Antibodies specifically reacting with prionic protein or its fragment, and their application

FIELD: medicine, biotechnology.

SUBSTANCE: the present innovation deals with elaborating diagnostic reagents for testing prionic protein in mammalian cerebral tissue due to IEA technique and refers to antibodies specifically reacting with prionic protein PrP or its fragment. The innovation includes polyclonal antibodies obtained to synthetic peptides including amino acid sequences of bovine prionic protein 143-168, 101-134 and 211-241, their conjugates with horseradish peroxidase and diagnostic reagents obtained upon their basis that enable to detect prionic protein in mammalian cerebral tissues.

EFFECT: higher specificity of detection.

15 cl, 8 ex, 8 tbl

 

The invention relates to the field of biotechnology and relates to antibodies specifically reactive with the prion protein PrP or its fragment, and their application. Prion diseases of humans and animals belong to the group of slow infections occur in degenerative changes in the Central nervous system and end in death. One of such diseases is spongy encephalopathy in cattle [GE]. Getting into the body in food or medicines derived from the tissues of infected animals, can cause prion disease - new variant disease Creutzfeld-Jakob disease. Vector-borne spongy encephalopathy or prion disease is an incurable neurodegenerative diseases affecting humans and some mammals. Currently four known human disease - the disease of creuzfeld-Jakob, Kuru, syndrome Gerstmann-Straussler-Sheinker, fatal familial insomnia and six animal diseases - scrapie of sheep and goats, GE cattle ("rabies cows"), transmissible mink encephalopathy, chronic wasting disease of deer and elk, GE cats and GE exotic ungulates [1]. Infectious agent of this group of diseases is the normal protein isoforms mammals, being insoluble and FR is sostoitsia glycoprotein, the resulting post-translational modification of normal mammalian glycoprotein. The mechanism of pathogenesis of this group of diseases is the conversion of normal PrP protein in its contact with the pathogenic isoform of the prion protein in sheep or cattle PrP-Sc with its subsequent accumulation in the nervous tissue. Due to the high interspecies homology of the prion protein, the development of methods for identifying pathogenic form allows you to diagnose prion diseases all of the above groups [2]. To develop sensitive methods of research and diagnosis of prion diseases is necessary antibodies specific to different parts of the prion. The problem of obtaining antibodies to the prion protein is associated with its low immunogenicity. For permission to develop different approaches, among which the most promising is immunization with synthetic peptide fragments of the prion.

Currently, methods for diagnosis of prion diseases is based on the use of enzyme-linked immunosorbent assay (ELISA) using monoclonal antibodies raised to different parts of the prion protein. Thus, the known monoclonal antibodies obtained to synthetic peptide 146-159 bovine prion protein, anywherefrom with KLH (hemocyanin snails) and used as immune the gene [3]. These monoclonal antibodies bind conservative epitope-Ile-His-Phe-Gli-PrP-Sc scrapie sheep. Next, using a combination of two monoclonal antibodies to the above-mentioned synthetic peptide and peptide 225-241, which bind more conservative epitope-Gln-Tyr-Gln-Arg-Glu-Ser-PrP-Sc, has improved the sensitivity and specificity of the determination of the pathogenic form of the prion protein by ELISA, as well as to extend its application for the diagnosis of prion diseases, ruminants, cats, mink, human and nonhuman primates [4]. There is a method of detection of prion protein for the diagnosis of GE by ELISA and immunohistochemical method using the same monoclonal antibodies that interact with the conservative epitope 150-153 bovine PrP or two pairs of the above monoclonal antibodies, including in vivo method preclinical determination of prion protein in lymphoid tissue and posthumous definition with pre-treatment of tissue with formic acid, trypsin digestion or exposure to autoclaving to activate pathogenic form of the protein [5]. The closest analogue is the source of information on obtaining antibodies against synthetic fragments of the bovine prion protein used for the diagnosis of GE grains is wow cattle [6]. To obtain polyclonal sera, the authors used 7 synthetic peptides by solid state method on n-alkoxybenzenes the polymer. To obtain protivoavarijnyh sera of rabbits were immunized three times with synthetic peptides or their conjugates with KLH. The first immunization was performed at a dose of 500 μg per animal in complete Freund's adjuvant (PAF), a second immunization was carried out in incomplete Freund's adjuvant (NAF) in the same dose and the third immunization was performed at a dose of 50 μg of peptide in the NAF. The most immunologically active peptides were peptides with the amino acid sequence 106-134 bovine PrP were then used in immunohistological method of determining prion protein in sections of paraffin blocks cows sick of GE. In this work revealed peptides allowed to obtain serum that communicates with the brain preparations of bovine animals, sick GE, and do not interact with drugs brain healthy animals. However, the proposed test method printarray protein is not quantitative, and the serum used for it have a relatively low specificity. In connection with the above, the technical result of the proposed invention is to develop obtaining highly specific polyclonal antibodies to multiple to nservative epitopes of the prion protein with the aim of creating a diagnostic for determining prion protein in brain tissue of mammals by ELISA.

