EP0533779A1 - Procede de determination de polypeptides et de proteines dans les solutions et les fluides biologiques - Google Patents

Procede de determination de polypeptides et de proteines dans les solutions et les fluides biologiques

Info

Publication number
EP0533779A1
EP0533779A1 EP91911244A EP91911244A EP0533779A1 EP 0533779 A1 EP0533779 A1 EP 0533779A1 EP 91911244 A EP91911244 A EP 91911244A EP 91911244 A EP91911244 A EP 91911244A EP 0533779 A1 EP0533779 A1 EP 0533779A1
Authority
EP
European Patent Office
Prior art keywords
antibodies
analyte
polypeptide
protein
peptide
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP91911244A
Other languages
German (de)
English (en)
Inventor
Francesco Della Valle
Lanfranco Callegaro
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Fidia SpA
Original Assignee
Fidia SpA
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Fidia SpA filed Critical Fidia SpA
Publication of EP0533779A1 publication Critical patent/EP0533779A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/48—Nerve growth factor [NGF]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4709—Amyloid plaque core protein
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/475—Assays involving growth factors
    • G01N2333/48—Nerve growth factor [NGF]

Definitions

  • the present invention relates to analytical methods for qualitatively and quantitatively determining the presence of polypeptides and proteins in solutions and biological fluids.
  • it relates to a competitive-type analytical assay system employing antibodies specific for a portion of the amino acid sequence of a polypeptide or protein, and the polypeptide, protein, or a portion thereof as an analytical reagent.
  • the presence or concentration of antigenic substances in biological fluids can be determined by immunoassay techniques. Such techniques are based on the formation of a complex between the antigenic substance being assayed and an antibody in which a member of the complex may be labelled to permit detection or quantitative analysis after separation of the complexed labelled antigen or antibody from uncomplexed labelled antigen or antibody.
  • the antigenic substance in a sample test fluid competes with a known quantity of labelled antigen for a limited quantity of antibody binding sites. Consequently, the amount of labelled antigen bound to the antibody is inversely proportional to the amount of antigen in the sample.
  • immunometric assays employ a labelled antibody, and the amount of labelled antibody associated with the complex is directly proportional to the amount of antigenic substance in the sample fluid.
  • hydrophilic regions are normally exposed on the surface of the protein molecule, they are likely to exercise a particular biological function. It may be expected, for example, that they could act as receptor binding regions, or that they represent antigenic sites, capable of inducing the formation of specific antibodies.
  • Hopp and Woods ((1961) Proc, Natl. Acad. Sci. USA 78:3824) developed an algorithm which is capable of producing the hydrophilic profile of a protein molecule, thus making it possible to assess whether a given peptide of the polypeptide chain tends to place itself on the surface or in the interior of the protein structure.
  • This method takes into consideration a peptide chain between 6 and 10 amino acid residues long, calculating its hydrophilic value by the coefficients assigned to the single amino acid residues. By shifting section by section along the sequence one amino acid at a time, a complete diagram can be obtained (Meier R.
  • NGF nerve growth factor
  • the "competitive" system is certainly the most sensitive in terms of specificity. Determination of an analyte in biological liquids by a competitive-type reaction system is performed by incubating the sample, with or without analyte, with a support to which anti-analyte antibodies are bound, usually defined as the solid phase. Subsequently, when the analyte which has not reacted with the antibodies has been eliminated, the solid phase is washed with suitable buffers and incubated with the analyte in question, which can be directly or indirectly labelled with a visualizing agent, for example with a biotin-avidin-enzyme system (Johnson B. et al., J. Immunol.
  • step (c) removing any unreacted biological fluid or solution and labelled peptide fragments from the reaction mixture of step (b);
  • step (e) qualitatively and quantitatively detecting or measuring said antibody complexes of step (d).
  • a disease such as a neuroimmunoendocrine disease, a neuroimmunodegenerative disease, a disease of the central nervous system, or a disease of the peripheral nervous system in a human or animal
  • kit for the qualitative or quantitative determination of a polypeptide or protein analyte present in a biological fluid or solution comprising:
  • anti-analyte antibodies specific for a peptide fragment of said polypeptide or protein analyte which are capable of recognizing said polypeptide or protein analyte; and a peptide fragment which is capable of being recognized by said antibodies, and which is also capable of being detected by a detection system.
