Antibodies and peptide antigens for producing antibodies having selective binding specificity to biologically active intact parathyroid hormone (pth) 1-84

FIELD: medicine.

SUBSTANCE: what is presented is a method for producing a polyclonal antiserum which is immunospecifically bound to a biologically active parathyroid hormone (PTH) in a part of three or four N-terminal amino acid residues of (1-84) PTH. The first peptide antigen is introduced in a host animal other than a human. A titre of produced antibodies is monitored. Antiserum is extracted. At least one antibody is separated and recovered from antiserum by affine chromatography with using the second peptide antigen. It is followed by removing at least one antibody possessing specificity with respect to the peptide antigen specified in a group consisting of (4-34) PTH, (5-34) PTH, (4-84) PTH and (5-84) PTH by affine chromatography with using the third peptide antigen specified in a group consisting of (4-34) PTH, (5-34) PTH, (4-84) PTH and (5-84) PTH respectively. There is recovered polyclonal serum possessing binding affinity with respect to no more than the first three or four amino acid residues of the N-terminal part of PTH and possessing no affinity with respect to any amino acid residues in the fifth amino acid residue or after the fifth amino acid residue of the N-terminal of PTH.

EFFECT: determining the level of biologically active intact PTH in serum, plasma or cell culture medium.

6 cl, 4 dwg

 

CROSS REFERENCES TO RELATED APPLICATIONS

This application is a partial continuation of Patent Application United States Serial Number 09/730164, filed December 5, 2000, entitled ANTIBODIES AND PEPTIDE ANTIGENS TO generate ANTIBODIES HAVING a SELECTIVE BINDING SPECIFICITY WITH BIOLOGICALLY ACTIVE PARATHYROID HORMONE (PTH) 1-84, and currently at the stage of issuance, the description of which is incorporated into this description by reference.

RESPONSE STATEMENT: RESEARCH/DEVELOPMENT, SPONSORED by the FEDERAL Not applicable

PRIOR art

Parathyroid hormone (PTH) and its important role in the regulation of the content of calcium ions in the blood are well known. In this regard, this hormone is produced by parathyroid glands and in combination with other factors acts, regulating the level of calcium ions in the blood, so that it is maintained in a stable concentration in the cells and in the surrounding fluid. Essentially, PTH acts, releasing reserves of calcium in the body, when the level of calcium in the serum is reduced. On the other hand, its secretion is suppressed by increasing the concentration of calcium in serum.

In its full form PTH includes a unique peptide consisting of 84 amino acids. The specific sequence of PTH, present the I in many species, namely, in humans, rats, mice, cattle, dogs, pigs, cats and monkeys, depicted in figure 1 and its variants in figure 2, which illustrate that a relatively permanent structure of this hormone is maintained among these types.

Because of its important role in the metabolism of calcium is not only for humans, but for many species, an accurate measurement of PTH does not cease to be of considerable clinical significance. As is well documented, the level of PTH in serum is an important option for patients who have, inter alia, diseases such as hypercalcemia, primary hyperparathyroidism, and osteoporosis. Similarly, PTH becomes significant clinical value in patients with chronic renal failure, which due to violations of the law may develop renal osteodystrophy.

Despite its important role in metabolism and the clinical significance continue to be significant challenges in determining the level of circulating biologically active PTH. First, it is well known that PTH normal is present in extremely low quantities, constituting from 10 PG/ml to 65 PG/ml moreover, it is known that PTH peptide may be present in many of circulating PTH fragments and, in particular, a large non-(1-84) circulating PTH fragments, which, veroia is but jointly migrate chromatography with a molecule PTH (7-84) and, as you know, are a special hindrance in normal tests in the measurement of PTH levels. Indeed, a large non-(1-84) PTH fragments can be about one-half (1/2) PTH levels measured in most of the existing analyses. Examples of existing deficiencies accurate measurement of PTH levels indicated in the international application PCT/US00/00855, international publication no WO/00/42437, entitled Methods of differentiation and monitoring of diseases associated with the state of the parathyroid glands and bones, as well as publications Lepage, Raymond et al., A non-(1-84) Assay Circulating Measurement In Uremic Samples, Clinical Chemistry 44:4, 1998, pages 805-809, descriptions of which are incorporated into this description by reference.

To overcome these shortcomings, a new analysis to determine the level of PTH researchers Scantibodies Laboratory, of Santee, California, which includes the antibody is a marker that has binding specificity with the extreme N-terminal of the Department of human PTH, and more specifically to the first six amino acid residues. As it turned out at the present time, this analysis seems minimize cross reactions with large non-(1-84) PTH fragments. However, to obtain such antibodies requires significant effort and cost to clean them. Moreover, such antibodies are markers as well RA who know only the first amino acid residue of PTH and have a significantly lower specificity with respect to subsequent residues, this eliminates the possibility of its use for some other species, where the first amino acid is different. Such shortcomings are discussed in the article John M.R. et al., called A Novel Immunoradiometric Assay Detects Full-Length Human PTH but not Amino-Terminally Truncated Fragments: Implications for PTH Measurements in Renal Failure, The Journal of Clinical Endocrinology and Metabolism, Vol. 84, No. 11, 1999, p. 4287-4290, the contents of which are incorporated into this description by reference.