The invention consists in the following: one aspect of the invention is a polyclonal antibody, specifically binding to the prion protein PrP or a fragment comprising amino acid sequence selected from the group:

a) a peptide or immunologically active fragment having the amino acid sequence:

SEQ ID NO:1

b) a peptide comprising the amino acid sequence:

143-168 SEQ ID NO:1

C) a peptide comprising the amino acid sequence:

101-134 SEQ ID NO:1

g) a peptide comprising the amino acid sequence:

211-241 SEQ ID NO:1

when this antibody is a purified immunoglobulin IgG protein content not less than 95% and the content of the isotype IgG1 at least 70%. The best option is an antibody, where the peptide 143-168 SEQ ID NO:1 has the following amino acid sequence:

SAMSRPLIHFGSDYEDRYYRENMHRY

An additional option is the antibody, where the peptide 101-134 SEQ ID NO:1 has the following amino acid sequence:

WGQLGTHWNKPSKPKTNMKKKHVAGAAAAGAVVG

Another aspect of the invention is an antibody, where the peptide 211-241 SEQ ID NO:1 has the following amino acid sequence:

ETDIKMMERVVEQMCITQYQRESQAYYQRGA

The invention also includes a method of obtaining polyclonal antibodies by immunization of rabbits with synthetic peptides, select the tion from the group includes: amino acid sequence: 143-168 SEQ ID NO:1, 101-134 SEQ ID NO:1 and 211-241 SEQ ID NO:1, with the primary immunization consists of 1 mg of protein per rabbit, mixed with a Bang, followed by 4 immunizations with peptides at a dose of 1 mg mixed with NAF, obtaining serum and purified by the affinity of the sorbent, representing the agarose with the immobilized peptide with obtaining purified antibodies. The invention also includes a conjugate for carrying out enzyme-linked immunosorbent assay (ELISA), which is an antibody obtained by the above method, conjugated to horseradish peroxidase with the degree of modification of 1.3 mol of horseradish peroxidase at 1 mol IgG1 rabbit.

The optimal conjugate is a conjugate, where the antibody specifically binds to a peptide 101-134 SEQ ID NO:1

Additionally, the conjugate comprises an antibody that specifically svyazyvaysya with peptide 211-241 SEQ ID NO:1

Diagnostic reagent for determining prion protein PrP or its fragments by ELISA, is a solution of the conjugate for conducting ELISA in 50% glycerol in 0.1 M phosphate buffer pH 7.4, containing BSA, with the following content of components, mg/ml:

Conjugate for conducting ELISA1
BSA5

The invention also includes PR is the application of a diagnostic reagent for determining prion protein PrP through direct and "sandwich method" ELISA.

Additionally, the invention includes the use of a diagnostic reagent for determining prion protein PrP in tissue of a mammal. The best aspect of the invention is the use of a diagnostic reagent for determination of pathogenic form of the prion protein PrP. The invention also includes a kit for determination of prion protein PrP in the brain tissue of mammals, including: diagnostic reagent, affiliazione antibody, excipients and the instructions for use. Set to "sandwich method" ELISA includes a pair of: the antibody specifically bind to the peptide 143-168 SEQ ID NO:1 and conjugated second antibodies raised against peptide 211-241 SEQ ID NO: 1.

The kit also includes excipients: lyse buffer, denaturing buffer, inhibiting reagent, reagent for cultivation proteinase K, proteinase K, buffer dilution, wash buffer, peroxidase substrate buffer, TMB stop reagent.

Examples of embodiment of the invention.

Example 1. Peptide synthesis and immunization of animals.

To obtain serum using the following peptides PrP: 143-168, 101-134 and 211-241. This choice of peptides due to the fact that the peptide 143-168 contains a conservative area of the prion protein 150-153, it was previously noted that monoclonal antibodies interacting the E. of this section, was successfully used to detect PrP-Sc protein and diagnostics GE [3-5]. Peptide 143-168 includes plot, forming βfolded structure, and the peptide 101-134 contains most of the third α-spiral plot. This choice of peptides due to the fact that it is necessary to obtain serum with high titer of antibodies for further separating pure antibodies and creation-based assays for ELISA. While peptides 101-134 and 211-241 contain 5 and 3 amino acids compared with peptides, published in the closest analogue.

