JP2002530650A - Immunoassay for determining infectious spongiform encephalopathy in cattle - Google Patents

Abstract

  (57) [Summary] The present invention relates to an immunoassay for measuring prion protein (PrP) in bodily tissue or body fluid samples from bovine animals, the method comprising the steps of: A) a capture antibody bound or bindable to a solid phase; and b) a detection antibody bound to the PrP obtained from the step; and a detection antibody bound to the solid phase. Quantifying the signal from The invention further relates to a method of diagnosing bovine spongiform encephalopathy (BSE) in bovine animals.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001] The present invention relates to an immunoassay for determining infectious spongiform encephalopathy in cattle. More specifically, the present invention relates to a novel method for detecting prion protein, which form is a diagnostic marker for bovine spongiform encephalopathy (BSE).

BACKGROUND OF THE INVENTION [0002] The publications and other materials used herein are hereby incorporated by reference, in order to elucidate the background of the invention, and in particular to provide further details of practice. .

[0003] TSE family scrapie of disease , Creutzfeldt-Jakob disease (CJD), Gerstmann-Straussler-Scheinker (GSS) syndrome and mink-related diseases (infectious mink encephalopathy), mule deer and elk-related diseases (chronic wasting) For infectious degenerative (or spongy) encephalopathy
(TSE). In recent years, cattle (bovine spongiform encephalopathy), cats (feline spongiform encephalopathy)
New species including cats and antelopes of various captive zoos have been affected, and new forms of CJDs in humans have recently been revealed. Iatrogenic infections of CJD have occurred in humans, and these diseases can be transmitted by inoculation or feeding of tissues from affected animals to healthy animals. The following text describing these diseases is taken from Hope's article, 1998 (see ref. 31).

[0004] Scrapies. Sheep scrapies have been known in Europe for hundreds of years and have spread to most parts of the world except Australia and Argentina due to the movement of humans and their livestock. This includes altered behavior,
It is characterized by sensitivity to sound or touch, loss of condition, pruritus and associated shedding and detachment of wool and incoordination of hind limbs. Diagnosis is confirmed by a histopathological triad of vacuolation, neuronal loss and gliosis at brain tissue necropsy (1).

[0005] Scrapies are mostly reported in sheep breeding and in sheep herds, which appear to occur in related animals. In natural clinical disease, the median developmental peak is 3.5 years in herds of animals, with the majority of cases occurring in the range of 2.5-4.5 years (2). For most of this period, the infected animals are clinically normal and cannot be distinguished from their uninfected herd. The incidence of clinical disease in sheep flocks is usually 1-2 cases per 100 sheep per year, although in some cases 40-50% of animals in the herd die from the disease within 1 year. It is. In recent years, many genetic markers have been identified as risk factors, and the introduction of genotyping in field studies on the occurrence of natural and experimental diseases has greatly facilitated elucidation (3).

[0006] Kreutzfeld-Jakob disease [0006] Kreutzfeld-Jakob disease is a progressive dementia with clinical signs indicative of dysfunction of the cerebellum, basal ganglia and lower motor neurons. This is associated with progressive degeneration of dementia and confusion, as well as progressive impairment of motor function. Most patients die within 6 months of the onset of clinical signs, and no case has been shown to recover. Pathologically, brain foci show vacuolar formation of various neural networks, astrocytes, and amyloid plaques in about 10% of CJD cases. Gerstmann-Straussler syndrome is a familial variant of CJD with a prolonged clinical time course.

[0007] The incidence of CJD-related diseases in humans is surprisingly constant at 0.5 to 1 case per million per year worldwide and is therefore not linked to the occurrence of any animal disease. This low incidence casts doubt on the role of infection in its transmission in the population (but see below). About one in seven cases is familial and is associated with a mutation in the open reading frame of the PrP (prion protein) gene. Numerous clinical and pathological studies have been performed on human cases of neurological disorders that appear to be associated with these rare mutations of the PrP gene (for verification, see (4)).

[0008] In some families, there is a complete penetrance of the phenotype, so this mutation is considered the cause of the disease. There is no epidemiological evidence of horizontal transmission of the disease, except in cases of iatrogenic disease induced by transplantation of infected tissue or inoculation of contaminated human-sourced drugs. The stochastic event associated with the conversion of the PrP protein to its disease-related isoform or the opportunity for mutation of a benign ubiquitous virus-like substance is indicated by two types of disease that have been shown to explain the sporadic disease incidence Mechanism (see section on the nature of the causative agent). Where genetic counseling is applicable, this can effectively prevent disease transmission from one generation to the next, but there is no cure for the clinical condition.

