EP1237927A2 - Anticorps, leur procede de production, leur utilisation, cocktails immunologiques, ensembles de dosage immunologique et peptides - Google Patents

Anticorps, leur procede de production, leur utilisation, cocktails immunologiques, ensembles de dosage immunologique et peptides

Info

Publication number
EP1237927A2
EP1237927A2 EP00985167A EP00985167A EP1237927A2 EP 1237927 A2 EP1237927 A2 EP 1237927A2 EP 00985167 A EP00985167 A EP 00985167A EP 00985167 A EP00985167 A EP 00985167A EP 1237927 A2 EP1237927 A2 EP 1237927A2
Authority
EP
European Patent Office
Prior art keywords
haptoglobin
protein
immunization
antibodies
peptides
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Withdrawn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
EP00985167A
Other languages
German (de)
English (en)
Inventor
Mark Hennies
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.)
Individual
Original Assignee
Individual
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 Individual filed Critical Individual
Publication of EP1237927A2 publication Critical patent/EP1237927A2/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • 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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4717—Plasma globulins, lactoglobulin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies

Definitions

  • Antibodies and processes for their preparation, their use, as well as immunization cocktails, immunoassay sets and peptides are included in the preparation, their use, as well as immunization cocktails, immunoassay sets and peptides.
  • the present invention relates to a method for producing protein-specific antibodies, protein-specific antibodies obtainable therewith, namely polyclonal, cross-reactive antisera with specificity for haptoglobin from at least two types of useful and domestic animals, and their use for determining haptoglobin in useful and domestic samples.
  • the invention further relates to immunization cocktails with protein and at least one protein fragment, peptides which are useful for producing the antisera, and immunoassay sets which are based on the antisera.
  • Plasma proteins the concentration of which varies with a host's response to infection, inflammation, or a traumatic event.
  • the increase in concentration in the plasma can be of different degrees, from a 100 to 500-fold very strong increase to a 3 to 4-fold increase in a moderate response to a 2-fold increase in the plasma level of a relatively weak response, cf. , Eckersall et al., Veterinary Medicine, Vol. 41, 643 (1999).
  • Haptoglobin belongs to the group of acute phase proteins. In cattle, the haptoglobin concentration rises up to 300 times during the acute phase response from a concentration of less than 0.01 g / 1 to 2-3 g 1 within 48 hours after infection. In pigs, on the other hand, there is only a moderate increase from about 1-2 mg / ml to about 5 mg / ml after turpentine injection (Eckersall et al, 1999).
  • haptoglobin Methods for the quantitative determination of haptoglobin are based on either biochemical or immunological methodology. Since the biochemical measuring method uses the binding of haptoglobin to hemoglobin, even the smallest traces of hemoglobin can impair the measuring accuracy.
  • Immunological measurement methods first require the production of suitable antibodies.
  • a monoclonal antibody directed against bovine haptoglobin as capture antibody and a rabbit directed against bovine haptoglobin as detection antibody Antiserum is used.
  • Haptoglobin concentrations in horse sera were determined immuno-turbidimetrically using a sheep antiserum directed against horse haptoglobin (Kent and Goodall, Equine Veterinary Journal, 23 (1), 59-66 (1991)).
  • a sheep antiserum directed against horse haptoglobin Kerbidimetrically using a sheep antiserum directed against horse haptoglobin (Kent and Goodall, Equine Veterinary Journal, 23 (1), 59-66 (1991)).
  • the immunoassays described so far are species-specific because it is generally assumed that only species-specific antisera and standards can meet the requirements for consistent and comparable immunochemical tests (Eckersall et al., 1999).
  • the production of a polyclonal antiserum usually starts from an active immunization, that is, the artificial generation of an immune response as a defense against a host organism.
  • the relevant antigen is administered to the host, and this stimulates antigen-reactive B cells to expand and differentiate.
  • Antibodies against a protein as an antigen are usually obtained by immunizing with the protein or else with synthetic polypeptides.
  • the pure antigen is ideally used to produce a specific antibody. If the high degree of purity required for this cannot be achieved or can only be achieved in a cumbersome manner, synthetic peptides with partial sequences of the protein can possibly also lead to antipeptide antibodies which cross-react with the complete protein.
  • the object on which the invention is based an immunological determination of proteins,
  • an immunological determination of proteins is achieved according to the invention by a special process for the production of protein-specific, in particular haptoglobin-specific antibodies.
  • This method uses at least one immunization cocktail that contains both protein and fragments of the protein.
  • the present invention therefore relates to a method for producing protein-specific antibodies by immunizing a host and in a manner known per se for obtaining the antibodies, characterized in that at least one immunization cocktail is administered to the host which contains protein and at least one protein fragment.
  • Protein-specific antibodies in the sense of the invention are antibodies with binding specificity for a specific protein.
  • the term protein includes polypeptides which can be essentially homogeneous structurally, but which also include a mixture of structurally different polypeptides, for example different phenotypes and isotypes of a protein, different species-specific variants of a protein due to evolutionary divergence or individual ones, mostly due to mutations Protein variants can be.
  • protein-specific antibodies in the sense of the invention can also be cross-reactive antibodies, polyclonal or monoclonal, with specificity for at least two structurally different proteins. Structural differences are to be understood in terms of antigen recognition and include different sequences as well as different spatial structures, such as conformities or derivatizations, e.g. Glycosylation.
  • Immunization means the generation of an immune response as an organism's defense against an antigen.
  • One or more antigens are administered to the host, preferably injected into the bloodstream or into a tissue.