Therefore, in this area continues to exist a need in the partner link for analysis and the method of obtaining such a binding partner that is specific in relation to biologically active intact PTH levels, which will help to determine the level of PTH with reduced cross-reaction with fragments of PTH peptide. There is also a need in this area in the superior binding partners PTH, which will help to measure the level of PTH way more cost effective, which have a greater affinity for PTH and which can be easily included in kits for immunological studies, etc. there is still a need in the binding partners, namely antibodies, which recognize the binding, which is specific in relation to PTH, which can be used to determine the level of PTH in many species. Finally, in this area there is a need for improved partners St. the statements, high affinity binding to PTH, which can be easily obtained using conventional techniques and which require minimal cleaning, leading to a better recognition of the binding of intact PTH, have minimal cross-reaction with large non-(1-84) PTH fragments and can be obtained using methods that give higher yields of antibodies than partners to link known in the prior art.

The INVENTION

The present invention specifically addresses alleviates the above shortcomings in this area. The present invention relates to certain antigens, antibodies and methods of producing antibodies that are applicable to determine the level of biologically intact PTH in the sample fluid, such as serum, plasma or culture medium of the cells. Antibodies and methods according to the invention have certain advantages, having a greater affinity for PTH, and, in particular, they are designed in such a way that they recognize some new amino acid residues starting from the first N-terminal residue of PTH, but preferably not beyond the fourth amino acid residue of PTH. In a most particularly preferred embodiments of the antibodies will be of specificity in respect of the first three amino acid the residue N-terminal PTH. In addition, antibodies are not cross-reacting with a large non-(1-84) molecular forms of PTH. Moreover, antigens, antibodies and methods for their preparation in accordance with the present invention have significant flexibility in terms of many different types and can be used to determine PTH levels not only in humans but also in rats, mice, cattle, dogs, pigs, cats and monkeys.

In accordance with a preferred embodiment of the invention, the antigen has the formula

VAL-SER-GLU-ILE-GLN-X-MET-HIS-ASN-LEU-GLY

where X is selected from the group consisting of LEU [SEQ ID NO.1] and PHE [SEQ ID NO.2]. Against such a variant embodiment of the invention such antigenic peptide represents amino acid residues 2-12 PTH, with his sixth amino acid residue selected from LEU and PHE, where the first is found in the law of man, rat, mouse, and pig, and the latter corresponds to the PTH ruminants and dogs. In an even more preferred embodiment of the invention, the antigen comprises a peptide having the formula

Y-VAL-SER-GLU-ILE-GLN-X-MET-HIS-ASN-LEU-GLY

where X is selected from the group consisting of LEU and PHE, as discussed above, and Y is an amino acid residue consisting of or SER or ALA [SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID No. 6, respectively], and reflects the first amino acid that is found in humans, dogs and pigs, the latter corresponds to the PTH rats mice and ruminants.

In additional embodiments of the invention the antigen has the formula

VAL-SER-GLU-ILE-GLN-X-MET-HIS-ASN-LEU-GLY-LYS-HIS-LEU

where X is selected from the group consisting of LEU [SEQ ID NO.7] and PHE [SEQ ID NO.8]. Such antigenic peptides are amino acid residues 2-15 PTH, with the sixth amino acid residue comprising either LEU or PHE, reflecting, thus, the corresponding amino acid residue found at the corresponding species mentioned above. In another embodiment of the invention the antigenic peptide represents amino acid residues 1-15 law and has the formula

Y-VAL-SER-GLU-ILE-GLN-X-MET-HIS-ASN-LEU-GLY-LYS-HIS-LEU

where X is selected from the group consisting of LEU and PHE and Y is an amino acid residue consisting of or SER or ALA [SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11 and SEQ ID No. 12, respectively], where the latter is chosen in accordance with the specific form disclosed above.

Regarding antibodies and methods for their preparation in accordance with the present invention, they are directed to antibodies that have affinity and specificity in relation to the above antigens. Preferably, if the antibodies are specific against PTH amino acid residues 2-12, 1-12, 2-15 and 1-15, respectively. In the most preferred embodiment of the invention the antibodies are specific only for the first t is ex PTH amino acid residues. Such antibodies preferably get through stages of introduction of one of the above peptide antigens, either alone or in combination with protein carrier animal to the owner, which preferably includes a goat, to obtain the production of antibodies to the peptide antigen. In an alternative preferred embodiment of the invention the production of antibodies induce through the introduction of a larger peptide fragments of PTH. Preferably, such a fragment of PTH may include (1-34) PTH, which optionally may further include a carrier protein covalently linked or merged with it to increase the antigenicity, or intact (1-84) PTH. In cases where antibodies are associated with determination of PTH in humans, the intact molecule PTH (1-84) preferably includes the intact rat PTH or, to a lesser extent, human PTH. Then monitor antibody titer obtained with the introduction of an antigen to an animal host. After this release anticigarette produced by the animal host and its antibodies are selected by affinity chromatography as having specificity against the desired antigenic site of PTH (i.e. amino acid residues of PTH 2-12, 1-12, 2-15 and 1-15, respectively). For additional stages of selected antibodies additionally cleaned for convenience, the population of antibodies, possessing affinity for amino acid residues that are located behind the fourth or fifth N-terminal amino acid residue of PTH using associated antigens corresponding to either (4-34) PTH and/or (4-84) law, or (5-34) PTH and/or (from 5 to 84) PTH, which keep unwanted antibodies, but allow the passage of antibodies that are specific only in respect of (1-3) or (1-4) PTH. Then the antibodies may be labeled or otherwise included in a variety of commonly used tests to determine PTH or a person, or any of the many types.

