SK67897A3 - Spongiform encephalopathy detection methods - Google Patents

Abstract

A method for detecting the presence of spongiform encephalopathy in an animal comprising determining the presence and/or amount of agent (e.g. by the use of 2DPAGE, followed by staining and densitometry readings of the stained agent) in a body fluid (e.g. cerebrospinal fluid) of test animal which cross-reacts with antibody raised against apoliprotein E, and has a molecular weight of between 34 and 38 kDa and a pI of between 5.4 and 5.7 comparing the concentration with a control value, and correlating the relationship between the two with the likely presence of spongiform encephalopathy in the animal.

Description

In vitro method for determining the disease state of spongiform encephalopathy in an animal

Technical field

The invention relates to a method for the in vitro determination of the disease state of spongiform encephalopathy in an animal, in particular to the determination of bovine spongiform encephalopathy (BSE) in bovine animals.

BACKGROUND OF THE INVENTION

Spongiform encephalopathies are a class of diseases that include scrapie in sheep and Creutzfeld-Jakob disease (CJD) in humans. BSE is a fetal neurodegenerative disease occurring in cattle that is subject to a reporting obligation. BSE is very important for the British farming industry.

Currently, BSE cases are identified based on clinical manifestations in animals. The cases are confirmed by post-mortem analysis of brain tissue, for example by histopathology, detection of scrapie-associated fibrils or proteinase K resistant protein.

These methods have the disadvantage that they require the slaughter of potentially infected animals which may prove not to be ill. Alternatively, clinical signs may be absent or undetectable and then the infected animals are left in the herd.

Herrington et al. (New England Journal of Medicine (1986), Vol. 315, No. 5, pp. 279-283) discovered the presence of four abnormal proteins in the cerebrospinal fluid of human patients affected by CJD by high-resolution two-dimensional polyacrylamide gel electrophoresis (2DPAGE). However, the exact identity of these proteins has not been established.

Thus, there is a need for a premature BSE test that can be used to diagnose potentially infected animals.

SUMMARY OF THE INVENTION

Further, the present invention provides a method for detecting spongiform encephalopathy in animals that addresses some, and preferably all, of these problems.

In one aspect, the present invention provides a method for detecting the presence of spongiform encephalopathy in an animal, comprising the step of determining in an animal's body fluid the presence and / or amount of an agent cross-reacting with an antibody raised against apolipoprotein E. in relation to the state of infection in the animal.

Preferably, the result of the assay is compared to a control value and its relationship is correlated to the animal's infectious condition.

Preferably, the method is used to detect BSE.

Apolipoprotein E is a cholesterol transporting protein that is produced in the peripheral and central nervous systems. In the cerebrospinal fluid or serum, its single or multiple form was distinguished.

The discovery that infection of an animal with spongiform encephalopathy can be correlated with the presence or increase in the concentration of one or more agents in the animal thus forms the basis of the invention.

body fluids of the method of the present invention

The reagent (s) that are cross-reactive with anti-apolipoprotein E have a molecular weight of about 34-38 kDa and a pI of about 5.4-5.7. This is consistent with their identification as apolipoprotein E. The term ApoE reagent, which is further used, includes any agent having these properties (including apolipoprotein E alone and its isoforms or multiple forms).

It should be emphasized that there is no requirement to accurately determine the concentration of ApoE, since spongiform encephalopathy can be detected by comparison with the control.

The control value can be derived from the concentration of ApoE in various animals (for which the infectious condition is known) and which are analyzed in parallel with the test animal. Alternatively, the control value may be from the same animal, or may be a known standard.

The control value can be determined using the same method used to test the animal or using a different analytical method.

Results from the control animal can be used to obtain a standard positive or negative control value or to calibrate the results of the test animal.

The preferred body fluid analyzed by the method of the invention is cerebrospinal fluid (CSF), since the major apolipoprotein found in the fluid is authentic apolipoprotein E. In addition, close CSF contact with the brain means that neurological diseases that produce changes in The composition of brain proteins can be manifested in CSF. Methods for extracting CSF samples are well known to those skilled in the art.

The invention encompasses any method of analyzing the concentration of ApoE in the animal body fluid that is now known in the art, and any method that will later become available.

Preferably, the presence and / or amount of ApoE in the body fluid of the animal is determined by PAGE or 2DPAGE, separating ApoE from other agents in the body fluid and then staining the gel and making densitometric measurements in the region of the gel of interest. to determine the presence and / or amount of ApoE.

Preferably, the identity of the ApoE reagent is confirmed using the ApoE immunogenic agent Technique for substances, for example, antibodies raised against or a synthetic peptide based on its sequence. the identification of the presence of cross-reacting agents based on an immunogen is well known in the art;

for example ELISA or Western blotting.

In an alternative embodiment of the invention, these immunogenic techniques can be used to both identify the ApoE agent and to estimate its concentration.

