IMMUNOGLOBULIN BINDING SUBSTANCE, SUBFRAGMENTS, A PROCESS FOR PREPARING THEREOF, REAGENT KIT AND IMMUNOGLOBULIN BINDING ORGANISMS

Abstract: The invention concerns an immunoglobulin binding, especially IgM binding, protein with an apparent molecular weight of about 160 kD on SDS-PAGE (sodium dodecyl sulphate-poly-acryl-amide gel) and subfragments thereof. It also concerns process for preparing an immunoglobulin binding substance and subunits thereof whereby immunoglobulin binding strains of Clostridium perfringens are cultivated in a medium, the microorganisms are separated from the medium and suspended in buffer whereafter the immunoglobulin binding substance is recovered from the medium and/or from the buffer immunoglobulin binding strains of Clostridium perfringens, especially Clostridium perfringens L540 deposited at Deutsche Sammlung von Mikroorganismen with deposition number DSM 4331 and a reagent kit for binding, separation and identification of immunoglobulins, comprising the protein, the subfragments or a Clostridium perfringens strain.

Description: Immunoglobulin binding substance, subfragments, a process for preparing thereof, reagent kit and immunoglobulin binding organisms 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 5 immunoglobulin binding substance. Some bacteria show immunoglobulin binding capacity. Thus, the ability of protein A from Staphylococcus aureus to bind IgG via the Fc region has made it a 0 valuable tool in immunological research (USP 3 850 798). In addition to this well-known protein from staphylococci, receptors that bind IgG in a non-immune manner have also been found in streptococci. Such streptococcal receptors have been purified and shown 5 to be powerful immunological tools. Bacterial receptors that preferentially bind other Ig classes than IgG would also be of value for immunological work. Receptors for IgA and IgM, in particular, could find a variety of applications. Bacterial proteins * 0 that bind IgA have been isolated, but an IgM receptor has not been described. 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 5 binds IgG. A large number of bacterial strains has now been screened for ability to bind IgM. The results show that certain strains of the anaerobic bacterium 0 Clostridium perfringens are able to bind a large fraction of polyclonal IgM but only a small fraction of polyclonal IgG. This binding, which seems to be mediated via the F(ab")2 part of the Ig molecules, represents a new type of Ig binding to bacteria. An immunoglobulin binding substance has also been isolated. Thus 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. 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 L15--0 has been deposited at Deutsche Sammlung von Mikro- organismen with deposition number DSM 4331. Growth conditions. 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 2-4 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). 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 L1540 of C. perfringens was 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. Characterization data 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„S, Sensitivity of the receptor structure to heat and proteolytic enzymes Heating of a suspension of L1540 to 80 C for 10 min did not affect its ability to bind IgM. Such a preparation also retained its ability to bind IgM after 6 months at 4°C. Binding of IgM was not affected by digestion with pepsin or trypsin, but was reduced ten-fold by digestion with papain (400 ,ug/ml, pH 8.0, 1 h at 37°). 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. Preferably 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. The data presented in the following examples show that a new type of non-immune binding of Ig can found in certain strains of the anaerobic bacterium C. perfringens. 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 di eric secretory IgA to monomeric IgG and serum IgA. The simplest explanation for this correlation appears to be that all Ig subunits contain a structure that can bind with low affinity to the bacterial receptor and that the avidity of an Ig molecule increases with increasing number of subunits. The finding that aggregated IgG binds to strain L1540, and that the fraction bound increases with increasing aggregate size, is in agreement with this explanation, but does not prove it. The binding structure in Ig is apparently located in the F(ab') 2 region, but it seems highly unlikely that the binding of Ig to the bacterial receptor represents a true antigen-antibody reaction, since at least 60 % of polyclonal IgM and several different myeloma proteins bind" to the bacteria. The location within the F(ab*)- 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. With