As is well known to specialists in this field, antigens, antibodies and methods according to the invention, is focused on amino acid residues 2-12, 1-12, 2-15 1-15 and PTH, respectively, focus on the part of the PTH molecule with N-terminal biological activity, and, therefore, as well identify such. Moreover, as for the more preferred embodiments of the invention, provide, using the antigens, antibodies and methods for their production, which include other amino acid residues located at the N-terminal biologically active area (i.e. up to and including the twelfth (12th) and the thirteenth (13th) amino acid residues of PTH), and, therefore, the specificity and affinity of these antibodies are higher and enable them to identify arovent with greater specificity and affinity, than the receptors of the prior art, when used in the analyses, etc. In the most preferred embodiment of the invention the antibodies have specificity to no more than the first three amino acid residues of PTH, which, as discussed, is achieved by removing or stage of "purification", which is why the antibodies obtained by the use of antigens according to the invention, selectively removed in those cases where any such antibodies have an affinity for (4-34) PTH or (4-84) PTH. As a consequence, the antibodies obtained in accordance with the present invention (as well as in methods and peptide antigens described in the present description to achieve such a result), virtually eliminated cross reactions with large non-(1-84) PTH peptide fragments, they do not possess the maximum recognition is only the first amino acid residue of PTH and, optionally, can be easily obtained cost-effective way to such an extent that the output of the antibodies obtained by the methods according to the invention must be more than methods of the prior technology.

Alternatively, such removal or stage "cleaning" is used for the selective removal of antibodies having affinity to (5-34) PTH or (from 5 to 84) PTH. Thus, ultimately selected antibodies will the be the affinity regarding no more than the first four amino acid residues N-terminal PTH. Therefore, the present invention is the provision of 1) selection of antigens for the acquisition and allocation of antibodies; 2) antibodies, and 3) methods of obtaining antibodies that have affinity binding and specificity in relation to PTH, which have a reduced cross-reaction with larger companies (1-84) PTH peptide fragments.

Another objective of the present invention is the provision of 1) selection of antigens to generate antibodies; 2) antibodies, and 3) methods for producing antibodies, which have a greater affinity and specificity in relation to PTH than the binding partners in the prior art, and, in addition, have a greater degree of cross-reaction between interspecific PTH, so that the antigens, antibodies and methods for their preparation in accordance with the present invention could easily be used to determine PTH many types.

Another objective of the present invention is the provision of 1) selection of antigens for the acquisition and allocation of antibodies; 2) antibodies, and 3) methods of obtaining antibodies that have affinity binding and specificity in relation to the more biologically active N-terminal part of the law and, therefore, are more efficient and accurate when determining the level of biologically intact PTH levels than directed at them binding partners in the previous from the Aries technology.

Another objective of the present invention is the provision of 1) selection of antigens for the acquisition and allocation of antibodies; 2) antibodies, and 3) methods of obtaining antibodies that do not have maximum affinity binding only in respect of the first N-terminal amino acid residue of PTH.

Another objective of the present invention is the provision of 1) selection of antigens for the acquisition and allocation of antibodies; 2) antibodies, and 3) methods of obtaining antibodies that are less expensive to obtain and give higher yield of antibodies than methods of the prior art generate antibodies having a binding affinity for the binding and specificity in relation to the N-terminal PTH.

Additional objectives of the present invention are to provide 1) selection of antigens for the acquisition and allocation of antibodies; 2) antibodies, and 3) methods of obtaining antibodies that are easy to make antibodies that can be easily included in any of the many commercially available tests and, in addition, can be modified (for example, to enable labels etc), as may be desirable for any of a multitude of applications in immunological research.

BRIEF DESCRIPTION of DRAWINGS

This and other features of the present invention will be clearer with reference to the drawings.

1 shows the amino acid posledovatel is of PTH for a variety of species, namely humans, rats, mice, cattle, dogs, pigs, cats and monkeys, and, in addition, shows the amino acid sequence determined in the present description as SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11 and SEQ ID NO. 12.

Fig. 2 is a variant of illustrations Fig. 1, depicting option 1-84 amino acid sequence of PTH levels above species, even depicting N-terminal PTH, where amino acid sequence of SEQ ID NO. 1-12 remain relatively constant.

3 shows the N-terminal portion of human PTH in the form of a diagram.

Figure 4 is a block diagram depicting stages of the antibodies in accordance with the preferred embodiment of the present invention.

DETAILED description of the INVENTION

The detailed description set out below is intended to describe preferred in the present variant embodiment of the invention and is not intended to represent the only forms in which the present invention may be construed or used. Description specifies the functions and sequence of steps for the creation and manipulation of the invention. You must understand, however, that the same or equivalent functions and sequences may be implemented in different and variants of the invention, which are also intended to be included in the scope of the invention.

The present invention encompasses antigens, antibodies and methods for producing antibodies, which are directed to antigenic site of PTH, located in its N-terminal section, and, more precisely, the first fifteen (15) amino acid residues located on the N-end is depicted as 10 in Fig. 1-3. In the most preferred embodiment of the invention the antibodies are specific only in respect of the first three (3) amino acid residues of PTH. As is well known, the N-terminal site of PTH consider necessary for binding to the PTH receptor/Pthrp, and, in addition, it is considered to be the most desirable epitope for level measurement of biologically intact PTH, which can be detected in the sample of biological fluid such as serum, plasma or environment for cell culture. Indicative of the current state of the technology associated with PTH levels and methods of its determination, discussed in detail in published international application to the patent cooperation Treaty (PCT no PCT/US00/00855, International Publication no WO/00/42437 named Methods for Differentiating and Monitoring Parathyroid and Bond Status Related Diseases and Lepage, Raymond et al., A Non-(1-84) Circulating Parathyroid Hormone (PTH) Fragment Interferes With Intact PTH Commercial Assay Measurement In Uremic Samples, Clinical Chemistry 44:4, 1998 pages 805-809, descriptions of which are included in this description is the ideal reference.

