A METHOD AND A KIT FOR THE DIAGNOSIS OF IgA NEPHROPATHY

Abstract: A method for the diagnosis of IgA nephropathy using an antigene-antibody interaction comprising the steps: a) preparing a substrate capable of binding fibronectin or IgA, b) contacting the substrate resulting from step a) with a sample of body fluid drawn from a patient subject to diagnosis to bind any fibronectin-IgA-complex present in said sample to the substrate, and c) determining the presence of complex bound to the substrate using the reaction between the exposed part of such bound complex and a corresponding antibody thereto; and a diagnostic kit for use in such diagnosis.

Description: A method and a kit for the diagnosis of IgA nephropathy. The present invention relates to a method for the dia¬ gnosis of IgA nephropathy using an antigen -antibody interac¬ tion, and the invention also includes a diagnostic kit for use in such a diagnosis. Patient* with primary IgA nephropathy, also known as Berger's disease, have been shown to have circulating IgA antibodies, binding to collagen IV prepared -.roe gloierular baseaent aeabrane (BBM) (1). It Mas also shown that the IgA antibodies bound equally well to collagen I, II and IV and that denatured collagens bound antibodies aost efficiently (1). In view of the wide distribution of the various collagens, it is of - interest to note the coexistence with IgA nephropathy of syβptoβs from extrarenal organs (2). Thus syβptoas froa skin, eye and joints are of particular interest since these structures contains collagen as a βajor coβponent. Indeed one study reports presence of vascular IgA deposits in skin froa patients with IgA nephropathy (3). The association of exacerbations of clinical disease with upper respira¬ tory tract or gastrointestinal infections and the finding of IgA depo¬ sits in the gloβerular aesangiua is well known. The finding of increased levels of IgA-bearing peripheral lγaphocytes (4) as well as decreased IgA-specific suppressor T cell activity (5) and increased IgA-specific helper T-alfa cells (6) suggests an iaaunological aechanisa. Fibronectin, also known as cold insoluble globulin (CIg), that is alte¬ red in aany disease processes, is present both as a plasaaprotein and as a cell surface protein. The two fores differ slightly in composition but share iaportant functions as binding to gelatin (collagen), heparin, fibrin and cell surface receptors (7). Fibronectin is the aajor plasβa coβponent binding to gelatin (S) and this property as well as its hepa¬ rin binding properties have been utilized in its isolation (9). Interes¬ tingly fibronectin is present also in baseaent aeabranes. It can be visualised by immunofluorescense not only in the glomerular basement membrane but also in the mesangium of healthy indi¬ viduals CIO). Furthermore a concomitant increase of mesangial fibronectin and mesangial matrix was observed in patients with IgA nephropathy and Henoch-Schdnleins purpura (10). The present invention is based on the surprising disco¬ very, that the IgA-antibodies are present in circulating immune complexes in patients with primary IgA nephropathy together with fibronectin, and this unexpected finding does, of course, explain the fact that the igA-containing complexes have the ability of binding to collagen. This new finding that the circulating immune complex contains, in addition to IgA, also fibronectin, enables the provision of both method for the diagnosis of IgA nephropathy and a diagnostic kit for use in such diagnosis. Accordingly, one object of the present invention is to provide a method for the diagnosis of IgA nephropathy using antigen-antibody interaction, and another object of the in¬ vention is to provide a diagnostic kit for use in such diag¬ nosis of IgA nephropathy. The method of the present invention is characterized by the following steps: a) preparing a substrate capable of binding fibronectin or IgA, b> contacting the substrate resulting from step a) with a sample of body fluid drawn from a patient subject to diag¬ nosis to bind any fibronectin-IgA-complex present in said sample to the substrate, and c) determining the presence of complex bound to the substrate using the reaction between the exposed part of such bound complex and a corresponding antibody thereto. In preparing such substrate capable of binding fibro¬ nectin preferred binding agents are collagen, heparin, fibrin or anti-fibronectin. According to this aspect of the inven¬ tion the substrate is prepared in such a way as to bind the ibronectin-IgA-complex through its fibronectin component. This in turn means that the IgA-component of the complex is