regard to possible iin vivo effects of the binding structure, it is interesting to note that 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. In agreement with this prediction, preliminary experiments show that strain L1540 is a strong lymphocyte mitogen. The binding characteristics of the new strains and purification of the immunoglobulin binding substance will now be described in the following examples with reference to the attached figures. FIGURE LEGENDS Figure 1_ Capacity of 204 bacterial strains representing 20 different species to bind a human IgM (V<) myeloma protein. The figures within parentheses give the number of strains tested for each species. Figure 2. Binding of polyclonal Ig preparations to C perfringens strain L1540 and L9593 and to Σ5 aureus Cowan I, at different bacterial concentrations. Figure 3_. Inhibition of binding of polyclonal IgM to perfringens L1540 by different Ig preparations. To increase the sensitivity of the tests, the bacterial concentration was reduced to 10^/ml in these experiments, as described in Materials and Methods. Figure _4. Binding of IgG-aggregates of different molecular weights to C_. perfringens L1540 at different bacterial concentrations. Figure 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 . To increase the sensitivity of the test, the bacterial concentration was reduced to 107/ml, as described in Materials and Methods. Figure 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. Example 1. Strain L1540 was streaked on 4 blood agar plates and incubated anaerobically at 37° for 24 h. From each plate a heavy inoculum was collected with a cotton-armoured swab and used to inoculate a 500 ml bottle containing 250 ml of sterile GLC broth. The cultures were incubated anaerobically at 37° for 24 h and the bacteria were then collected by centrifugation, resuspended in PBSA, and heat inactivated, as described above. These bacteria were used for testing immunoglobulin bindin'g. In the examples 2 - 5 below the following materials and methods were used. Material and Methods Bacterial strains, media and growth conditions. Unless otherwise stated, the bacterial strains used for binding test (Fig. 1) were isolated from routine clinical specimens of human origin and typed by standard techniques. 53. aureus Cowan I (NCIC 8530) served as the reference protein A strain, and a strain of 5. epidermidis (L603) was used as a non-binding control strain. For binding studies, bacterial were grown in the following media: B. catarrhalis, S. aureus, S ^ . epidermldis, streptococci, diphteroid rods, H . fortuitum: Todd-Hewitt broth; II. influenzae: brain heart infusion broth supplemented with hemin, NAD and L-histidine (10 ug/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. Immunoglobulin preparations All preparations used were of human origin and were polyclonal, unless otherwise stated. IgG was purchased from AB Kabi, Stockholm, Sweden, and contained less than 1 % IgM, as shown by rocket electrophoresis with specific antisera. IgM was from Calbioche 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-produkter AB, Brom a, Sweden). Both IgM preparations contained less than 1 % IgG, as shown by rocket electrophoresis. Fc fragments of IgG, light chains (K plus Λ ) and V 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. The preparations of 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. Radiolabelling Unless otherwise stated, proteins were labelled with carrier-free 125ι (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 Ci/mg protein) were stored at 4°C and used within 1 month. Binding assays All assays were carried out in plastic tubes at room temperature. Duplicate samples containing 2-5 ng (about 10 4 cpm) of radiolabelled protein in 25 /ul PBSAT were mixed with 2 x 10 * 3 bacteria suspended in 200 /ul PBSAT. After incubation for 60 min, 2 ml of PBSAT was added to each tube and the bacteria were spun down (2000 x g for 15 min). The supernatant was discharged and the radioactivity in the pellet was measured in a gamma counter. The quantity bound is expressed in per cent of added radioactivity. Non-specific uptake recorded with non-binding bacteria (less than 5 % ) has been deduced. In some experiments, 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 (concentration 109 /ml), to keep the total number of bacteria in the mixture constant. The non-binding strain used was in all cases f5. epidermidis L603. Preparation of radiolabelled IgG aggregates 1.0 mg of IgG (dissolved in 150 /ul of 0.05 M phosphate buffer, pH = 7.5) was radiolabelled by the chloramine-T method (Greenwood, F.C., W.M. Hunter, and j.s. Glover. 