In accordance with a preferred embodiment of the invention, the antigenic peptide according to the invention includes amino acid residues corresponding to amino acid residues 2-12 PTH, together defined as 12 in Fig. 1-3. In particular, the antigenic peptide will have the formula

VAL-SER-GLU-ILE-GLN-X-MET-HIS-ASN-LEU-GLY

where X is an amino acid residue selected from either LEU [SEQ ID NO. 1], or PHE [SEQ ID NO. 2]. The person skilled in the art it is known that the sixth (6th) amino acid residue of this peptide fragment of PTH does not differ between these species on the basis of which this balance includes the LEU in humans, rats, mice and pigs and PHE in ruminants and dogs. Expert it is clear that despite the differences in single amino acid residue, such antigenic peptide remains in other respects constant between these species, which, as discussed in more detail below, may allow obtaining and precise application of antibodies (cross and, therefore, effective in determining the level of PTH many such species.

In a more preferred embodiment of the invention the peptide antigen reflects the first twelve (12) amino acid residues of PTH, defined as 14, has the formula

Y-VAL-SER-GLU-ILE-GLN-X-MET-HIS-ASN-LEU-GLY

where X is an amino acid residue selected from LEU or PHE, AU is an amino acid residue, selected from SER or ALA [SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6, respectively]. In relation to the options in the first amino acid residue that is easily understandable that such antigenic peptide can be obtained so that was included SER that is found in humans, dogs and pigs, or ALA, which is found in rats, mice and ruminants. In this regard, the options presented to the antigenic peptide in the present invention and, in particular, in the preferred embodiment, its implementation, have the capacity, i.e. antibodies derived from such antigenic peptides can be obtained in such a way that they possessed higher affinity binding, which may be desirable to determine the PTH data types.

In more high-precision variants of implementation of the present invention antigenic peptide includes the sequence that correspond to amino acid residues of PTH 2-15 and 1-15, respectively. In regard to the first, indicated in Fig. 1-3 as 16, such antigenic peptide will have the formula

VAL-SER-GLU-ILE-GLN-X-MET-HIS-ASN-LEU-GLY-LYS-HIS-LEU

where X is an amino acid residue selected from LEU [SEQ ID NO. 7] or PHE [SEQ ID NO. 8]. In the latter variant of the invention, corresponding to amino acid residues 1-15 PTH indicated as 10, such antigenic peptide will have the formula

p> Y-VAL-SER-GLU-ILE-GLN-X-MET-HIS-ASN-LEU-GLY-LYS-HIS-LEU

where X is an amino acid residue selected from LEU or PHE, and Y is an amino acid residue selected from SER or ALA [SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11 and SEQ ID NO. 12, respectively]. Was presented the CD-ROM that provides machine-readable form SEQ ID NO. 1-12 in accordance with the requirements of 37 CFR 1.821-1.825, which is identical to the recorded list presented in the present description sequences for compliance with statutory requirements. Despite the above formula for the above peptides, it is obvious that the above will apply to all of their functional derivatives, which means the inclusion of functionally comparable peptides derived from the same plot PTH, is reflected in the sequences of Fig. 1-3, has a similar ability to induce specific anti-PTH antibodies and, more specifically, antibodies that are specific against N-terminal amino acid residues of PTH. In this respect, such a functional derivative may be similarly constructed peptide or a peptide derived from the sequence discussed above and as reflected in figure 1-3, with substitutions, additions or deletions of amino acids, provided that such substitutions, additions or deletions will not change the ability of the peptide antigen to induce antibodies reactive against PTH.

You must additionally recognize that peptide antigens according to the invention include peptides whose amino acid sequence can be shifted within a few amino acids up or down along the chain peptides discussed above in figures 1-3, as well as peptides having conservative amino acid changes, so that substitutions, additions or deletions of amino acids or modifications do not materially affect the ability of the peptide antigen to induce antibodies with high affinity and specificity in respect of the first twelve amino acid residues of PTH or any of its subcomponents.

In obtaining antibodies, Fig. 4 illustrates a method 20 of their receipt. Source, provide the peptide 22 above set, which fully or partially matches the first twelve (12)fifteen (15) amino acid residues of PTH-defined types. In accordance with the preferred embodiment of the invention, however, the antigenic peptide used to generate antibodies that will include larger fragments of PTH, including, but not limited to, (1-34) PTH and fully intact (1-84) PTH peptide. Along with this, while the resulting antibodies will have specificity in respect of such amino acid residues corresponding to videopom is by antigenic peptides (i.e. amino acid sequence 2-12, 1-12, 2-15 and/or 1-15 PTH), in the framework of the method according to the invention, at least in relation to the initial stage of obtaining antigen 22 and sequential introduction of such, by stage 24, as discussed below, it will be obvious that the whole PTH peptide and its N-terminal fragment can preferably serve as a suitable antigenic peptide to induce the production of antibodies, which will ultimately have a perfect affinity binding and specificity in relation to biologically active part of PTH on its N end. In the most preferred embodiment of the invention and provide input antigenic peptide will include the PTH fragment, corresponding to amino acid residues 1-34 PTH, which can be not necessarily connected with protein carrier. Alternative such peptide fragment may preferably be in the form of (1-84) PTH full length, which may take the form characteristic of any type, specified in Fig. 1-2. Preferably, in the context of the development of antibodies specific against human PTH, application (1-34) rat PTH or (1-84) rat PTH full length is preferred.