Peptides receive solid-phase method on n-alkoxybenzenes the polymer. Protective groups of the side functional groups of amino acid residues chosen from the calculation for the final release TFA [triperoxonane acid]. To protect the side features Thr, Tyr, Ser use But-the group of Asp, Glu-OBut-rpynny for Lys-Boc-for His Trt. As a temporary Nα -protection is a Fmoc group. For increasing polypeptide chains on the polymer used TBTU (tetrafluoroborate 2-(1H-benzotriazol-1-yl)-N,N,N,N-tetramethylrhodamine) method, using a five-fold excess of amino acids. Cleavage of the synthesized peptide from the polymer with simultaneous release is carried out with a mixture of TFA with additives that prevent the occurrence of side reactions. After the release of the peptides absoluut gel-filtracia purified using reverse-phase HPLC. The yield of the final product after purification is from 11 to 41% in the calculation of the C-terminal amino acid. The individuality of the compounds obtained confirmed by amino acid analysis, mass spectrometry and reverse-phase HPLC.

Obtaining immune sera. Immunization of rabbits produce the above peptides in parallel on 2 rabbit on the peptide. The primary introduction of antigen animals is carried out in the form of an emulsion in PAF. Subsequent 4-fold injection of antigen is carried out in the NAF according to the standard scheme of immunization. Throughout the course of immunization (2.5 months) exercise control fences blood samples for carrying out ELISA tests. Output rabbit antisera: about 20 ml of each blood of the animal.

The standard scheme of immunization of animals are presented in table 1.

Table 1.

Scheme immunization of rabbits.
AnimalRabbitNo.The method of immunization: subcutaneous injection along a ridge
DateStageThe number of withdrawn bloodAntigenAdjuvant
start controlling blood5 mlweightvolumevolume
the first immunization1 mg1 mlfull beta-blockers1 ml
after 6 weeksthe second immunization1 mg1 mlincomplete beta-blockers1 ml
8 weeks after the startthird immunization1 mg1 mlincomplete beta-blockers1 ml
through 9 weeks after the startcontrolling blood40 ml
10 weeks after the beginningthe fourth immunization1 mg1 mlincomplete beta-blockers1 ml
after 11 weeks after the startcontrolling blood40 ml
analysis of antisera

The primary stage of obtaining serum includes the processing of the bunch, centrifugation antisera and analyzed by ELISA using the peptide, which immunized animals (see below). Characteristics of the obtained sera are presented in table 2.

Table 2

Breeding protivoavarijnyh antibodies polyclonal sera.
PeptideBreeding serum
143-1681:100000
101-1341:150000
106-134 (known)1:64000
211-2411:250000
214-240 (known)1:120000

Example 2. Purification of serum.

Preparation of the affinity of the sorbent. To 5 ml of the freshly prepared CNBr-activated agarose add 5-6 mg of peptide in 5 ml borate-salt buffer (0.1 M NaCl, 0.05 M Na-borate, pH 8.0), incubated for 2 hours with continuous stirring on the rotator. The ligand concentration determined spectrophotometrically at a wavelength of 280 nm. The reaction is stopped by adding 0.2 M ethanolamine, pH 8.0,incubated for 30 min at room temperature. Washed sorbent in the filter 100 ml of distilled water, 20 ml of eluting buffer (0.1 M NaCl, 0.1 M Na-acetate, pH 2.5) and 50 ml of working (0.15 M NaCl, 0.01 M Na-phosphate, pH 7.5) buffer. The contents of the ligand on the sorbent is 0.9-1.0 mg/ml Sorbents stored in borate buffer in the presence of 3 mm of sodium azide at a temperature of 4°C.

Clearing serum and chromatograph conditions were as follows.

1. Put 20 ml of the antisera to the sorbent with the immobilized peptide.

2. Collect the breakthrough fractions (5 ml) - not less than 5 factions.

3. Washed with 10 volumes (volume of the column) assay buffer (0.15 M NaCl, 0.01 M Na-phosphate, pH 7.5).

4. Elute adsorbed material 5 volumes of eluting buffer (0.1 M NaCl, 0.1 M Na-acetate, pH 2.5).

5. Bring the pH of the eluates dry sodium tetraborate to a pH of 7.2 - 7.5.

6. Measure the optical density at 260, 280 and 310 nm in all fractions of the eluate.

The resulting antibodies are immunoglobulin rabbit IgG containing not less than 95% of total protein content and IgGl not less than 70%. The solution affination antibodies diluted to a content of 1 mg/ml in 0.15 M sodium chloride solution containing 0.01 M phosphate pH 7.4. Table 3 presents the concentration of purified antibodies.

For the final concentration protivoavarijnyh antibodies adopt an antibody concentration at which the ELISA developed staining of not less than 0. optical density at 450 nm, exceed the background level by more than two times.

Table 3.