1963. The preparation of 131l-labelled human growth hormone of high specific radioactivity. Biochem. J_. 89:114) which is incorporated as a reference, using 1.0 mCi of 125i. After extensive dialysis against 0.15 M phosphate buffer, pH = 7.5, the labelled protein was aggregated by the method of Avrameas (Avra eas, S. 1969. Coupling of enzymes to proteins with glutaraldehyde. Use of conjugates for the detection of antigens, and antibodies. Immunochemistry 6:43) : the preparation (volume 320 /ul) was mixed with 30 /ul of 0.1 % glutaric dialdehyde and incubated for 20 hours at room temperature. The reaction was terminated by the addition of 300 /ul 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 ) to the position of monomeric IgG. Fractions corresponding to different molecular weights were used in binding assays. Example 2. Binding of IgM to Clostridium perfringens In initial experiments, 204 bacterial strains representing 20 different species of aerobic and anaerobic bacteria were tested for ability to bind an IgM myeloma protein. As shown in Fig. 1, binding was observed only for Staphylococcus aureus and for Clostridium perfringens. The strains of S_^ aureus all showed a high level of binding, whereas the binding to different strains of C^ perfringens varied from a high level to no binding at all. Binding tests with a second IgM myeloma protein gave very similar results, except that no binding was observed with strains of S^ aureus (data not shown). The binding of the first myeloma protein to S^ aureus is most likely due to protein A, which is known to bind a minority of IgM myeloma proteins. ' Example 3. Comparison of different strains of C. perfringens: binding of IgM, IgG and IgA Different strains of ____ perfringens were compared in more detail with regard to their ability to bind IgM, IgG and IgA. To avoid possible pitfalls due to unusual properties of myeloma proteins, these experiments and all subsequent experiments were performed with polyclonal Ig preparations, unless otherwise stated. The strains of C^ perfringens were compared in experiments where decreasing numbers of bacteria were tested for ability to bind a constant amount of IgM or IgG. Results for two strains are shown in Figs. 2A and 2B, and for comparison data for similar experiments with S^ aureus Cowan I, the standard protein A strain, are shown in Fig. 2C. With regard to binding of IgM, it is clear that both strains of C^ perfringens bind a large fraction of the polyclonal IgM at the highest bacterial concentration, but when the number of bacteria is reduced the binding capacity of strain L9593 decreases strongly whereas that of strain L1540 remains almost unchanged. This difference is not due to different binding rates, since further incubation did not increase the fraction of Ig bound (data not shown). Experiments of this type showed that strain L1540 binds more IgM than any other strain of C^ perfringens tested here. Virtually identical results were obtained with two different preparations of polyclonal IgM. Both strains of C^ perfringens are also able to bind polyclonal IgG, but the fraction bound is much smaller than for IgM (Figs. 2A and 2B). The reverse is true for S. aureus Cowan I, as expected (Fig. 2C). All other strains of C^ perfringens used in this work were also tested for their ability to bind IgG, and in no case was the fraction of IgG bound higher than for strain L1540. Among all strains, the capacity to bind I G was actually correlated to the capacity to bind IgM, suggesting that both classes of immunoglobulin bind to the same bacterial surface structure in each strain (data not shown). The two strains of C^ perfringens described above, L1540 and L9593 > were chosen for more detailed characterization. Only results obtained with strain L1540 will be reported below, but qualitatively similar results were in all cases obtained with strain L9593. A strain of C^ perfringens that does not bind IgM was always included as a control, and no binding was observed in any case with this strain. Binding of IgA 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). The ability of 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. Exemple 4. Inhibition tests. The simplest explanation for the data presented above is that a single receptor in C^ perfringens L1540 is able to bind Ig molecules of different classes. This question was further analysed in inhibition experiments where different immunoglobulins were tested for ability to inhibit the binding of labelled polyclonal IgM to strain L1540. Inhibition of binding was tested by mixing 50 ,ul of labelled protein with 50 /ul 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. To increase the sensitivity of the inhibition test, the concentration of