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WO1993009142A1 - Proteines reagissant au moment des poussees evolutives pour la detection et le pronostic d'infections et de lesions tissulaires - Google Patents

Proteines reagissant au moment des poussees evolutives pour la detection et le pronostic d'infections et de lesions tissulaires Download PDF

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Publication number
WO1993009142A1
WO1993009142A1 PCT/CA1992/000476 CA9200476W WO9309142A1 WO 1993009142 A1 WO1993009142 A1 WO 1993009142A1 CA 9200476 W CA9200476 W CA 9200476W WO 9309142 A1 WO9309142 A1 WO 9309142A1
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acute phase
kda
protein
bovine
phase protein
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PCT/CA1992/000476
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English (en)
Inventor
Manuel Campos
Mark J. Redmond
Richard Harland
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University Of Saskatchewan
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    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • C07K14/811—Serine protease (E.C. 3.4.21) inhibitors
    • 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

Definitions

  • the present invention relates generally to the detection and prognosis of infection and tissue damage. More particularly, the invention pertains to proteins present during the acute phase response and antibodies reactive with these proteins.
  • Major production losses would be prevented if infection could be detected early in the disease state or before disease occurs. Meat inspection would also be more efficient if infection and injury could be determined before slaughter, or in some cases, confirmed after slaughter.
  • the immune system of mammals reacts to tissue injury and infection at an early stage in what is known as the acute phase response.
  • acute phase response see, Kushner, I., Ann . N. Y. Acad. Sci . (1982) , pp. 39-48.
  • One of the main characteristics associated with this response is a change in the concentration of certain plasma proteins, mostly of hepatic origin. These proteins are collectively known as acute phase proteins.
  • the serum profile of acute phase proteins varies among species and relates to the type of inflammatory stimuli.
  • Haptoglobin, ceruloplasmin, ⁇ -1 proteinase inhibitor, fibrinogen, amyloid A, and ⁇ -1 acid glycoprotein (seromucoid) have all been shown to increase during the acute phase response caused by infections in cattle. For example, infection with Pa ⁇ teurella haemolytica has been shown to raise the levels of haptoglobin, ⁇ -1 protein inhibitor, and seromucoid. Conner, J.G., et al. , Res . Vet . Sci . (1989) 42:203-207. (See Morimatsu, et al. , J. Biol . Chem .
  • the present invention provides a method for detecting the presence of infection or injury in a mammalian subject during the acute phase response.
  • Three acute phase proteins have been identified herein in bovine subjects suffering from infection or artificially induced tissue inflammation. These proteins have been found to be indicative of both the presence and the severity of tissue damage or the disease state. Accordingly, identification of the proteins in a subject provides a convenient mode for detecting and/or monitoring the progress of the inflammatory response or the disease state. Based on these discoveries, the present invention can take several embodiments.
  • the present invention is directed to isolated 23 kDa, isolated 17.5 kDa and isolated 37 kDa acute phase proteins. In particularly preferred embodiments these proteins are derived from a bovine subject.
  • the invention is directed to polyclonal and monoclonal antibodies reactive with these proteins.
  • the subject invention pertains to a method for determining the presence or absence of infection or tissue damage in a mammalian subject.
  • the method comprises assaying a biological sample from the mammalian subject for the presence or absence of at least one acute phase protein, wherein the acute phase protein is selected from the group consisting of 17.5 kDa, 23 kDa and 37 kDa acute phase protein, thereby determining the presence or absence of infection or tissue damage in the mammalian subject.
  • the invention is directed to a method for determining the presence or absence of bovine respiratory disease in a bovine subject.
  • the method comprises:
  • the invention is directed to a method for monitoring the course of infection or tissue damage in a mammalian subject.
  • the method comprises
  • kits for determining the presence or absence of infection or tissue damage in a mammalian subject comprise antibodies reactive with the 17.5 kDa, 23 kDa and/or 37 kDa acute phase proteins, packaged in a suitable container.
  • Figure 1 is a representation of an SDS-PAGE gel, showing the 17.5 kDa, 23 kDa and 37 kDa acute phase proteins of the present invention.