available for determining the presence of complex bound to the substrate . On the other hand the binding of the fibronectin-IgA- -complex to the substrate can be directed to the IgA-part of the complex. In such case there can be used as a binding a- gent either anti-IgA or a lectin, both of which are capable of binding IgA. In this case the fibronectin part of the com¬ plex will be available for determining the presence of com¬ plex bound to the substrate. The determination under step c) of the method of this invention can be of a quantitative nature and can be based on a conventional detection system. Among such detection systems there may be mentioned those based on enzymatic activity, or systems based on radiation emitted by a radioactive isotope or based on fluorescence. The substrate used in binding the fibronectin-IgA-com¬ plex present in a sample subject to diagnosis can be consti¬ tuted by different objects, such as icrotiter plates, labo¬ ratory test tubes, nitro-celluloβe paper, plastic spheres or any other object suitable for the purpose. Samples of body fluid drawn from a patient subject to the diagnostic method of the present invention may be consti¬ tuted by for example human blood, serum, plasma or saliva. The invention also provides for a diagnostic kit for use in the diagnosis of IgA nephropathy, and such kit com¬ prises : a) an object capable of binding fibronectin or IgA, and b) reagent comprising a component which is capable of binding IgA or fibronectin and a detection component enabling deter¬ mination of fibronectin-IgA complex bound to the said object. In the embodiments wherein the IgA part constitutes exposed part of the complex the object carries attached to the surface thereof heparin, collagen, fibrin or anti-fibro- nectin. In such case the reagent preferably comprises lectin or antibody directed against IgA. In the oppoβit* cait, wherein fibronectin is the ex¬ posed part of the complex the object carries attached to the surface thereof ant i- IgA or lectin, the reagent comprising heparin, collagen, fibrin or anti-f ibronectin . In βuch diagnoβtic kit according to the invention the function of the detection component may be baaed on enzymatic activity, r&diation emitted by a radioactive isotope or fluo¬ rescence . The Invention will be further illustrated in the follo¬ wing by non-limiting specific examples. This illustration is associated with references to the appended drawing, wherein: Fig. 1 shows the binding of IgA, fibronectin (FN) and a complex to a heparin-Sepharoβe column; Fig. 2 shows a diagram on the elution of proteins from, an ordinary ion exchanger using a salt gradient; Fig. 3 shows the binding of IgA, fibronectin and a com¬ plex to a jacalin-Sepharoβe column; and Fig. 4 shows the separation of proteins using SDS-FAGE and immunoblotting. EXAMPLE 1 Antisera^ Patient sera were from patients with primary IgA nephropathy ( glomerulonephritis and dominating mesangial IgA deposits detected by immuno luorescense microscopy of renal biopsy speciaens). None of the patients had signs of systemic lupus erythematosus (5LE), Henoch-Schδn- leins purpura or cirrhotic liver disease. Sera from healthy blood donors were used as controls. EXAMPLE 2 IS5LitiθQ.__.5__i__.lt5ϋ!lt_!__.SELi5.tlj. Antisera ( 1 ml ) were dialysed against 0.05 1 * 1 Tris, 0.05X sodium azide pH 7.4 before applied to a 10 ml Hepa- rin-Sepharose CL-66 column (Pharmacia, Uppsala, Sweden). Material eluted -from the column with 0.5 M NaCl in the same buffer was dialysed against 0.1 K sodium phosphate, 0.05X sodium azide pH 7.5 before chromatographed on a 1 al Mono 0 coluan (Pharaacia). Chroaatography was pβrforaβd uiing a LKB (Bromma, Sweden) HPLC system with low pressure mixing. Absorbance was measured at 280 nm with a LKB 2151 variable wavelength detector. Fractions binding to collagen I and reacting with anti human IgA (Oako- patts, Hagersten, Sweden) were pooled and applied to a jacaline agarose ( Pierce chemicals, Rockford, Illinois ) . IgA containing complexes were eluted with 0.2 M elibiose in a buffer containing 0.05 M Tris, 0.15 M NaCl, 0.05X sodium azide H 7.5. EXAMPLE 3 Ecej___catLQQ_Qi_.CQUftaeQ.l_. Collagen I was prepared by pepsin extrac¬ tion from bovine flexor tendon as described by Vogel et al ( 12 ) . EXAMPLE 4 Ilfc roB oresiii SDS-PAGE was performed as described by Laemmli ( 13 ) using 3-16X linear gradient gels. Bels were stained with silver accor¬ ding to the method of Morrisey (14) omitting glutaraldehyde. Reduction was performed by adding 2-mercaptoethanol to 0.2/. v/v to samples prior to electrophoresis and boiling for two minutes. EXAMPLE 5 ikL§__. Antigen