MONOCLONAL ANTIBODIES AGAINST S100

Abstract: The present invention relates to novel monoclonal antibodies against the calcium binding protein S100, the use of these antibodies for the development of immunoassays for specific determination of total S100 or the different isoforms of S100 in serum, plasma, cerebrospinal fluid and other body fluids.

Description: MONOCLONAL ANTIBODIES AGAINST SI 00 FIELD OF THE INVENTION The present invention relates to novel monoclonal antibodies against the calcium binding protein S 100, the use of these antibodies for the development of immunoassays for determination of total SI 00 or specific determination of the different isoforms of SI 00 in serum, plasma, cerebrospinal fluid and other body fluids. BACKGROUND OF THE INVENTION. S I 00 is a 21kDa protein belonging to the SlOO/calmodulin/troponin C super family of EF-hand calcium binding proteins (for review see Zimmer et al, Brain Res Bull 37: 417 - 429, 1995). S 100 was originally isolated by Moore and co- workers from human brain, and was originally considered as a brain specific protein (Moore, Biochem Biophys Res Commun 19: 739 - 744, 1965). There has today been identified A 16 members of the SI 00 family based upon amino acid sequence and functional similarities (Zimmer et al. Brain Res Bull 37: 417 - 429, 1995). S100 in brain tissue consists of homo- and heterodimers of α and β subunits. The α and β subunits show a high degree of sequence and species homology (Isobe et al, Eur J Biochem., 89: 379 - 388, 1978; Isobe et al. Eur J Biochem. 1 16: 79 -86, 1981). The S lOOββ exists in high concentration in glial cells and Schwann cells, while the SlOOαβ is expressed in glial cells but not in Schwann cells (Isobe et al, J Neurochem 43: 1494 - 1496, 1984). SlOOαα is present in neurones, but is mainly expressed in extra cerebral tissues such as skeletal muscle, kidney and heart (Kato and Kimura, Biochim. Biophys Acta 842: 146 - 152, 1985). Small amounts of SI 00 is present in different normal human tissues: skin, chondrocytes, spleen, adipose tissue, kidney, heart, skeletal muscle and in different malignant tissues; malignant melanoma, glioma, and soft tissue tumours (Zimmer et al. Brain Res Bull 37: 417 - 429, 1995). Determination of S 100 in serum and CSF has shown to be useful in the management of patients with brain trauma and to evaluate neurological complications after cardiopulmonary bypass surgery (Michetti et al. J Neurol. Sci. 44: 731 - 743 1980; Persson et al. Stroke 18: 91 1 - 918, 1987; Ingebrightsen et al. J Neurol, Neurosurg. Psych. 59: 103 - 104, 1995; Westaby et al. Ann Thorac Surg 61 : 88 - 92, 1996). Serological determination of SI 00 has also been shown to be useful for follow-up and in order to obtain prognostic information in patients with malignant melanoma (Henze et al. Dermatology 194: 208 - 212, 1997; Buer et al. Br J Cancer, 75: 1373 - 1376, 1997). Different disorders may be associated with changed total amount of SlOOαβ and SlOOββ or changed ratio between the different isoforms of S100. Therefore accurate methods for determining the total amount of SlOOαβ and SlOOββ as well as for determining the different isoforms of SlOOαβ and SlOOββ are needed. WO 98/01471 discloses a method for determining the total amount of S 100. This method comprises one step with the major drawback that information of the amount of the individual isoforms SlOOαβ and S lOOββ is not given, and the response for SlOOαβ and SlOOββ is not identical, hence the assay according to the invention gives a more accurate determination of total S100 (i.e. the sum of SlOOαβ and S lOOββ). The method according to the present invention gives a more accurate determination of the total amount SI 00 since the SlOOαβ and SlOOββ are determined in two separate steps with specific determination of the isoforms SlOOαβ and SlOOββ in the individual steps. The method according to the present invention also gives accurate information of the concentration of the two isoforms SlOOαβ and S lOOββ. SUMMARY OF THE