  • the generation of an immune response is artificial, one speaks therefore of an active immunization.
  • the immunization is usually carried out according to a defined immunization scheme.
  • the primary immunization advantageously is followed by further immunizations to produce secondary immunological reactions.
  • the number of immunizations to be carried out depends primarily on the host and the respective development of the antibody titer. It is preferred according to the invention to carry out more than two and in particular three to five immunizations. According to a particular embodiment of the present invention, four immunizations are carried out.
  • the individual immunizations are usually carried out at intervals of several weeks, which should advantageously not exceed two months. Immunization intervals of two to seven and in particular three to six weeks are preferred. According to an advantageous embodiment of the present invention, the interval between the individual immunizations is about five weeks.
  • the immunization cocktails administered for the individual immunizations can be the same or preferably different.
  • at least one immunization cocktail contains protein and at least one protein fragment, while with the others in the conventional manner either protein or at least one
  • Protein fragment can be administered. However, it is advantageous if all immunization cocktails used within a method contain protein and at least one protein fragment, although these can differ in their respective composition, which is preferred in certain cases.
  • a higher vertebrate usually serves as the host.
  • Rodents in particular rabbits, mice, rats and hamsters, as well as goats and sheep, are preferred. According to an advantageous embodiment of the present invention, rabbits serve as the host.
  • the antibodies formed can be made in a manner known per se, for example from spleen,
  • full serum consists only partially of immunoglobulins, so that further purification steps for enriching the desired antibodies can follow.
  • salting-out e.g. by means of fractionated ammonium sulfate precipitation, ultrafiltration, re-buffering, dialysis, precipitation by dialysis against acidic buffers, various chromatographic methods such as ion exchange chromatography, gel permeation chromatography, hydrophobic chromatography, AfFinity chromatography, etc. can be used.
  • High-titered full sera are advantageously obtained over a reasonable period of time, usually several weeks or months, with the host finally bleeding out.
  • spleen tissue from the host and to fuse the splenic lymphocytes contained therein, which are antibody-producing B cells, with myeloma cells, then to select and clone suitable hybridomas which produce a desired antibody and then to produce monocional antibodies use.
  • the respective work steps are known to the person skilled in the art.
  • the antibodies obtained in this way in particular the monoclonal antibodies, can be modified, fragments of these antibodies or chimeric antibodies can be generated, depending on the task and area of use. Suitable techniques are known to the person skilled in the art.
  • an immunization cocktail is understood to be a mixture of antigen and, if necessary, adjuvants, in particular adjuvant.
  • adjuvants are substances in which the antigen is incorporated or at least injected together with the antigen and which enhance the immune response. These include depot-forming,
  • Macrophage activating and / or influencing adjuvants usually contain oils such as paraffin oil, squalene or fatty acid esters such as mannide monooleate, sorbitan monooleate etc., for the formation of oil-in-water or water-in-oil emulsions of the antigen.
  • Adjuvants can also contain killed pathogens, toxins or components thereof, such as killed Mycobacterium tubercolosis, pertusis vaccine, detoxified monophosphoryllipid A from S.
  • Freund's adjuvant is preferred according to the invention, in particular complete for the start immunization and incomplete for further immunizations.
  • Antigen according to the invention is composed of protein and at least one protein fragment.
  • the protein expediently has at least one antigenic determinant against which the protein-specific antibodies to be produced are directed.
  • at least part of the protein to be used as antigen corresponds to at least part of the protein against which the protein-specific antibodies are directed.
  • the protein to be used in accordance with the process can be a structurally essentially homogeneous polypeptide fraction, but a mixture of structurally different polypeptides is preferred, in particular a mixture of species-specific variants of a protein.
  • Protein suitable according to the invention can be obtained from natural sources. Another possibility is recombinant production. In both cases, the person skilled in the art is familiar with the relevant techniques from protein biochemistry and molecular biology. Pure protein is preferred.
  • Protein fragments which can be used according to the invention are preferably derived from the protein against that the protein-specific antibodies are directed. At least one antigenic fragment is advantageously used. As a rule, these are optionally derivatized peptides whose amino acid sequences comprise at least parts which are part of one or more polypeptides of the protein. Peptides with a length of 10 to 30 amino acids, preferably of 15 to 25 amino acids, are advantageous. For reasons of antigenicity, peptides which are particularly useful according to the invention are derived from, preferably glycosylation-free, surface areas and have a high antigenicity index. Particularly preferred are peptides whose sequence differs from homologous sequences that may be present in the host.
  • the immunization cocktail contains at least one type of fragment, advantageously 2 or more different fragments.
  • Protein fragments which can be used according to the invention can be prepared in a manner known per se.
  • peptides can be obtained from natural sources or synthesized from corresponding amino acids.
  • a natural source that can be used according to the invention is protein that can be digested enzymatically, for example.
  • the resulting fragments can be worked up and fragments of interest can be obtained and modified if necessary.
  • Peptide synthesis is preferably carried out chemically, in particular using known solid-phase synthesis systems.
  • functionalized resins are usually used, to which the peptides to be synthesized can be linked. Wang resins for FMOC solid phase peptide synthesis or Merrifield resins for BOC solid phase peptide synthesis are common examples.