Such antigenic peptides can be obtained by any of a variety of ways well known in this field,including synthesis by conventional means, such as solid-phase chemical synthesis or recombinant technology. As will become apparent hereinafter, synthetic peptides can optionally be chemically linked to a protein carrier or, alternatively, recombinant peptides can be obtained in the form of a fused protein to increase antigenicity. As will be further obvious to the person skilled in the art, such antigenic peptides can be subjected to screening on the basis of their ability to induce anti-PTH antibodies. In this regard, such screening methods may include, without limitation, immunoprecipitation or immunological analysis.

After receipt of such peptide antigens can then be used to generate antibodies according to the invention using conventional techniques. For this purpose, the peptide antigen(s), preferably in combination with the additive, introducing the animal to the carrier 24, which preferably includes a goat. You must understand, however, that other species such as rabbits, mice, sheep, chickens, etc. can optionally be used as animal hosts media. To this end, the introduction of such antigens may be any of a variety of ways, including, without limitation, subcutaneous or intramuscular injection. As will be obvious, the dose of injected peptide antigen will be, respectively, argirova the change in the peptide, as well as the animal host. As will be noted, however, in order to obtain antibodies with the highest affinity and specificity, it is possible for these species, the individual antibodies should be obtained against the respective compatible peptide from each type.

After the introduction of the monitor 26 of the outcomes of education titers of antibodies in an animal host, which may be any of a variety of techniques well known in the field, using routine blood test etc., emitting antisera (e.g., by centrifugation) at stage 28, and then screened for the presence of antipeptide antibodies with affinity of their binding. In addition, note that in the presence of the above-mentioned conventional immunological methods for producing antibodies according to the invention, such antibodies may be monoclonal or polyclonal in nature. In accordance with the usual practice, it is preferable that anticigarette received from a variety of animal hosts.

The resulting antisyware obtained from an animal host, can be purified by affinity to generate antibodies according to the invention. As is well known in this field, anticavity can be purified by conventional techniques, such as drawing on the separation column with wiseup is mentioned antigenic peptides corresponding to the sequence of amino acid residues 2-12, 1-12, 2-15 and/or 1-15 PTH levels associated with the solid phase (for example, granules, etc.). Then anticavity can be washed to remove antibodies not having specificity for the antigenic peptide or peptides, with the remainder being bound by antibodies specific for the antigenic peptide or peptides, ultimately, buervenich from it. Such antibodies can then be stored in accordance with conventional techniques, well known specialist in this field.

Additional stage 29 antibodies obtained through such methods of isolation and purification, will be subjected to a subsequent purification process, whereby any antibodies present in anticigarette having affinity to (4-34) and (5-34) PTH and/or (4-84) or (from 5 to 84) PTH selectively removed or hatshepsuts". For this purpose it is envisaged that after the selection of antisera on stage 28 it can be cleaned to remove any antibodies having specificity against (4-34) and (5-34) PTH and/or (4-84) or (from 5 to 84) PTH for any of the species mentioned in the present description, through the separation column, or other routine methods by which antigenic peptides corresponding to each of the amino acid sequence (4-34) and (5-34) PTH or (4-84) or (from 5 to 84) PTH, associated with solid f is zo, and passed through anticigarette for cleaning the latter. Any antibody having specificity against specific (4-34) and (5-34) PTH and/or (4-84) or (from 5 to 84) PTH peptide will remain connected, and the remaining antibody, having specificity for (4-34) and (5-34) PTH and/or (4-84) or (from 5 to 84) PTH peptide, will be separated. Mainly in this way, therefore, you can only select antibodies having affinity mainly to the first three (3) or four (4) amino acid residues of PTH and therefore no affinity binding to any amino acid residue at the fifth position from the N-terminal PTH or after the fifth position from the N-terminal PTH. This method, therefore, provides that any antibody that is ultimately selected will actually specific for N-end - (1-84) PTH and, therefore, will not in any way make contact with however it was not a PTH fragment, which does not include at least the first three or four N-terminal amino acid residue of PTH.

Because these antibodies produce such antigenic peptides corresponding to the sequences of amino acid residues 2-12 and 2-15 PTH, respectively, therefore, will be selected antibodies that do not necessarily will have affinity binding, significantly less the maximum is on affinity binding with the first amino acid residue of PTH, what is the problem of the antibodies obtained by the methods of the prior art. In this respect, the destruction or significant suppression of antibodies having an affinity to the first amino acid residue of PTH, will similarly be achieved using peptide sequences corresponding to 1-12 and 1-15 PTH, respectively, provided that such sequences selected from those species in which the first amino acid residue different from that of species which can be used ultimately antibodies. For example, to generate antibodies suitable for detecting levels of PTH in humans that have no specificity with regard to the first amino acid residue of human PTH, it is obvious that anti-rat PTH serum obtained from the animal host may be cleared of peptides corresponding to the sequences of amino acid residues 1-12 and 1-15 of human PTH. It is clear that since the first N-terminal amino acid residue of rat PTH corresponds to ALA, in contrast to SER, find a person, any of the antibodies secreted in the end, will necessarily have an affinity binding to amino acid residues located in the sequence after the first amino acid residue.

