EP0444174A1 - Test de detection d'anticorps anti-recepteurs de la fibronectine staphylococcique - Google Patents

Test de detection d'anticorps anti-recepteurs de la fibronectine staphylococcique

Info

Publication number
EP0444174A1
EP0444174A1 EP90913666A EP90913666A EP0444174A1 EP 0444174 A1 EP0444174 A1 EP 0444174A1 EP 90913666 A EP90913666 A EP 90913666A EP 90913666 A EP90913666 A EP 90913666A EP 0444174 A1 EP0444174 A1 EP 0444174A1
Authority
EP
European Patent Office
Prior art keywords
fibronectin
receptor
aureus
antibodies
test
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Withdrawn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
EP90913666A
Other languages
German (de)
English (en)
Other versions
EP0444174A4 (en
Inventor
Richard A. Proctor
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.)
Wisconsin Alumni Research Foundation
Original Assignee
Wisconsin Alumni Research Foundation
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 Wisconsin Alumni Research Foundation filed Critical Wisconsin Alumni Research Foundation
Publication of EP0444174A1 publication Critical patent/EP0444174A1/fr
Publication of EP0444174A4 publication Critical patent/EP0444174A4/en
Withdrawn legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/566—Immunoassay; Biospecific binding assay; Materials therefor using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G01N33/56938—Staphylococcus