  • the desired peptide After the desired peptide has been built up, it is then detached from the carrier, it being possible for protective groups which may be present to be split off simultaneously or subsequently. Any subsequent purification that may be required, for example via HPLC, provides the peptide in the desired purity, which can be determined, for example, by means of mass spectroscopy or NMR.
  • Immunization cocktails which can be used according to the invention can contain protein and protein fragments as a physical mixture, ie protein and fragments and any adjuvants present are mixed with one another in any order. However, it is preferred that at least some of the fragments are coupled to at least some of the protein. Coupling is understood according to the invention to mean any molecular interaction which leads to a binding of the fragments to the protein, so that a certain spatial arrangement with respect to one another is achieved. The interactions can be based on covalent, metal complex-like coordinative or hydrophobic bonds, hydrogen bonds, electrostatic attractive forces, van der Vaals forces and the like. Preferred according to the invention are covalent bonds. There can be direct or indirect interactions between proteins and protein fragments.
  • Indirect interactions are usually mediated via linkers, which can be homo- or heterobifunctional.
  • linkers which can be homo- or heterobifunctional.
  • Glutaraldehyde is preferred.
  • the handling of these reagents is well known to the person skilled in the art, for example protein and peptide can be reacted with an aqueous solution of glutaraldehyde at room temperature, an effective coupling being achieved after only a few hours.
  • the protein fragments themselves are not immunogenic, it is advantageous to also couple those fragments which are not coupled to the protein used as antigen according to the invention to other carriers, in particular carrier proteins. This is primarily the case when peptides with less than about 30 amino acids are to be used as protein fragments. If the carriers are also proteins, the above statements regarding the coupling of protein and protein fragments apply mutatis mutandis. If carriers of a different constitution are used, the coupling naturally depends on the functional groups present on the carrier. Homo- or heterobifunctional linkers are also available for these cases.
  • Strongly immunogenic carriers which can bring about an effective induction of antibodies are preferred as carriers. These include, for example, various albumins, such as bovine serum albumin, ovalbumin or human serum albumin, globulins, such as thyroglobulin, etc.
  • the hemocyanin of the horseshoe crab limulus polyphemus also called keyhole limpets hemocyanin (KLH), is preferred.
  • all protein fragments that are used for such an immunization cocktail are included in the coupling reaction with the protein used as an antigen in the immunization cocktail. Provided that the coupling reaction is complete, all fragments are thus coupled to at least a part of the protein.
  • the part of the fragments that is not coupled to the protein can be coupled to a further carrier, so that all protein fragments are coupled to a carrier, namely a part to the protein and another part to a further carrier, preferably also a protein ,
  • a carrier namely a part to the protein and another part to a further carrier, preferably also a protein
  • the protein component with regard to concentration, degree of coupling, type of protein, for example protein from only one animal species or from several animal species , different selection of animal species, and for the protein fragments with regard to the type and possibly number of different fragments, their concentration and the type of coupling partner as well as the ratio of fragment (s) to protein.
  • immunization cocktails which are administered in an early phase of the immunization contain uncoupled protein, conjugates of protein and at least one protein fragment, and conjugates of at least one protein fragment with other carrier proteins.
  • Immunization cocktails that are administered in a late phase of the immunization preferably contain uncoupled protein and conjugates of the protein with at least one protein fragment, but no conjugates of protein fragments with another carrier phrotein in this embodiment.
  • the early phase of the immunization can mean, for example, the start immunization and the second immunization, while the late phase of the immunization includes, for example, the third and the fourth immunization.
  • Immunization cocktails administered in the early phase of the immunization cocktails higher amounts of protein fragments than the immunization cocktails administered in the late phase of the immunization cocktail.
  • immunization cocktails are administered to the host which contain protein from at least two different animal species and whose protein components differ with regard to the number and / or type of animal-specific protein variants used.
  • a particular embodiment of the present invention relates to a method for manufacturing Haptoglobin-specific antibodies by immunizing a host and obtaining formed antibodies, which is characterized in that the host is administered at least one immunization cocktail which contains haptoglobin and at least one haptoglobin fragment.
  • Haptoglobin (short: Hp) is a group of chemically related glycoproteins from
  • haptoglobin thus stands for all haptoglobin phenotypes, isotypes and variants. Haptoglobins from different animal species are included as well as mutated or otherwise modified haptoglobins. Species-specific haptoglobins of humans and all types of useful and domestic animals, that is to say mammals, in particular rodents, for example mice, rats, hamsters, rabbit-like mammals, for example rabbits, dogs, cats, ungulates, for example pigs and cattle, unpaired horses, for example horses, are particularly included , and primates.
  • Haptoglobin which can be used according to the invention can be of natural or recombinant origin.
  • Haptoglobins of natural origin can be obtained, for example, from blood components such as sera or plasma.
  • haptoglobin can be obtained from human plasma.
  • Such a product is commercially available as an essentially salt-free lyophilized powder.
  • haptoglobins can also be obtained from other animal species; suitable cleaning processes for this purpose are known.
  • Non-hemolytic sera can advantageously be used, resulting in particularly high yields. The following procedural measures have proven to be particularly suitable:
  • the optimal concentration for the selective precipitation of haptoglobin can be determined in a preliminary experiment.
  • hemoglobin preferably bovine hemoglobin
  • a solid phase for example to activated CNBr-Sepharose.