Upon receipt the stage 30 such antibodies can be used in immunological techniques to correlate the presence of biologically intact PTH, that may be detected in a given sample (e.g. serum or plasma). In this regard, the antibodies according to the invention can be used separately or in combination for screening a given sample to determine the presence of biologically intact PTH, while they primarily will be no cross-reaction with large non-(1-84) PTH fragments. As an example, such antibodies can be included in a kit for immunological analysis. Examples of such applications include ELISA kits for human biologically active intact PTH and rat bioactive intact PTH produced by Immutopics, Inc., San Clemente Californis, which provide enzyme-linked immunosorbent assay (ELISA) for the quantitative determination of the level of biologically active intact PTH in serum, plasma or the environment of the cultivation of cells. Concerning the applicability of the obtained antibodies specific against PTH many species, it is obvious that the kits for immunological analysis, such as provided Immutopics, Inc., can be specifically created so that the affinity and specificity of these antibodies are applicable to a wide variety of species or, alternatively, created against the respective comparable peptide data types so that sets specifically treated is anomo mind.

Additional modifications and improvements of the present invention may also be apparent to the ordinary person skilled in the art. Therefore, a particular combination of parts described and illustrated in the present description, are intended only to illustrate specific implementation of the present invention and is not intended to be limitations of alternative devices within the volume covered by this invention.

1. The way to obtain polyclonal antisera, which immunospecificity associated with biologically active parathyroid hormone in the N-terminal part (1-84) PTH, with the indicated N-terminal part consists of: (i) three N-terminal amino acid residues (1-84) PTH or (ii) four N-terminal amino acid residues (1-84) PTH, providing stage:
a) introducing a first peptide antigen of the animal to the owner, not a person, to induce the production of antibodies against the indicated first peptide antigen specified in an animal host that is not a person, where the first peptide antigen selected from the group consisting of SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, (1-34) PTH and (1-84)PTH;
b) monitoring antibody titer produced by the specified introduction of this first antigen indicated animal to the owner, not a man;
c) extracts and antisera, produced in specified an animal host that is not a person specified by the entry into force of this first peptide antigen;
d) allocation and selection of at least one of the antibodies of the indicated antisera extracted at the stage (C), using affinity chromatography with the use of the second peptide antigen selected from the group consisting of SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6;
e) remove the specified at least one antibody selected in stage d), having specificity against a peptide antigen selected from the group consisting of (4-34) PTH, (5-34) PTH, (4-84) PTH and (from 5 to 84) PTH by affinity chromatography using a third peptide antigen selected from the group consisting of (4-34) PTH, (5-34) PTH, (4-84) PTH and (from 5 to 84) PTH, respectively; and
f) allocation of polyclonal antisera in stages d) and e)with a binding affinity in respect of not more than the first three or four amino acid residues N-terminal part of the law, and provided the polyclonal anticavity does not have a binding affinity for any amino acid residues in the fifth amino acid residue or after the fifth amino acid residue N-terminal PTH, by collecting the eluate obtained in stage e).

2. The method according to claim 1, where in stage a) specified animal-host selected is from the group consisting of mouse and rabbit.

3. The method according to claim 1, where the phase and group these animal hosts includes at least one goat.

4. The method according to claim 1, where in stage a) specified (1-34) PTH derived from a species selected from the group consisting of human, rat, mouse, some animal, dogs, pigs, cats and monkeys.

5. The method according to claim 1, where in stage a) said first peptide antigen is associated with a carrier protein.

6. The method according to claim 1, where in stage a) specified (1-84) PTH derived from a species selected from the group consisting of human, rat, mouse, some animal, dogs, pigs, cats and monkeys.



 

Same patents:

FIELD: medicine, leprology.

SUBSTANCE: the present innovation deals with predicting exacerbation of leprous neuropathies in patients at leprosy and neuropathies of nonleprous etiology. In patients at leprosy one should detect antibodies to antigen of peripheral nerves in blood serum. According to the availability of antibodies to ceramide in blood serum and at their increased level from 0.26 U optic density and higher one should predict the exacerbation of leprous neuropathies in patients at leprosy.

EFFECT: higher accuracy of prediction.

4 dwg, 4 ex, 5 tbl

FIELD: medicine.

SUBSTANCE: method involves dividing cells by using agglutination. Required reagents comprise dextrane solution, anti-Glycophorin A-antibody, anti-CD15-antibody, anti-CD9-antibody, and antibodies selected from a group composed of anti-CD3-antibody, anti-CD72-antibody, anti-CD16-antibody, anti-CD4-antibody, anti-CD8-antibody and anti-CD2-antibody.

EFFECT: enhanced effectiveness in selecting blood cells of rare cell types with high output amount.

33 cl, 17 tbl

FIELD: childhood and infection diseases.

SUBSTANCE: invention relates to infection diseases and laboratory diagnostics. Patient's coprofiltarte is sampled during early periods of disease, which samples are consecutively applied onto slide-antigens Campylobacter jejuni and Campylobacter coli preliminarily fixed on object-plate in the form of two rows. Each antigen row is designed to reveal secretory specific M and A class immunoglonuline, respectively. After incubation in moist chamber, one drop of substance is added to each row: fluorescein diisocyanate-labeled monospecific diagnostic serum to reveal specific SigM to the first-row preparations and the same to reveal SigA to the second-row preparations. When bright specific luminescence is only observed in first-row drops (SigM), acute period of campylobacteriosis infection is stated and prolonged recurring course of the sickness is predicted. When bright specific luminescence is simultaneously observed in first and second rows of drops, presence of SigM and SigA is stated, which is indicative of favorable campylobacteriosis output.

EFFECT: increased accuracy and rapidity of prediction.

1 tbl, 3 ex

FIELD: medicine, oncology.

