better leukocyte ^preparations ), an ELISA could also be developed that ^detects E ^rns in the serum or plasma of infected pigs (Depner et al., 1995). However, detection is only possible until antibodies against virus proteins are produced in the infected animal. These antiviral antibodies interfere with the detection of E ^rns . Table 1 shows that the detection of E0 or E ^{rns is} increasingly inhibited with an increasing amount of antibodies specific for swine ^fever in the serum. The increase in anti-swine pest antibodies was measured in the neutralization test (NT). As soon as a clear neutralization ^titer can be measured, the measured ^{Enns is reduced} by 21% (in this example four weeks after the infection) to 72% (thirteen weeks after the infection).

The starting situation for cattle is more complicated. First, the detection of free ^Erns in bovine blood has not been carried out so far, so that it could not be assumed that free ^Erns can be found in bovine serum or plasma. Second, probably more than 60% of cattle worldwide have had BVDV infection in their lifetime and have antibodies to the BVD virus. Thirdly, it is not about the detection of transiently infected animals, but primarily the detection of persistent viremic animals, which do not develop antibodies against their endogenous BVD virus, but are able to produce antibodies against a heterologous BVD virus.

Anti-E ^rns antibodies can occur in persistent viremic cattle for several reasons:

• 1. After birth, the animal ^ingests maternal antibodies against E ^rns via the colostrum.
• 2. There is a super infection with an antigenically heterologous BVD virus.
• 3. The cattle population is vaccinated with a BVD vaccine.

So there were two main reasons against an E ^rns detection in cattle. First, no free ^{Ens has been detected} in bovine plasma or serum. Secondly, disturbing anti-E ^rns antibodies can also be expected in persistent viremic animals.

Surprisingly, an E ^rns detection system was also developed for the BVD virus.

The subject of the invention is accordingly a method for the detection of bovine virus diarrhea, which is characterized in that the presence or absence of E ^{rns is} detected in samples of the cattle to be examined. The detection method according to the invention has the following advantages over the detection methods for a BVD virus infection of the prior art:

• 1. E ^rns in various body fluids and / or body tissues serves as an indicator of the contact of the corresponding animal with the BVD virus.
• 2. In contrast to methods using virus isolation, no infectious virus in be included in the samples.
• 3. Cell-bound virus proteins are not required, so there is no need the extraction of the leukocyte fraction from whole blood samples, whereby the Implementation of the verification procedure is significantly simplified.
• 4. The sample material is also for further standard investigations by Infectious diseases in cattle can be used.

The detection ^{method according to} the invention in all its embodiments is particularly suitable for the detection of free, ie not cell-bound, E ^rns . The method according to the invention can be carried out in different ways. It can be carried out as a homogeneous process or as a heterogeneous process. Suitable homogeneous processes are e.g. B. Agglutination tests with turbinimetric or nephelometric measurement, enzyme immunoassays such as the "cloned enzyme donor immunoassay" (CEDIA, Henderson, DR, Friedman, SB, Harris, JB et al. 1986, "CEDIA", a new homogeneous immunoassay system. Clin. Chem 32, p. 1637), RIAs or "luminiscent oxygen channeling assays" (LOCI, Ullman, EF et al. 1996; Clinical Chemistry 42 (9), 1518-1526. Preferred are heterogeneous methods, such as, for example, solid phase immunoassays or MIA, modular immunoassay (Aventis, Frankfurt am Main), solid-phase immunoassays are particularly preferred.

The solid phase of the solid phase immunoassay can be designed differently be like B. a gel electrophoresis plate, latex or plastic particles or a Microtiter plate, a microtiter plate is preferred.  

The detection reaction of the examination procedure can be based on different Principles are based. Z are particularly suitable. B. the detection of a radioactive Labeling (RIA), a chemiluminescent or fluorescent reaction or the Detection of an enzyme-catalyzed reaction (ELISA).

The reaction for the detection of E ^rns can be carried out in various ways known in the literature (Tijssen, P. 1985. Practice and theory of enzyme immunoassays in: Laboratory techniques in biochemistry and molecular biology (RH Burdon and PH von Knippenberg, Eds) Vol. 15, Elsevier, Amsterdam, New York, Oxford). In the preferred embodiment, the solid phase immunoassay, the detection takes place e.g. B. instead of:
the immunological test for the detection of the causative agent of bovine viral diarrhea

• a) coating a solid phase with an antibody against E ^rns .
• b) bringing a sample to be examined into contact with the one under a) described solid phase under conditions that the formation of a Allow antibody-antigen complex,
• c) adding an anti-E ^rns antibody labeled with a marker under conditions which allow the labeled anti-E ^rns antibody to bind to the antibody-antigen complex described under b) and
• d) triggering a reaction associated with the marker mentioned under c) and measuring or measuring a physical property of the marker to detect the presence or absence of ^Erns in the sample described under b).

