WO1989007649A1 - Substance de liaison d'immunoglobuline, sous-fragments, leur procede de preparation, kit de reactif et organismes de liaison d'immunoglobuline - Google Patents

Substance de liaison d'immunoglobuline, sous-fragments, leur procede de preparation, kit de reactif et organismes de liaison d'immunoglobuline Download PDF

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Publication number
WO1989007649A1
WO1989007649A1 PCT/SE1988/000054 SE8800054W WO8907649A1 WO 1989007649 A1 WO1989007649 A1 WO 1989007649A1 SE 8800054 W SE8800054 W SE 8800054W WO 8907649 A1 WO8907649 A1 WO 8907649A1
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WO
WIPO (PCT)
Prior art keywords
binding
igm
immunoglobulin binding
strains
protein
Prior art date
Application number
PCT/SE1988/000054
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English (en)
Inventor
Göran Kronvall
Gunnar Lindahl
Original Assignee
Hightech Receptor Ab
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 Hightech Receptor Ab filed Critical Hightech Receptor Ab
Priority to EP19880902958 priority Critical patent/EP0398872A1/fr
Priority to PCT/SE1988/000054 priority patent/WO1989007649A1/fr
Priority to JP50284988A priority patent/JPH03503117A/ja
Publication of WO1989007649A1 publication Critical patent/WO1989007649A1/fr

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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
    • G01N33/6854—Immunoglobulins
    • G01N33/6857—Antibody fragments
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/33—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Clostridium (G)
    • 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
    • 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
    • G01N33/6854—Immunoglobulins

Definitions

  • Strain L1540 (and other strains of Clostridium perfringens) can be grown in GLC broth (Lab M Ltd, Salford, England) under anaerobic conditions, using standard bacteriological techniques. After growth for 24 h at 37°, in GLC broth, the bacteria are harvested by centrifugation at 5000 - 10 000 g for 30 minutes and resuspended in phosphate-buffered saline "PBSA": 120 mM NaCl, 30 mM phosphate, 0.02 % sodium azide, pH 7.4) to a concentration of 10 % (v/v).
  • PBSA phosphate-buffered saline
  • the bacterial concentration was determined by centrifugation at 10.000 x g for 5 min in capillary tubes in a miniature centrifuge (Microfuge Model 152, Beckman Instruments, Inc., Fullerton, CA). The bacteria were stabilized by heat-treatment at 80° for 5 min, washed once in PBSA and again resuspended to a concentration of 10 % in PBSA. Such bacterial preparations can be kept at 4°C for several months without loosing binding activity.
  • strain 1540 One of the strains studied, strain 1540, has a particularly high binding capacity and is therefore of special interest.
  • the ability of this strain to bind polyclonal Ig varies for different classes of Ig, the highest degree of binding being obtained with IgM.
  • the ability of polyclonal Ig preparations to bind to L1540 is actually correlated to the number of subunits in the Ig: the fraction that is able to bind decreases from pentameric IgM via dimeric secretory IgA to monomeric IgG and serum IgA.
  • bacterial strains used for binding test were isolated from routine clinical specimens of human origin and typed by standard techniques.
  • S. aureus Cowan I (NCIC 8530) served as the reference protein A strain, and a strain of S. epidermidis (L603) was used as a non-binding control strain.
  • bacterial were grown in the following media: B. catarrhalis, S. aureus, S. epidermldis, streptococci, diphteroid rods, M . fortuitum: Todd-Hewitt broth; H. influenzae: brain heart infusion broth supplemented with hemin, NAD and L-histidine (10 ⁇ g/ml of each); E .
  • IgM preparations contained less than 1 % IgG, as shown by rocket electrophoresis.
  • Fc fragments of IgG, light chains (K plus ⁇ ) and ⁇ heavy chains were purchased from Calbiochem AG, Lucerne, Switzerland.
  • F(ab') 2 fragments of IgG, serum IgA and secretory IgA were from Cappel Laboratories, Pennsylvania, USA.
  • myeloma IgM used for binding tests (Fig. 1) and inhibition tests (Fig. 3) were isolated from the serum of patients with macroglobulinemia; the sera used contained monoclonal myeloma components of high concentration and had reduced levels of background IgG.
  • the myeloma IgM was purified by zone electrophoresis in 0.6 % agarose (Johansson, B.G. 1972. Agarose gel electrophoresis. Scand. J. Clin. Lab. Invest. 29 (suppl. 124) :7) which is incorporated as a reference, followed by gel filtration.
  • Non-specific uptake recorded with non-binding bacteria (less than 5 % ) has been deduced.
  • Immunochemistry 6:43 the preparation (volume 320 ⁇ l) was mixed with 30 ⁇ l of 0.1 % glutaric dialdehyde and incubated for 20 hours at room temperature. The reaction was terminated by the addition of 300 ⁇ l of PBSAT containing 0.1 M L-lysine. No precipitate was formed during the aggregation. To fractionate aggregates according to sizes, they were applied to a calibrated column of Sepharose 4B (Pharmacia, Uppsala, Sweden). The column was eluted with PBSAT containing 0.1 M L-lysine and fractions were assayed for radioactivity. The aggregated IgG was present in a broad peak from the void volume (V o ) to the position of monomeric IgG. Fractions corresponding to different molecular weights were used in binding assays.
  • Strain L1540 is also able to bind polyclonal IgA, as shown in Fig. 2D.
  • a comparison with Fig. 2A shows that the bindning of serum IgA, which is a monomer, is similar to that of IgG, while the binding of secretory IgA, a dimer, is intermediate between that of IgG and that of IgM. (This pattern was observed repeatedly, in experiments where the binding of IgA, IgG and IgM was studied in the same experiment).
  • polyclonal Ig to bind to strain L1540 is apparently correlated to the number of subunits in the Ig: the fraction that is able to bind decreases from pentameric IgM via dimeric secretory IgA to monomeric IgG and serum IgA.
  • Inhibition of binding was tested by mixing 50 ⁇ l of labelled protein with 50 ⁇ l of the inhibitor (of known protein concentration), both preparations being diluted in PBSAT. Bacteria (200 /ul) were then added and the assay was subsequently performed as described above.
  • the concentration of binding bacteria was reduced hundredfold, as compared to the binding assay described above.
  • the total number of bacteria in the test mixture was kept unchanged by diluting the binding bacteria in a suspension of non-binding L603 bacteria.
  • binding of IgM could be completely inhibited both by polyclonal IgM and by polyclonal IgG as well as by an IgM myeloma protein. Similar results were obtained when the binding of polyclonal IgG was inhibited by the same three types of Ig.
  • polyclonal IgA could also inhibit binding of IgG or IgM to strain L1540 (data not shown).
  • the procedure is based on the fact that the receptor structure 'is liberated from the bacteria at elevated pH.
  • the suspension (40 ml) was incubated at 37° for 5 hours.
  • the bacteria were then removed by centrifugation, and the supernatant was subjected to affinity chromatography on IgM-Sepharo'se (5 ml; 1 mg IgM/ml).
  • the Sepharose was washed with 1000 ml of PBS and then eluted with 3 M KSCN.
  • the eluate (6 ml) was concentrated to 1 ml and analyzed by Western blotting (H. Towbin, T. Stachelin and J. Gordon 1979 Proc. Natl. Acad. Sci. USA 76, 4350) which is incorporated as a reference, using radiolabelled IgM as the probe ( Figure 6).
  • the immunoglobulin receptor has an apparent molecular weight of about 160 kD.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Cell Biology (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biotechnology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Food Science & Technology (AREA)
  • Organic Chemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Genetics & Genomics (AREA)
  • Biophysics (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Virology (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