  • Figure 1 shows the reactivity of human NGF antipeptide 20-36 polyclonal antibodies compared to that of polyclonal antibodies produced by immunization with the 2.5S form of NGF against human NGF in a Western blot: a) polyclonal antibodies produced in rabbit by immunization with NGF peptide 20-36; b) polyclonal antibodies produced in rabbit by immunization with the 2.5S form of NGF and purified by affinity chromatography on peptide 20-36 immobilized on a solid support and on immobilized NGF, respectively. The presence of polyclonal antibodies was detected by means of a commercially available amplification system.
  • Detection system blotted proteins were visualized with anti-rabbit AuroProbe BL plus secondary antibodies (Janssen) following the manufacturer's instructions.
  • Figure 2 is a standard curve showing absorbance readings at 492 nm for samples treated as described in
  • Example 1 infra, disclosing a linear correspondence between the analyte, human NGF, and optical density in the presence competitive immunological assay system.
  • Bo absorbance value (in nm) of the standard concentration of the tracer (peptide 20-36 labelled with biotin);
  • the purpose of the present invention is to provide an assay for a soluble polypeptide or protein or their fragments based on the calculation of the hydropathic profile derived from its gene sequence and the consequent amino acid sequence.
  • the invention is illustrated hereafter by Examples 1 and 2, and exemplifies the assay of the biologically active form of human NGF obtained by recombinant DNA methodology (Italian patent application No. 48564A89). Moreover, within the context of the present invention, the use of NGF must be considered illustrative and not limiting in any way, since the experimental approach on which the invention is based can be applied to all polypeptides and proteins.
  • NGF from mouse glands behaves as a 7S-type protein complex (molecular weight about 140,000 Daltons) formed by three different subunits ( ⁇ , ⁇ , ⁇ ) coordinating a Zn + atom.
  • the most interesting part of the 7S molecule consists of two polypeptide chains, each with a molecular weight of 13,250 Daltons and formed by 118 amino acids. Each chain or monomer has three disulfide bridges, which form covalent bonds between two cysteine residues, and which confer great stability to the three-dimensional structure of the protein.
  • the amino acid portion identifiable as 20-36 i.e., the amino acids at positions 20-36 of the NGF polypeptide, was chosen to illustrate the utility of the present invention.
  • the synthetic peptide 20-36 of NGF can be used to produce polyclonal antibodies capable of selectively recognizing and interacting not only with the peptide itself, but also with the entire NGF molecule. This fragment is common to a variety of animal species, facilitating analysis of the NGF protein therein.
  • anti-NGF antibodies used in the solid phase, infra i.e., adsorbed onto the support, were produced by immunizing rabbits via multiple site intradermal injections of the free peptide, absent a carrier, in complete Freund's adjuvant, with a specific NGF fragment, a peptide comprising the amino acid sequence corresponding to portion
  • NGF in its biologically active form, 2.5S or ßNGF, cloned and expressed in eukaryotic cells (Italian patent application No. 48564A89).
  • Assessment of the reactivity of these NGF anti-peptide 20-36 polyclonal antibodies is shown in Figure 1, where it can be seen that rabbit polyclonal antibodies produced by immunization with either NGF peptide 20-36 or the 2.5S form of NGF exhibit the same reactivity with human NGF upon Western blotting.
  • the concentration of antibody used to adsorb to the wells ranged between 0.1-10 ⁇ g/ml, preferably 1 ⁇ g/ml, in carbonate buffer, 0.1M, pH 8.5.
  • Wells were coated with 20 ⁇ l-100 ⁇ l, preferably 50 ⁇ l, of this solution.
  • the value of 50 ⁇ l corresponds to 50 ng/well, indicated in Fig. 2.
  • analyte sample 50 ⁇ l of analyte sample, at different concentrations, were placed in the wells of the microtitration plate in the presence of peptide 20-36 labelled with biotin (hapten-peptide), and left to incubate for 24 hours at room temperature.
  • the concentration of NGF ranged between 0.1-100 ng/ml.
  • the buffer employed was PBS, containing 0.1% bovine serum albumin.
  • the quantity of labelled peptide 20-36 ranged from 2-20 ng/ml, preferably 10 ng/ml. This corresponded to 0.5 ng/well.
  • the wells prepared as described in Section A.2. were filled with 50 ⁇ l of a commercially obtained tracer composed of avidin-labelled enzyme, such as horseradish peroxidase diluted in 0.15M phosphate buffer, pH 7.2, containing 0.05 % Tween 20 and 2% bovine albumin. This was incubated for 4 to 6 hours at room temperature or at 37oc. Any excess reagent was removed by washing with 0.15M phosphate buffer, pH 7.2.