  • haptoglobin is then used according to the invention for immunization. In addition, it is used as a standard in immunoassays, preferably in the form of the haptoglobin that is specific for the species on which haptoglobin is to be determined.
  • the immunization cocktails contain mixtures of haptoglobins from different animal species, preferably from pork, cattle, horses, dogs and / or humans. It is preferred to administer immunization cocktails whose haptoglobin composition differs in the course of a method.
  • immunization cocktails can be administered which contain a selection of three or four of the aforementioned haptoglobins on the one hand, and all of the aforementioned haptoglobins on the other hand.
  • the former are preferably administered at a comparatively early point in time, the latter preferably towards the end of the immunization.
  • the selection can also be varied with regard to the number or type of haptoglobins used.
  • the first immunization cocktail contains haptoglobins from pork, cattle, horses and humans, the second and the third immunization cocktail haptoglobins from pigs, dogs, horses and humans, and the fourth immunization cocktail haptoglobins from pigs, cattle, dogs, horses and Man.
  • Haptoglobin peptides are generally used as protein fragments. Particularly suitable peptides have a relatively high degree of homology to corresponding sequences of haptoglobins from different animal species and in particular those animal species whose haptoglobins are to be specifically recognized by the antibodies to be produced. A is preferred
  • Peptide sequences useful in the present invention include conserved regions of haptoglobins, i.e. their sequence is essentially nonspecific. Peptides that are not part of the hemoglobin binding site are beneficial.
  • suitable peptides have a length of from 10 to 30 amino acids, preferably from 15 to 25 amino acids.
  • Examples of such peptides that have proven to be particularly suitable are the amino acid sequences SEQ ID NO: 1 and SEQ ID NO: 2. These peptides are also the subject of the present invention. If the protein fragments to be used are not or only weakly immunogenic, their immunogenicity can be increased by coupling them to carriers. For this purpose, a number of coupling options are available to the person skilled in the art, some of which have been explained above.
  • reaction with glutaraldehyde is preferred, for example by incubating the haptoglobin or haptoglobin mixture with the peptide or peptide mixture in water or an aqueous solvent.
  • This reaction can conveniently be carried out at ambient temperature, usually room temperature. However, it can also be expedient to cool or to warm slightly. As a rule, the reaction leads to the desired result within a few hours; a reaction time of, for example, 2 hours is in the usual range.
  • the glutaraldehyde concentration is generally in the ppm to% range, advantageously from 10 ppm to 1%, preferably from 100 ppm to 0.5%.
  • the optimization of the reaction parameters lies in the area of professional skill.
  • the haptoglobin fragments are coupled to at least some of the haptoglobin. If a haptoglobin mixture of haptoglobins from different animal species is used, the haptoglobin fragments can be coupled to haptoglobin of all animal species used or to a selection of haptoglobins of individual animal species. If a mixture of different haptoglobin fragments, for example a peptide mixture, is used, these can be coupled without differentiating according to the type of fragment, or certain types of fragments can be assigned to specific coupling partners.
  • haptoglobin and haptoglobin fragments thereof can be obtained according to the invention by reacting haptoglobin, preferably a mixture of haptoglobins from different animal species, with one or more haptoglobin fragments, preferably one or more haptoglobin peptides. If different fragments are used, this conversion is carried out for each type of fragment, preferably separately.
  • haptoglobin fragments it is not absolutely necessary to couple the entire amount of haptoglobin fragments to haptoglobin. If the fragments are not or only weakly immunogenic, it is advantageous in such a case to couple the fragments not coupled to haptoglobin to a further carrier, preferably to a carrier protein. KLH is preferred in connection with haptoglobin. Reference is made analogously to the above statements on the coupling reaction.
  • immunization cocktails contain the In an early phase of immunization, uncoupled haptoglobin, at least one haptoglobin-peptide and at least one KLH-peptide conjugate are administered.
  • Immunization cocktails administered in a late phase of the immunization contain uncoupled haptoglobin and at least one haptoglobin-peptide conjugate, but no KLH-peptide conjugate.
  • the haptoglobin-peptide conjugates are preferably mixtures of conjugates with haptoglobins from different animal species. It is also preferred to use different peptides, so that a conjugate mixture should also be used in this regard.
  • immunization cocktails containing haptoglobin-peptide and KLH-peptide conjugate can be administered in the first and in the second immunization, whereas the immunization cocktails administered in the third and fourth immunizations, however, haptoglobin-peptide conjugate do not contain a KLH-peptide conjugate.
  • the immunization cocktails preferably additionally contain uncoupled haptoglobin or a mixture of uncoupled haptoglobins from various animal species.
  • the antigen used in the immunization cocktails is composed of several components, for example uncoupled haptoglobin or a mixture of uncoupled haptoglobins from different animal species, one or more haptoglobin-peptide conjugates and / or one or more KLH-peptide conjugates.
  • the components to be used are first combined. It is advantageous to incubate the resulting component mixture first. This is conveniently done at ambient temperature, usually at room temperature. However, it may be appropriate to cool the mixture or to warm it slightly. The incubation period is usually a few minutes to a few hours, an incubation time of 1 hour has proven to be advantageous.
  • immunization cocktails usually contain other auxiliary substances, in particular adjuvants that are usually used for immunization.
  • adjuvants that are usually used for immunization.
  • the use of Freund's adjuvant is preferred.
  • complete Freund's adjuvant is used for the first immunization, whereas all further immunizations are carried out with incomplete Freund's adjuvant.
  • the antigen preferably as a component mixture described above, is added to the auxiliary material or components. As a rule, the antigen is emulsified.
  • Rabbits are particularly suitable as hosts for this embodiment.