L'invention concerne une protéine de liaison d'immunoglobuline, notamment de liaison d'IgM, d'un poids moléculaire apparent d'environ 160 KD sur SDS-PAGE (gel de sodium dodécyl sulfate-poly-acryl-amide), et ses sous-fragments. Elle concerne également un procédé de séparation d'une substance de liaison d'immunoglobuline et de ses sous-unités, au moyen desquelles on cultive des souches de liaison d'immunoglobuline de Clostridium perfringens dans un milieu. On sépare les microorganismes du milieu puis on les met en suspension dans un tampon, après quoi on récupère la substance de liaison d'immunoglobuline à partir du milieu et/ou des souches de liaison d'immunoglobuline de tampon de Clostridium perfringens, notamment de Clostridiun perfringens L540 déposé auprès du registre allemand des microorganismes sous le numéro de dépôt DSM 4331. L'invention se rapporte en outre à un kit de réactif permettant la liaison, la séparation et l'identification d'immunoglobulines, comprenant la protéine, les sous-fragments ou une souche de Clostridium perfringens.
PCT/SE1988/000054 1988-02-12 1988-02-12 Substance de liaison d'immunoglobuline, sous-fragments, leur procede de preparation, kit de reactif et organismes de liaison d'immunoglobuline WO1989007649A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
EP19880902958 EP0398872A1 (fr) 1988-02-12 1988-02-12 Substance de liaison d'immunoglobuline, sous-fragments, leur procede de preparation, kit de reactif et organismes de liaison d'immunoglobuline
PCT/SE1988/000054 WO1989007649A1 (fr) 1988-02-12 1988-02-12 Substance de liaison d'immunoglobuline, sous-fragments, leur procede de preparation, kit de reactif et organismes de liaison d'immunoglobuline
JP50284988A JPH03503117A (ja) 1988-02-12 1988-02-12 免疫グロブリン結合性物質そのサブフラグメント、それらの製造方法、試薬キットおよび免疫グロブリン結合性物質

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/SE1988/000054 WO1989007649A1 (fr) 1988-02-12 1988-02-12 Substance de liaison d'immunoglobuline, sous-fragments, leur procede de preparation, kit de reactif et organismes de liaison d'immunoglobuline

Publications (1)

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WO1989007649A1 true WO1989007649A1 (fr) 1989-08-24

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PCT/SE1988/000054 WO1989007649A1 (fr) 1988-02-12 1988-02-12 Substance de liaison d'immunoglobuline, sous-fragments, leur procede de preparation, kit de reactif et organismes de liaison d'immunoglobuline

Country Status (3)

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EP (1) EP0398872A1 (fr)
JP (1) JPH03503117A (fr)
WO (1) WO1989007649A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992000763A1 (fr) * 1990-07-03 1992-01-23 Akzo N.V. Compose immunoreactif
US7220415B1 (en) 1999-05-05 2007-05-22 Gunnar Lindahl Vaccine composition

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4604234A (en) * 1983-06-30 1986-08-05 Sanwa Kagaku Kenyusho Co., Ltd. Protein having cell growth stimulating action, composition thereof and method for producing the same

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4604234A (en) * 1983-06-30 1986-08-05 Sanwa Kagaku Kenyusho Co., Ltd. Protein having cell growth stimulating action, composition thereof and method for producing the same

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992000763A1 (fr) * 1990-07-03 1992-01-23 Akzo N.V. Compose immunoreactif
US7220415B1 (en) 1999-05-05 2007-05-22 Gunnar Lindahl Vaccine composition

Also Published As

Publication number Publication date
EP0398872A1 (fr) 1990-11-28
JPH03503117A (ja) 1991-07-18

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