The final working concentration aminoacidemia antibodies when tested against peptides in IFA.
PeptideThe final concentration of the antibody mcg/mlWorking antibody concentration, µg/ml
143-1680.0080.1
101-1340.010.2
211-2410.0020.1

Next, perform the conjugation of horseradish peroxidase with affiliazione antibodies rabbit method controllable periodic destruction oxidation [7], with the degree of modification, component 1.3 mol of horseradish peroxidase at 1 mol IgG rabbit.

Diagnostic reagent for determining prion protein PrP is prepared by dissolving the conjugate in a concentration of 1 mg/ml of solution containing 5 mg/ml bovine serum albumin (BSA), 50% glycerol in 0.1M phosphate sodium, pH 7.4. Diagnostic reagent stored at -20°within 2 years.

Example 3. The procedure for determining prion in a sample of brain by ELISA.

Preparation of samples. Make a 1.5 ml lyse buffer in a test tube. Insert the filter into the test tube until it clicks into place and put 200±20 mg tissue vagus nerve brain cows (sheep) on f is ltr. Next, the tissue homogenized with a pestle light polukriminalnymi movements. After removal from the tube holder filter the supernatant liquid is decanted and dried test tube for a few seconds on filter paper. Separate the precipitate from the supernatant liquid by centrifugation, dried, treated with proteinase K and the reaction stopped by PMSF (phenylmethanesulfonyl). Then add denaturing buffer and incubated for 10 min at 100°C.

Example 4. Comparison of conjugates of horseradish peroxidase with affiliazione a rabbit antibodies by direct ELISA.

Conjugates compare and evaluate their specificity to the prion in a sample of brain prepared as described in example 3, treated and not treated with proteinase K. the Sample of the brain, not treated with proteinase K, consider opportunistic, because, as mentioned earlier, the pathogenic form of the prion unlike non-pathogenic resistant to proteases [2].

On a 96-well plate as antigen put a sample of the brain treated with proteinase K, the sample of the brain, not treated with proteinase K, positive and negative controls. As a positive control, use of recombinant prion in a concentration of 2.5 µg/ml as a negative control (background) using PBS with 0.05% tween-20 (PBSt) and 1% BSA. Posle hours incubation of the antigen in the wells add conjugates of horseradish peroxidase with affiliazione by rabbit antibodies to the peptides: 143-168, 101-134, 211-241. Then incubated for 1 hour in each well add peroxidase substrate tetramethylbenzidine (TMB). After development in the wells with positive control blue staining reaction is stopped with 10% H2SO4and measure the optical density (OD) at a wavelength of 450 nm. Absorbance values are presented in table 4.

The selection criterion of the conjugate to determine prion in a sample of the brain by direct ELISA specificity is calculated by the formula:

Specificity=OD (sample)/OD (sample+proteinase),

where OD (sample) - value of optical density in the wells with the sample of the brain, not treated with proteinase K (signal);

OD (sample+proteinase) is the value of optical density in the wells with the sample marrow, treated with proteinase K (noise).

That is, the specificity is characterized by the ratio of values of optical density in the wells with the sample of the brain, not treated with proteinase K, and the sample of the brain treated with proteinase K. the higher the ratio, the more specific the conjugate to the prion in the sample of the brain.

The values of optical density in the wells with positive control were the maximum in the hole with the negative control is minimal, which confirms the correctness of the conducted experiments.

On the basis of the received results to determine prion in the sample m is ZGA direct ELISA the most specific was conjugated antibodies to peptide 101-134. When using this conjugate ratio of optical densities in the wells of a sample not treated with proteinase and the sample treated with proteinase was the greatest.

Table 4

Comparison of the specificity of the conjugates
AntigenThe values of optical density OD450nm
conjugate 143-168conjugate 101-134conjugate 211-241
the positive control110018002200
negative control455065
sample+proteinase555075
sample80016002000
specificity14.53226.6

Example 5. Cross-interaction with foreign proteins in direct ELISA.

An important parameter of conjugates is their selectivity, i.e. the ability to communicate only with prion and not interact with other proteins and peptides. To test the selectivity of 96-hole plate as antigen put different about what einy and peptides, positive and negative controls. As a positive control using synthetic peptide corresponding to the conjugate and recombinant prion in a concentration of 2.5 µg/ml as a negative control (background) using PBS with 0.05% tween-20 (PBSt) and 1% BSA. After 1 hour incubation of the antigen in the wells add conjugates of horseradish peroxidase with affiliazione by rabbit antibodies to the peptides: 143-168, 101-134 and 211-241, then incubated for 1 hour and added to each well of peroxidase substrate TMB. After development in the wells with positive control blue staining reaction is stopped with 10% H2SO4and measure the optical density (OD) at a wavelength of 450 nm. Absorbance values are presented in table 5.

