IT9041626A1 - METHOD FOR THE QUANTITATIVE AND QUALITATIVE DETERMINATION OF POLYPEPTIDES IN PROTEIN LIQUIDS - Google Patents

Description

BACKGROUND OF THE INVENTION

It is known that a certain sequence of DNA codons corresponds to a certain and defined sequence of amino acids, which after their union form a polypeptide. DNA ultimately collects the genetic code of information within itself, but does not fulfill the prescription. These are satisfied through ribonucleic acids (RNA) in which the uracil base (U) stands in place of thymine (T). There will therefore be couplings A-U and G-C. The first messenger RNA or m-RNA: it consists of a single strand that forms on the DNA template strand in an operation called transcription: the DNA double helix partially unwinds and RNA is formed on the template strand. Thus, with a double transcription of the coding strand, the RNA will repeat the sequence of codons found on the coding strand of the DNA, with the only variant of having U instead of T.

The DNA, before it reaches the coding sequence, still contains a sequence of bases called operator which is essential in the regulatory processes, and a short sequence of bases called the ribosonic binding site. After the ribosomal binding site is the start codon which is always ATG (AUG in the m-RNA), corresponding to the amino acid and, at the end of the coding sequence, one of the three stop codons. It is therefore clear that m-RNA is called a messenger precisely because it carries the genetic message of the DNA to the cellular site where protein synthesis takes place, a site made up of ribosomes. Arrived at the stop codon, the m-RNA detaches itself from the DNA strand on which it was formed and goes to place itself in a ribosome and precisely in the smaller of the two ribosomal units.

Another type of RNA is involved, called transport or transfer and therefore commonly referred to as t-RNA. In one point of the t-RNA chain there is an amino acid bound and in another point, more or less opposite, there is the anticodon that corresponds to that amino acid. Expressing itself in very elementary terms, the anticodon then reads the codon that corresponds to it on the m-RNA, thus placing the relative amino acid in the desired sequence. It is therefore clear that there are at least as many t-RNAs as amino acids or, better still, as many codons that correspond to them. The amino acids are therefore placed in the desired sequence and in the largest part of the ribosome. They bind one after the other, as the m-RNA chain flows through the ribosome and its codons are read by different t-RNAs. The evolution of knowledge has led to the possibility, among other things, of being able to isolate and sequence the DNA, identify the m-RNAs, trace the amino acid sequence and, recently, associate and identify how the polypeptides have different characteristics and sites in terms of hydrophilicity and hydrophobicity, which may correspond to situations of different antigenicity of proteins. Knowledge of biology has always been driven by the availability of analytical methodologies, which necessarily had to be specific and sensitive. Many technologies for dosing analytes in biological liquids have been developed to date (Levi Montalcini R. et al., J. Exp. Zool. 116: 321, 1951; Varon S. et al., Biochemistry 6: 2202, 1967; Angeletti R. H., Proc. Nati. Acad. Sci. USA 65: 668, 1970; Harper G. P. et al., Nature 279: 160, 1979; Goldstein L. D. et al., Neurochem. Res. 3: 175, 1978; Walker P. et al. al., Life Science 26: 195, 1980; Calissano P. et al., Hormonal Prot. Peptides, XII: 2, 1984; Italian patent application n. 47745A88; Callegaro et al., Proceedings of the Satellite to the 20th Annual Meeting of the American Society for Neurochemistry "Trophic Factors and the Nervous System", Columbus, Ohio, March 12-14, 1989, Raven Press, Fidia Research Series - in press; Kingsbury D.T., Falkow S., "Rapid Detection and Identification of Infectious Agents ", Academic Press; Levi Montalcini R., Science 1237, 1154, 1987).

All these methods are based on the availability of one or more monoclonal and / or polyclonal antibodies, or on the availability of the analyte that must carry the signaling system.

In all cases it was and is necessary to have discrete quantities of the analyte to obtain antibodies by immunization and cloning methods or to develop labeling systems. Currently, the evolution of technologies is such that it is necessary to develop analytical methodologies to dose polypeptides, even those for which the biological implications are not fully known. One of them is the nerve growth factor (NGF).

The in vitro and in vivo studies on animal models are numerous and at a level of advancement that can hypothesize a potential use of NGF in the treatment of Alzheimer's disease (Science 243: 11, 1989). Recently, further extremely important hypotheses are being advanced relating to a mediating action of NGF between the central nervous system and the immune system (Aloe L. et al., Proc. Nati. Acad. Sci. USA 1983, 6184: 1986; Spillantini M.G. et al., Proc. Nati. Acad. Sci. USA 1986, 8555: 1989). From the above considerations derives the importance of having a quantitative analytical methodology available for the determination of human NGF in biological liquids or culture media. Consequently, it is necessary to have an analytical method and the appropriate specific reactants available according to the molecule to be analyzed.