The method described is particularly suitable, in which the marker is an enzyme is with which a corresponding enzyme-catalyzed measurable reaction, e.g. Legs  Color reaction, is catalyzed. Particularly suitable enzymes are peroxidases, whole Horseradish peroxidase is particularly suitable.

The procedure described is carried out under conditions (Use of solvents, buffer systems, washing steps, etc.) that the Are known to those skilled in the art (Tijssen, P. 1985. Practice and theory of enzyme immunoassays in: Laboratory techniques in biochemistry and molecular biology (R. H. Burdon and P. H. von Knippenberg, Eds) Vol. 15, Elsevier, Amsterdam, New York, Oxford, US Patent USRE 31006 reissue of US 3791932). A particularly preferred one The method is described in the exemplary embodiment, in which, of course certain reagents, reaction conditions and reaction steps Alternatives known in the art can be replaced.

The anti- ^Erns antibodies required for the method ^according to the invention can be prepared in different ways (see, for example, Harlow, E. and Lane, D. (1988). Antibodies: A laboratory manual, Cold Spring Harbor Laboratory, p. 53-318.). These antibodies are preferably produced by producing test animals (for example chickens, rabbits, etc.) with the pestivirus E ^rns - preferably recombinantly using methods of the prior art - and preferably using methods of the prior art (for example chromatographic) cleaned. There is also the possibility of using monoclonal antibodies which can be produced by methods of the prior art. The conjugation of, or the antibody with a labeling substance is carried out according to methods of the prior art (Wong, SS (1993). Chemistry of protein conjugation and cross-linking. CRC Press, Boca Raton, Ann Arbor, Boston, London).

The actual detection method for the presence or absence of ^Er is carried out by methods of the prior art, colorimetrically, fluorimetrically or by luminescence measurement, preferably by colorimetric measurement after an enzymatic conversion of a substrate. Suitable enzymes are the alkaline phosphatase, β-galactosidase or peroxidase, preferably horseradish peroxidase, which are coupled to anti-E ^rns antibodies. The coupling of the enzymes to the antibodies takes place according to methods of the state of the art (e.g. according to Wong op. Cit.), Preferably by reacting the antibodies, activated with GMBS (N- (y-maleimidobutyryloxy) succinimide ester), with enzyme which is associated with SATA (N-succinimidyl-S acetylthioacetate) was reacted. Suitable substrates are water-soluble chromogenic substances such as p-nitrophenyl phosphate (PNP), o-nitrophenyl-β-D-galactopyranoside (ONPG) or H ₂ O ₂ with the chromogens 3,3 ', 5,5'-tetramethylbenzidine ( TMB), o-phenylenediamine (OPD) or particularly preferably 2,2'-azino-di (3-ethyl-benzothiazolinesulfonic acid (ABTS).

The inventive method can with any body fluids and partly also with body tissues, e.g. B. material from skin biopsies performed become. It is particularly preferred to use whole blood, serum, plasma, Semen, milk and nasal secretions. The is very particularly preferred Detection in whole blood, plasma or serum samples.

The subject of the present invention also includes a test kit for carrying out the method according to the invention. The test kit preferably contains anti-E ^rns antibodies for coating a solid phase, enzyme-labeled anti-E ^rns antibodies and optionally substances for triggering the detection ^reaction associated with the enzyme.

Through the following embodiment and the content of The present invention is to be explained in more detail.

example 1 Execution of the test

Microtiter plates (Nung, Maxisorp) are coated with antibodies that are directed against the glycoprotein E ^rns . The antibodies are generated by repeated immunization of rabbits or other experimental animals such as chickens with recombinantly produced pestivirus E ^rns and purified by chromatography.