SUBSTANCE: one should carry out morphological testing of therapy results, moreover, in the course of therapy it is necessary to conduct immunohistochemical study for the levels of Ki-67 antigen expression, markers to protein-negative regulator of apoptosis: p53 and Bcl-2 and the level of melanoma marker expression HMB-45 and at decreased levels against the values in untreated tumors it is possible to state upon therapy as efficient. The innovation enables to achieve high clinical efficiency of therapy due to applying neoadjuvant autohemochemotherapy in combination with radiation therapy.

EFFECT: higher accuracy of detection.

2 ex

The invention relates to medicine, namely to pulmonology, and can be used to address the question about the effectiveness of therapy glucocorticoid hormones and selection of adequate doses of glucocorticoid hormones in patients with bronchial asthma (BA)

The invention relates to means for determining the status and classification of biological material

FIELD: medicine.

SUBSTANCE: for the purpose of diagnosing metabolic compensation disturbances and predicting a risk of developing complications in the patients suffering type 2 diabetes mellitus (type 2 DM), peripheral venous blood is examined for a percentage of apoptotic lymphocytes An+/Pl-. The lymphocyte An+/Pl- value below 3.5 % enables diagnosing a condition of carbohydrate metabolism compensation and predicting a favourable clinical course of the disease for the following year. The lymphocyte An/PI- content within the range 3.5 to 6% provides diagnosing a condition of subcompensation and a moderate risk of developing complications. The An+/Pl- value exceeding 6 % described a condition of decompensation and a high risk of developing chronic complications of type 2 diabetes mellitus.

EFFECT: higher accuracy of prediction and reduction of the risk of developing complications in diabetic patients.

4 ex

FIELD: medicine.

SUBSTANCE: method involves preparing a biochip containing immobilised antibodies to various antigens, incubating the biochip with analysed cell suspension for 1-2 hours at room temperature, washing off the biochip from unconjugated cells and detecting an antigen-antibody reaction with using an UV light source. A bacterial cell suspension is taken in the concentration of min. 1x105 microbial cell/ml inactivated in any method not destroying the analysed antigen and resuspended in a buffer solution containing Tris HCl 50 mM, 0.02 % Tween 20 and NaCl 50 mM and propidium iodide in the concentration of 1.0-2.0 mcg/ml and used to coat the biochip surface in an amount sufficient to coat all its segments.

EFFECT: invention provides more simple and cheap method to detect the presence of bacterial antigens.

3 tbl, 3 ex

FIELD: medicine.

SUBSTANCE: analysed sample is studied simultaneously by two methods: the first one is a indirect immunofluorescence (IIMF) reaction, while the second one involves a polymerase chain reaction (PCR); the IIMF provides using monoclonal antibodies "BCKK" (P-384D and 434D). The antibodies interact with the capsular antigen F1 specific for Y.pestis species, or the plasmid-temperature-independent surface protein PFV which is found in all Y.pestis strains and rare R-form Y.pseudotuberculosis strains. The bacteria luminescence shows the presence of native or fraction-less Y.pestis bacteria, or typical and PFV-atypical Y.pseudotuberculosis strains in the sample. The PCR is conducted by two pairs of primers vlm33for/ISrevl754 - specific for Y.pestis species, and JS - specific for Y. pseudotuberculosis species. The values derived with the first pair of the primers are estimated as positive if observing the amplicons in 400 bps, and with the second pair if observing the amplicons in 223 bps; the analysed sample is identified by the matching the IIMF and PCR values with the reference strains.

EFFECT: method provides quick identification and high-reliability differentiation of all strains.

7 tbl, 6 ex

FIELD: medicine.

SUBSTANCE: invention refers to medicine, particularly to infectious diseases. It involves simultaneous analysis of blood serum for reaginic and treponema-specific antibodies to cardiolipin antigen and recombinant T pallidum protein complex with molecular weight 15, 17, 39, 41, 42, 44.5, 47 kDa, immobilised on microscope aldehyde slides to detect antibodies by a conjugate solution containing Cy5 phosphor tagged human IgG antibodies and Cy3 phosphor tagged human IgM antibodies; slide scanning in a multichannel biochip scanner; automatic data analysis in a program used to convert fluorescent signals to digital positiveness coefficients; and qualitative presentation. Reaginic and treponema-specific antibodies found in a sample indicate syphilis, while no reaginic and treponema-specific antibodies found in the sample shows the absence of syphilis in a patient.

EFFECT: method allows for differentiated detection of antibodies to the most immunogenic T Pallidum antigens produced at the different stages of disease, for the objective automated data record, for cutting examination and diagnosing time.

4 cl, 2 ex

FIELD: biology, gene engineering.

SUBSTANCE: invention can be used for marking of biological objects. The molecule of nucleic acid which codes the fluorescing protein chosen from fluorescing proteins of representatives of kind Phialidium sp. are both suborder Anthomedusae and fluorescing mutants of the specified proteins allocated. By means of the allocated nucleic acid are obtained cloning and expressing vectors, fluorescing protein, the protein of merge capable to fluorescence, and also the expressing cartridge. The cell and the stable cellular line, containing such express ionic cartridge, produce fluorescing fiber. The fluorescing protein, nucleic acid coding it and the express ionic genetic designs containing this nucleic acid, use in a set for marking of a biological molecule. Fluorescent protein is also used in methods of marking of a biological molecule, a cell or a cellular organella.

EFFECT: invention application allows dilating an arsenal of agents for marking of biological objects.

13 cl, 12 dwg,12 ex

FIELD: biology, medicine.