[0009] There is considerable clinical and pathological heterogeneity with respect to human prion disease,
Despite the genotyping and sequencing of the P ORF (ORE = open reading frame) providing some unifying concepts, the PrP protein mutation does not seem to be a complete explanation. Other genetic factors, including the association of the ApoE gene with the E4 allele, have implications as risk factors for developing CJD.

Emerging human TSE lethal familial insomnia (FFI) is becoming a problem as a new member of human infectious encephalopathy. This includes disorders of the autonomic nervous system that usually present with insomnia,
It is characterized by temperature and blood pressure regulation problems. The brain pathology at necropsy is mostly neurological deprivation and thalamic degeneration with little or no vacuolation of the neural network. Its classification as a prion disease was initially based on the association of an asparagine (N) to aspartic acid (D) mutation at codon 178 of the PrP gene, and this mutation was a variant of the conventional form of CJD. Is also relevant. Which of these two phenotypes prevail seems to depend on the amino acid encoded at codon 129 of the same PrP allele: in FFI, codon 129 encodes methionine. In CJD, codon 129 encodes valine. Homozygotes at codon 129,
Although appearing to be risk factors for developing sporadic CJD, each polymorphism at this codon is quite common and does not appear to be pathogenic in itself. The classification of FFI has been confirmed by disease transmission to laboratory mice (5).

[0011] In March 1996, the UK government announced concerns about new variants of CJD, and details of these cases are becoming increasingly clear (6). New neuropathological and clinical profiles in the UK to date-extensive PrP adhesion, cerebellar amyloid plaques,
Spongy changes most prominent in the basal ganglia and thalamus, prolonged (up to 2 years), atypical EEG
Twenty-eight CJDs with early ataxia, behavioral and mental disorders have been identified. Similar cases have not been identified in archived patient files or elsewhere in Europe (except one in France (7)), and the risk factors for this variant appear to be unique to the UK. Although the new bovine TSE (BSE) and its co-occurrence, which are largely restricted within the UK, lead to the presumption that this new CJD type shows cross-species transmission from cattle, This has not been proven.

Bovine Spongiform Encephalopathy Bovine spongiform encephalopathy (BSE) has disrupted the cattle industry in the UK for the past decade (8). 19
From the first reported solitary case in 1986 and several cases identified in retrospect in May 1985, a major epidemic was ongoing until 1988, at which time 180,000 people were in the British Isles. More than a cattle's life had been taken at that time. Cases have been confirmed in other countries: Switzerland (450+), Ireland (700+), Portugal (300).
+), 1 in France and Germany or 2 in Italy, Denmark, Canada, Netherlands, Oman and Falkland Islands.

[0013] The disease causes progressive degeneration of the central nervous system, and BSE-
It is so called because brain tissue has a spongy appearance (9). Warning signs of the disease include changes in cattle behavior and body temperature. Affected animals should be
In particular, hind limb concerns begin to grow and become problematic. Cows (or bulls) become more paralyzed as they become more sensitive to touch and sound, lose weight, and suffer from disease in their nervous systems. This clinical phase of BSE lasts from two weeks to more than six months. Although the majority of affected animals are dairy cows, the neurological disease can occur in both genders with a mode of onset age of 4-4.5 years (range 1.8-18 years). Most cases of BSE occur in cattle aged 3-5 years and most of the time of onset show no evidence of their presence (10).

The neurological lesions of the bovine brain affected by BSE are virtually the same as those seen in sheep affected by scrapie, including spongy changes that account for the BSE name. BSE is readily speculated from its clinical and neuropathological manifestations to belong to the scrapie family of infectious spongiform encephalopathies. This is a biochemical study (11),
And has been confirmed by experimental transmission of BSE to mice (12), sheep and goats (13), among other species.

Prion Protein (PrP) The conversion of the normal membrane glycoprotein, cellular prion protein or PrP ^C , to the aggregated insoluble isoform PrP ^Sc has been demonstrated in BSE, scrapie and other infectious spongiform encephalopathies (TSE). It is an important process of developmental pathology. Detection of specific PrP ^Sc forms the basis of biochemical diagnosis of these diseases. Differences in the folding of the aberrant isoform of the prion protein (PrP ^Sc ) can be attributed to whether normal protein is destroyed or dramatically reduced in amount before detection by SDS-PAGE / immunoblot or ELISA techniques. Proteolysis under any given condition allows one to search for and examine the configuration of this protein in diseased tissue.