binding bacteria was reduced hundredfold, as compared to the binding assay described above. However, the total number of 14 bacteria in the test mixture was kept unchanged by diluting the binding bacteria in a suspension of non-binding L603 bacteria. As shown in Fig. 3A, binding of IgM could be completely inhibited both by 5 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. Furthermore, polyclonal IgA could also inhibit binding of IgG or IgM to strain 0 L1540 (data not shown). These results clearly indicate that IgG, IgM and IgA bind to one and the same receptor structure. Exemple 5. Binding of immunoglobulin fragments 5 To further characterize the binding of immunoglobulins to C^ perfringens L1540, the ability of this strain to bind F( b')2 and Fc fragments was tested (Fig. 2E). Fragments derived from IgG were used for these 0 studies. A comparison with Fig. 2A shows that the binding of F(ab')2 fragments to L1540 is similar to that of whole IgG, but Fc fragments do' not bind. Binding therefore appears to take place in the F(ab') 2 region of IgG. This conclusion is supported by 5 inhibition experiments: binding of IgG was effectively inhibited by F(ab') 2 fragments but not by Fc fragments (data not shown). The same two IgG fragments were also tested for ability to inhibit the binding of IgM (Fig. 3B). Only F(ab')2 fragments were 0 effective inhibitors, which is in agreement with the previous conclusion that the mode of binding is similar for IgM and IgG. Separated heavy and light chains were also tested in 5 both binding and inhibition experiments with strain L1540. No binding was observed with polyclonal V- chains or with polyclonal light chains ( X. plus A), 15 nor could any of these proteins inhibit the binding of whole IgG or IgM (data not shown). This suggests that the binding structure in the F(ab') 2 part of the molecule is only present when heavy and light chains are combined. Example 6. Purification of the immunoglobulin binding substance The procedure is based on the fact that the receptor structure 'is liberated from the bacteria at elevated pH. Eight 500 ml bottles, each containing 250 ml of GLC broth, were inoculated with strain L1540 and incubated at 37° for 24 hours, under anaerobic conditions. The bacteria were harvested by centrifugation, washed twice with PBS, heat-inactivated at 80° for 5 min, washed once more in PBS and resuspended to 10 % (v/v) in 0.5M Tris buffer, pH = 9.0. 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 chro atography 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. Similar results were obtained when an IgM myeloma protein was used as the probe. When a non-binding strain of C. perfringens was analyzed in the same way, no IgM binding substance was found. These data confirm that the bands observed in Figure 6 are indeed due to the immunoglobulin receptor on strain L1540, which therefore has been purified. Intsmstiont! Application No: PCT/ SE88-' 00054

Claims: Claims 1. A protein characterized in that it has an apparent molecular weight of about 160 D on SDS-PAGE (sodium dodecyl sulphate-polyacryl-amide gel) and binds to the F(ab " )2 part of immunoglobulins, especially to IgM. 2. Subfragments of a protein according to Claim 1. 3. A process for preparing an immunoglobulin binding substance and subunits thereof, characterized in that immunoglobulin binding strains of Clostridium perfringens are cultivated in a medium, the micro¬ organisms are separated from the medium and suspended in buffer whereafter the immunoglobulin binding substance is recovered from the medium and/or from the buffer. 4. A process according to Claim 3, characterized in that the strains are cultivated in GLC-medium. 5. A process according to Claims 3 and 4, characterized in that the immunoglobulin binding substance is recovered by affinity chromatography. 6. A process according to Claim 5, characterized in ity chromatography is performed with IgM- 7. Clostridium perfringens strains for use as an immunoglobulin binding and lymphocyte mitogenic material. 18 8. Clostridium perfringens strains according to Claim 7 for use as an IgM binding or precipitating material. 9. Clostridium perfringens L1540 deposited at Deutsche Sammlung von Mikroorganismen with deposition number DSM 4331. 10. Reagent kit for binding, separation and identification of immunoglobulins, characterized in that it comprises a protein or a subfragment according to anyone of Claims 1 - 2 or a microorganism according to anyone of Claims 7 - 9.

Inventors: KRONVALL GOERAN, LINDAHL GUNNAR, KRONVALL, GOERAN, LINDAHL, GUNNAR
Applicants: HIGHTECH RECEPTOR AB
Application year: 1988
Main classification: C12P21
IPC codes: C07K1, C07K14, C12N1, C12P21, C12R1, G01N33