DETAILED DESCRIPTION OF THE INVENTION NGF from murine glands acts as a protein complex of type 7S (molecular weight about 140,000 daltons) composed of three different subunits (a, B, Y) which coordinate a Zn <+> atom.

The most interesting part of the 7S molecule, relative to biological activity, is made up of two polypeptide chains, each having a molecular weight of 13,250 and made up of 118 amino acids. Each chain or monomer has three disulfide bridges, which form covalent bonds between two residues of the amino acid cysteine, which give a strong stability to the three-dimensional structure of the protein. The two monomers of NGF joined together by weak bonds form a dimer with a molecular weight of 26,500. It has been shown that biological activity is associated with the dimer called 2.5S, or conventionally BNGF. A strategy currently in use for the identification of the active sites of a protein molecule involves the determination of its hydrophilic and hydrophobic regions. In fact, these regions, being normally exposed to the surface of the protein molecule, exercise a particular biological function, for example of binding with the receptor, or represent antigenic sites, ie regions capable of inducing the formation of specific antibodies. The amino acid sequence of NGF was analyzed on the computer using an algorithm developed by Hopp and Woods (Proc. Nati. Acad. Sci. 78, 3824, 1961). This method of analysis provides the hydrophilicity profile of the molecule, thus allowing to evaluate whether a given peptide of the polypeptide chain has a tendency to localize to the surface or within the protein structure. The method in question considers a polypeptide chain with a length between 6 and 10 residues and calculates the hydrophilicity value for this portion by averaging the coefficients assigned to the individual amino acid residues. By shifting the range of interest of one amino acid at a time along the NGF sequence, the complete diagram is obtained (Meier R. et al., EMBO J., 5: 1489, 1986). The most hydrophilic sequences correspond to the positive apexes of the Hopp and Woods diagram and identify the areas most exposed to the solvent. These portions of the molecule could therefore represent the potential antigenic sites and also the active site, that is, the exposed area of the molecule capable of interacting with other biological macromolecules.

Among the possible hydrophilic regions of the Hopp and Woods diagram of the NGF, the amino acid portion identifiable as 20-36 was chosen.

The 20-36 synthetic peptide of NGF can be used for the production of polyclonal antibodies capable of selectively recognizing and interacting not only with the peptide itself, but also with the entire NGF molecule. The application possibilities of these anti-peptide antibodies are many, for example they can be used for the development of immunochemical assays that can be used in the quantitative assay of NGF in biological fluids and for the realization of immunoaffinity columns, making possible an easier purification of the NGF often present in very low concentration and in very complex biological liquids. (Corona G.et al., "Synthetic Peptides: Approaches to biological problems", UCLA Symposia Colorado USA 1980)

Among the analytical methods available, the competitive system is certainly the most advanced in terms of specificity. The determination of an analyte in biological liquids, by means of a competitive type reaction system, is performed by incubating the sample, with or without analyte, with a support to which antianalyte antibodies, generally defined as solid phase, are bound. Subsequently, after the elimination of the analyte not reacted with the antibodies, the solid phase is washed with suitable buffers and incubated with the analyte in question. The latter can be directly or indirectly labeled with a visualizing agent, for example with a biotin-avidin-enzyme system (Johnson B. et al., J. Immunol. Meth.

115, 219, 1988).

To date, the methods of detection, that is of visualization of an interaction between analyte and antibody, in the competitive system, are numerous, as well as the supports where antibodies can be absorbed. Beyond the modifications of the supports and tracer systems, which are part of the general technological advance, a quantitative or qualitative dosage of an analytical type with a competitive system bases its fundamental properties of use on its specificity, that is, on the ability to identify in the liquid unambiguously and exclusively the presence of the analyte. It is evident that recognizing the analyte only when it is in a well-defined structural form, such as that associated with its biological activity, increases the specificity characteristics of the analytical assay and consequently the biological significance in the quantitative identification of the analyte in question. . The specificity in a competitive analytical assay is given predominantly by the antianalyte antibodies and by the labeled analyte used as tracer.