INVENTION The present invention provides monoclonal antibodies against S 100, and the design of immunoassays for determination of the total amount of SlOOαβ and S lOOββ as well as for specific determination of the different isoforms of S100, i.e. specific for either SlOOαβ or S lOOββ. The invention will be useful for serological determination of total S100 and for determination of the different isoforms of SI 00 in the management of patients with brain damage, neurodegenerative diseases, and in the follow-up of patients with malignant melanoma, gliomas and certain soft tissue tumours. DESCRIPTION OF THE SPECIFIC EMBODIMENTS The present invention provides novel monoclonal antibodies against S 100, and the design of immunoassays for determination of SI 00 in serum, plasma, cerebrospinal fluid or other body fluids. The method comprises the establishment of monoclonal antibodies against specific epitopes of S100, the use of these antibodies for the design of immunoassays for determination of total SI 00, immunoassays specific for SlOOαβ and immunoassays specific for SlOOββ. The immunosassays are designed for, but not restricted to, immunometric (non- competitive) assay format. The antibodies according to the invention may also be used for competitive assay format. The immunoassays employ, but are not restricted to, monoclonal antibodies against specific epitopes of SI 00 β for catching and detection of SI 00. According to the invention monoclonal antibodies are preferred, but the assay according to the invention may employ polyclonal antibodies, peptides, proteins, protein or DNA fragments with essentially the same binding specificity as the monoclonal antibodies provided by the invention. Furthermore the immunoassays employ, but are not restricted to, a solid-phase containing the S I 00 monoclonal antibodies for the adsorption of the SI 00 from the sample. The solid-phase may be wells of microtiter plates, plastic tubes, beads, magnetic particles or the like. The solid-phase may be produced from glass, polypropylene, polystyrene, and the like. The catching antibody may be bound to the solid phase by, but is not restricted to, physical adsorption, covalent binding or through biotin - avidin bridges, or the like, using methods generally known to those skilled in the art. In the present invention a wide variety of labels are suitable for detection of the S100. Enzymes are of particular interest, including hydrolases particularly esterases and glycosidases, and oxidoreductases, particularly peroxidases. Suitable are also radioactive labels, such as 1251, 32P, 14C, 3H and the like, fluorescent compounds such as fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, lanthanid derivatives, and the like, and chemiluminencers such as luciferin, luminol, acridinium esters and the like. The assays according to the invention can be performed in any double epitope assay format; either two-step forward sandwich assay, one-step sandwich assay or backward sandwich assay. In performing the assays according to the present invention, a sample (preferably 1 - 1000 μL) is incubated in the test receptacle containing a catching anti S100 MAb. In the preferred configuration the sample consists of serum, plasma or cerebrospinal fluid. According to the invention the sample and S100 MAb is incubated under conditions allowing binding of the SI 00 antigen to the catching S100 MAb. In the preferred configuration the catching antibody is immobilized to the solid-phase through biotin - streptavidin bridges. For the detection of the SI 00 antigen adsorbed to the solid-phase bound antibody a labeled monoclonal antibody directed against independent epitopes of S I 00 is added, and incubated in the test receptacle under conditions allowing the antibody to bind to the S lOO-antibody complex. After washing the label is detected in a system suitable for the label used. The present invention provides antibodies specific for the different isoforms of SI 00 that are used as catching antibodies and antibodies, whose