Primary Structure of PrP The amino acid sequences of mouse, human and bovine PrP are shown below using the IUPAC amino acid single letter code:

[0017]

[Table 1]

[0018]

[Outside 1]

Although prion proteins are expressed in many different cells, they are associated with neurons of the mid-autumn nervous system and have the greatest abundance. Therefore, the abnormal form of PrP is
It accumulates preferentially in the brain, but is also detected in extraneural tissues such as tonsils and spleen early in the onset. Thus, the statement that total PrP concentration is greater in diseased tissue compared to healthy tissue is correct.

In cell culture, the PrP protein is circulated to and from the cell surface via the endosomal-lysosomal system; during this process, the protein is proteolytically between residues 109 and 112 Appears to be severed. Although the extent to which this cleavage occurs in vivo is unknown, a C-terminal fragment of PrP similar to that expected to result from the cleavage of this peptide bond has been identified in mouse and human P.
It is observed by the attachment of rP ^Sc . The exact location of the cut is related to the disease phenotype or the infectious agent lineage; this is the scope of current research.

Prior Art for PrP Detection Since prion protein (PrP) has been discovered, measurement of prion and PrP
This could be done in relation to the number of protein molecules per infectious particle. This is the case in allogeneic rodent bioassays; for example, in hamsters.
As measured by intracerebral inoculation using agents of the 263K strain or the ME7 strain in mice, it is usually estimated to be 100,000 PrP molecules per infectious particle. Detection of PrP ^Sc as fibrils (14,15) or immunochemical methods (16-22) can be used as a TSE diagnostic test in either necropsies or surviving animals. Preliminary validation of this method using the ICC of fibrils or PrP (ICC = immunocytochemistry) to track the oral pathogenesis of BSE in cattle has recently been published (23). Western blot or dot blot is now able to detect PrP ^{S c} of 10~100pg (10 ⁸ ~10 ⁹ mol), these methods are substantially the heterologous bioassay of bovine or human tissue for BSE infectivity in mice Same sensitivity. For example, typically, BSE-
1 g of affected brain contains 1 μg of PrP ^Sc (11) and has an infectivity of 10 ^3.5 LD ₅₀ units / g.
(24). This is equivalent to the specific infectivity of one infectious particle / ¹⁰⁹ molecules. Higher specific infectivity is observed in allogeneic transmission (25,26), but from these calculations, the mouse bioassay does not provide greater sensitivity than the PrP immunochemical assay for detecting BSE infectivity It is clear. Improving the sensitivity of this assay system is that horizontal or vertical transmission of BSE may increase the infectivity (and PrP ^Sc ) in milk (27), blood, placenta and other peripheral tissues of BSE-infected cattle. (28) can shed light on the scientific challenge of why it occurs in non-existent situations (26) (28).

Specificity of FH11 and 3F4 Monoclonal Antibodies The epitope specificity of FH11 and 3F4 has been mapped as follows (Birke
tt et al., unpublished; (29)). The use of FH11 for immunocytochemical detection of PrP in sheep brain tissue sections has been reported (30); this paper describes its N-terminal specificity and PrP ^Sc
The use of trypsin to enhance the detection of E. coli.

FH11 (also BG4)

[Table 2] The essential parts of these epitopes are shown in uppercase. The "core" of these epitopes is shown in italics.

SUMMARY OF THE INVENTION In one aspect, the present invention relates to an immunoassay method for measuring prion protein (PrP) in bodily tissue or body fluid samples obtained from bovine animals. In the present invention, the method comprises the steps of treating PrP to promote its extraction and binding to the antibody, and to PrP obtained from the preceding step, a) for capture or binding to a solid phase. And b) applying an antibody for detection, and quantifying a signal from the antibody for detection bound to a solid phase.

In another embodiment, the present invention relates to a method of diagnosing bovine spongiform encephalopathy (BSE) in a bovine animal, the method comprising the steps of: Measuring the prion protein (PrP) in a body fluid sample. PrP
Is used as an indicator of BSE in the bovine animal.

In yet another aspect, the invention relates to a method for managing an autopsy of a post-slaughter bovine body to identify individuals exhibiting a BSE infection, the method comprising the steps of: Measuring prion protein (PrP) in a body tissue or body fluid sample obtained from the bovine body. The presence of PrP is used as an indicator of BSE in the bovine body.