Table 5.

The selectivity of binding of the conjugates.
AntigenConjugates
Conjugate 143-168Conjugate 101-134Conjugate 211-241
BSA454055
casein555260
IgG rabbit706678
211-24180702200
65180070
143-16816505562
PBSt505565
Recombinant PrP110018002200

As can be seen from the table, the binding of conjugates with other proteins and peptides is minimal, since the values of optical density in the wells containing foreign proteins and peptides that are close to the values of optical density in the wells containing PBSt.

Example 6. The definition of prion in a sample of brain "sandwich method" ELISA. Comparison of the affinity-purified antibodies to the peptides: 143-168, 101-134, 211-241, and their conjugates.

To determine prion in a sample of brain "sandwich method" ELISA, you must choose from 6 pairs "antibody-conjugate pair with the highest specificity to the prion. In a preliminary experiment to determine the specificity of a pair of "antibody conjugate" to the recombinant prion. For this purpose, 96-well plate with immobilized thereto antibodies to peptides 143-168, 101-134, 211-241 cause of recombinant prion in a concentration of 2.5 µg/ml as a negative control (background) using PBS with 0.05% tween-20 (PBSt) and 1% BSA. After 1 hour incubation of antigen added to the wells conjugates of horseradish peroxidase with affiliazione by rabbit antibodies to the peptides 143-168, 101-34, 211-241, then incubated for 1 hour and added to each well of peroxidase substrate TMB. After development in the wells of the staining reaction is stopped with 10% H2SO4and measure the optical density (OD) at a wavelength of 450 nm.

Next expected specificity pairs "antibody conjugate" by the formula:

Specificity=OD (prion)/OD (background)

where OD (prion) is the value of optical density in the wells with recombinant prion in a concentration of 2.5 µg/ml (signal)

OD (background) - the value of optical density in the wells with PBS (noise)

The results are shown in table 6.

Table 6.

Determination of specificity pairs "antibody-conjugate antibodies against OD (prion) to OD (background).
Antibodies immobilized on the flatbedConjugates
Con-168Con-134Con. 211-241
AT 143-1684048
AT 101-1341040
AT 211-2411320
Based on these results we chose three pairs of "antibody conjugate":

1) AT 143-168 - Con 101-134: 40-fold excess of OD (prion) above OD (PBSt)

2) AT 101-134 - To the n 211-141: 40-fold excess of OD (prion) above OD (PBSt)

3) AT 143-168 - Con 211-241: 48-fold excess of OD (prion) above OD (PBSt), as the most specific to the protein.

Example 7. The choice of the optimal pair "antibody conjugate" in determining prion "sandwich method" ELISA in samples of brain.

Determination of specificity and selection of optimal pairs "antibody conjugate" is conducted according to the previously described method, and then treated or not treated him with proteinase K. the Sample of the brain, not treated with proteinase K, consider opportunistic.

On the 96-well plate with immobilized thereto antibodies to peptides 143-168, 101-134 put a sample of the brain treated with proteinase K, and the sample of the brain, not treated with proteinase K. as a positive control, use of recombinant prion in a concentration of 2.5 µg/ml as a negative control (background) using PBS with 0.05% tween-20 (PBSt) and 1% BSA. After 1 hour incubation of antigen added to the wells conjugates of horseradish peroxidase with affiliazione by rabbit antibodies to the peptides 143-168 and 211-241. Then incubated for 1 hour and added to each well of peroxidase substrate TMB. After development in the wells of the staining reaction is stopped with 10% H2SO4and measure the optical density (OD) at a wavelength of 450 nm.

Next expected specificity pairs "antibody conjugate" by the formula:

Specificity=OD (sample)/OD (sample+is proteinase),

where OD (sample) - value of optical density in the wells with the sample of the brain, not treated with proteinase K (signal);

OD (sample+proteinase) is the value of optical density in the wells with the sample marrow, treated with proteinase K (noise).

The results of the calculations are shown in table 7.

Table 7

The choice of the optimal pairs of reagents for determination of prion protein in brain tissue "sandwich method" ELISA.
AntigenThe values of optical density OD450 nm
AT 143-168 - Con 101-134AT 143-168 - Con 211-241AT 101-134 - Con 211-241
the positive control180022002000
negative control454550
sample+proteinase604585
sample5001300800
specificity8.328.89.4

The values of optical density in the wells with positive control were the maximum in the hole with the negative control is minimal, which confirms correctly is to be conducted experiments.

On the basis of the received results to determine prion in a sample of brain "sandwich method" ELISA pair "antibody conjugate" AT 143-168 - Con 211-241.

Conclusions.