epitopes are not overlapping the epitopes of said catching antibodies, that are used as detecting antibodies (see Example 2.3b). Depending on the selection of catching and detecting antibodies immunoassays with the preferred specificity may be designed, i.e. assays for specific determination of SlOOαβ, and assays for specific determination of SlOOββ. The hybridoma cell lines S 10:3, S21 :4, S23:2, and S35:2, producing the monoclonal antibodies according to the invention, were deposited at the ECACC (European Collection of Cell Cultures, Centre for Applied Microbiology & Research, Salisbury, Wiltshire SP4 0JG, UK) and assigned ECACC Accession Nos. 98082619, 98082618, 98082617, and 98082616, respectively. These deposits were made on the 26:th of August, 1998. Also included in the invention are antibodies, antibody fragments or peptides with the same binding specificity as antibodies produced by the hybridoma cell lines S10:3, S35:2, S23:2, and S21 :4 as shown by that they inhibit the binding of the antibody in question by > 75 % as measured by inhibition studies as outlined in Example 2.3b. This invention will now be described in greater detail by reference to the following non-limiting examples and the attached figures, wherein: Figure 1 shows reactivity with purified S lOOββ, SlOOαβ, and S lOOαα of selected S100 MAb in immunometric assays. The reactivity was determined as described in Example 2.2. Figure 2 shows the inhibition of selected S 100 MAb:s as described in Example 2. 3b. Figure 3 shows the step of the assay that is specific for SlOOββ, designed as described in Example 3.1 , using Biotin S10 MAb as catching antibody and HRP S23 MAb as detecting antibody. Figure 4 shows the step of the assay that is specific for S lOOαβ, designed as described in Example 3.2, using Biotin S35 MAb as catching antibody and HRP S23 MAb as detecting antibody. EXAMPLE 1 Establishment of hyb ή domas and production of monoclonal antibodies specific for SIOO. SlOOββ is commercially available from several suppliers, e.g. Affinity Research, UK; Calbiochem, US; Sigma Chemical Co, US; HyTest, Finland. The commercially available S lOOββ may be used as immunogen for establishment of monoclonal antibodies. S lOOββ may also be isolated from human or bovine brain following methods available in the literature (Moore, Scand J Immunol 9: 53- 74, 1982). In the preferred procedure Balb/c mice were immunised intra peritoneally with 10 - 50 μg of S lOOββ in Ribi adjuvant. After the immunisation and 3 - 4 booster doses during 60 - 90 days, with the same antigen or mixture of SlOOββ/ S lOOαβ, spleen cells from the immunised mice were fused with P3 x 63Ag 8 myeloma cells as described (Lindholm et al. Int. Arch Allergy and Appl. Immunol. 71 : 178 - 181, 1983). Hybridomas producing antibodies reacting with S100 were selected by screening of hybridoma supernatants in microtiter wells coated with affinity purified polyclonal antiserum against mouse IgG + M, (Jackson Immuno Res Lab, US). The wells were then incubated with SlOOββ antigen, and after washing the bound SIOO antigen was detected by incubation with polyclonal Rabbit Anti SIOO and HRP labelled Swine Anti Rabbit Ig (Dako AS, Denmark). Clones producing antibodies reacting with SlOOββ and SlOOαβ, but negative for SlOOαα were cloned twice by limiting dilution. Monoclonal antibodies were produced by in vitro cultivation of the hybridoma clones by inoculation of 10 4 cells/mL in DMEM, 5 % Fetal Calf Serum in roller bottles and allowed to grow for 10 - 14 days. The monoclonal antibodies were then purified from the culture medium by Protein A (Bioprocessing Ltd, Durham, UK) affinity chromatography according to the manufacturers recommendation. The S10, S21 , S23, and S35 monoclonal antibodies (MAb), produced by hybridoma cell-lines S10:3, S21:4, S23:2, and S35:2, respectively, were selected for the design of immunoassays according to the invention. EXAMPLE 2. Characterization of the antibodies 2. 