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WO1991018290A1 - Immuno-analyse de peptides d'elastine dans l'urine - Google Patents

Immuno-analyse de peptides d'elastine dans l'urine Download PDF

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Publication number
WO1991018290A1
WO1991018290A1 PCT/US1991/003252 US9103252W WO9118290A1 WO 1991018290 A1 WO1991018290 A1 WO 1991018290A1 US 9103252 W US9103252 W US 9103252W WO 9118290 A1 WO9118290 A1 WO 9118290A1
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WO
WIPO (PCT)
Prior art keywords
elastin
derived peptides
urine
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.)
Ceased
Application number
PCT/US1991/003252
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English (en)
Inventor
Umberto Kucich
Joel Rosenbloom
George Weinbaum
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.)
GRADUATE HOSPITAL FOUNDATION RESEARCH Corp
Original Assignee
GRADUATE HOSPITAL FOUNDATION RESEARCH Corp
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Filing date
Publication date
Application filed by GRADUATE HOSPITAL FOUNDATION RESEARCH Corp filed Critical GRADUATE HOSPITAL FOUNDATION RESEARCH Corp
Publication of WO1991018290A1 publication Critical patent/WO1991018290A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6884—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids from lung

Definitions

  • This invention relates generally to a method for th immunological identification of elastin-derived peptides (EDP) i urine to assess the existence of lung tissue damage and/or chronic obstructive pulmonary disease (COPD) .
  • EDP elastin-derived peptides
  • Chronic obstructive pulmonary disease usually develops over many years, and it is not until lung structure and function have been significantly compromised that COPD can be diagnosed with certainty by radiological and pulmonary function tests.
  • the connective tissue protein, elastin is largely responsible for maintaining the elasticity of major blood vessels and lung tissue.
  • the major emphasis in research has been the study of the destruction of mature elastin fibers by administration of selective proteases by aerosol or intratracheal instillation.
  • a sample of urine containing an unknown amount of elastin-derived peptides is obtained and analyzed in accordance with the above steps.
  • the results of such analysis are corrected for creatinine excretion and compared to the standardization curve. From these results one can determine whether the amount of elastin-derived peptides in the unknown urine sample is sufficiently in excess of a standard value in the urine of a group of control individuals not having lung damage or chronic obstructive pulmonary disease, to establish the existence of lung disease or chronic obstructive pulmonary disease.
  • the kit of the above invention for carrying out a method for immunologic detection of elastin-derived peptides in urine comprises a synthetic peptide or natural peptide, and is employable with elastin-derived peptides prepared from the amorphous component of human lung elastin.
  • the kit includes a method to use the synthetic peptide in an indirect ELISA to quantify the elastin-derived peptides in a urine sample, and to compare the results obtained with an established standard.
  • Microtiter plates may be obtained from Scientific Accessories of
  • a synthetic amino acid sequence such as GFPGGACLGKACG-
  • RKRK may be coupled to keyhole limpet hemocyanin (KLH) using glutaraldehyde. This complex may then be used to produce antibodies for use in the method of the present invention as described below. Baron, M.H., and Baltimore, D. , Antibodies
  • the amorphous component of human lung elastin may be digested with, human neutrophil elastase at a 1:500 ratio of enzyme to elastin (w/w) for 24 hours at 37°C.
  • the antibodies to elastin-derived peptides isolated from urine may be prepared by the following procedure, in accordance with the preferred method of the present invention. Preparation and use of antibodies against the antigens isolated from urine should result in increased sensitivity of the present invention, as these anti- bodies are believed to be more specific to the EDP antigen in an junknown sample of urine.
  • BSA bovine serum albumin
  • SBTI soy trypsin inhibitor
  • CYTO C cytochrome C
  • the desired fractions can then be collected and the sample may then be placed on a Superose 12 liquid chromatography column (FPLC) which separates materials according to molecular weight. It is expected that desired column effluent fractions will contain peptides having a molecular weight in the range of 13,000 daltons. Peptides having a molecular weight in this range exhibit the desired activity when subjected to peptide detection analysis. These purified EDP from urine can then be used to generate antibodies in accordance with the method of Section IV below. IV. Generating Antibodies to the Synthetic or Elastin-Derived Peptides
  • Standard curves for the indirect ELISA may be generated by incubating 5 ⁇ g/ml primary antibody (for the elastin-derived peptides) or 7 ⁇ g/ml primary antibody (for the synthetic peptide) with variable concentrations of competing antigen for 16 hours at 4°C. These reaction mixtures (200 ⁇ l per well) may be transferred to the coated wells and incubated at 4°C for 1 hour.
  • the wells may be washed with phosphate buffered saline (PBS-Tween 20) , and afterwards 200 ⁇ l per well of goat antirabbit serum at a dilution of 1:2000 may be added to the wells and incubated for 1 hour at room temperature. After washing, 200 ⁇ l per well of the peroxidase-antiperoxidase complex may be added to the wells at a dilution of 1:2000 in PBS- Tween 20, and incubated for 30 minutes at room temperature.
  • PBS-Tween 20 phosphate buffered saline
  • a 50-100 ⁇ l sample of human urine containing an unknown amount of elastin-derived peptides is obtained after centrifuga- tion of the collected urine sample in a Sorvall refrigerated centrifuge, DuPont- Company, RC-5B sized rotor, at 12,000 rpm for 10 minutes to remove solids. The supernatant may then be removed and frozen at -20°C, as the elastin-derived peptides may be unstable at 4°C. The urine is then concentrated 10-fold by ultrafiltration in an Amicon cell Model 52, and it is believed that there will be a 60-70% recovering of elastin-derived peptides.
  • Multiple samples from the same patient revealed that urinary EDP levels varied less than 20-25%.
  • COPD patients appeared to excrete lower molecular weight peptides into the urine, since dialysis through a 3500 M.W. cut-off membrane resulted in the loss of 80% of the immunoreactivity, while the urine of a non-smoker lost less than 35% immunoreactivity.
  • EDP antigen may be detected in any urine using an elastin carboxy-terminus antibody, which detects EDP in plasmas of non- smokers, smokers and COPD individuals. Am.Rev.Resp.Dis. 137:A372 (1988) . These data suggest considerable antigen processing o EDP prior to urinary excretion, particularly in COPD patients. The easy sampling and the presence of low molecular weight ED make the urine ideal to use for isolation and characterization o such elastin antigen(s) . Identifying the in vivo - generated elastin antigen(s) may provide tools for developing more sensi ⁇ tive probes of lung destruction and for monitoring therapy.