As a result of the experiments selected conjugate 101-134 to determine prion in a sample of brain direct ELISA. In the case of the "sandwich method" of determining prion in a sample of brain most specific pair "antibody conjugate" AT 143-168 - Con 211-241.

Example 8. The definition of prion in a sample of brain sheep in the direct ELISA assays and "sandwich method" using selected antibodies and conjugates.

Use treated and not treated with proteinase K samples of sheep brain. A sample of the brain, not treated with proteinase K, consider opportunistic. Prion protein in the brain of sheep determine direct ELISA according to the above method, using the most specific conjugated antibodies to peptide 101-134. Also determine the prion in the brain sheep "sandwich method" ELISA using a pair of "antibody conjugate" AT 143-168 - Con 211-241.

The measurement results are presented in table 8.

Exceeded more than 2 times the optical density of the wells with the sample not treated with proteinase over optical density of the wells with the sample treated with proteinase is true.

OD (sample)/OD (sample+proteinase)≥2

As can be seen from table 8, the definition of prion all is brazzo brain of sheep is reliable as in the direct ELISA, and the "sandwich method", as higher than the optical density of the wells with the sample not treated with proteinase over optical density of the wells with the sample treated with proteinase, in all cases more than twice.

Table 8.

The determination of prion in the brain samples of sheep.
AntigenThe values of optical density 00450 nm
direct ELISA"sandwich method"
the positive control15501400
negative control4874
sample 1+ proteinase5270
sample 1550820
sample 2+ proteinase80114
sample 210721020
sample 3+ proteinase56130
sample 37801278

Next, on the basis of the obtained in example 8 data prepare a diagnostic kit for determination of prion protein in brain tissue. For the direct ELISA method as the best option in the set is diagnostically the reagent conjugate antibodies to peptide 101-134. For the "sandwich method" ELISA in the set included a pair of "antibody conjugate": antibody to the peptide 143-168 - conjugated antibodies to peptide 211-241. Conjugates as diagnostic reagents represent a solution affination antibody in 0.1M phosphate buffer pH 7.4 with addition of 50% glycerol and BSA. Conjugate antibodies with a concentration of 1 mg/ml contains 5 mg/ml BSA.

The kit also includes excipients: lyse buffer, denaturing buffer, inhibiting reagent, reagent for cultivation proteinase K, proteinase K, buffer dilution, wash buffer, peroxidase substrate buffer, TMB, stop solution.

Thus, in the present invention developed methods to create a diagnostic kit for determination of prion protein PrP-Sc in the brain tissue of mammals and the subsequent diagnosis of prion diseases of mammals.

Optimization of the choice of unconjugated synthetic peptides to obtain polyclonal antisera, the allocation of affinity-purified UN-denatured antibodies and their conjugation with peroxidase allows you to create specific diagnosticum to determine GE produced on an industrial scale.

Sources of information

1. Prusiner S.B., Prion diseases and the BSE crisis. Science, 1997, okt, 10, 278 (5336), pp.245-251. Review.

2. Harmeyer, S., Pfaff, E., Groshup M.H., Synthetic peptide vaccines yield monoclonal antibodies to cellular and pathological prion proteins of ruminants, J.Gen. Virol., 1998, apr, 79, Pt 4, pp.937-945.

3. US 6165784.

4. US 6261790.

5. US 6514707.

6. Volpin O.M., I. G. zhmak M.N., Denoted by MB and others, Antibodies against synthetic fragments of the prion protein for the diagnosis of a spongy encephalopathy of cattle, Bioorganic chemistry, 2001, v.27 (5), str-358.

7. Nakane P.K., A. Kawaoi, Buffer-labeled antibody. A new method of conjugation, J. Histochem. Cytochem., 1974, dec 22 (12), pp.1084-91.

1. Polyclonal antibody specifically binding to the prion protein PrP or a fragment comprising amino acid sequence selected from the group:

a) a peptide or immunologically active fragment having the amino acid sequence

SEQ ID NO:1

b) a peptide comprising the amino acid sequence

143-168 SEQ ID NO:1,

C) a peptide comprising the amino acid sequence

101-134 SEQ ID NO:1,

g) a peptide comprising the amino acid sequence

211-241 SEQ ID NO:1,

and representing the immunoglobulin IgG protein content not less than 95% and the content of IgG1 at least 70%.

2. The antibody according to claim 1, characterized in that the peptide 143-168 SEQ ID NO:1 has the following amino acid sequence:

SAMSRPLIHFGSDYEDRYYRENMHRY.

3. The antibody according to claim 1, the nature of Zoumana fact, the peptide 101-134 SEQ ID NO:1 has the following amino acid sequence:

WGQLGTHWNKPSKPKTNMKKKHVAGAAAAGAVVG.