  • the figure depicts the gel profiles of serum from both healthy calves and calves with bovine respiratory disease (BRD) .
  • BTD bovine respiratory disease
  • Figure 2 depicts an analysis of the appearance of the 37 kDa protein in relation to the disease state after challenge in the bovine respiratory disease (BRD) model with bovine herpes virus-l (BHV-1) , as described in Example 3.
  • the numbers on the Y-axis correspond to a scale of 1-4 based on the visual intensity of the stain.
  • Figure 3 shows the results of an ELISA wherein levels of the 23 kDa protein were measured in the serum of calves challenged with BHV-l and Pasteurella haemolyt ⁇ ca, as described in the Examples.
  • the assay was done using anti-23 kDa, produced as described in Example 7.
  • protein and “polypeptide” are used in their broadest sense, i.e., any polymer of amino acids (dipeptide or greater) linked through peptide bonds.
  • polypeptide include oligopeptides, protein fragments, analogs, muteins, fusion proteins and the like.
  • An “isolated” protein or antibody is one which is devoid of other substances (either in whole or part) with which the protein or antibody would be associated in its natural environment.
  • an "acute phase protein” is defined herein as a protein which is present during the acute phase response, i.e., in the initial phases of tissue damage, including damage caused by an inflammatory response due to trauma, infection, disease, malnutrition etc. Typically, these proteins will be present in detectable quantities within 48-72 hours of the onset of the inflammatory response or other tissue damage. Such a response will not necessarily occur immediately after exposure to the source of trauma, but may be delayed. The proteins may persist as long as inflammation persists. For a review on the acute phase response, see, Kushner, I., Ann . N. Y. Acad . Sci . (1982), pp. 39-48.
  • a "17.5 kDa" acute phase protein is an acute phase protein, as defined above, with a molecular mass of approximately 17.5 kDa, determined by SDS-PAGE, as described in the examples.
  • Bovine 17.5 kDa acute phase protein has an N-terminal sequence of (X)LTAEDTGNRT(X) (X)DGK, where X specifies an undetermined amino acid.
  • a "23 kDa" acute phase protein is an acute phase protein, as defined above, with a molecular mass of approximately 23 kDa, determined by SDS-PAGE, as described in the examples.
  • Bovine 23 kDa acute phase protein has an N-terminal sequence of VETGSEATDDGR(X)KAD(X)IANSDV wherein X specifies an undetermined amino acid.
  • a "37 kDa” acute phase protein is an acute phase protein, as defined above, with a molecular mass of approximately 37 kDa, determined by SDS-PAGE, as described in the examples.
  • Bovine 37 kDa acute phase protein has an N-terminal sequence of IIGGSLDA(X)GSFP(X)QAEMD, where X specifies an undetermined amino acid.
  • Native proteins or polypeptides refer to proteins or polypeptides recovered from a source occurring in nature. Thus, a native acute phase protein would include naturally occurring acute phase proteins and fragments thereof.
  • Two polypeptide sequences are "substantially homologous" when at least about 80% (preferably at least about 90%, and most preferably at least about 95%) of the amino acids match over a defined length of the molecule.
  • mammalian subject any member of the class Mammalia, including humans and all other mammary gland possessing animals (both male and female) , such as ruminants, including, but not limited to, bovine, porcine and Ovls (sheep and goats) species.
  • a "biological sample” refers to a sample of tissue ' or fluid isolated from a mammalian subject, including but not limited to, for example, blood, plasma, serum, stool, urine, bone marrow, bile, spinal fluid, lymph fluid, the external secretions of the skin, respiratory, intestinal, and genitourinary tracts, tears, saliva, milk, blood cells, organs, and also samples of in vitro tissue culture constituents (including but not limited to conditioned medium resulting from the growth of tissues in culture medium, recombinant cells, and cell components) .
  • BTD bovine respiratory disease
  • infectious bovine rhinotracheitis infectious bovine rhinotracheitis
  • pneumonic pasteurellosis fibrinous pneumonia.