1. Determination ofisotype The isotype of the antibodies were determined after adsorption of the established antibodies in microtiter plats coated with antibodies against mouse IgG+A+M antibodies (Zymed Lab. Inc, CA, US). The isotype were then detected by incubation with HRP labeled antibodies specific for Mouse IgGl , Mouse IgG2a, Mouse IgG2b, Mouse IgG3, Mouse IgA and Mouse IgM antibodies (Zymed Lab. Inc., CA, US), and incubation with OPD substrate and determination of OD at 450 nm. All antibodies according to the invention were found to be of the IgGl isotype. 2. 2 Reactivity with Sl OOββ, Sl OOcxβ and Sl OOaa Reactivity with SlOOββ, SlOOαβ and SlOOαα of the established S100 MAb:s was determined in enzyme immuno-metric assay (EIA) with the SIOO MAb adsorbed to the wells of microtiter plates, incubation with purified SIOO antigen, and detection of bound SIOO with polyclonal Rabbit Anti S 100 followed by HRP Swine Anti Rabbit Ig (Dako AS, Denmark). S100 MAb were coated in the wells of Nunc MaxiSorp Immunoplate (Nunc AS, Denmark), by incubation of S100 MAb solution over night at room temperature (200 μL/well, 10 μg MAb/mL in 0.2 M NaH2PO4). After washing unspecific binding were blocked by incubation with 300 μL/well of 50 mM Tris, pH 7.8, 0.1 g/L BSA, 60 g/L Sorbitol. In the EIA 50 μL of SlOOββ, SlOOαβ, or S lOOαα (50 μg/L), (Affinity Res. Inc. UK), and 100 μL of Assay Buffer (50 mM Tris, 0.15 M NaCl, 10 g/L BSA, 0.5 g/L bovine Ig, 0.1 g/L methylisothiazolone, pH 7.75) were incubated in duplicates in the S100 MAb coated microtiter wells. After 1 h incubation with constant shaking the wells were washed twice, and 100 μL of Rabbit Anti Human S100 (Dako AS, Denmark), diluted 1/3000 in PBS- P/oBSA, 0. 1 % Tween 20 was added, and the incubation continued for 1 h. After additional washings bound Rabbit Anti S100 was determined by incubation with HRP Swine Anti Rabbit Ig, Dako AS, diluted 1 /500 in PBS- 1%BSA, for 1 h, and after washing determination of OD 620 nm after incubation with 100 μL 3, 3', 5, 5' - terra methyl benzidine (TMB) substrate (Elisa Technologies, Lexington, KY, US). The reactivity of selected S100 MAb:s in immunometric assay format are shown in Figure 1. These results show that: S35 MAb of Group A (see Example 2.4) recognises an epitope accessible only in SlOOαβ, but not in SlOOαα and SlOOββ. S21 MAb of Group B recognises an epitope accessible both in S lOOββ and SlOOαβ, but not accessible in S lOOαα. S23 MAb of Group C recognises an epitope equally accessible in S lOOββ and SlOOαβ, but not accessible in SlOOαα. S10 MAb of Group E recognises an epitope accessible only in SlOOββ, but not accessible in SlOOαα or SlOOαβ. 2. 3. Epitope characterisation The structural relation between the antigenic domains of the different S100 MAb was characterised by determination of possible sandwich combinations (Example 2. 3a) and inhibition of binding of SlOOββ and S lOOαβ (Example 2. 3b). 2. 3a. Dose-response curves for heterologous SI OO EIA SIOO EIA:s were designed with SIOO MAb coated in microtiter wells as described above, and horse radish peroxidase (HRP) labeled S IOO MAb. The SIOO MAb:s were labeled with HRP (Type V, Sigma Chemical Co., US) according to the procedure of Nakone (Nakone and Kawaoi, J Histochem Cytochem, 22: 1084 - 1089, 1974). The dose-response curves for SlOOββ and SlOOαβ were determined as follows: 50 μL of the S lOOββ or SlOOαβ (0 - 100 μg/L) + 100 μL of assay buffer was incubated in duplicates in the MAb coated wells for 1 h during constant shaking. After washing 100 μL of HRP S 100 MAb, 2 μg/mL, was added, and the incubation continued for 1 h. After washing and incubation with 100 μL TMB substrate OD was determined at 620 nm in an ELISA reader. 2. 