The object of the invention is to verify the possibility of developing a dosage for a soluble polypeptide or its fragments in the face of a prediction of its hydropathicity profile derived from its gene sequence and the consequent amino acid sequence. As an example, the NGF polypeptide is used to carry out this check. The anti-NGF antibodies used in the solid phase, i.e. absorbed to the support, were produced by immunization of rabbits with a defined fragment of NGF, a peptide consisting of the amino acid sequence corresponding to the 20-36 portion and purified by affinity chromatography on the same peptide , immobilized on a solid support belonging to a synthesis preparation different from that used in immunization. These 20-36 antipeptide polyclonal antibodies have been evaluated as cross-reactivity with human NGF in its biologically active form of 2.5S or BNGF, cloned and expressed in eukaryotic cells (Italian patent application n.

48564A89).

The evaluation of the reactivity of these anti-peptide 20-36 polyclonal antibodies of the NGF molecule is shown in Figure 1.

Figure 1

Example of Western Blot after reaction of human NGF with polyclonal antibodies, a) produced in rabbit by immunization with peptide 20-36, b) produced in rabbit by immunization with the 2.5S form of NGF and purified by affinity chromatography respectively on peptide 20-36 immobilized on a solid support and on the immobilized NGF. The presence of the polyclonal antibodies was detected by means of a commercial amplification system.

The present invention also relates to a kit containing some of the components used for the development of the method object of the invention itself. This kit must contain at least the anti-peptide antibodies bound or not to a support and the analyte polypeptide or a specific segment thereof, the latter bound to a marker agent. As support it is possible to use, for example, tubes, microtiter plates or their strips, balls or discs obtained from glass, plastic, or other substances usable for these applications. Given the type of assay, it is not limiting that the antibody is bound to the support. If the antibodies are not bound to the support, it is necessary to have a chemical or biological precipitating or complexing agent available. Enzymes, fluorophores, colored compounds, metals or their derivatives can be used as the marker agent. If desired, a kit may also contain adjuvants for the determination of marker agents, such as a substrate for the enzyme, wash or dilution buffers, whether or not they contain detergents. The invention is illustrated below by examples 1 and 2 and relates to the dosage of the biologically active form of human NGF obtained by recombinant DNA methodology (Italian patent application no. 48564A89). Moreover, in the spirit of the patent, the use of NGF must be considered entirely exemplary and not limiting since the experimental approach underlying the patent is applicable for all proteins.

- Determination of human NGF in culture medium in polycarbonate microtiter plates.

A. Methods

To the. Coating of the microtiter plates The wells of the polycarbonate microtiter plate were absorbed inside with the previously characterized polyclonal anti-peptide -20-36 of the polypeptide NGF, diluted in a bicarbonate buffer pH 9.0 by incubation for 24 hours at temperature environment. The wells were then washed with 0.15 mol / liter of phosphate buffer, pH 7.2.

A.2. Incubation of the microtiter plates with the sample to be analyzed

50 μi of sample to be analyzed, at different concentrations, were distributed in the well of the microtiter plate in the presence of the peptide 20-36 labeled with biotin and allowed to incubate for 24 hours at room temperature. Subsequently, the excess of unreacted reactive was removed and the wells of the microtiter plate were washed with 0.15 mol / liter of phosphate buffer, pH 7.2, containing 2% bovine albumin and a non-ionic detergent such as Tween. 20 at 0.05%.

A. 3. Incubation with labeled avidin

The wells obtained with the procedure described in point A.2. were filled with 50 μl of a commercial tracer consisting of enzyme-labeled avidin, such as horseradish root peroxidase diluted in 0.15M phosphate buffer, pH 7.2, containing 0.05% Tween 20 and 2% bovine albumin. The incubation took place for 6 to 4 hours at room temperature or 37 ° C. Subsequently, the excess of reactive was removed by washing with 0.15M phosphate buffer, pH 7.2.

A.4. Substrate incubation

The wells obtained with the procedure described in point A.3. were filled with 50 μl of a mixture of 0.4 mg / ml of orthophenylenediamine and 0.2 mg / ml of hydrogen peroxide in a buffer at pH 5.0 After incubation for 15 minutes, 50 μl of sulfuric acid was added 2.5 moles / liter in the wells of the microtiter plate.

A.5. Measurement of absorbency

The absorbances of these solutions present in the wells of the microtiter plates were performed with a photometer at a wavelength of 492 mm (OD 492 mm).

B. Results

The absorbances corresponding to the wells where the solutions of human NGF were distributed, at different concentrations, present in the culture medium, examined with the method described above are shown in Figure 2.

EXAMPLE 2 - Test kit

For the determination of human NGF in accordance with example 1, a kit consisting of several dosages and consisting of:

- polycarbonate microtiter plates with 20-36 polyclonal antipeptide antibodies absorbed inside the wells that recognize NGF in its form called 2.5S or flNGF and its non-dimeric forms;

- ampoules containing specific tracings made up of amino acid sequences that recognize the 20-36 antipeptide antibody;

- bottles containing phosphate buffer pH 7.2, containing bovine albumin;

bottles containing Tween 20 detergent solution;

- substrate bottles containing orthophenylenediamine and hydrogen peroxide in buffer at pH 5.0;

- ampoules containing solutions of different concentrations of human NGF, including also a negative control solution;

- an instruction booklet to perform the test.

