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

• This invention relates to immunoglobulin (Ig) binding especially IgM binding strains of Clostridium perfringens, an immunoglobulin binding protein, subfragments thereof and a process for preparing an immunoglobulin binding substance.
• Ig immunoglobulin
• Protein A is known to show some reactivity with IgM, but this reactivity is of very limited use since only a minority of IgM molecules bind, and since protein A preferentially binds IgG.
• the invention concerns a protein with an apparent molecular weight of about 160 kD on SDS-PAGE (sodium dodecyl sulphate-polyacryl-amide gel), which protein binds to the F(ab') 2 part of immunoglobulins, especially to IgM.
• SDS-PAGE sodium dodecyl sulphate-polyacryl-amide gel
• the invention also concerns subfragments of this protein that binds to immunoglobulins.
• the immunoglobulin binding strains of Clostridium perfringens can according to the invention be used as immunoglobulin binding and especially IgM binding material for analytical and separating purposes.
• the microorganisms can also be used in buffer suspensions for precipitation reaction with immunoglobulins.
• the immunoglobulin binding strains of Clostridium perfringens bind to the F(ab') 2 region of IgM and as might be expected they therefore act as strong lymphocyte mitogens.
• the invention also concerns use of the strains as lymphocyte mitogens.
• the invention especially concerns the new strains Clostridium perfringens L1540 and L9593 of which L1540 has been deposited at Deutsche Sammlung von Mikro-organismen with deposition number DSM 4331.
• 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.
• C. perfringens were found among many strains of C. perfringens that were isolated from routine clinical specimens at the Laboratory of Clinical Microbiology, University Hospital, Lund, Sweden It was characterized as C. perfringens by standard bacteriological techniques (see: Holdeman, L.V., Cato, E.P. and Moore, W.E.C., 1977, Anaerobe Laboratory Manual. Virginia Polytechnic Institute, Blacksburg, Va). All strains of C. perfringens are anaerobic, gram-positive, spore-forming rods.
• strain L1540 When strain L1540 is cultured in peptone yeast extract with glucose (PYG) or in GLC broth, gas chromatographic analysis of the medium shows production of acetic, propionic, butyric, lactic and succinic acids.
• the strain utilizes fructose, glucose, glycerol, inositol, lactose, maltose, mannose, melibiose, raffinose, starch, sucrose, and trehalose. There is curd formation in milk, digestion of meat and formation of hemolysis double zones. There are subterminal spores and the lecithinase reaction is positive.
• the strain produces NH 3 and H 2 S. Sensitivity of the receptor structure to heat and proteolytic enzymes
• the invention also concerns a process for preparing an immunoglobulin binding substance especially an IgM binding substance, characterized in that immunoglobulin binding strains of Clostridium perfringens are cultured in a medium, the medium is freed from microorganisms and the substance recovered from the medium or from the microorganisms by suspending them in a buffer and recovering the substance from the buffer.
• Suitable strains of Clostridium perfringens can be selected as is described in example 2.' Preferably strains L1540 and L9593 are used.
• the medium is preferably GLC and the strains are grown preferably 24 hours at 37°C.
• the microorganisms can be separated from the medium e.g. by centrifugation at about 5000 - 10.000 g for about 30 minutes and the substance recovered from the medium with affinity chromatography, preferably IgM-Sepharose® (Pharmacia, Sweden).
• the substance can also be produced by suspending the microorganisms after they have been separated from the medium in a buffer.
• the pellet after centrifugation is suspended in 0,5M Tris buffer, pH 9.0, and left at 37°C for about 5 hours whereafter the substance is recovered as above by separating the buffer from the microorganism and purifying the substance from the buffer especially with affinity chromatography.
• 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.
• the location within the F(ab') 2 region of the binding structure is not yet known, but the finding that isolated heavy or light chains do not inhibit binding indicates that the binding structure may be generated through the combination of heavy and light chains.
• the ability of the bacteria to bind to the F(ab') 2 region of IgM might allow them to bind to the surface of B lymphocytes.
• preliminary experiments show that strain L1540 is a strong lymphocyte mitogen.
• Figure 1 Capacity of 204 bacterial strains representing 20 different species to bind a human IgM (4) myeloma protein. The figures within parentheses give the number of strains tested for each species.
• FIG. 5 Inhibition of binding of aggregated IgG to C. perfringens L1540 by different Ig preparations.
• the IgG-aggregates used for this experiment had a molecular weight of about 1.3 x 10 6 .
• the bacterial concentration was reduced to 10 7 /ml, as described in Materials and Methods.
• FIG. 6 Western blot analysis of immunoglobulin receptor purified from strain L1540.
• the purified receptor was subjected to SDS-PAGE (sodium dodecyl sulphate-polyacryl-amide gel) electrophoresis and the proteins were then electroblotted onto an Immobilon membrane, which was probed with radiolabelled IgM, followed by autoradiography. The autoradiograph is shown.
• SDS-PAGE sodium dodecyl sulphate-polyacryl-amide gel
• Example 1 Strain L1540 was streaked on 4 blood agar plates and incubated anaerobically at 37° for 24 h.
• Bacterial strains media and growth conditions.
• 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 .
• coli, K. pneumoniae, P. mirabilis, P. aeruginosa tryptone broth; anaerobic bacteria: GLC broth (Lab M Ltd, Salford, England). After overnight incubation at 37° C, the bacteria were harvested, washed twice with PBSAT (0.12 M NaCl, 0.03 M phosphate, 0.02 % NaN 3 , 0.05 % Tween 20, pH 7.2) and resuspended in PBSAT to 10 9 organisms/ml.
• PBSAT 0.12 M NaCl, 0.03 M phosphate, 0.02 % NaN 3 , 0.05 % Tween 20, pH 7.2
• IgG was purchased from AB Kabi, Sweden, and contained less than 1 % IgM, as shown by rocket electrophoresis with specific antisera. IgM was from Calbiochem AG, Lucerne, Switzerland; unless otherwise stated this IgM preparation was used for the experiments with IgM reported here.
• a second preparation of IgM was the kind gift of Dr A. Grubb; this preparation had been purified by repeated gel filtrations on Ultrogel AcA22 (LKB- ister AB,
• 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.
• proteins were labelled with carrier-free 125 I (Amersham International, England) using a modified lactoperoxidase method (Myhre, E.B., and P. Kuusela. 1983. Binding of human fibronectin to group A, C, and G streptococci. Infect. Immun. 40:29) which is incorporated as a reference. Free unreacted isotope was removed by extensive dialysis against PBSAT. Labelled proteins were further purified by gel filtration and fractions corresponding to the proper molecular weight were pooled and used for binding experiments. Labelled preparations (specific activity about 4 mCi/mg protein) were stored at 4°C and used within 1 month.
• Non-specific uptake recorded with non-binding bacteria (less than 5 % ) has been deduced.
• the binding capacity of different bacterial strains was studied by varying the number of test bacteria in the incubation mixture (Figs. 2 and 4). In these experiments the test bacteria were diluted in a suspension of non-binding bacteria
• 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.

Landscapes

• 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)

Applications Claiming Priority (1)

Publications (1)

Family

ID=20371026

Family Applications (1)

Country Status (3)

Families Citing this family (2)

Family Cites Families (1)

• 1988
  • 1988-02-12 JP JP50284988A patent/JPH03503117A/ja active Pending
  • 1988-02-12 EP EP19880902958 patent/EP0398872A1/de not_active Withdrawn
  • 1988-02-12 WO PCT/SE1988/000054 patent/WO1989007649A1/en not_active Application Discontinuation

Non-Patent Citations (1)

Also Published As

Similar Documents

Legal Events