SUBSTANCE: invention relates to nano-size composite material for DNA/RNA adsorption and desorption in form of nano-particles comprising of 80-99.5 mass % of tin oxide (SnO2) with tetragonal crystal lattice and 20-0.5 mass % of substance selected from group containing Fe, Fe2O3, Fe3O4 or mixture thereof. Said material makes it possible to increase specific capacity of DNA/RNA adsorption and desorption at desired magnetization of composite material, in particular being necessary for magnetic deposition.

EFFECT: new nano-size composite material for DNA/RNA adsorption and desorption.

1 tbl

FIELD: medicine, in particular oncology and hematology.

SUBSTANCE: claimed method includes determination of blood cell composition wherein absolute lymphocytes and CD34-lymphocytes in peripheral blood and percent ratio of CD34-cells to lymphocytes amount is calculated according to the next equation: CD34 % = CD34 abs./lymphocytes abs. x 100 %, wherein CD34 % is relative amount of CD34-lymphocytes; CD34 abs.; lymphocytes abs. is absolute lymphocyte amount. When patient treated by chemotherapy has CD34-lymphocyte content from to 0-7 %, infective complication will not be observed, and when said value is 7.1 % or more, there is danger of generalized infection.

EFFECT: earlier and more perfect determination of chemotherapy infective complications in patients.

3 ex, 1 tbl

FIELD: medicine, laboratory diagnostics.

SUBSTANCE: one should isolate neutrophilic leukocytes, form immune complexes, measure background luminescence of incubation medium (A), add neutrophils, measure the level of spontaneous chemiluminescence (B), introduce immune complex and detect the value of stimulated chemiluminescence (C), calculate the coefficient of stimulation (CS) and at CS≥0.3 it is possible to diagnose allergic reaction. The innovation provides higher accuracy and specificity in detecting the presence of allergic sensitization to certain antigen, the chance for screening serial assay, excludes additional body sensitization associated with introducing allergens in case of skin sample.

EFFECT: higher accuracy of allergodiagnostics.

1 cl, 5 ex, 6 tbl

FIELD: medicine, microbiology.

SUBSTANCE: method involves preparing smears from clinical material taken in inspected patients and carrying out analysis for the presence chlamydia, mycoplasma and ureaplasma in smears by method of fluorescent antibodies. The total amount of cells damaged by these pathogens is determined. In 2-7 days from the treatment onset smears are taken again and analysis of clinical material is repeated by the same method. The high sensitivity to antibiotic in this microorganism is estimated in detection <5% of epithelial cells in smear containing bright-green granules of the corresponding agent, and the prescribed treatment course with the same antibiotic is continued. The low sensitivity to antibiotic is estimated in detection ≥5% of above indicated specifically luminescent granules of antigen, and in this case a medicinal preparation is replaced. Method provides carrying out determining the effectiveness of antibiotics effect for the first days after their prescription and to correct their prescription operatively. Method is simple and doesn't require the preliminary isolation of the pathogen culture.

EFFECT: improved method for express-control.

5 ex

The invention relates to medicine, in particular to obstetrics, and can be used for prediction of preeclampsia in women undergoing severe preeclampsia

FIELD: medicine, biotechnology.

SUBSTANCE: invention proposes variants of antibodies showing specificity to peptide domain located by both side of hinged site R76S77 in pro-BNP(1-108). Indicated antibodies recognize specifically also circulating pro-BNP(1-108) in human serum or plasma samples but they don't recognize practically peptides BNP(1-76) or BNP(77-108). Also, invention describes variants of peptides used in preparing antibodies. Amino acid sequence is given in the invention description. Also, invention discloses methods for preparing indicated antibodies and among of them by using indicated peptides. Also, invention describes methods for preparing antibody-secreting hybridoma, and hybridoma is disclosed prepared by indicated method. Also, invention describes a monoclonal antibody secreted by hybridoma 3D4 and deposited at number CNCM I-3073. Also, invention discloses variants for diagnosis of cardiac insufficiency in vitro and by using antibodies proposed by the invention. Also, invention describes a set used for detecting pro-BNP(1-108) in a biological sample. Using this invention simplifies detection of pro-BNP(1-108) circulating in human serum or plasma samples and provides specific detection of pro-BNP(1-108) that can be used in early diagnosis of human cardiac insufficiency.

EFFECT: valuable medicinal properties of antibodies.

24 cl, 16 dwg, 5 tbl, 20 ex

FIELD: biotechnology, in particular production of corticotropin-releasing factor 2 receptor (CRF2R) agonists.

SUBSTANCE: CRF2R agonist protein is produced as derivative of CRF, urocortine I, urocortine II urocortine III, sauvagine, urotensine I and other connate peptides by amino acid displacement in protein sequences. Obtained CRF2R agonist is useful in pharmaceutical composition for prophylaxis and treatment of disorders associated with CRF2R in host.

EFFECT: effective agent for prophylaxis and treatment of disorders modulated with CRF2R, such as muscle dystrophy.

25 cl, 3 tbl, 3 ex

FIELD: biotechnology, medicine, pharmacy.

SUBSTANCE: invention proposes peptides as agonists of corticotropin-releasing factor CRF2R prepared by solid-phase synthesis. Synthesized agonists of CRF2R are used as components of pharmaceutical composition used in prophylaxis or treatment of diseases modulated by CRF2R. Proposed invention provides enhancing effectiveness of therapeutic effect and avoiding by-side responses in treatment of disorders modulated by CRF2R. Invention can be used in synthesis of novel peptides possessing activity of agonists of corticotropin-releasing factor-2 (CRF2R).

EFFECT: improved method of synthesis, valuable medicinal properties of agonists.

3 tbl, 3 ex

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