Since the invention is thus described, it is clear that these methods can be modified in various ways. These modifications are not to be considered as divergences from the spirit and perspectives of the invention and all those modifications that would appear evident to an expert in the field are included in the following / claims

Claims (31)

CLAIMS 1. A method for the determination of a polypeptide, present in a biological liquid, characterized by the fact that the reagents to be used in the assay were originated not having available the polypeptide, considered as an analyte in its whole or partial form, but its nucleotide sequence. 2. A method according to claim 1, characterized in that the sample to be examined is incubated with antibodies specific for a suitable polypeptide present in the analyte, bound or absorbed to a support or in solution under defined concentration conditions, which recognize the whole molecule, that the solid phase is separated from the liquid one, that the complex is separated from the free analyte by means of precipitating agents, that the bond between the reactants and the labeled analyte or a portion thereof gives a quantitative or qualitative measure of the analyte present in the biological liquid. 3. A method according to claims 1 and 2, in which the incubation with antibodies that recognize the analyte, is developed with antibodies bound to a support, in solution and with a peptide reactive of an amino acidic nature specific for the antibody, labeled with an agent, and the possible binding of the analyte to antibodies is visualized by the binding of a labeled agent bound to the analyte's competitor and a signal. 4. A method according to claims 1 to 3, characterized in that the competitor of the analyte is a peptide. 5. A method according to claims 1 to 4, characterized in that the antibody is specific for the analyte. 6. A method according to claims 1 to 5, characterized in that the antibody was obtained by immunization with a peptide provided in the peptide sequence of the anality. 7. A method according to claims 1 to 6, characterized in that the antibody was obtained by immunization with a peptide without the use of adjuvants. 8. A method according to claims 1 to 7, characterized in that the analyte is a soluble cytokine. 9. A method according to claim 8, characterized in that the analyte is a growth factor. 10. A method in accordance with claims 8 and 9, characterized by the fact that the analyte is the nerve growth factor of human origin. 11. A method according to claims 1 to 3, characterized in that a support is used wherein polyclonal antibodies specific for a peptide portion of the analyte recognize the analyte of human origin. 12. A method according to claims 8 to 11, characterized in that the analyte is the human nerve growth factor. 13. A method according to claims 1 to 12, characterized in that the peptide antibodies are in solution. 14. A method according to the preceding claims, characterized in that a hapten-peptide system, labeled with an enzyme, is used as a competition and visualization agent. 15. A method according to claims 1 to 3, characterized in that the incubation with antibodies directed against human NGF takes place with antibodies bound to a solid support. 16. A method according to claim 3, characterized in that the solid support is given by microtiter plates. A method according to claims 1 to 7, characterized in that the detergent is non-ionic. 18. A method according to the preceding claims, where the non-ionic detergent is Tween 20. 19. A method according to claim 1, which can be configured in a kit comprising anti-analyte antibodies bound to a solid support directed against a determined amino acid site of the analyte, prepared by immunization with a peptide whose amino acid sequence is within the analyte is the peptide recognized by antibodies that carries a signal system. 20. A kit according to claim 19 characterized in that it comprises: - a solid support to which the antibodies that recognize an amino acid portion of the human peptide, for example NGF, are bound. - an amino acid sequence recognized by antibodies and included in the human polypeptide sequence, for example NGF, which carries the signal directly or indirectly. 21. A kit according to claims 19 and 20, where the tracer is an enzyme. 22. A kit according to claim 21, where the kit contains a substrate for the enzyme. 23. A kit according to claims 19 to 22, where the solid support are polycarbonate microtiter plates. 24. A kit according to claims 19 to 23, where a standard human NGF curve is present at different concentrations. 25. A kit according to claims 19 to 24, where a protein solution containing a detergent is present. 26. A method to visualize in the serum polypeptides and their fragments whose decrease or increase has been ascertained by the analysis of genes for example through subtraction libraries. 27. A method that can be used for the diagnosis and monitoring of neuroimmunoendocrine diseases. 28. A method that can be used for the diagnosis and monitoring of neuroimmunodegenerative diseases. 29. A method that can be used for the diagnosis and monitoring of diseases of the central nervous system. 30. A method that can be used for the diagnosis and monitoring of diseases of the peripheral nervous system. 31. An analytical method for use in human diagnostics.