  • interstitial pneumonia enzootic pneumonia
  • pleuropneumonia acute bovine pulmonary emphysema
  • tracheal edema syndrome Haemophilus infections
  • Haemophilus infections among others.
  • Shipping fever is a general term describing an affliction common to feedlot cattle. The disease is associated with pathogenic microorganisms, particularly Pasteurella , and various stresses, such as transportation and overcrowding.
  • Pasteurella particularly P. haemolytica and P.
  • bovine herpes virus type 1 (BHV-1)
  • parainfluenza-3 virus bovine respiratory syncytial virus
  • Chlamydiae and Mycoplasma species For a general background on BRD and shipping fever see, Yates, W.D.G., Can . J. Comp. Med . (1982) 46:225-263.
  • central to the present invention is the discovery of several acute phase proteins in the serum of bovine subjects following bacterial and viral challenge, as well as following tissue trauma. More specifically, these proteins have been found to be present in response to varying stimuli, including during the acute phase response in bovine subjects presenting with symptoms of bovine respiratory disease (BRD) (both naturally acquired and artificially induced) .
  • BRD bovine respiratory disease
  • the three acute phase proteins described herein appear at elevated levels beginning at approximately 48 hours following challenge with Pasteurella haemolytica , a causative agent of BRD, significantly prior to the onset of clinical symptoms.
  • the proteins have also been observed in the serum of bovines administered turpentine oil, a tissue irritant. Accordingly, these proteins are indicative of tissue inflammation generally, whether caused by infectious agents or direct trauma.
  • the proteins are also predictive of the course and prognosis of disease. Specifically, the proteins are present in greater amounts during the later, more severe phases of infection. Accordingly, quantitation of the proteins can help determine the prognosis of the infected subject, as well as the course of treatment that should be followed.
  • the proteins described herein have molecular masses of approximately 17.5 kDa, 23 kDa and 37 kDa, respectively, as determined by SDS-PAGE chromatography ( Figure 1) . These proteins can be isolated directly from a desired subject, using standard protein purification techniques. For example, the proteins can be conveniently isolated from serum using gel electrophoresis, which provides for the purification of a sufficient quantity of protein for the production of antibodies thereto. Such antibodies are useful in detection assays, as described further below.
  • the proteins can be isolated from serum by e.g. salting out extraneous proteins in the sample and further purifying the supernatant using techniques such as column chromatography, HPLC, immunoadsorbent techniques, electrophoresis, or other conventional methods well known in the art. See, e.g. Protein Purification Methods, supra.
  • the proteins can also be isolated directly from tissue samples, for example, by first preparing a crude extract which lacks tissue components and several extraneous proteins. The desired proteins can then be further purified as described above. Purification of the above proteins as described herein also permits the sequencing of the same by any of the various methods. For example, the amino acid sequences of the subject proteins can be determined from -li ⁇
  • the bovine 17.5 kDa protein has been found to have an N-terminal sequence of (X)LTAEDTGNRT(X) (X)DGK, where X specifies an undetermined amino acid.
  • This protein sequence has partial (55%) homology with a rat serine protease inhibitor.
  • the bovine 23 kDa protein has the N-terminal sequence
  • VETGSEATDDGR(X)KAD(X)IANSDV which shows 74% homology to the ⁇ subunit of bovine haptoglobin and less than 50% homology to the ⁇ subunit of human haptoglobin.
  • the bovine 37 kDa protein has the N-terminal sequence IIGGSLDA(X)GSFP(X)QAEMD, which shows 95% homology to the ⁇ subunit of human haptoglobin.
  • these proteins can be used to produce antibodies, both polyclonal and monoclonal. If polyclonal antibodies are desired, a selected mammal, (e.g., mouse, rabbit, goat, horse, etc.) is immunized with a protein of the present invention, or an epitope bearing fragment, or an immunogenic mutein. Serum from the immunized animal is collected and treated according to known procedures. If serum containing polyclonal antibodies are desired, the polyclonal antibodies can be purified by immunoaffinity chromatography, using known procedures.
  • a selected mammal e.g., mouse, rabbit, goat, horse, etc.