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a novel two-site (two-site) for measuring prion protein (PrP).
site) For immunometric assays. This method involves the use of a capture antibody that recognizes the epitope, preferably in the N-terminal region of the molecule. Preferably, the detection antibody recognizes an epitope in the protease-resistant core of the PrP molecule that is occuluded when PrP is aggregated. The use of a proteolytic enzyme at a limited concentration promotes the disaggregation of PrP. This leads to increased binding of the detection antibody. "N-terminal region" refers in particular to the region defined by amino acids 55-95 of the PrP molecule.

However, it must be noted that one or both of the capture and detection antibodies are directed against an epitope in the N-terminal region, protease resistant core, or C-terminal region of the PrP molecule. The treatment of PrP may include, for example, any of the following: (i) limited proteolysis; (ii) addition of detergent; (iii) addition of chaotropic reagents and / or solvents; (iv) temperature. And (v) sonication. These processes can be used individually or in combination.

“Protease resistant core of a PrP molecule” is that portion of a PrP molecule that is not degraded by proteolysis. The normal metabolic cleavage of this molecule involves amino acid region 1
It is within 09-112. This cleavage is caused, for example, by proteolytic enzymes that vary depending on the tissue, cell type, mammal, and the like. Regarding proteinase K,
Cleavage occurs in the 90 region of the LVG hamster 263K line. It is preferred that both the detection antibody in addition to the capture antibody are monoclonal antibodies.

[0030] The detection antibody can be labeled with any detectable label. However, in a preferred embodiment, the label is a lanthanide chelate, wherein detection is based on time-resolved fluorescence measurements.

The specimen is, for example, brain tissue, spinal cord, lymph tissue, spleen, tonsils, whole blood or blood fraction. For post-necropsy analysis, brain tissue is a particularly preferred specimen. For the study of live bovine individuals, less invasive tissue or body fluid samples should be used. An example of a bodily fluid sample can mean whole blood or a fraction thereof.

The use of antibody FH11 as a capture reagent is novel and unclear in any assay system for the detection of PrP ^Sc using proteinase K (PK). Conventional assay systems (eg, Western blot analysis) use enough PK to digest PrP to its protease resistant core. This protease
The resistant core does not have a primary epitope for FH11 or any other antibody in the N-terminal region of the molecule.

[0033] The 3F4 antibody is widely used to detect PrP in hamster and human tissue samples. However, cross-reactivity of sheep or bovine PrP in Western blot analysis is not shown. Therefore, its use in assays for the measurement of PrP in sheep or bovine tissue is novel and unclear. It has proven to be a very useful reagent because of the increased sensitivity obtainable in DELFIA, which can explain the low cross-reactivity of 3F4 for sheep and bovine epitopes.

[0034] Another novel aspect of the use of this antibody is the finding that the 3F4 epitope is in the "core" region of the molecule involved in the aggregation process. When PrP is in an aggregated state, the 3F4 epitope is occluded and is not available for antibody binding. PrP
If is disaggregated by limited PK digestion, treatment with chaotropic reagents (eg,> 2M guanidinium chloride) and / or the addition of detergent exposes the 3F4 epitope, binds the antibody and increases the signal.

Low doses of PK can facilitate the measurement of highly aggregated PrP for the following reasons: 1. By proteolytic cleavage of PrP from its GPI anchor that binds the molecule to the cell membrane PrP solubilization achieved. 2. Limited proteolysis and protein removal associated with aggregated PrP. These proteins include PrP itself and non-PrP binding proteins that have not yet been identified. 3. Removal of binding proteins ideally promotes disaggregation of PrP, but does not require the presence of detergents with simultaneous exposure of epitopes within the core region of the molecule.

The PK effect can be achieved by adding a limited concentration of PK. Increasing enzyme concentration will result in cleavage of PrP, resulting in eventual loss of the primary FR11 epitope. The results of the immunoassay method of the invention can be used for various purposes. These can be used for the diagnosis of bovine spongiform encephalopathy (BSE) in bovine animals, which can be living or dead individuals.

A particularly important area of application can refer to the routine management of bovines in slaughterhouses. Specimens can be obtained from each carcass and analyzed. Body showing BSE infection will be destroyed. This is an effective way to prevent the sale of infected meat and other bovine-based products. On the other hand, unnecessary disposal of healthy bodies can be avoided. Therefore, this management method has great economic value.

The present invention is described in more detail by the following non-limiting examples. (Experimental Section) Materials and Methods 1. DELFIA® reagent was purchased from EQ & G Wallac (20 Vincent Avenue, Cr
Available from ownhill Business Centre, Crownhill, Milton Keynes MK8 OAB). 1.1 Assay buffer Cat. No. 1244-106 Tris-HCl buffer (pH 7.8) salt solution containing bovine serum albumin, bovine globulin, Tween 40, inert red dye, and <0.8% sodium azide as preservative Was prepared before use. Stored at 2-8 ° C. until expiration date marked on vial label.