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

Abstract

Test non intrusif quantitatif d'urine présentant une sensibilité améliorée, capable d'identifier la présence d'une altération pulmonaire chez des patients asymptomatiques. L'invention est utile dans le contrôle de la progression de maladies pulmonaires. Le procédé comprend les étapes consistant à préparer un peptide synthétique ou à produire des peptides dérivés d'élastine et à utiliser soit le peptide synthétique soit les peptides dérivés d'élastine afin de produire des anticorps. Ensuite, on incube les anticorps à l'aide de quantités croissantes du peptide synthétique ou de peptides derivés d'élastine dans un ELISA indirect. On applique un mélange d'anticorps et de peptides à des plaques de microtitrage enrobées d'un peptide dérivé d'élastine ou synthétique de manière que les anticorps et les peptides se combinent afin de former un premier complexe. On lave les plaques de microtitrage contenant le premier complexe afin d'éliminer les anticorps non liés excédentaires. Ensuite, on utilise une quantité connue d'un conjugué d'enzyme-anticorps et on prépare une courbe de normalisation. On analyse un échantillon inconnu d'urine à l'aide de peptides dérivés d'élastine selon les étapes précitées. Ensuite, on corrige les données en fonction de l'excrétion de créatinine et on les compare à la courbe de normalisation afin de déterminer si la quantité de peptides dérivés d'élastine se trouvant dans l'échantillon d'urine inconnu permet d'établir l'existence d'une maladie pulmonaire.
PCT/US1991/003252 1990-05-11 1991-05-10 Immuno-analyse de peptides d'elastine dans l'urine Ceased WO1991018290A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US52244790A 1990-05-11 1990-05-11
US522,447 1990-05-11