  • Serum from the immunized animal is collected and treated according to known procedures.
  • serum containing polyclonal antibodies are desired, the polyclonal antibodies can be purified by immunoaffinity chromatography, using known procedures.
  • Monoclonal antibodies to the proteins of the present invention, and to the fragments thereof, can also be produced. Generally, such antibodies are made using hybridoma technology. Immortal antibody-producing cell lines can be created by cell fusion, and also by other techniques such as direct transformation of B lymphocytes with oncogenic DNA, or transfection with Epstein-Barr virus. See, e.g., M. Schreier et al., Hybridoma Techniques (1980) ; Hammerling et al. , Monoclonal Antibodies and T-cell Hvbrido as (1981); Kennett et al.. Monoclonal Antibodies (1980); see also U.S. Patent Nos.
  • Panels of monoclonal antibodies produced against the protein of interest, or at least one epitope thereof, can be screened for various properties; i.e., for isotype, epitope, affinity, etc.
  • Monoclonal antibodies are useful in purification, using immunoaffinity techniques, of the individual proteins which they are directed against, as well as in detection assays for the same.
  • the acute phase proteins of the invention provide convenient markers for identifying the presence of infection prior to the onset of observable symptoms, i.e. in feedlot animals. Thus, serious illness can be thwarted and economic losses averted.
  • the quantification of the proteins provides a method of monitoring the disease state and aids in determining the level of treatment necessary.
  • proteins can be detected post-mortem to identify contaminated meat.
  • the proteins can be identified for these purposes using any of several standard identification techniques. For example, the presence of acute phase proteins in a biological sample can be determined using standard electrophoretic techniques, described herein. Immunodiagnostic techniques also provide convenient methods for the detection and quantitation of the proteins. Antibodies (both polyclonal and monoclonal) , raised to one or more of the acute phase proteins, can be used in immunoassays, such as competition, direct reaction, or sandwich type assays, for identifying the presence or absence of the proteins by forming complexes therewith. Such assays include, but are not limited to.
  • the reactions generally include revealing labels such as fluorescent, chemiluminescent, radioactive, or enzymatic labels or dye molecules.
  • an immunoassay for detecting one or more of the acute phase proteins will involve selecting and preparing the test sample, such as serum, and then reacting it with antibodies directed against one or more of the acute phase proteins, under conditions that allow acute phase protein-antibody conjugates to form.
  • Solid supports can be used such as nitrocellulose, in membrane or microtiter well form; polyvinylchloride, in sheets or microtiter wells; polystyrene latex, in beads or microtiter plates; polyvinylidine fluoride; diazotized paper; nylon membranes; activated beads and the like.
  • the solid support is first reacted with the biological sample, washed and then antibodies (either polyclonal or monoclonal) are applied.
  • a sandwich type format such as a sandwich ELISA assay
  • a commercially available anti-immunoglobulin i.e. anti- rabbit i munoglobulin conjugated to a detectable label, such as horseradish peroxidase, alkaline phosphatase or urease, can be added.
  • a detectable label such as horseradish peroxidase, alkaline phosphatase or urease
  • a "two antibody sandwich” assay can be used to detect the proteins of the present invention.
  • the solid support is reacted first with anitbodies (either monoclonal or polyclonal) directed against one or more of the acute phase proteins, washed and then exposed to the test sample.
  • Monoclonal or polyclonal antibodies are again added and the reaction visualized using either a direct color reaction or using a labeled second antibody, such as an anti-immunoglobulin labeled with horseradish peroxidase, alkaline phosphatase or urease.
  • Assays can also be conducted in. solution, such that the acute phase protein and antibody thereto form complexes under precipitating conditions.
  • the precipitated complexes can then be separated from the test sample, for example, by centrifugation.
  • Necessary reagents to conduct the assays described above can be provided in kits, with suitable instructions.
  • antibodies to the acute phase proteins can be provided in a diagnostic immunoassay test kit.
  • the kit can also contain, depending on the particular immunoassay used, suitable labels and other packaged reagents and materials (i.e. wash buffers and the like) .