3b. Inhibition of binding of Sl OOββ and Sl OOccβ. Inhibition studies were made to estimate the three dimensional relationship between the epitopes of the different antibodies according to the invention. If the binding of a second antibody to the complex between a first antibody and the antigen is inhibited, the epitopes of the two antibodies are overlapping or close in three dimensional structure. If the binding of a second antibody to the complex between a first antibody and the antigen is unaffected, the epitopes of the two antibodies are separated from each other. Inhibition of binding of SlOOββ and SlOOαβ to the S100 MAb was tested by incubation for 30 min of 50 μL S lOOββ or S lOOαβ, 50 μg/L, + 100 μL of S100 MAb, 5 μg/mL, in microtitre plates, as control 50 μL S100 antigen + 100 μL PBS-1% BSA was pre-incubated in the same way. After the pre-incubation 100 μL was transferred to another microtiter plate coated with the S 100 MAb to be tested, and incubated for 1 h. After washing bound S 100 antigen was detected by incubation with Rabbit Anti Human S100 and HRP SwAR. Inhibition was calculated as percent decrease in binding of S100 antigen pre- incubated with S 100 MAb compared to S 100 antigen pre-incubated with PBS- 1% BSA. The inhibition was performed with all antibodies as catching and inhibiting antibody. Some of the results of inhibition of selected MAb:s are shown in Figure 2. S35 MAb (Figure 2A) was inhibited only by itself, S34, and S72. S21 MAb (Figure 2B) was inhibited by itself, S5, S16, S 18, S21, S49, S50, S53, S54, S55, S59, S60, S62, S67, S66, S77, and S83. S23 MAb (Figure 2C) was inhibited by itself only, i.e. recognizing a unique epitope, not recognized by any of the other MAb:s established. S25 MAb (Figure 2D) was inhibited by itself, Sl l, S25, S28, S36, S43, S44, S58, and S65. S36 MAb (Figure 2E) was inhibited by itself, S28, S36, S43, S58, and S65. Further S36 MAb was partially inhibited by Sl l, S25, and S44. S10 MAb (Figure 2F) was inhibited only by itself, S12, and S13. 2. 4 Antigenic domains Based on reactivity with the purified SIOO iso-forms (Example 2.2), inhibition of binding of SIOO (Example 2.3b), and the dose-response of different antibody combinations (Example 2.3a) the antibodies were separated into different epitope groups, Group A - E, Table 1. Antibodies belonging to the same epitope group, i.e. antibodies having overlapping epitopes, inhibited each other >75%. Antibodies belonging to different epitope groups, i.e. antibodies having epitopes separated from each other, inhibited each other <40%. Separation into different subgroups was made when the inhibition differed depending on which antibody was first reacted with the antigen and which antibody was reacted with the formed antigen-antibody complex, i.e. when partial inhibition (40 - 75%) occurred. Table 1. Epitope groups in SIOO. Epitope group A B C D E 1 2 1 2 3 S34 S16 S5 S23 S25 Sll S28 S10 S35 S21 S18 S44 S36 S12 S72 S50 S49 S43 S13 S53 S54 S58 S60 S55 S65 S62 S59 S67 S66 S77 S83 EXAMPLE 3. The specificity of the assays were confirmed by determination of dose-response curves with purified SlOOββ, SlOOαβ and SlOOαα obtained from Affinity Research Ltd, UK. The lyophilised antigens were reconstituted with distilled water to a concentration of 1 μg/μL. The purity of the antigens were tested by SDS-PAGE of reduced and non- reduced samples on 8 - 25 % polyacryl amide gels using Pharmacia Phast system according the manufacturers instructions, and stained with Comassie Brilliant Blue. Based on the evaluation of the staining the purity of the antigens were estimated as > 95 % pure. 3. 1 Assays for specific determination of SlOOββ Assays specific for SlOOββ without significant reactivity with S lOOαβ and SlOOαα were designed by using antibodies of Group E in combination with antibodies of Group C or Group B l. In the preferred configuration SIO MAb was used as catching antibody and S23 MAb or S 21 MAb were used as detecting antibody. Example 3.1. Development of EIA specific for SlOOββ. SIO MAb was biotinylated with BiotinNHRS caproate ester (Sigma Chemical Co, US) using standard procedures, and used as catching antibody. S23 MAb or S21 MAb were conjugated with HRP, Type V (Sigma Chemical Co, US) according to a modification of the Nakone procedure. The biotinylated S IO MAb and HRP conjugated S23 or S21 MAb were used in two-site EIA according to the following protocol. Assay procedure: 1. Add 50 μL of S100 standards (0 - 100 μg/L in PBS, 60 g/L BSA, pH 7.2) + 100 μL of Biotin S10 MAb, 2 μg/mL, in Assay Buffer in Streptavidin coated microtiter plates (Labsystems Oy, Helsinki, Finland). 