4. The antibody according to claim 1, characterized in that the peptide 211-241 SEQ ID NO:1 has the following amino acid sequence:

ETDIKMMERVVEQMCITQYQRESQAYYQRGA.

5. The method of obtaining polyclonal antibodies according to claims 1-4 by immunization of rabbits with synthetic peptides selected from the group comprising amino acid sequences 43-168 SEQ ID NO:1, 101-134 SEQ ID NO:1 and 211-241 SEQ ID NO:1, with the primary immunization consists of 1 mg of protein per rabbit, mixed with a Bang, with the subsequent 4 immunizations with peptides at a dose of 1 mg mixed with NAF, obtaining serum and purified by the affinity of the sorbent, representing the agarose with the immobilized peptide.

6. Conjugate for conducting ELISA, representing the antibody obtained according to the method of claim 5, conjugated to horseradish peroxidase with the degree of modification of 1.3 mol of horseradish peroxidase at 1 mol IgG1 rabbit.

7. The conjugate according to claim 6 where the use of the antibody according to claim 3.

8. The conjugate according to claim 6 where the use of the antibody according to claim 4.

9. Diagnostic reagent for determining prion protein PrP or its fragments by ELISA, representing the conjugate solution for conducting ELISA for PP-8 in 50%glycerol in 0.1 M phosphate buffer pH 7.4, containing BSA, in the following compound is: conjugate for conducting ELISA 1 mg/ml; BSA, 5 mg/ml

10. The application of diagnostic reagent according to claim 9 for determining prion protein PrP through direct and "sandwich method" ELISA.

11. The application of diagnostic reagent of claim 10 for determining prion protein PrP in tissue of a mammal.

12. Use diagnostic reagent for PP-11 to determine the pathogenic form of the prion protein PrP.

13. Kit for determination of prion protein PrP in the brain tissue of mammals which includes a diagnostic reagent according to claim 9, affinity-purified antibody obtained by the method according to claim 5, excipients and instructions for use.

14. Set in item 13, where the "sandwich method" ELISA uses a pair antibody according to claim 2 and the conjugate of claim 8.

15. Set in item 13, characterized in that as excipients set contains lyse buffer, denaturing buffer, inhibiting reagent, reagent for cultivation proteinase K, proteinase K, buffer for dilution of sample, wash buffer, peroxidase substrate buffer, a solution of TMB and stop solution.



 

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EFFECT: high sensitivity and specificity of estimation method.

1 tbl

FIELD: medicine.

SUBSTANCE: method involves determining cerebral neurotrophic factor concentration BDNF in blood serum by applying immunoenzyme assay techniques. The value being less than 34.54 pg/l, brain dysfunctions of minimum intensity degree in children are to be diagnosed.

EFFECT: high specificity of method.

1 tbl

FIELD: veterinary science.

SUBSTANCE: the present innovation deals with selecting blood serum to detect average-weight molecules in it, where at concentration of the above-mentioned molecules being above 0.134 units of optic density at wave length being 254 and 280 nm, correspondingly, it is necessary to diagnose lead intoxication. The present method enables to simplify the technique for detecting lead intoxication in animals, shorten the terms of laboratory testing using blood serum against the well-known ones.

EFFECT: higher efficiency of detection.

1 ex

FIELD: medicine, immunology.

SUBSTANCE: the present innovation deals with detecting immunoglobulin binding with allergen in a sample. Moreover, one or more allergens should be immobilized upon a structural chip, then the sample should be incubated at the presence of immobilized allergens. Immunoglobulins having got specificity towards allergens are bound with specific allergens and after that one should detect immunoglobulins.

EFFECT: increased efficiency of immunodiagnostics.

37 cl, 4 dwg, 5 ex, 5 tbl

FIELD: medicine.

SUBSTANCE: method involves determining PP14 content in menstrual blood sample in pregravidic period. The value being below 180 ng/ml, early stage placenta insufficiency development is to be predicted on pregnancy onset.

EFFECT: high accuracy in evaluating functional endometrium activity and predicting placenta insufficiency in women carrying mono- and mixed Chlamidia infection.

FIELD: medicine.

SUBSTANCE: method involves determining low and medium molecular mass substances availability in plasma, on/in erythrocytes, in urine, effective albumin concentration in blood plasma, antiprotease plasma activity index, birefringent crystal number in citrated plasma with proteolysis inhibitor and orthophenanthroline test. Next, three integral characteristics, showing level of endogenous intoxication syndrome gravity as light, moderate and severe, are to be calculated. The endogenous intoxication syndrome gravity degree is determined from which of the indices under measurement reaches maximum value.

EFFECT: higher diagnosis accuracy.

FIELD: medicine.