  • avidin-labelled enzyme such as horseradish peroxidase diluted in 0.15M phosphate buffer, pH 7.2, containing 0.05 % Tween 20 and 2% bovine albumin.
  • the wells treated as described in Section A.3. were filled with 50 ⁇ l of a mixture of 0.4 mg/ml of orth ⁇ phenylenediamine and 0.2 mg/ml of hydrogen peroxide in 0.1M citrate buffer at pH 5.0. After incubation for 15 minutes, 50 ⁇ l of sulfuric acid, 2.5 mol./litri, were added to the wells of the microtitration plates. A.5. Absorbance measurement.
  • the present invention also concerns a kit containing components necessary to detect the protein or polypeptide of interest.
  • This kit must at least contain anti-peptide antibodies unbound or bound to a support, and the analyte polypeptide or protein, or one of their specific segments, bound to a labelling agent.
  • Human NGF was determined as described in Example 1 using a multi-assay kit. This kit contained the following:
  • NGF in its form known as 2.5S or BNGF, and its non-dimeric forms
  • a support for example, tubes, microtitration plates or their strips, beads or disks made of glass, plastic, or other substances suitable for this purpose.
  • the antibody does not necessarily have to be bound to a support, and can be present free in solution. Should the antibodies not be bound to a support, it is necessary to utilize a chemical or biological precipitating agent such ae polyethylene glycol, or a complexing agent such as protein-A or protein-G.
  • labelling/detection agents it is possible to use enzymes such as horseradish peroxidase, ⁇ -galactosidase, or alkaline phosphatase, fluorescent substrates, fluorogenic, chemiluminescent, bioluminescent, radioactive, or metallic molecules, or biotin.
  • a kit can contain aids for the determination of labelling agents, for example a substrate for the enzyme, and washing or dilution buffers which may or may not contain detergents.
  • Any polypeptide or protein analyte can be qualitatively and/or quantitatively detected by the method of the present invention.
  • proteins which can be detected by the method of the present invention include, but are not limited to, for example, soluble cytokines and growth factors, including the nerve growth factor of human origin.
  • soluble cytokines and growth factors including the nerve growth factor of human origin.
  • antibodies can be raised against such selected peptide fragments.
  • the extent of cross-reactivity of these antibodies with the entire polypeptide or protein molecule can be determined by routine immunological methods.
  • peptide sequences that result in the production of antibodies that effectively cross-react with the entire analyte molecule are useful in the present method and kit, and can be employed in the qualitative and/or quantitative detection of polypeptide or protein analytes as described herein.
  • the qualitative and quantitative detection of proteins of medical and veterinary significance via the instant method provides a valuable rapid diagnostic tool permitting the diagnosis and monitoring of a wide variety of diseases and therapies.
  • the latter include, but are not limited to, diseases of the neuroimmunoendocrine type, the neuroimmunodegenerative type, such as Alzheimer's disease, multiple sclerosis, Huntington's disease, motoneuron disease, Guillain Barre syndrome, and Parkinson's disease, diseases of the central nervous system, such as those of traumatic, anoxic, degenerative, or toxic-infective origin, diseases of the peripheral nervous system, such as those of traumatic-compressive, degenerative, dysmetabolic, or toxic-infective origin, etc.
  • a further advantage of the present inventive method derives from the fact that the peptide sequence employed to produce the antibodies necessary to carry out the analysis of an analyte-containing fluid can be generated from the nucleotide sequence of the target protein or polypeptide. Knowledge of the amino acid sequence of the protein or polypeptide, or availability of the analyte itself, which may be very expensive or in limited supply, is not required. Furthermore, such antibodies can be obtained by immunization with a peptide without the use of adjuvants. Finally, the length of the peptide fragment required is short, varying between 8-30, and preferably about 17, amino acids.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Engineering & Computer Science (AREA)
  • Hematology (AREA)
  • Biomedical Technology (AREA)
  • Urology & Nephrology (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Biochemistry (AREA)
  • Physics & Mathematics (AREA)
  • Food Science & Technology (AREA)
  • Organic Chemistry (AREA)
  • Analytical Chemistry (AREA)
  • Microbiology (AREA)
  • Pathology (AREA)
  • Biotechnology (AREA)
  • Cell Biology (AREA)
  • General Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Genetics & Genomics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Zoology (AREA)
  • Toxicology (AREA)
  • Endocrinology (AREA)
  • Peptides Or Proteins (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