2. Incubate for 1 h ± 10 min with shaking 3. Wash 3 times with 5 mM Tris buffer, 0.05 % Tween 40, pH 7.75. 4. Add 100 μL HRP S23 MAb (or 100 μL HRP S21 MAb), 2 μg/mL, in Assay Buffer. 5. Incubate for 1 h ± 10 min with shaking. 6. Wash 6 times with 5 mM Tris buffer, 0.05 % Tween 40, pH 7.75. 7. Add 100 μL TMB (ELISA Technology, US) 8. Incubate 30 min± 5 min 9. Determine OD 620 nm in ELISA reader. Dose-response curves for SlOOββ, SlOOαβ and SlOOαα are shown in Figure 3. Based on the dose-response curves the assay according to this example is specific for S lOOββ, with a cross-reactivity for S lOOαβ and SlOOαα of < 5% and < 2%, respectively. 3. 2. Assays for specific determination of SlOOαβ Assays specific for SlOOαβ without significant reactivity with SlOOββ and SlOOαα were designed by using antibodies of Group A in combination with antibodies of Group C or Group Bl. In the preferred configurations S35 MAb was used as catching antibody and S23 MAb or S21 MAb were used as detecting antibody. Example 3.2. Development of EIA specific for SlOOαβ. S35 MAb was biotinylated with BiotinNHRS caproate ester (Sigma Chemical Co, US) using standard procedures, and used as catching antibody. S23 MAb or S21 MAb were conjugated with HRP (Type V, Sigma Chemcal Co., US) according to a modification of the Nakone procedure. The biotinylated S35 MAb and HRP conjugated S23 or S21 MAb were used in two-site EIA according to the following protocol. Assay procedure: 1. Add 50 μL of S100 standards (0 - 100 μg/L in PBS, 60 g/L BSA, pH 7.2) + 100 μL of Biotin S35 MAb, 2 μg/mL, in Assay Buffer in Streptavidin coated microtiter plates (Labsystems Oy, Helsinki, Finland). 2. Incubate for 1 h ± 10 min with shaking 3. Wash 3 times with 5 mM Tris buffer, 0.05 % Tween 40, pH 7.75. 4. Add 100 μL HRP S23 MAb (or 100 μL HRP S21 MAb), 2 μg/mL, in Assay Buffer. 5. Incubate for 1 h ± 10 min with shaking. 6. Wash 6 times with 5 mM Tris buffer, 0.05 % Tween 40, pH 7.75. 7. Add 100 μL TMB, ELISA Technology, US. 8. Incubate 30 min± 5 min 9. Determine OD 620 nm in ELISA reader. Dose-response curves for SlOOββ, S lOOαβ and S lOOαα are shown in Figure 5. Based on the antibodies according to 3.3 immunoassays specific for SlOOαβ, with < 10 % and < 2 % cross-reactivity for SlOOββ and SlOOαα, respectively, may be designed. The results of the assays of Examples 3.1 and 3.2 are summerized to give total S 100 i.e. the sum of S lOOαβ and SlOOββ. REFERENCES Buer J, Probst M, Franzke A, Duensing A, Duensing S, Haindl J, Volkenandt M, Wittke F, Hoffman R, Ganser A, Atzpodien J. 1997. Br J Cancer 75: 1373 - 1376. Henze G, Dummer R, Joller Jemelka H, Bδni R, Burg G. 1997. Dermatology 194: 208 - 212. Ingebrigtsen T., Romner B, Kongstad P., Langbakk B., 1995. J Neurol. Neurosurg. Psych. 59: 103 - 104. Isobe T, Okuyama T., 1978. Eur J Biochem. 89: 379 - 388. Isobe T., OkuyamaT. 1981. Eur J. Biochem. 1 16: 79 - 86. Isobe T, Takahashi K, Okuyama T. 184. J Neurochem. 43 : 1494 - 1496. Kato K., Kimura S. 1985. Biochim. Biophys. Acta 842: 146 - 150. Lindholm L., Holmgren J, Svennerholm L, Fredman P, Nilsson O, Persson B, Myrvold H, Lagergard T. 1983. Int Arch Allergy and Appl. Immunol. 71: 178 - 181. Michetti F., Massaro A, Russo G., Rigon O. 1980. J Neurol 44: 731 - 743. Moore BW. 1965. Biochem Biophys. Res. Comm. 19: 739 - 744. Moore BW. 1982. Scand J Immunol 9: 53 - 74. Nakane PK, Kawaoi A. 1974. J Histochem. Cytochem., 22 1084 - 1090. Persson L., Hardemark H-G., Gustafsson J., Rundstrom G., Mendel-Hartvig I., Esscher T., Pahlman S. 1987. Stroke 18: 911 - 918. Westaby S, Johnson P, Parry AJ, Blomquist S, Solem JO, Ailing A, Pillai R., Taggart DP, Grebenik C, Stahl E. 1996. Ann Thorac Surg 61 : 88 - 92. Zimmer DB, Cornwall EH, Landar A and Song W. 1995. Brain Res. Bull 37: 417 - 429.