  • the Immunization cocktails are injected, preferably subcutaneously. Four injections at five-week intervals have proven to be advantageous.
  • the antibody titers can be determined with an immunoassay, for example competitively with a sheep antiserum directed against host IgG and labeled haptoglobin.
  • the antibody titer can be determined after the third immunization and then antibodies can be obtained from animals which have a sufficient antibody titer.
  • Serum can be obtained from the blood obtained in a manner known per se, which contains the desired antibodies.
  • the full serum obtained can, if necessary, be further purified in a professional manner in order to enrich the antibody fraction contained therein and in particular the haptoglobin-specific antibodies.
  • Monoclonal haptoglobin-specific antibodies can also be obtained in this way. For this purpose, however, it is preferred to remove spleen tissue from the hosts and, based on the splenic lymphocytes obtained in this way, to establish hybridomas in the usual way which produce monoclonal antibodies.
  • the present invention also relates to the immunization cocktails described in connection with the method according to the invention. These contain, optionally in addition to other auxiliary substances, protein and at least one protein fragment and, if necessary, adjuvant, preferably in the configurations described above.
  • the present invention further relates to antisera which can be obtained by the above processes.
  • These can be full serums, i.e. blood obtained from the host after separation of the cellular and coagulable constituents, or fractions of this serum, in which in particular the immunoglobulin fraction and preferably the haptoglobin-specific immunoglobulin fraction is enriched.
  • Such fractions can be obtained using the methods described above in connection with the antibody purification.
  • the antisera according to the invention are polyclonal, ie they contain antibodies of different specificity, as a rule different classes and subclasses, normally all L chain isotypes are represented and several protein epitopes are recognized.
  • Antisera according to the invention are cross-reactive. In particular, they are specific for more than one haptoglobin, preferably for haptoglobin from at least two animal species, in particular useful and domestic species. Examples of particular embodiments are antisera with specificity for haptoglobin from pigs and horses or for haptoglobin from horses, dogs and cattle. In this context, specificity means the possibility of being able to detect a certain haptoglobin with sufficient sensitivity.
  • the required sensitivity can vary from animal species to animal species, depending on the concentration in which haptoglobin occurs in the healthy or sick animal.
  • the antisera according to the invention advantageously have sensitivities of less than 1 mg / ml sample, preferably less than 100 ⁇ g / ml sample and particularly preferably less than 10 ⁇ g / ml sample. This means that at least the specified concentration of haptoglobin per ml of sample can be detected with the antiserum according to the invention, advantageously also lower concentrations.
  • Another object of the present invention is the use of such a cross-reactive antiserum for haptoglobin determination in useful and domestic samples.
  • the samples are usually body fluids, for example blood, plasma, serum, saliva, milk and the like.
  • haptoglobin can also be determined in other samples derived from farm and domestic animals. Meat juice should be mentioned in this context.
  • the determination is carried out immunologically. In principle, this can be done with any analytical or diagnostic test method in which antibodies are used. These include agglutination and precipitation techniques, immunoassays, immunohistochemical procedures and immunoblot techniques, e.g. Western blotting.
  • immunoassays are preferred according to the invention. Both competitive immunoassays, ie antigen and labeled antigen (tracer) compete for antibody binding, are suitable, as are sandwich immunoassays, ie the binding of specific antibodies to the antigen is detected with a second, usually labeled antibody.
  • sandwich immunoassays ie the binding of specific antibodies to the antigen is detected with a second, usually labeled antibody.
  • assays can be both homogeneous, ie without separation into solid and liquid phases, and also heterogeneous, ie bound labels are separated from unbound labels, for example via antibodies bound to solid phases.
  • the various heterogeneous and homogeneous immunoassay formats can be assigned to specific classes depending on the labeling and measurement method, for example RIAs (radioimmunoassays), ELISA (enzyme linked immunosorbent assay), FIA (fluorescence immunoassay), LIA (luminescence immunoassay), TRFIA (temporal dissolved FIA), IMAC (immune activation), EMIT (enzyme multiplied immune test), TIA (turbodimetric immunoassay).
  • RIAs radioimmunoassays
  • ELISA enzyme linked immunosorbent assay
  • FIA fluorescence immunoassay
  • LIA luminescence immunoassay
  • TRFIA temporary dissolved FIA
  • IMAC immunoassay
  • EMIT enzyme multiplied immune test
  • TIA turbodimetric immunoassay
  • haptoglobin determination is preferred for the haptoglobin determination according to the invention.
  • Labeled haptoglobin (tracer) competes with the haptoglobin of the sample to be quantified for binding to the antiserum used.
  • the amount of antigen in the sample can be determined from the amount of tracer displaced using a standard curve.
  • enzymes have proven to be advantageous.
  • systems based on peroxidases in particular horseradish peroxidase, alkaline phosphatase and ⁇ -D-galactosidase, can be used.
  • Specific substrates are available for these enzymes. can be followed photometrically.
  • Suitable substrate systems are based on p-nitrophenyl phosphate (p-NPP), 5-bromo-4-chloro-3-indolyl phosphate / nitro blue tetrazolium (BCIP / NPT), fast red / naphthol-AS-TS phosphate for alkaline phosphatase; 2,2-azino-bis- (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), o-
  • these substrate systems are commercially available in ready-to-use form, for example in the form of
  • Tablets which can also contain other reagents, such as appropriate buffers and the like.
  • Labeled haptoglobin is used as the tracer.
  • the haptoglobin to be determined can be marked for the determination of haptoglobin of a specific animal species and used as a tracer.
  • an antigen for the production of the tracer which can be used in the determination of haptoglobin from at least two types of useful and domestic animals.
  • the antigen can be described as non-species-specific and a tracer based on it can be called a multi-species tracer.