Definitions

  • the invention relates to diagnostic tests or assays for the detection of staphylococcal fibronectin-receptor antibodies in body fluids, such as sera.
  • Staphylococcus aureus an infectious microorganism
  • adherence is the first step in the develop ⁇ ment of an infection. If bacteria are unable to adhere to a surface, they will be swept away by the body fluids that normally bathe the tissues and an infection will not occur. Hence, adherence is the crucial first step in initiating and spreading infections. It is generally accepted that microorganisms adhere to surface components of the host tissues.
  • Fibronectin is a large glyco- protein which is a major component of the material found on the surfaces of cells, in clots and also in the inter ⁇ cellular matrix. Fibronectin is essential to the well-being of the host as it serves as the "glue” which links one cell to another cell and plays a major role in wound healing. There are several lines of evidence which suggest that fibronectin plays an important role in the adherence of S. aureus to host tissues. Supporting this concept are the following observations: (i) S. aureus has a specific receptor for fibronectin, (ii) the S.
  • aureus fibronectin- receptor is expressed on the surface of the bacterium where it can interact with host tissues
  • the fibro ⁇ nectin-receptor is expressed in greater numbers on clinical isolates of S>. aureus that have invaded the host as compared to noninvasive isolates
  • purified FN-R decreases Si. aureus interactions with fibronectin
  • antibodies directed against FN-R reduce fibronectin binding to S. aureus
  • fibronectin is found at sites frequently infected by S. aureus
  • fibronectin enhances S. aureus adherence
  • specific removal of fibronectin from a complex mixture of host proteins decreases S. aureus binding while removal of other proteins does not
  • anti ⁇ bodies against fibronectin inhibit S . aureus binding to host tissues.
  • fibronectin- receptor polysaccharide plays a major role in the initiation of S. aureus infections: (i) S. aureus mutants that are isogenic with the parent strain except for the ability to express fibronectin-receptor colonize the heart valves of rats 120-fold less effectively than the parent strain and (ii) a monoclonal antibody which recognizes the most invasive strains of S. aureus interacts preferentially with the fibronectin-receptor.
  • exopolysaccharide which is expressed on S . aureus and which specifically binds fibronectin. It can be highly purified and it functions as a fibronectin-receptor.
  • an antigen based diagnostic test or assay for the detection of staphlococcal fibronectin- receptor antibodies can be prepared using as the antigen a purified fibronectin-receptor exopolysaccharide which is expressed on Staphylococcus aureus and which specifically binds to fibronectin.
  • the exopolysaccharide contains no lipids, less than 2% protein and the sugars making up the exopolysaccharide are aminohexoses.
  • the purified exopolysaccharide cross-reacts with monoclonal antibody directed against the type 8 capsular polysaccharide of S. aureus, but the fibronectin- receptor polysaccharide of the present invention contains, galacturonic acids and thus is distinct from type 8 capsular material.
  • the fibronectin receptor exopolysaccharide of the present invention has an average molecular weight of -1000 kdal and it competes with intact organisms for fibronectin binding.
  • the fibronectin-receptor exopolysaccharide is harvested from intact Staphylococcus aureus cells by gently releasing the surface material, including the expressed fibronectin- receptor polysaccharide, on the cells without rupturing or killing the cells by non-destructive procedures, such as gentle sonication.
  • the exopolysaccharide is then purified by DEAE ion exchange and fibronectin affinity chroma- tography.
  • the preferred S . aureus is designated strain 6850.
  • the exopolysaccharide which serves as a ligand, is bound to a solid phase immunosorbent support.
  • the test sera is reacted with the bound exopolysaccharide.
  • fibronectin is added, and the mixture incubated and washed.
  • an alkaline phosphatase-conjugated, anti-fibronectin-antibody is added.
  • the test concludes with a detection step, compatible with the label used, designed to measure the amount of antibodies bound to the exopolysaccharide in the test sera.
  • Monoclonal antibodies against the fibronectin- receptor exopolysaccharide are used as standards in the ELISA test employing the fibronectin-receptor polysaccharide as an antigen.
  • the monoclonal antibodies employed can be the previously described monoclonal antibodies directed against type 8 capsular polysaccharide or antibodies obtained by injecting the fibronectin-receptor exopolysaccharide into mice, removing the spleens of the mice showing antibody that blocked fibronectin binding to FN-R, cloning the antibodies, preparing hybridomas by the method of Kohler and Milstein and selecting the monoclonal antibodies that demonstrate blocking activity and inhibit fibronectin binding to FN-R.
  • Other objects and advantages of the invention will be apparent to those skilled in the art.
  • the diagnostic test is an ELISA test in which fibronectin- receptor exopolysaccharide (FN-R) is absorbed on the polystyrene walls of microtiter plates, washed and dried.
  • FN-R fibronectin- receptor exopolysaccharide
  • the plates are incubated at room temperature.
  • the test sera is added and the mixture is incubated for about 60 minutes.
  • polyclonal rabbit anti-human fibronectin antibody conjugated to alkaline phosphatase (1:2000 dilution) is added and incubated for 2 hours and washed.
  • An enzyme substrate is added and the mixture is incubated for 2 hours. It is then read with an automated fluoresence spectrophotometer to determine the amount of Staphylococcal fibronectin-receptor antibodies present in the test sera.