  • Example 1 Identification of Acute Phase Proteins in Calves Suffering from BRD Serum samples were collected from feedlot calves presenting with typical signs of the early stages of BRD, as well as from apparently healthy calves. All serum samples were diluted 1:10 in reducing IX Laemmli buffer comprising jS-mercaptoethanol/bromphenol blue, and boiled for 5 min before polyacrylamide gel electrophoresis. The system used was as follows. Resolving gel, stacking gel and the buffers used for the mini gel system (BioRad) were made up based on the method described by Laemmli, Nature (1970) 227:680.
  • Resolving gels consisting of 7.5, 10, 12.5 and 15% acrylamide and stacking gels consisting of 3% acrylamide were used in preliminary experiments. Subsequent to these experiments, it was concluded that a 15% acrylamide resolving and a 3% acrylamide stacking gel provided the best resolution for the three acute phase proteins and hence was adopted for all protein separations unless otherwise indicated. A 0.75 mm thick resolving gel was cast at 15% acrylamide and a 3% acrylamide stack was applied using a 10-well comb.
  • the gel was destained in 20:10:70 MeOH:acetic acid:water destain.
  • at least three acute phase proteins having molecular masses of approximately 37 kDa, 23 kDa and 17.5 kDa, respectively, were identified in sera obtained from field cases of BRD. These proteins were either absent or present in very low concentration in sera from normal animals.
  • Polyclonal antibodies specific for acute phase proteins were produced by immunizing rabbits with protein bands obtained by SDS-PAGE (Example 1) of serum from sick animals. Electrophoresis was performed on serum sa ples loaded into all of the wells of the gel, as described in Example 1. A strip containing a representative lane of the gel was excised and stained for 15 min with Coomassie blue. The gel was then destained and allowed to swell back to the original size in double-distilled water. The stained gel was aligned with the unstained portion of the gel and used to indicate the location of the acute phase proteins. The areas of the gel containing the acute phase proteins were excised and washed in double-distilled water for 2 min.
  • the gel pieces were diced and emulsified with adjuvant and used as immunizing antigens for the production of rabbit antibodies.
  • the specificity of the antibodies was examined by western blots, using standard techniques. Antibodies were produced which were able to identify the 37 kDa, 23 kDa proteins and 17.5 kDa proteins.
  • Acute Phase Proteins Electrophoretic procedures for the preparation of samples to be sequenced were performed as described by Moos, et al., J . Biol . Chem . (1986) 363:6005-6008.
  • a serum sample containing the acute phase proteins was run using standard procedures. After electrophoresis, the area of the gel containing proteins of molecular weights of approximately 10,000-58,000 daltons, was cut out and washed in glycine-Tris-CH 3 OH blot buffer for 5 min. The washed gel was blotted for 1 hr at a constant voltage of 100 V onto Immobilon-PVDF transfer paper (Millipore, PVDF, 0.45 urn) .
  • the blot was stained in Coomassie blue for 4 min and destained just adequately to observe the relevant protein bands.
  • the identified protein bands were cut from the blots, sealed in vials, and submitted for amino acid sequence analysis. Edman degradation analysis is described in Edman, P. et al., Eur . J. Biochem . (1967) 1:80. Recent improvements in this technology are also known to those skilled in the art (Hunkapiller, et al., In: Methods of Protein Microcharacterization (1986) J.E. Shiveley, ed., Humana Press, NJ, p.223).
  • the 23 kDa protein had an N-terminal sequence as follows:
  • VETGSEATDDGR(X)KAD(X)IANSDV This sequence is 74% homologous to the ⁇ subunit of bovine haptoglobin.
  • the 17.5 kDa protein had the following N- terminal sequence:
  • the 17.5 kDa protein has partial (55%) homology with a rat serine protease inhibitor.
  • Example 6 Purification of the 23 kDa Acute Phase Protein
  • the 23 kDa acute phase protein was purified from 380 ml of serum (acute phase serum) obtained from a calf undergoing acute disease caused by Haemophilu ⁇ somnu ⁇ .