Procédé de détermination qualitative et quantitative d'une substance polypeptidique ou protéique soumise à l'analyse et présente dans une solution ou un fluide biologique. On utilise un système d'analyse du type à compétition dans lequel le réactif compétitif est un fragment peptidique marqué et présent dans le séquence d'aminoacides de ladite substance. Par exemple, le procédé consiste à déterminer le facteur de croissance nerveuse chez l'homme. On a prévu un kit destiné à la détermination des substances polypeptidiques ou protéiques soumises à l'analyse, à l'aide de ce procédé, ainsi qu'un procédé utile dans le domaine de la médecine humaine et vétérinaire lors du diagnostic et/ou de la surveillance de diverses maladies et de traitements correspondants dans lesquels un polypeptide ou une protéine caractéristique peut servir de moyen de diagnostic efficace.
EP91911244A 1990-06-12 1991-06-12 Procede de determination de polypeptides et de proteines dans les solutions et les fluides biologiques Withdrawn EP0533779A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
IT04162690A IT1243281B (it) 1990-06-12 1990-06-12 Metodo per la determinazione quantitativa e qualitativa di polipeptidiin liquidi proteici
IT4162690 1990-06-12

Publications (1)

Publication Number Publication Date
EP0533779A1 true EP0533779A1 (fr) 1993-03-31

Family

ID=11252188

Family Applications (1)

Application Number Title Priority Date Filing Date
EP91911244A Withdrawn EP0533779A1 (fr) 1990-06-12 1991-06-12 Procede de determination de polypeptides et de proteines dans les solutions et les fluides biologiques

Country Status (6)

Country Link
EP (1) EP0533779A1 (fr)
CN (1) CN1058846A (fr)
AU (1) AU7969091A (fr)
IL (1) IL98466A0 (fr)
IT (1) IT1243281B (fr)
WO (1) WO1991019982A1 (fr)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1050757A1 (fr) * 1999-05-03 2000-11-08 Evotec BioSystems AG Méthodes de diagnostic ou de traitement de maladies neuropsychiatriques basées sur des taux accrus du NGF (Nerve Growth Factor) dans la fluide cérébrospinale
AU4753900A (en) * 1999-05-03 2000-11-17 Evotec Biosystems Ag Methods of diagnosing or treating alzheimer's disease
US11918936B2 (en) 2020-01-17 2024-03-05 Waters Technologies Corporation Performance and dynamic range for oligonucleotide bioanalysis through reduction of non specific binding

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1989003430A1 (fr) * 1987-10-13 1989-04-20 Terrapin Diagnostics, Ltd. Procede de production de reactifs pour diagnostics d'immunite
DE68925923T2 (de) * 1988-06-10 1996-11-14 Merck & Co Inc Antigene der einzigen Spaltungsstelle von Fibrinogen durch Elastase
SE465573B (sv) * 1989-03-14 1991-09-30 Lope Medicine Ab Nervtillvaextfaktorpeptider, motsvarande antikroppar och foerfarande foer bestaemning av nativ nervtillvaextfaktor
ATE160288T1 (de) * 1989-08-28 1997-12-15 Takeda Chemical Industries Ltd Antikörper, ihre herstellung und verwendung

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9119982A1 *

Also Published As

Publication number Publication date
WO1991019982A1 (fr) 1991-12-26
CN1058846A (zh) 1992-02-19
IT9041626A1 (it) 1991-12-12
IL98466A0 (en) 1992-07-15
AU7969091A (en) 1992-01-07
IT9041626A0 (it) 1990-06-12
IT1243281B (it) 1994-05-26

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