  • S. aureus (strain 6850) is grown in chemically defined or dialyzed media for 16 hours. Bacteria are harvested by centrifugation, washed twice in Hank's balanced salt solution, and then once in sterile distilled water. The organisms are then resuspended in distilled water and gently sonicated (two, 2.5 minute bursts in a cup horn Heat Systems sonicator at 18-20% output). The sonication removes cell surface material without killing the S . aureus, as determined by no change in the viability counts and by lack of DNA release. The sonicated mixture is then centrifuged
  • FN-R fibronectin receptor polysaccharide
  • the FN-R has an average molecular weight of -1000 kdal. This polysaccharide cross-reacts with monoclonal antibody directed at the type 8 capsular polysaccharide of
  • bacteria can adsorb complex media components onto their surface.
  • the media used in the previous reports are made from crude digests of
  • fibronectin such as collagen and fibrin
  • the nonchemically defined edia contain many macromolecules which could stick to the surface of the £. aureus and be purified as if they were produced by the bacteria.
  • broken open S . aureus many intracellular proteins are being released, some of which may interact with fibronectin, but they may not be expressed on the surface of the microorganism. It has been observed that broken open S . aureus contain many more fibronectin receptors than do intact bacteria. The fact that antibodies produced against the protein fibronectin receptor do not block fibronectin binding to S .
  • aureus suggests that these protein materials are not the critical ligands for promoting fibronectin-S. aureus interactions. This is in contrast to antibodies which react with the carbohydrate FN-R of the present invention, wherein the binding of fibronectin is almost completely blocked. Hence, breaking open the bacteria may release fibronectin receptor materials which are not expressed normally. These "released" materials are different from the FN-R expressed on the outside of the bacteria. Consequently, the novel technique of harvesting the surface material without breaking open the bacteria prevents the harvesting of potentially nonexpressed, nonsurface fibronectin receptor material. Thus, the harvesting techniques are unique and have led to the isolation of a carbohydrate FN-R polysaccharide which is important in the pathogenesis of S_. aureus infections.
  • the FN-R polysaccharide obtained by the above method was used as an antigen to prepare monoclonal antibodies.
  • One hundred micrograms of the antigen was mixed with complete Freund's adjuvant and injected subcutaneously into multiple sites on BALB/c mice.
  • the mice were then injected with 100 yg of antigen intraperitoneally after 2 and 4 weeks. At 6 weeks, the mice were injected with 100 ⁇ g intravenously. Two and three days later, the mice were given 100 ⁇ _g of antigen intraperitoneally.
  • mice were bled and those showing blocking antibody (antibody which blocked fibronec ⁇ tin binding to the FN-R in the ELISA test) had their spleens removed.
  • blocking antibody antibody which blocked fibronec ⁇ tin binding to the FN-R in the ELISA test
  • NS-1 cells cancerous tumor cells derived from myeloma tumors of bone marrow after the method of Kohler and Milstein.
  • the hybridomas were screened to select ones producing the desired antibody and grown in culture to produce monoclonal antibodies. Seven monoclonal antibodies were produced which demonstrated blocking activity. Two monoclonal antibodies gave 70-92% inhibition of fibronectin binding to the FN-R in an ELISA assay when these antibodies were diluted 1:100.
  • the FN-R exopolysaccharide employed was harvested from the surface of a strain expressing large numbers of FN-R (S_. aureus strain 6850) (ATCC No. 53657). This strain was discovered by screening several hundred strains of S . aureus.
  • the FN-R polysaccharide was purified as described above. The lyophilized polysaccharide was resuspended in distilled water to a concentration of 1 mg/ml. It was then tested for inhibitory activity in a standard binding assay and adjusted to a concentration that gave 50% inhibition of 3 ug 125 I-labeled fibronectin binding to 5 x 10° S.
  • aureus ATCC 25923 (this is generally about 1 yg of polysaccha- ride.) Then 10 ⁇ g of this FN-R polysaccharide which was to serve as the antigen was placed in each well of a 96 well microtiter polystyrene plate and allowed to dry overnight. Antibody, either from the patient's serum or added as a control, was then added to the well. Sera were pretreated with gelatin-Sepharose to remove plasma fibronectin. One microgram of fibronectin was then added to each well. After a 60 minute incubation at room temperature, the wells are washed and an alkaline phosphatase conjugated to rabbit anti-fibronectin monoclonal antibody is added. After incubation, the wells are washed again and the appropriate substrate is added.
  • the test is read fluoroscopically. If the patient's serum has no antibody, then the maximal value will be recorded. However, if there is specific antibody present, then this will interfere with fibronectin binding to the FN-R polysaccharide and will give a lower optical fluor ⁇ escent reading.
  • a direct inverse relationship between antibody concentration and fluorescence obtains which can be used to determine antibody titer. It has been found that antibodies to the FN-R displace the fibronectin binding to the well and that fibronectin concentrations of 1 yg are sufficient to saturate all the receptors in the well.
  • ELISA Enzyme-linked Immunoadsorbant Assay
  • buffer A 50 mM Tris, 150 mM NaCl, 20 mM MgCl 2 , 20 yM ZnCl 2 / 3% bovine serum albumin, and 10 yg per ml polyclonal rabbit IgG [Sigma Chemical Co., St. Louis, MO] at pH 7.4) for 30 minutes. All incubations were at room temperature, reagent dilutions done using buffer A, and washes performed using buffer A minus BSA and rabbit IgG.