Publications (1)

Publication Number Publication Date
WO1991018290A1 true WO1991018290A1 (fr) 1991-11-28

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Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1991/003252 Ceased WO1991018290A1 (fr) 1990-05-11 1991-05-10 Immuno-analyse de peptides d'elastine dans l'urine

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AU (1) AU8088191A (fr)
WO (1) WO1991018290A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005029090A1 (fr) * 2003-09-25 2005-03-31 Astrazeneca Ab Empreintes peptidiques de l'elastine et procedes d'analyse pour les mmp12 associees aux broncho-pneumopathies chroniques obstructives (bpco)

Non-Patent Citations (8)

* Cited by examiner, † Cited by third party
Title
AMERICAN REVIEW RESPIRATORY DISEASE, Volume 131, issued 1985, U. KUCICH et al., "Utilization of a Peroxidase Antiperoxidase Complex in an Enzyme-Linked Immuno-Srobent Assay of Elastin-Derived Peptides in Human Plasma", pages 709-713. *
BIOCHEMISTRY, Volume 25, 1986, D.S. WRENN et al., "Characterization of Biologically Active Domains on Elastin: Identification of a Monoclonal Antibody to a Cell Recognition Site", pages 5172-5176. *
CHEMICAL ABSTRACTS, Volume 106, No. 19, issued 11 May 1987, J. ROSENBLOOM et al., "Newly Determined Carboxy Terminal Sequences in Tropoelastin: Immunologic Identification Insoluble Elastin", see abstract 151824; & COLLAGEN RELATES RESEARCH, 6(5), 423-33. *
CHEMICAL ABSTRACTS, Volume 107, No. 21, issued 23 November 1987, J. ROSENBLOOM et al., "Immunologic Identification of Carboxy Terminal Sequences of Elastin in Human Plasma Using Monospecific Antibodies", see abstract 194478; & PULM. EMPHYSEMA PROTEOLYSIS, 1986, (Conf.) 1986, 245-54. *
CHEST, Volume 96, No. 2, Supplement, issued August 1989, E.E. SCHRIVER et al., "Elastin Fragment Levels in Human Plasma, Urine, and Bronchoalveolar Lavage Fluid (BALF)", page 153S. *
JOURNAL OF CLINICAL INVESTIGATION, Volume 61, 1978, R.A. GOLDSTEIN et al., "Uriniary Ecretion of Elastin Peptides Containing Desmosine After Intratracheal Injection of Elastase in Hamsters", pages 1286-1290. *
JOURNAL OF IMMUNOLOGICAL METHODS, Vol. 107, No. 1, issued 1988, P. LAURENT et al., "Quantitation of Elastin in Human Urine and Rat Pleural Meothelial Cell Matrix by a Sensitive Avidinbiotin ELISA for Desmosine", pages 1-11. *
U. KUCICH et al., "Urine from Emphysema Patients Contains Elevated Levels of Elastin-Derived Peptides", 1990 World Conference on Lung Health, see abstract, AM. REV. RESPIR. DIS. 141, (4 Part 2), A232. *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005029090A1 (fr) * 2003-09-25 2005-03-31 Astrazeneca Ab Empreintes peptidiques de l'elastine et procedes d'analyse pour les mmp12 associees aux broncho-pneumopathies chroniques obstructives (bpco)
US8012692B2 (en) 2003-09-25 2011-09-06 Astrazeneca Ab Elastin peptide fingerprints and analysis methods for MMP12 related to COPD

Also Published As

Publication number Publication date
AU8088191A (en) 1991-12-10

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