Thus, the invention provides methods for detecting the presence of spongiform encephalopathy in test animals which can solve many, and preferably all, problems of the prior art. The balance between the reliability of the assay and its ease of use will depend on the particular ApoE reagent analysis method chosen for the method of the present invention. In any case, the pre-mortem diagnosis of BSE in bovine animals opens up the possibility of mass testing of herds, thus reducing the likelihood of slaughtering uninfected animals or missing slaughter of infected animals.

The process according to the invention will now be described, by way of illustration only, by way of the following example and figures. Other embodiments within the scope of the invention will be apparent to those skilled in the art in light of these examples.

DETAILED DESCRIPTION OF THE INVENTION

Identification of BSE in bovine animals

Sample Preparation: CSF samples from BSE positive bovine and BSE negative bovine were collected. In any case, the diagnosis was confirmed by post-mortem histopathology and electron microscopy. CSF samples were collected by cistern magna after death and were concentrated 10-15 times. CSF volumes containing a total of 40 µ9 protein were mixed 4: 1 with a denaturing solution (1 g sodium dodecyl sulfate (SDS) and 0.232 g titiotreitol in 10 ml water) and heated to 95 ° C for 5 minutes. The samples were then subjected to pulse centrifugation.

Electrophoresis: Prepared samples were two-dimensional (2D) electrophoresed using the Millipore Investigator 2D electrophoretic system according to the method described in the manual. One-dimensional isoelectric focusing of the first dimension was performed in 26 cm 1 mm threaded glass tubes, threaded, in a pH gradient of 3-10 using 18,000 volthodin after gel pre-focusing for 1 hour at 1,500 V. Second dimension SDS-PAGE was made using 1 mm thick large size gels (23 cm x 23 cm) with 12.5% acrylamide without vertical gel.

Staining and image analysis: 2D gels were stained with silver according to Millipore manual. The gels were scanned using the Omnimedia scanner XRS and analyzed using Bioimage software and Investigator Database (Millipore) using a sunSPARC station.

ApoE: 2D gels identity verification was electroblotted onto Immobilon-P membranes overnight at 30 V using a Bio-Rad Trans-Blot cell. The blots were blocked using a Tween 80 soak for 1 hour and then incubated for 90 minutes with a sheep antiserum containing a polyclonal antibody raised against authentic apolipoprotein E. Binding of sheep antibodies was detected using a rabbit anti-sheep IgG and horseradish peroxidase detection system. The number of agents of interest (approximately 34 to 38 kDa molecular weight range and 5.4 to 5.7) that cross-react with anti-apolipoprotein E antibody is significant.

Comparison of BSE negative and BSE positive bovine: A comparison of stained gels derived from a typically BSE positive and typically BSE negative sample is shown in FIG. 1a and FIG. Ib. As can be seen, the number and intensity of the silver stained spots in the region corresponding to reagents with an approximate molecular weight of 34-38 kDa and a pI of about 5.4-5.7 (substantial ApoE) are higher for the BSE positive sample.

A comparison of the mean optical density of those silver stained gels on the gels that also cross-react with the anti-apolipoprotein E antibody is given below:

reagent no. BSE - negative BSE- positive 1 0.13 0.47 2 0.42 0.84 3 0.45 0.64 4 0.45 0.99 5 0.46 1.02 6 0.41 0.69 7 0.87 1.12

Comparison of the two sets of optical density values shows that each reagent is consistently present in a higher

BSE-positive animals (n = 31) as in BSE-negative animals (n = 27), a number of these agents indicating that the presence or can be used to detect the likelihood of BSE being present in potentially infected animals.

Claims (8)

PATENT CLAIMS What is claimed is: 1. A method for the in vitro determination of a disease state of spongiform encephalopathy in an animal comprising: a) determine the amount of apolipoprotein E in the cerebrospinal fluid of the animal, (b) compares the result of this determination with the control value; and B c) the increased concentration of apolipoprotein E correlates with the positive disease state of the animal. Method according to claim 1, characterized in that the spongiform encephalopathy is bovine spongiform encephalopathy. The method of claim 1 or 2, wherein the amount of apolipoprotein E in the cerebrospinal fluid of the animal is determined by estimating the amount of cross-reactant reacted with the antibody raised against apolipoprotein E contained in the cerebrospinal fluid of the animal. 4. The method of claim 1 or 2, characterized in characterized in that the amount of apolipoprotein E in CSF _Λ of the animal is determined by the apolipoprotein E is separated from the other substances present in the CSF electrophoresis' polyacrylamide gel, the gel was stained. Apolipoprotein E is identified and its amount is estimated from the dye density. The method of claim 4, wherein the amount of apolipoprotein E is determined by estimating the amount of agent having a molecular weight ranging from 34 to 38 kDa and a pI value ranging from 5.4 to 5.7 contained in the cerebrospinal fluid. Method according to claim 5, characterized in that the identity of apollpoprotein E on the gel is confirmed using an antibody raised against apolipoprotein E. Method according to any one of claims 4 to 6, characterized in that the polyacrylamide gel electrophoresis is performed as a two-dimensional electrophoresis. Method according to any one of the preceding claims, characterized in that the control value is obtained in the same manner as that used in the determination of the animal to be examined, but using another animal known to be either negative or positive for spongiform encephalopathy. .