  • buffer A 50 mM Tris, 150 mM NaCl, 20 mM MgCl 2 , 20 yM ZnCl 2 / 3% bovine serum albumin, and 10 yg per ml polyclonal rabbit IgG [Sigma Chemical Co., St. Louis, MO] at pH 7.4
  • fibronectin binding and control assays 100 yl of fibronectin (0.01, 0.1, 1.0, 10, and 100 yg per ml of buffer A), the 180 kilodalton fibronectin fragment (10 yg per ml), and the 27 kilodalton fibronectin fragment (10 yg per ml) were added to wells and incubated 60 minutes.
  • wells were preincubated 60 minutes with 100 yl of serial dilutions of serum or fibronectin fragments prior to adding 40 yl of fibronectin (2.0 yg per ml) with the incubation period continued for 60 minutes.
  • polyclonal rabbit anti-human fibronectin antibody conjugated to alkaline phosphatase (1:2000 dilution) was added, incubated for two hours, and washed off.
  • the substrate, 4- methylumbelliferyl phosphate (0.2 mg per ml in 1 M 2-amino- 2-methyl-l-propanol, pH 9.9, supplemented with 25 mM ZnCl 2 and 1.0 mM MgCl 2 ), was added to each well, and the plates incubated two hours prior to reading in an automated fluoresence spectrophotometer. All samples were run in triplicate.
  • High concentrations of rabbit Ig result in only a slight reduction in FN binding, indicating specificity of the inhibition by the MAb's.
  • Type 8 antiserum was more active than type 5 antiserum.
  • relatively high concentrations of antibody protein were needed, suggesting that the common epitopes were only partially cross-reactive. This is consistent with our data concerning the chemical composition of the FN-R.
  • the ELISA assay can recognize antibodies that can inhibit FN binding to the FN-R on S. aureus which may prove useful in 3 clinically related areas: (i) The ELISA may be valuable in screening for antibodies that will protect against staphylococcal adhesion to host tissues. (ii) If these antibodies prove to be protective in animal models, then they can be tested in humans to prevent disease (vaccine or passive antibodies are possibilities). (iii)
  • the ELISA might also be useful in predicting which patients are at higher risk for the development of invasive staphylococcal infections.
  • Human plasma normally contains between 300 and 600 yg of FN per microtiter. Although clotting consumes a large amount of the FN, enough FN remains in the serum to compete with anti-FN-R antibodies for FN-R sites in the microtiter well. When the highly purified FN is used, 100 ng of FN can be detected by the assay. The sensitivity of the ELISA can be increased by treating the serum with gelatin-Sepharose which specifically removes FN via its interaction thru its gelatin-binding domain but leaves the antibodies.
  • capsular serotype and FN-R expression can be separated in some strains.
  • the FN-R and encapsulation are independent variables in the pathogenesis of S. aureus.
  • the FN-R may be important for adhesion to host tissues whereas capsules may be important for resisting host defenses, a concept supported by the finding that anti-capsular antibodies enhance phagocytosis of S. aureus by human neutrophils.
  • anti-FN-R antibodies might be active against a very wide range of S . aureus, not only for anti- adhesive properties, but also as opsonic antibodies.
  • the described competitive ELISA test and similar diagnostic tests employing the FN-R exopolysaccharide as an antigen, or the monoclonal antibodies to the FN-R can be useful in predicting which patients are at high risk for developing invasive S. aureus infections.
  • S_. aureus The microorganism, S_. aureus, is a potent infectious pathogen. Interactions between host fibronectin and the S. aureus FN-R play an important role in the initiation of infections. Because antibodies directed against the FN-R polysaccharide block binding of fibronectin to S. aureus, monoclonal or polyclonal antibodies directed against the polysaccharide FN-R can be employed in methods to protect against infection caused by S. aureus. The patients at highest risk for developing a S_. aureus infection can be predicted by use of the previously described diagnostic tests. Therefore, monoclonal antibodies directed against the FN-R may be administered in safe and effective amounts in methods which produce passive immunization in high risk patients. Patients with the following clinical situations would benefit from the passive immunization with anti-FN-R monoclonal antibodies:
  • Immunosuppressed patients especially those patients who are likely to have prolonged periods of neutropenia.
  • Patients who will have indwelling intravascular catheters e.g., patients receiving hyperalimentation or chemotherapeutic agents.
  • FN-R polysaccharide as an antigen, such as described in the ELISA test, to measure the pre-existing levels of antibody direct ⁇ ed against the FN-R polysaccharide can help determine which patient will benefit from this passive immunization. How ⁇ ever, the monoclonal antibody also can be of value in the treatment of S. aureus infections. Because the FN-R poly ⁇ saccharide is expressed in the largest numbers on invasive S . aureus and because the FN-R polysaccharide is on the surface of the bacteria, the monoclonal antibody directed against this immunogen also can act as an opsonin as well as a blocking antibody.
  • the amount to be injected is an amount which is safe and effective as determined by the size and condition of the animal and the severity of its infection, if any.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Molecular Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Cell Biology (AREA)
  • Pathology (AREA)
  • Biotechnology (AREA)
  • Food Science & Technology (AREA)
  • General Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Virology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