  • the binding affinity of such multi-species tracers to the antiserum used in the immunoassay is expediently chosen so that haptoglobins of the animal species to be examined can displace the multi-species tracer.
  • Bovines, especially haptoglobin labeled with biotin can be used according to the invention as a multi-species tracer.
  • the coupling of markings to haptoglobin for the production of tracers can take place in a manner known per se. On the above statements on the coupling of haptoglobin Protein fragments are referred to accordingly.
  • suitably modified labels available for conjugation to proteins, for example biotin, avidin, extravidin or streptavidin-conjugated enzymes, maleimide-activated enzymes and the like.
  • These markings can be implemented directly with haptoglobin or - if necessary - with correspondingly derivatized haptoglobin to form the tracer. If, for example, a streptavidin-peroxidase conjugate is used, this first requires biotinylation of haptoglobin.
  • haptoglobin can be reacted with biotin amidocarboxylic acid N-hydroxysuccinimide ester, for example the caproate ester, biotin hydrazide, N-hydroxysuccinimidobiotin or 3- (N-maleimidopropionyl) biocitin
  • a bovine, biotinylated and haptoglobin coupled to a streptavidin-peroxidase conjugate is used as the tracer.
  • the antigen-antibody complex can be bound to the carrier, for example, via an anti-idiotypic antibody coupled to the carrier, for example an antibody directed against rabbit IgG, for the purpose of separation.
  • Carriers in particular microtiter plates, which are coated with corresponding antibodies, are known and some are commercially available.
  • hemoglobin is added to the test system.
  • the amount is usually such that all hemoglobin binding sites on saturated haptoglobin are saturated. This is an advantage when determining haptoglobin in strongly hemolytic samples and in whole blood.
  • the haptoglobin determination is used in particular to diagnose infections, inflammations, trauma (tissue injuries) or immunological stress. It can be indicated in acutely ill animals, can be used for therapy control and in stock management, can help in optimizing husbandry conditions and can be used as an early indicator of a successful vaccination, since the haptoglobin concentration is already there after a short time, usually already 24 hours after vaccination, correlated with the antibody titer in the plasma of the vaccinated individual.
  • the method according to the invention is suitable for determining haptoglobin quantitatively even in long-term samples (eg more than 2 years at -18 ° C), improperly stored samples (eg 5 days at room temperature) and heat-inactivated samples (eg 30 minutes at 56 ° C) ,
  • kits usually contains several containers for the separate arrangement of components. All components can be provided in ready-to-use dilution, as a concentrate for dilution or as dry matter or lyophilisate for dissolving or suspending; some or all of the components may be frozen or kept at ambient temperature until use. Sera are preferably snap frozen, for example at -20 ° C, so that in these cases an immunoassay should preferably be kept at freezing temperatures before use.
  • immunoassay Other components added to the immunoassay depend on the type of immunoassay. As a rule, standard protein, any necessary tracer and control serum are included with the antiserum. Furthermore, microtiter plates, preferably coated with antibodies, buffers, for example for testing, washing or for converting the substrate, and the enzyme substrate itself can be added.
  • the sample was stirred again at room temperature for one hour. The subsequent centrifugation corresponded to the above information.
  • the pellet obtained was dissolved in 10 ml of Tris buffer (0.1 M Tris HCl, 0.5 M NaCl, pH 7.0) and dialyzed against Tris buffer at 4 ° C. overnight.
  • Tris buffer 0.1 M Tris HCl, 0.5 M NaCl, pH 7.0
  • bovine hemoglobin (Sigma H 2500) was covalently coupled to activated CNBr-Sepharose (Amersham Pharmacia Biotech). The resulting material was converted into a
  • haptoglobin mainly depended on the haptoglobin concentration in the sera used for cleaning.
  • haptoglobin could be obtained from pig serum 4.8 mg, from bovine serum 0.6 mg, from horse serum 1.5 mg and from dog serum 1.6 mg.
  • Immunization cocktails were made with a mixture of haptoglobin from various animal species and two synthetic peptides.
  • the following stock solutions were made up: Origin and concentration of the stock solutions
  • haptoglobin mixtures were prepared with these stock solutions:
  • the antibody titer was determined using a competitive ELISA. Microtiter plates were coated with 5 ⁇ g ovine IgG / ml coating buffer (Na carbonate pH 9.6) and incubated at 4 ° C. overnight. The antibodies were obtained from sheep immunized against rabbit IgG. The antibodies contained in the sheep serum were obtained by affinity chromatography. The plates were then saturated with 2.5% casein solution at room temperature for two hours. After five washes with wash buffer, the antiserum to be tested could be applied. The antiserum was applied in various dilution levels from 1: 10,000 to 1: 100,000 in test buffer containing hemoglobin (1 mg / ml hemoglobin).
  • biotin-labeled haptoglobin was diluted of 8 ng ml pipetted onto the plates. After incubation at room temperature for one hour, the plates were washed three times. In the next step, the plates were incubated with streptavidin peroxidase for 30 minutes. The plates were then washed five times. TMB solution served as substrate; the color reaction was stopped after 20 minutes with 2 MH 2 S0 4 . The absorbance was determined at 450 nm in a plate photometer. The antibody titer was considered to be the one
  • a rabbit was withdrawn from the experiment because of low titer development. High-titre antisera could be obtained from the other rabbits. The weekly decrease was started one week after the third immunization.
  • Example 2 The binding of the antisera obtained in Example 2 to haptoglobin from pigs, cattle, horses, dogs and humans was investigated. The antisera were found to cross-react with all of the haptoglobins tested. In addition, haptoglobin could also be determined in cat serum, although the immunization cocktail used did not contain purified cat haptoglobin.
  • Pig haptoglobin could be sensitively detected in a concentration range from 30 to 1000 ng / ml serum.
  • the haptoglobin concentrations can also be determined on saliva or meat juice samples.
  • the values are at significantly lower concentrations than in serum, about 1000 times lower in saliva and about 10 times lower in meat juice.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Immunology (AREA)
  • Toxicology (AREA)
  • Zoology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Peptides Or Proteins (AREA)