SUBSTANCE: method involves determining cytokine of tumor necrosis factor-α (TNF-α) in umbilical cord blood serum of newborn babies born by mothers aggravated with obstetric-gynecologic anamnesis. The TNF-α level being equal to or greater than 50 pg/ml, hypoxic ischemic brain lesion is considered to take place.

EFFECT: accelerated prognosis method.

FIELD: medicine, obstetrics.

SUBSTANCE: in pregnant women at pregnancy terms being after 39 wk in average portion of morning urine one should detect due to a solid-phase immunoenzymatic assay the concentration of pregnancy-associated protein-A (PAPP-A). At the level of PAPP-A being 768.2 ng/ml and more it is possible to conclude upon physiological mature pregnancy. The innovation is noninvasive and enables to increase accuracy in predicting true over-mature pregnancy.

EFFECT: higher efficiency and accuracy of diagnostics.

2 ex, 1 tbl

FIELD: biochemistry.

SUBSTANCE: one should test blood serumal samples in immunoenzymatic assay by applying a test system ELI-N-1, the components of which are being antigens of nervous tissue or their immunochemical analogs specifically binding antibodies with tendency to antigens of nervous tissue, and, also, idiotypical antibodies to them, or their variable parts. Prediction should be performed according to the level of idiotypical and anti-idiotypical autoantibodies to antigens of nervous tissue and, also, according to their ratio. Thus, a new test system has been elaborated that enables to predict the flow of available nervous-psychic diseases.

EFFECT: higher accuracy of prediction.

2 cl, 7 ex

FIELD: medicine, cardiology.

SUBSTANCE: one should detect the level of activity of IgM and IgG immunoglobulins to cytomegalovirus on the 5th and 15th d of large-focus myocardial infarction. At increased diagnostically valuable result of specific immunoglobulins of type M by 0.10-0.20 times and for type G by 0.73-2.09 times it is possible to predict favorable clinical flow of large-focus myocardial infarction without exudative pericarditis. At the value of specific immunoglobulins exceeding diagnostically valuable result for type M - by 0.86-1.67 times and for type G by 2.42-3.01 times one should predict early clinical flow of post-infarction pericarditis. The method enables to carry out prophylactic measures in due time.

EFFECT: higher accuracy of prediction.

5 ex, 2 tbl

FIELD: medicine, surgery.

SUBSTANCE: one should carry out virological testing patient's blood serum and hepatic bioptates. At detecting TTVDNA and HGVRNA it is necessary to perform ultrasound survey, and at availability of biliary sludge one should conclude upon early stage of cholelithiasis.

EFFECT: higher accuracy of diagnostics.

3 ex

FIELD: medicine, ophthalmology.

SUBSTANCE: one should detect the content of tumor necrosis factor alpha in acute period, moreover, the above-mentioned factor should be determined in lacrimal liquid on the 11th - 15th d against the onset of herpetic ophthalmoinspection, at its content ranged 176-250 pcg/ml prediction is considered to be favorable, and from the value of 300 pcg/ml and higher - as unfavorable.

EFFECT: higher accuracy of prediction.

2 ex

FIELD: medicine, ophthalmology.

SUBSTANCE: in lacrimal liquid one should detect the content of interleukin 8 (IL-8) and that of interleukin 1 beta (IL-1β) to calculate prognostic coefficient (PC) due to dividing the first value by the second one by the following formula: At PC value being below 10.0 one should predict favorable disease flow, and at PC value being above 10.0 - unfavorable flow.

EFFECT: higher accuracy of prediction.

2 ex

The invention relates to medicine, in particular to obstetrics

The invention relates to medical Virology and Microbiology, and in particular to identification of a new strain of measles virus NovO/96 to obtain the antigen component of the diagnostic test system and the test system for the diagnosis of antibodies to measles virus

The invention relates to biotechnology, relates to a method of conjugating luciferase with chemical particle, in particular antibody, providing (a) mixing luciferase with one or more components, such as D-luciferin, magnesium ions and ATP, and (b) carrying out the reaction of covalent binding between luciferase and linking reagent using covalently bonding agent, where D - luciferin, magnesium ions and/or adenosine triphosphate present in a quantity sufficient to protect the luciferase activity from inhibition of covalently binding agent

The invention relates to veterinary Virology and biotechnology

FIELD: biotechnology.

SUBSTANCE: invention relates to proteins and polynucleotides, which stimulate enzyme cleavage and releasing of TNF receptors. Also disclosed are methods for identification of additional agents, which influence on TNF receptor releasing from cells. Products of present invention containing in pharmaceutical compositions as active ingredients increase or decrease TNF signal transduction and consequently abate disease pathology.

EFFECT: new pharmaceutical compositions.

27 cl, 8 ex, 3 tbl, 5 dwg

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