  • Haemoglobin free serum was precipitated with a saturated solution of ammonium sulphate (40% vol./vol.). After centrifugation (10,000 rpm for 30 min at 4°C) the supernatant was collected and dialysed against several changes of double distilled water. This sample was lyophilized and resuspended in 200 ml of double distilled water.
  • the reconstituted sample was supplemented with 75 ml of 3.5 M guanidine, pH 5.0 and incubated for 1 hr at room temperature with continuous rocking.
  • the guanidine was removed from the sample through dialysis against double distilled water and by passage through an Amicon filter (PM of 30,000) to about one fifth of the original volume (75 ml) .
  • the solution was further clarified by centrifugation (3,500 rpm for 20 min).
  • the resulting supernatant consisted of a complex of the 37 and the 23 kDa proteins.
  • the 23 kDa protein appears to be partially homologous to the ⁇ subunit of the haemoglobin-binding protein haptoglobin. Therefore, in order to enrich for the 23 kDa acute phase protein, an affinity purification procedure was performed as follows. The 75 ml sample resulting from the ammonium sulphate precipitation and guanidine treatment was loaded onto an agarose- haemoglobin column (Sigma) previously equilibrated with double distilled water. After further equilibration with double distilled water, the bound material was eluted using 3.5 M guanidine pH 5.0. The guanidine was then removed as described above.
  • the 23 kDa acute phase protein was further purified by Reversed Phase HPLC on a C4 column.
  • 20 ml of the affinity purified material were mixed with 7 ml of Tris buffer pH 6.8 and 1.06 ml of 2- mercaptoethanol (2ME) .
  • This material (in two ml batches) was passed through the C4 Reversed Phase HPLC column HPLC equilibrated with 0.05 KH 2 P0 4 / 0.07 % 2ME buffer.
  • the sample was eluted with an elution gradient of 9:1 acetonitrile:water with 0.07% 2ME. Using an elution gradient of 5% to 95% over 55 min, the 23 kDa protein was recovered in fractions 22, 23, and 24 as demonstrated by SDS-PAGE and western blots of the eluted fractions.
  • the material recovered after affinity purification was subjected to a size exclusion step using a Sephacryl S200 HR column. In this system, the 23 kDa acute phase protein eluted in the early protein peaks (fractions 21- 29) .
  • Monoclonal antibodies (MAbs) against the 23 kDa acute phase protein were produced following standard procedures. The selection of hybridomas producing antibodies specific for the 23 kDa acute phase protein was done by screening supernatants using an ELISA method. Six out of 175 supernatants specifically reacted with the 23 kDa protein. These six hybridomas were subcloned by limiting dilution. After cloning, all hybridoma supernatants were tested for the presence of specific antibodies. The cloned hybridoma cell line designated 1D1 was selected as high antibody producer and was used to produce ascites fluid. This hybridoma cell line was cloned once again using limiting dilution and to ensure that the line was monoclonal.
  • a sandwich ELISA method was developed using the antibodies described above. This method allows for the measurement of serum levels of 23 kDa.
  • the sandwich ELISA employed the MAB 1D1 to capture the 23 kDa protein present in serum samples. The captured 23 kDa protein was then detected with the rabbit anti-23 kDa protein described in example 7 or with a rabbit anti-whole haptoglobin.
  • This reaction was measured colorimetrically by adding a commercially available anti-rabbit immunoglobulin conjugated to horse radish peroxidase and the appropriate substrate to develop a color reaction.
  • the sensitivity of this method allowed the differentiation between sick and healthy animals (Fig. 3 i.e. before and after infection) .
  • Example 9 Use of Sandwich ELISA for the Detection of Sick Animals at Different Stages of Disease
  • the sandwich ELISA method described in example 8 was used to measure the levels of 23 kDa present in the serum of five calves (numbered 1, 2, 3, 7 and 8, Figure 3) that were challenged with BHV-1 and P. haemolytica as described in example 3.
  • this assay it was demonstrated that the serum levels of the 23 kDa protein increased 3 days after BHV-1 challenge. Serum levels of 23 kDa protein remained elevated until the termination of the experiment (Fig. 3) .