1.2 Wash Concentrate Cat. No. B117-100 A 25-fold concentrate of Tris-HCl buffered (pH 7.8) salt solution is Tween 20 and Ge as a preservative
nnall II. Stored at 2-8 ° C until expiration. Wash solution with distilled water 25
Prepared by diluting the fold wash concentrate (ie, dilute 40 mL of concentrate to 1 liter). 1.3 Enhancement solution Cat. No. 1244-105 Prepared before use with Triton X-100, acetic acid and chelating agent. Until the expiration date, 2 ~ 25 ℃
Stored at Avoid direct light.

1.4 Eurobium-Labeling Reagent Kit Cat. No. 1244-302 Each kit contains 0.2 mg of labeling reagent, Eu-standard, enhancer, stabilizer to increase stability of labeled protein (purified BSA) , Uncoated microtiter strip plates, assay buffer and wash concentrate. The kit contents were sufficient to label up to 1 mg of protein. Stored at 2-8 ° C until expiration. 1.5 Uncoated microtitration plate with low fluorescence background Cat. No. 1244-550 DELFIA plates are 8 x 12 strips and were manufactured by NUNC from plastic with low fluorescence background. These had a high immunoglobulin binding surface (MAXISORB).

2. Other Reagents 2.1 ImmunoPure (A) IgG Purification Kit Available from: Pierce Wallina (UK) (44 Upper Northgate Street, Cheste
r, Cheshire CHL 4EF), Cat. No. 44667 Contents: 1. From the highest quality buffered salt by filtration using ultrapure reagent grade water (0.2
Micron) prepared buffer for binding ImmunoPure IgG (1000 mL; pH 8.0). This buffer contains EDTA as a preservative. 2. ImmunoPure IgG elution buffer (500 ml; pH 2.8). The buffer was purged with nitrogen to remove oxygen. The low pH in the absence of oxygen resulted in a reagent with good stability without the use of preservatives. 3. Protein A affinity pack column, 5 x 1 ml column

2.2 Amicon Microconcentrator (Microcon 30) The Microcon concentrator used a low-binding, non-uniform hydrophilic YM membrane manufactured by Amicon. Microcon-30 used a membrane with a molecular weight cutoff of 30,000 KDa. The device can be used in any Eppendorf centrifuge (e.g., MSE Micro-Centaur Microfuge) and simply and efficiently concentrate proteins before or after labeling with lanthanide chelates (or biotin), It provides a means for desalination and purification.

The low absorption properties of the YM membrane, and the components of the device, are combined with inverted collection spins to obtain unusually high recovery-typically 95% of the analyte with a high concentration factor of 100 ×. Available from: Millipore, UK (The Boulevard, Blackmore Lane, Watford WD1 SYW)
, Catalog No. 42409

2.3 Labeling Buffer Sodium bicarbonate buffered saline (50 mmol / L) was added to 4.2 g NaHCO ₃ (Sigma S-8
875) and 9.0 g NaCl (Sigma S-9625) in 1 liter of ultrapure water (Milli-Q or equivalent)
Prepared by dissolving in. The pH of the resulting solution was adjusted between 8.5 and 9.0 with NaOH (2N; Aldrich 22,146-5). This solution was stored at 2-8 ° C.

2.4 Coating Buffer Phosphate buffered saline (Sigma 1000-3) prepared by dissolving the contents of the sachet in 1 liter of distilled water contains NaCl (120 mmol / L), KCl (2.7 mmol / L) and a phosphate buffer (10 mmol / L) at 25 ° C. and pH 7.4. Sodium azide (
(Sigma S-2002, 0.1%) was added. This solution was stored at 2-8 ° C. 2.5 Blocking buffer Sodium azide (Sigma S-2002; 0.1%) and bovine serum albumin (Sigma)
A-7888; 2%) in phosphate buffered saline (Sigma 1000-3). Stored at 2-8 ° C.

3. Materials provided by the Agency for Animal Health (IAH) 3.1 Monoclonal Antibodies At the time of the present invention, the most useful monoclonal antibodies available are: (i) Capture antibody-FH11 approx. Concentration: 3.9 mg / mL (purified IgG) (ii) Detection antibody-3F4 Approximate concentration: 1 mg / mL (purified IgG)

3.2 Recombinant PrP Standard 3.2.1. GST-Hamster PrP Fusion Protein This stock solution was stored at −20 ° C. in chaotropic buffer estimated to be 0.48 mg / mL and containing 4 M guanidinium hydrochloride. . Preparation of Standards The stock PrP-GST fusion protein was serially diluted 10-fold in assay buffer to give the following series of diluted standards: (i) buffer blank; (ii) 0.48 ng / mL; (iii
) 4.8 ng / mL; (iv) 48 ng / mL; (v) 480 ng / mL; and (vi) 4800 ng / mL. These standards were prepared fresh before each experiment.