Abstract

La présente invention concerne un procédé de production d'anticorps spécifiques de protéine, les anticorps spécifiques de protéine obtenus selon ce procédé, plus précisément des antisérums à réactivité croisée, polyclonaux, à spécificité pour l'haptoglobine, issus d'au moins deux espèces d'animal d'élevage et d'animal domestique, ainsi que leur utilisation pour le dosage d'haptoglobine dans des échantillons d'animaux d'élevage et d'animaux domestiques. La présente invention concerne également des cocktails immunologiques et des peptides correspondants, qui sont utiles à la production des antisérums, ainsi que des ensembles de dosage immunologique, qui se basent sur les antisérums. Aussi bien des protéines que des fragments de protéine sont utilisés pour l'immunisation.
EP00985167A 1999-12-15 2000-12-15 Anticorps, leur procede de production, leur utilisation, cocktails immunologiques, ensembles de dosage immunologique et peptides Withdrawn EP1237927A2 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DE19960500 1999-12-15
DE19960500A DE19960500A1 (de) 1999-12-15 1999-12-15 Antikörper und Verfahren zu ihrer Herstellung, deren Verwendung sowie Immunisierungscocktails, Immunoassay-Sets und Peptide
PCT/EP2000/012798 WO2001044299A2 (fr) 1999-12-15 2000-12-15 Anticorps, leur procede de production, leur utilisation, cocktails immunologiques, ensembles de dosage immunologique et peptides

Publications (1)

Publication Number Publication Date
EP1237927A2 true EP1237927A2 (fr) 2002-09-11

Family

ID=7932766

Family Applications (1)

Application Number Title Priority Date Filing Date
EP00985167A Withdrawn EP1237927A2 (fr) 1999-12-15 2000-12-15 Anticorps, leur procede de production, leur utilisation, cocktails immunologiques, ensembles de dosage immunologique et peptides

Country Status (5)