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Abstract

L'invention se rapporte à des protéines réagissant au moment des poussées évolutives et à des anticorps de ces protéines, ainsi qu'à des procédés pour déterminer la présence et la gravité d'infections et de lésions tissulaires, en testant ces protéines. Des protéines de 17,5 kDa, de 23 kDa et 37 kDa ont été identifiées dans des sujets bovins souffrant à la fois d'infections et de lésions tissulaires induites par voies chimiques. Ces protéines fournissent une indication de la gravité et du pronostic de la maladie. Des anticorps de ces protéines sont utiles dans des dosages diagnostiques.
PCT/CA1992/000476 1991-11-08 1992-11-06 Proteines reagissant au moment des poussees evolutives pour la detection et le pronostic d'infections et de lesions tissulaires WO1993009142A1 (fr)

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US79039491A 1991-11-08 1991-11-08
US07/790,394 1991-11-08
US79867091A 1991-11-26 1991-11-26
US07/798,670 1991-11-26
US97155992A 1992-11-05 1992-11-05
US07/971,559 1992-11-05

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Cited By (3)

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ES2128948A1 (es) * 1996-07-26 1999-05-16 Univ Zaragoza Metodo de diagnostico de las enfermedades del cerdo y en la evaluacionde la calidad de su carne.
WO2001044299A3 (fr) * 1999-12-15 2002-05-16 Giesing Michael Anticorps, leur procede de production, leur utilisation, cocktails immunologiques, ensembles de dosage immunologique et peptides
ES2200611A1 (es) * 2000-11-14 2004-03-01 Univ Zaragoza Metodo de diagnostico de procesos patologicos en mamiferos, asi como de la calidad de la carne, leche y productos derivados en mamiferos no humanos.

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993009437A2 (fr) * 1991-10-31 1993-05-13 Matritech, Inc. Analyse de proteines matricielles nucleaires solubles dans les fluides corporels

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ELECTRONIC DATABASE MEDLINE, U.S. NATIONAL LIBRARY OF MEDICINE (NLM) BETHESDA, US; AN 91012656 P.E. BANKEY ET AL. 'HEPATIC ACUTE PHASE PROTEIN SYNTHESIS IS INDIRECTLY REGULATED BY TUMOR NECROSIS FACTOR' *
GASTROENTEROLOGY vol. 100, no. 3, March 1991, NEW YORK, US pages 775 - 782 V. KEIM ET AL. 'Characterization of a Rat Pancreatic Secretory Protein Associated With Pancreatitis' *
JOURNAL OF BIOLOGICAL CHEMISTRY. (MICROFILMS) vol. 266, no. 18, 25 June 1991, BALTIMORE, MD US pages 11833 - 11837 M. MORIMATSU ET AL. 'Isolation and Characterization of Bovine Haptoglobulin from Acute Phase Sera' cited in the application *
RESEARCH IN VETERINARY SCIENCE vol. 47, 1989, OXFORD, UK pages 203 - 207 J.G. CONNER ET AL. 'Acute phase response in calves following infection with Pasteurella haemolytica, Ostertagia ostertagi and endotoxin administration' cited in the application *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ES2128948A1 (es) * 1996-07-26 1999-05-16 Univ Zaragoza Metodo de diagnostico de las enfermedades del cerdo y en la evaluacionde la calidad de su carne.
WO2001044299A3 (fr) * 1999-12-15 2002-05-16 Giesing Michael Anticorps, leur procede de production, leur utilisation, cocktails immunologiques, ensembles de dosage immunologique et peptides
ES2200611A1 (es) * 2000-11-14 2004-03-01 Univ Zaragoza Metodo de diagnostico de procesos patologicos en mamiferos, asi como de la calidad de la carne, leche y productos derivados en mamiferos no humanos.
ES2200611B1 (es) * 2000-11-14 2005-05-01 Universidad De Zaragoza Metodo de diagnostico de procesos patologicos en mamiferos, asi como de la calidad de la carne, leche y productos derivados en mamiferos no humanos.

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