3.2.2 Histidine-tagged hamster PrP protein The subsequent recombinant hamster PrP protein contained an extract of histidine-tagged hamster PrP protein in chaotropic buffer. The protein concentration of these extracts ranged from 0.78 to 8 mg / mL. Stock solutions were stored at -20 ° C.

3.2.3 Recombinant Bovine PrP Recombinant bovine PrP (4 mg / mL) stock solution was stored at −20 ° C. Stock solutions were serially diluted in assay buffer to give the following series of diluted standards: (i) buffer blank; (ii) 4 ng / mL; (iii) 40 ng / mL; ( (iv) 400 ng / mL; (v) 4000 ng / mL; and (v
i) 40,000 ng / mL. These standards were prepared fresh before each experiment.

Purification of Antibodies Using ImmunoPure (A) IgG Kit Approximately 0.5 mL aliquots of the following: (i) mouse ascites (3F4); or (ii) culture supernatant (FH11) in 1 mL of ImmunoPure IgG binding buffer And loaded onto a 1 mL column of AffinityPak pre-filled with immobilized Protein A, previously equilibrated with binding buffer. Chromatography was performed at room temperature and the eluent was monitored at 280 nm using a UVICORD flow detection monitor connected to a flatbed recorder.

[0051] Once all of the protein had flowed off the AffinityPak column, the specific IgG was eluted, followed by elution buffer. The presence of protein in the eluate was detected by a monitor, and the buffer was collected in glass vials. The concentration of the eluted protein is estimated by measuring the absorbance at 280 nm using a spectrophotometer. The column was regenerated by washing with 4 column volumes of 0.1 M citric acid and 6 N NaO
Adjusted to pH 3.0 with H. For storage, the column was further washed with 5 ml of water containing 0.02% sodium azide.

Label Buffer Exchange of Selected Antibodies with Europium A 500 μL aliquot of the monoclonal antibody for 3F4 detection purified on protein A was added to a Microcon-30 sample storage container and inserted into an Eppendorf vial holder. The tubes were capped and centrifuged at 6000 rpm for 15 minutes. Thereafter, the eluate was removed and discarded, and a 400 μL aliquot of the labeling buffer was added to the sample container. The procedure was repeated two more times after rotating the tube as described above and adding 400 μL of labeling buffer.

After the final preparative centrifugation, the vial was separated from the sample container and placed upside down in a new vial. The device was capped and spun by short pulses to transfer the concentrate to a vial. The concentrated solution of purified 3F4 IgG was diluted with a labeling buffer to obtain an approximate concentration of 2-3 mg / mL IgG.

Labeling Procedure Approximately 1 mg of the concentrated and purified specific 3F4 IgG preparation was combined with the lyophilized labeling reagent (0
.2 mg). The vial was stoppered and the contents were vortexed gently and left overnight at room temperature.

Purification The europium-labeled 3F4 monoclonal antibody from the unreacted labeling reagent was separated and acted on using a Microcon-30 microconcentrator in the same procedure as described above. However, the buffer used in this purification was Tris buffered saline (TBS; 50 nmol / L).
Tris-HCl buffer, pH 7.75, containing NaCl (0.9%) and NaN ₃ (<0.1%).
Purified europium-labeled antibodies were stored in glass vials.

For more elaborate separations, the bound label was removed by gel filtration on a Sephadex G-50 column. Elution was performed with TBS (described above), and contaminants were removed by washing the column with 10 mmol / L (pH 4) of a phosphate buffer containing 0.001% DTPA. Aggregates, which may be present in the protein fraction or often formed during labeling, often cause an increase in background in solid phase assays. These agglomerates are separated by a suitable gel filtration medium (eg, Sepharose 6B or Sephaczyl S
400).

Characterization of Labeled Protein The europium content of the labeled antibody was determined by diluting the labeled product in the enhancer (1: 10,00
0 v / v), and determined by measuring the signal obtained with a time-resolved fluorometer. This count was compared to the signal generated from a 1: 100 dilution of the Europium standard in the enhancer (equivalent to ¹ nmol / L Eu3 ⁺ ). The protein concentration of the labeled antibody was determined at 280 nm using a spectrophotometer. This concentration must be adjusted by correcting for the absorbance generated by the thiourea binding (0.008 A / 1 μmol / Eu L at levels below 20 Eu / IgG).