Country Link
US (1) US20030158391A1 (fr)
EP (1) EP1237927A2 (fr)
CA (1) CA2392423A1 (fr)
DE (1) DE19960500A1 (fr)
WO (1) WO2001044299A2 (fr)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB0423196D0 (en) * 2004-10-19 2004-11-24 Nat Blood Authority Method

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2890492A (en) * 1991-11-08 1993-06-07 University Of Saskatchewan Acute phase proteins for detection and prognosis of infection and tissue damage
WO1994004563A1 (fr) * 1992-08-14 1994-03-03 Shino-Test Corporation PEPTIDES CONTENANT DES SEQUENCES D'ACIDES AMINES RESPECTIVES SELECTIONNEES PARMI CELLES DE LA LIPOPROTEINE(a) ET DE L'APOLIPOPROTEINE(a), ANTICORPS RECONNAISSANT CES SEQUENCES D'ACIDES AMINES ET PROCEDE D'ANALYSE UTILISANT CES ANTICORPS
US5552295A (en) * 1994-03-02 1996-09-03 The United States Of America As Represented By The Secretary Of Agriculture Monoclonal antibodies to bovine haptoglobin and methods for detecting serum haptoglobin levels

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO0144299A2 *

Also Published As

Publication number Publication date
US20030158391A1 (en) 2003-08-21
CA2392423A1 (fr) 2001-06-21
DE19960500A1 (de) 2001-07-12
WO2001044299A3 (fr) 2002-05-16
WO2001044299A2 (fr) 2001-06-21

Similar Documents

Publication Publication Date Title
DE3587687T2 (de) Monoklonale Antikörper für HbA1c.
DE69432629T3 (de) Antikörper gegen beta-amyloid oder derivative davon und seine verwendung
DE3751997T2 (de) Aktives protein in humoraler hyperkalzemia, durch bösartigkeit verursacht
EP1110970B1 (fr) Anticorps anti-procalcitonin, procédé de production et utilisation
DE3785038T2 (de) Monoklonale antikoerper gegen nicht reduzierte, nicht enzymatisch glykolysierte proteine.
DE69531311T2 (de) Monoklonale antikörper spezifisch für endprodukte der fortgeschrittenen glycosylation in biologischen proben
EP0013930B1 (fr) Immunogène, anticorps contre l'hormone thymique, hormone thymique marquée et procédé de détermination de cette hormone thymique
EP1111050A2 (fr) Procalcitonine humaine, sa production et son utilisation
DE60027542T2 (de) Methoden zur herstellung immunologischer reagentien positionsspezifisch für phosphorylierungen
DE3751316T2 (de) Materialzusammensetzung, welche ein Polypeptid aus Pankreas-Insel-Amyloid und/oder Antikörpern gegen dieses Polypeptid enthält, und Anwendung dieser Zusammensetzung.
DE3879085T2 (de) Snrnp-a-antigen und fragmente davon.
DE19961350A1 (de) Verfahren zur Bestimmung der Parathormon-Aktivität in einer menschlichen Probe
DE3714634A1 (de) Verfahren zur selektiven immunologischen bestimmung von intaktem prokollagen peptid (typ iii) und prokollagen (typ iii) in koerperfluessigkeiten und mittel zu dessen durchfuehrung
DE69733957T2 (de) Verfahren zur bestimmung der gegenwart des gehirnspezifischen proteins s-100 beta
DE69629976T2 (de) Typ I Prokollagen aminoterminales Propeptid, dessen Antikörper und Testverfahren unter Verwendung davon
Abrass Evaluation of sequential glomerular eluates from rats with Heymann nephritis.
DE3806198A1 (de) Immunogen und seine verwendung zur gewinnung von antikoerpern gegen hba(pfeil abwaerts)1(pfeil abwaerts)(pfeil abwaerts)c(pfeil abwaerts)
DE69028784T2 (de) Testverfahren von glykosylierten proteinen unter verwendung eines antikörpers gerichtet gegen reduzierte glykosylierte n-terminal-aminosäuren
EP0547059A1 (fr) Anticorps specifique a la pancreas-elastase-1, un procede pour son obtention et un kit de test contenant de tels anticorps.
DE3883145T2 (de) Verfahren zur Messung von humanen Insulin.
EP1237927A2 (fr) Anticorps, leur procede de production, leur utilisation, cocktails immunologiques, ensembles de dosage immunologique et peptides
EP0289930B1 (fr) Anticorps monoclonal pour la détermination immunologique sélective de procollagène peptide intact (type III) et procollagène (type III) dans les fluides corporels
DE69233673T2 (de) Protein S polypeptide und deren Verwendungen
DE69110067T2 (de) Monoklonale Antikörper gegen nicht-A 1C glykosyliertes Hämoglobin.
EP0420043B2 (fr) Anticorps contre des séquences très conservées d'amino-acides de substances immunogènes, procédé de préparation de ces anticorps et leur utilisation dans des immunoessais

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20020523

AK Designated contracting states

Kind code of ref document: A2

Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE TR

RIN1 Information on inventor provided before grant (corrected)

Inventor name: HENNIES, MARK

17Q First examination report despatched

Effective date: 20040818

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20041229