Claims: CLAIMS 1. A monoclonal antibody, or antigen binding fragment thereof, which recognizes an epitope of SIOO that is accessible in SlOOββ, but that is only accessible to an extent of less than 5% in SlOOαα and SlOOαβ. 2. Antibody according to claim 1, which is produced by hybridoma cell line S10:3 given ECACC Accession No. 98082619, or an antigen binding fragment t ereof. 3. Antibodies, antibody fragments or peptides with the same binding specificity as the antibody according to claim 2 as shown by that they inhibit the binding of said antibody by > 75 %. 4. The hybridoma cell line S10:3 given the ECACC Accession No. 98082619. 5. A monoclonal antibody, or antigen binding fragment thereof, which recognises an epitope of S100 that is accessible in S lOOαβ, but that is only accessible to an extent of less than 5% in SlOOαα and SlOOββ. 6. Antibody according to claim 5, which is produced by hybridoma cell line S35:2 given ECACC Accession No. 98082616, or an antigen binding fragment thereof. 7. Antibodies, antibody fragments or peptides with the same binding specificity as the antibody according to claim 6 as shown by that they inhibit the binding of said antibody by > 75 %. 8. The hybridoma cell line S35:2 given the ECACC Accession No. 98082616. 9. Antibody, or antigen binding fragment thereof, which is produced by hybridoma cell line S23:2 given ECACC Accession No. 98082617. 10. Antibodies, antibody fragments or peptides with the same binding specificity as the antibody according to claim 9 as shown by that they inhibit the binding of said antibody by > 75 %. 1 1. The hybridoma cell line S23:2 given the ECACC Accession No. 98082617. 12. The antibody, or antigen binding fragment thereof, which is produced by hybridoma cell line S21:4 given ECACC Accession No. 98082618. 13. Antibodies, antibody fragments or peptides with the same binding specificity as the antibody according to claim 12 as shown by that they inhibit the binding of said antibody by > 75 %. 14. The hybridoma cell line S21:4 given the ECACC Accession No. 98082618. 15. A method of quantitatively determining the total amount of SlOOαβ and SlOOββ in a sample which comprises: a) contacting an aliquot of the sample with an antibody of claim 1 under conditions permitting the antibody to bind to SlOOββ and form a complex therewith; b) determining the amount of antigen bound to said antibody, thereby determining the amount S lOOββ in the sample. c) contacting an aliquot of the sample with an antibody of claim 5 under conditions permitting the antibody to bind to SlOOαβ and form a complex therewith; d) determining the amount of antigen bound to said antibody, thereby determining the amount SlOOαβ in the sample. 16. Method according to claim 15 wherein step b and/ or d comprise the use of any antibodies according to claims 9 and 12. 17. A kit for assaying the total amount of SlOOαβ and SlOOββ comprising any antibodies of claims 1, 5, 9, and 12. 18. A method of quantitatively determining the amount of SlOOββ in a sample which comprises step a and b according to claim 15. 19. A kit for assaying the amount S lOOββ comprising any antibodies of claims 1, 9, and 12. 20. A method of quantitatively determining the amount of SlOOαβ in a sample which comprises step c and d according to claim 15. 21. A kit for assaying the amount of SlOOαβ comprising any antibodies of claims 5, 9, and 12.

Inventors: NILSSON OLLE, NILSSON, OLLE
Applicants: CANAG DIAGNOSTICS AB, NILSSON OLLE, NILSSON, OLLE
Application year: 1999
Main classification: C07K16
IPC codes: C07K16, C12N5, C12N15, C12P21, C12R1, G01N33