Storage For good stability, the labeled antibodies were stored at high concentrations in the absence of competing metals (or chelators) in buffer. The concentrated solution (0.050 mg / mL) was stored at + 4 ° C. A small amount (about 0.1%) of a stabilizer contained in a labeling kit comprising BSA purified so as not to contain heavy metal impurities was added to increase the stability.

Measurement of Prion Protein Using Purified Monoclonal Antibody FH11 as Capture Antibody and Europium -Labeled 3F4 as Detector Coating of Capture Antibody The day before the DELFIA assay, the required number of microtiter plates were diluted with coating buffer. (1: 1000 v / v) coated with purified monoclonal antibody FH11. 200 μL of the diluted antibody solution was added to each well using a multichannel pipette. The plates were coated and incubated overnight at 4 ° C. next day,
Plates were washed three times using a WALLAC plate washer. Block of plate 200 μL of blocking buffer was added to each well using a multi-channel pipette. These plates were incubated for 1 hour at room temperature on a plate shaker.

Addition of Standards or Samples 200 μL of diluted recombinant standards or tissue extracts were pipetted into appropriate microtiter wells. These plates were incubated for 1 hour at room temperature on a plate shaker. The plate was then washed three times with the plate washes and patted dry on filter paper.

Addition of Europium-labeled detection antibody 200 μL of europium-labeled 3F4 detection monoclonal antibody (1: 5000 v / v) diluted in assay buffer was added to each well using an Eppendorf repeat pipette. . Plates were incubated for 1 hour at room temperature on a plate shaker. Plates were washed six times and patted dry on filter paper.

Addition of Enhancer 200 μL of Enhancer was added to each well using a predetermined Socorex dispenser. Plates are incubated for 5 minutes at room temperature on a plate shaker and
Transferred to a 4-plate fluorimeter. Calibration curve The hamster calibration curve is shown in FIG. The bovine calibration curve is shown in FIG.

Brain Tissue Extraction Protocol Homogenate of Tissue Brain tissue was carefully weighed and transferred to a sterile Dounce homogenizer. 1.5 mL of buffer for homogenate was added per 1 g of brain tissue. Homogenate buffer A: PBS (without detergent) or B: 1M GdnCL (3-14) in 50 mM Tris-HCl (pH 7.5) containing 0.5% sulfabetaine. Brain tissue was homogenized for 10-15 passes using a "loose" pistol. 1.5 mL of buffer for homogenization was added and the brain tissue was further homogenized 10-15 times using a "tight" pistol. The resulting mixture was a creamy emulsion, equivalent to 25% brain homogenate, based on the assumption that 1 g of brain tissue was equivalent to 1 mL. An aliquot of this emulsion was transferred to a polycarbonate ultracentrifuge tube (Beckman).

Proteolytic digestion Proteinase K (Sigma or Boehringer) was carefully weighed and dissolved in PBS to obtain a suitable stock solution. An aliquot of the PK solution was added to the emulsion in the polycarbonate tube to give a final PK range of 0-100 μg / mL. The tubes were sealed with PVC tape and incubated at 37 ° C. for 30 minutes with gentle agitation. Digestion was stopped by adding 10 μL Pefabloc SC (0.5 M solution in PBS).

First ultracentrifugation The polycarbonate tube was transferred to a Beckman TL100.3 rotor and TL1
Centrifuge at 100,000 rpm for 8 minutes at 22 ° C. in a 00 ultracentrifuge. The supernatant was removed and saved (primary supernatant). Extraction buffer was added in an amount equal to the amount of supernatant removed. Extraction buffer: 6M GdnHC in 50 mM Tris-HCl (pH 7.5) containing 0.5% sulfabetaine
l (3-14). The pellet was crushed using a plastic disposable Pasteur pipette and resuspended. Specimens were incubated at room temperature for 5 minutes. Second Ultracentrifugation The polycarbonate tube was centrifuged at 100,000 rpm for an additional 8 minutes at 22 ° C. in a TL100 ultracentrifuge. The supernatant was called the secondary supernatant.

Results Time-resolved fluorescence immunoassay of PrP in brain tissue extracts from scrapie-infected and normal hamsters is shown in FIG. 3 and shows the PrP in brain tissue extracts from BSE-infected and normal cattle. The time-resolved fluorescence immunoassay of FIG. 4 is shown in FIG. 4 and the time-resolved fluorescence immunoassay of PrP in brain tissue extracts from scrapie-infected and normal sheep is shown in FIG.