Depending on the number of samples, 50 µl sample and Conjugate diluent submitted. The sample and conjugate thinner consists of a part of supplement A [one third fetal calf serum (biochrom, Berlin, Germany) and two thirds rabbit serum (biochrom) and 0.1% sodium azide as a preservative] and a part of additive B [10% Brij 700 (Sigma, Buchs, Switzerland) and 0.5% phenol (Roth, Reinach, Switzerland)] diluted with three parts Water. In addition to Brij 700, other non-ionic or zwitterionic detergents such as CHAPS, n-octyl-β-D-glucopyranoside, NP-40 or Triton X-100 can be used.

Then 150 µl of the serum, plasma, whole blood or milk samples are added and mixed by shaking the plates. E ^rns -free bovine serum and recombinantly produced E ^rns serve as ^controls . which is diluted in bovine serum. Samples and controls are incubated overnight at 4 ° C or one hour at 37 ° C in a humid chamber.

After this incubation phase, the test plate is knocked out, and the Reaction wells three times for three minutes with 300 µl surfactant buffered Saline solution (e.g. CHEKIT washing & thinning solution from Dr. Bommeli AG, Bern, Switzerland) per reaction well. With every wash the plates knocked out well.  

Then 200 μl of an anti-E ^rns- peroxidase conjugate, 1/100 diluted in sample and conjugate diluent, are added to each reaction well. In turn, sera or egg yolks with recombinant ^Erns immunized test animals serve as the IgG source for the peroxidase conjugate. The anti-E ^rns- peroxidase conjugate is prepared by adding N-succinimidyl-sacetylthioacetate (Calbiochem, La Jolla, USA) to horseradish peroxidase and adding IgG to the reaction product, which is purified on a protein G column and treated with N (-y maleimidobutyryloxy) succinimide ester (Calbiochem) was activated. The test plates are incubated at 37 ° C for one hour. The test plates are then washed again as described above.

Bound E ^rns is ^detected by adding H ₂ O ₂ and 2,2'-azino di (3-ethyl-benzothiazolinesulfonic acid-6 (ABTS, e.g. CHEKIT-Chromogen from Dr. Bommeli AG) by measuring a Color reaction at a wavelength of 405 nm (reference wavelength: 492 nm) The enzymatic reaction is terminated before the measurement by adding sodium dodecyl sulfate (eg SDS, stop solution from Dr. Bommeli AG). The results are evaluated on the basis of the values of the positive and negative controls. In the case of duplicate determinations, the mean value of the negative control is subtracted from the mean value of the positive control and from the mean values of the individual samples Sample values are each divided by the corrected value of the positive control, and the result is multiplied by 100.

If the value is less than 20, the sample is to be rated as negative. Show values over 30 a positive result. For results between 20 and 30, the result is questionable. The results of a typical series of tests are shown in Table 2. The true value of the samples shown in the table was determined using  flow cytometric measurement of the samples after incubation with fluorescence-labeled anti-NS-2,3 antibodies (FACS).

Table 1

Detection of CSFV-E0 in the presence of swine fever-specific antibodies

Table 2

Claims (10)

1. A method for the detection of bovine viral diarrhea, characterized in that the presence or absence of E ^{rns is} detected in samples of the cattle to be examined. 2. The method according to claim 1, characterized in that the method is performed as a solid phase immunoassay. 3. The method according to claim 2, characterized in that as a solid phase a microtiter plate is used. 4. The method according to one or more of claims 1-3, characterized characterized in that an enzyme or a Isotope connection is used. 5. An immunological test for detecting the causative agent of bovine viral diarrhea, comprising

• a) coating a solid phase with an antibody against E ^rns .
• b) bringing a sample to be examined into contact with the solid phase described under a) under conditions which permit the formation of an antibody-antigen complex,
• c) adding an anti-E ^rns antibody labeled with a marker under conditions which ^permit the binding of the labeled anti-E ^rns antibody to the antibody-antigen complex described under b) and
• d) triggering a reaction associated with the marker mentioned under c) and measuring or measuring a physical property of the marker to detect the presence or absence of E ^rns in the sample described under b).

6. The method according to claim 5, characterized in that the solid phase is a microtiter plate. 7. The method according to claims 6 or 7, characterized in that the Enzyme labeling using horseradish peroxidase. 8. The method according to one or more of claims 1-7, characterized characterized in that it was run as a sample using whole blood, plasma or serum becomes. 9. Test kit for the detection of bovine viral diarrhea according to a method such as described in one or more of claims 1-7. 10. Test kit according to claim 9, containing anti-E ^rns antibodies for coating a solid phase, enzyme-labeled anti-E ^rns antibodies and optionally substances for triggering a detection ^reaction .