Test visant à déterminer la présence d'anticorps anti-récepteurs de la fibronectine Staphylococcique dans un échantillon de test, employant un exopolyoside récepteur de la fibronectine purifié, dérivé de Staphylococcus aureus utilisé comme antigène. Un kit de mise en oeuvre d'un test ELISA est également décrit.
EP19900913666 1989-09-08 1990-09-07 Test for the detection of staphylococcal fibronectin-receptor antibodies Withdrawn EP0444174A4 (en)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US40446389A 1989-09-08 1989-09-08
US404463 1989-09-08
US50874690A 1990-04-12 1990-04-12
US508746 1990-04-12

Publications (2)

Publication Number Publication Date
EP0444174A1 true EP0444174A1 (fr) 1991-09-04
EP0444174A4 EP0444174A4 (en) 1992-09-16

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EP19900913666 Withdrawn EP0444174A4 (en) 1989-09-08 1990-09-07 Test for the detection of staphylococcal fibronectin-receptor antibodies

Country Status (3)

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EP (1) EP0444174A4 (fr)
JP (1) JPH04503114A (fr)
WO (1) WO1991003572A1 (fr)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
IT1298539B1 (it) 1998-02-03 2000-01-12 Bracco Spa Metodo per la determinazione di infezioni da protesi
EP1838341B1 (fr) 2005-01-20 2013-08-14 Isconova AB Composition vaccinale comprenant une proteine ou un peptide de liaison a la fibronectine

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5034515A (en) * 1987-09-22 1991-07-23 Wisconsin Alumni Research Foundation Staphylococcal fibronectin receptor, monoclonal antibodies thereto and methods of use

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5034515A (en) * 1987-09-22 1991-07-23 Wisconsin Alumni Research Foundation Staphylococcal fibronectin receptor, monoclonal antibodies thereto and methods of use

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
BIOLOGICAL ABSTRACTS, vol. 86, no. 4, 1988 (Philadelphia, Pa., USA) G. F. A. MOTA et al. "Monoclonal antibodies to Staphylococcus aureus laminin-binding proteins cross-react with mammalian cells." page AB-466, column 1, abstract no. 37939. *
BIOLOGICAL ABSTRACTS, vol. 89, no. 5, 1990 (Philadelphia, Pa., USA.) J. M.-C. LUK et al. "Detection in rabbit sera of blocking antibodies against staphylococcal fibronectin-binding protein by ELISA.", page AB-448, column 2, abstract no. 48717. *
CHEMICAL ABSTRACTS, vol. 111, no. 13, 25 September 1989, Columbus, Ohio, US; abstract no. 112817, S. K. AKIYAMA ET AL.: 'Analysis of fibronectin receptor function with monoclonal antibodies: roles in cell adhesion, migration, matrix assembly, and cytoskeletal organization.' page 446 ;column 2 ; *
JOURNAL OF BIOLOGICAL CHEMISTRY. vol. 264, no. 24, 25 August 1989, BALTIMORE US pages 14566 - 14570; R. PASQUALINI ET AL.: 'Determination of the putative binding site for fibronectin on platelet glycoprotein IIb-IIIa complex through a hydropathic complementarity approach' *
See also references of WO9103572A1 *

Also Published As

Publication number Publication date
JPH04503114A (ja) 1992-06-04
EP0444174A4 (en) 1992-09-16
WO1991003572A1 (fr) 1991-03-21

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