A typical PK titration experiment involved the production of a total of 10 mL of brain homogenate. This was equivalent to 2.5 g of brain tissue (approximately 2.5 brains). Ten aliquots (0.8 mL) were transferred to individual polycarbonate tubes and equal volumes of PK were added at ten different concentrations to ensure equality of each tube. This complete experiment was repeated four times on normal and scrapie hamster brains and yielded essentially the same results with respect to concentration and PK titration profile. The method was adapted for bovine and sheep brain tissue and gave similar results.

In this extraction method, PK had a dual effect. This not only digested PrPc, but also facilitated the measurement of PrP in primary and secondary extracts. For example, PK 5μg / mL, in hamster brain tissue, without PK or PK 100μg / mL
"Releases" PrP up to 100 times more than analytes treated in the presence of

It will be appreciated that the methods of the present invention are described herein only in part, and may incorporate various aspects. It will be apparent to those skilled in the art that other embodiments exist without departing from the spirit of the invention. The described embodiments are therefore illustrative and not intended to be limiting.

[0070]

[Table 3]

[0071]

[Table 4]

[0072]

[Table 5]

[0073]

[Table 6]

[0074]

[Table 7]

[Sequence list]

[Brief description of the drawings]

FIG. 1 shows a calibration curve of hamster PrP.

FIG. 2 shows a calibration curve of bovine PrP.

FIG. 3 shows a PrP assay in hamster brain tissue, in which the detected PrP concentration is plotted against proteinase K concentration (solid bars: scrapie, and shaded bars: normal ).

FIG. 4 shows a PrP assay in bovine brain tissue, in which the PrP detected was
P concentration is plotted against proteinase K concentration (black bar: BSE, and hatched bar: normal).

FIG. 5 shows a PrP assay in sheep brain tissue, which was detected in this experiment.
PrP concentration is plotted against proteinase K concentration (black bar: scrapie, and hatched bar: normal).

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) // C12P 21/08 C12P 21/08 (72) Inventor Hope, James United Kingdom, Edinburgh EH106 6J, Midrossian, Buckstone Loan 45 (72) Inventor Bernard, Joffrey John Lassell United Kingdom, Dorset B.H.R. 220 United Kingdom, West Moores, Pinehurst Road 88 (72) Inventor Barquette, Christopher Robin, United Kingdom, Berkshire OEX 100 Kwai, Oxon, Wallingford, Brightwell-Kum-Sottwell, Kings Orchard 9 F-term (reference) 4B063 QA01 QA18 QA19 QQ02 QQ03 QQ79 QQ96 QR16 QR48 QR56 QS28 QS33 QX01 4B064 AG27 CA19 CC24 DA13 4H045 AA00 AA11 CA45 DA76 EA50

Claims (9)

[Claims] 1. An immunoassay for measuring prion protein (PrP) in bodily tissue or bodily fluid samples obtained from bovine animals, comprising the following steps:-To facilitate its extraction and binding to antibodies, Applying PrP to the following steps: a) capturing antibody bound to or bindable to a solid phase, and b) applying a detection antibody to the PrP obtained from the above step; and Quantifying the signal from the antibody for use. 2. The method of claim 1, wherein said treatment is limited proteolysis. 3. The method according to claim 2, wherein the PrP is treated with a proteolytic enzyme, for example, proteinase K. 4. The method according to claim 1, wherein the capture antibody and the detection antibody are monoclonal antibodies. 5. The capture antibody is FH11 and the detection antibody is 3F4.
The method of claim 4. 6. The method according to claim 1, wherein the detection antibody is labeled with a lanthanide chelate. 7. The method according to claim 1, wherein the specimen is brain tissue, spinal cord, lymph tissue, spleen, tonsils, whole blood, or a blood fraction. 8. A method for diagnosing bovine spongiform encephalopathy (BSE) in a bovine animal, wherein the method according to any one of claims 1 to 7 comprises the steps of: Such a method, comprising examining prion protein (PrP), wherein the presence of PrP is used as an indicator of BSE in said bovine animal. 9. A method for managing an autopsy of a bovine body after slaughter for identifying a bovine body showing BSE infection, wherein the bovine body is obtained from the bovine body by the method according to any one of claims 1 to 7. Such a method, comprising measuring prion protein (PrP) in body tissue and body fluid samples, wherein the presence of PrP is used as an indicator of BSE in said bovine body.