was coated to polystyrene 96-well βicrotiter plates (NUNC immunoplate I, NUNC, Roskilde, Denmark). Fractions from the columns were coated over night under non denaturing conditions using 0.05 M sodium carbonate buffer at pH 9.6 containing 0.05X sodium azide. This was followed by incubation for 1 hour with the same buffer also containg 2X bovine serum albumin (blocking buffer ) to prevent non speci¬ fic binding. Collagen I was coated under denaturing conditions using 6 M guanidine-HCl , 0,05 M Tris-HCl pH 7.4 over night. In this case incuba¬ tion with blocking buffer was not needed. Sera were diluted in 0.01 M phosphate pH 7.5, 0.15 M Na l , 0.05X Twtm 20 and 0.25 H guanidint-HCl and incubated for 1 hour in the coated βicrotiter plate. IgA antibodies were detected by incubation with affinity purified anti huaan IgA alka¬ line phosphatase conjugate (Dako) for one hour. Fibronectin was detected using rabbit anti huaan fibronectin antiserum (Dako) followed by anti rabbit-IgG alkaline phophatase conjugate (Dako). Enzyme activity was determined using p-nitrophenyl phosphate as the substrate. Microtiter plates were rinsed between each step with 0.15 M NaCl containing 0.05.4 Tween 20. All samples were analysed as triplicates. Absorbance was moni¬ tored at 405 nm using a Titertek Multiskan photometer. EXAMPLE 6 Immungbl.otti.ngj. Proteins were separated using SDS-PAGE and βlectropho- retically transferred to nitrocellulose paper (Schleicher and Schull,- Dassel ,W.Germany) at 0.5 A for 4 hours. Non specific binding was preven¬ ted by incubation with blocking buffer for 1 hour. IgA antibodies were detected using peroxidase conjugated anti human IgA antiserum (Dako) and fibronectin was detected using rabbit anti human fibronectin followed by peroxidase conjugated anti rabbit antiserum. Enzyme activity was deter¬ mined with HsOz/diaminobenzidine (0.5mg/ml) (Fluka) in 0.05 M sodium phosphate pH 7.5 containing cobalt chloride and ammonium nickel sulfate (15). EXAMPLE 7 iSSlitign_gf_CNBr;fragments.. Since we have previously found that IgA antibodies from patients with IgA nephropathy bind to collagen I,II and IV (1) an attempt was made to isolate the specific epitope of bovine collagen I responsible for the binding. Pepsin extracted collagen I was fragmented by the use of CNBr and the fragments were separated by cation exchange chro atography on a Mono S column followed by gel chromatograp- hy on a TSK 63000SH coluan. Binding of antibodies to collagen I could bt completely inhibited by fragments identified as the CB7 fragment of the alfa » chain and the CB3,5 fragaent of the alfβa chain. These fragments are known to contain the fibronectin binding domain (16). Thus, it appears that fibronectin is invol¬ ved in the binding of IgA antibodies to collagen. Indeed, as we found for patient IgA antibodies, Engvall et al (8) have previously shown that fibronectin binds to both collagens I and II and that binding is enhan¬ ced when denatured collagens are used. In, further support we could demonstrate by ELISA that anti fibronectin antibodies coated to a icro- titer plate caused binding of IgA antibodies as well as fibronectin when incubated with serum from patients with primary IgA nephropathy. When using serum froa healthy blood donors as control no IgA antibodies were bound to the anti fibronectin antibody coat. He therefore decided to purify the complexes, in principle using affi¬ nity columns for fibronectin and IgA, respectively, in sequence. EXAMPLE 8 SέS9CEti.on_gf_fL&rgne ti.n_to_ eβarin_SeBhargse 5 . Heparin-Sepharose (9) was used as the initial step for the purification of ibronectin-IgA complexes from human serum. As demonstrated in ELISA (fig I), using the fractions as coat, expectedly most of the IgA antibodies were not retai¬ ned on the heparin column. The fibronectin bound to the heparin column, and was eluted using 0.5 K NaCl (fig 1). It is known that fibronectin binds to collagen by its collagen bin¬ ding domain located in the (.-terminal part of the molecule (7,17 ) . The collagen binding properties of fibronectin were utilized in an ELISA to demonstrate the presence of .ibronectin-IgA immune complexes in the eluate from the heparin column. Denatured collagen I (17) was used as coat. Fractions froa the heparin-Sβpharost coluan were incubated in tht collagen coated aicrotiter wells. Bound IgA antibodies were detected using a specific antibody conjugate (fig 1). Only the fraction eluted from the heparin coluan by the use of NaCl contained antibody reactivity in the assay, apparently representing fibronectin-IgA iaaune complexes in which the IgA antibodies are bound to fibronectin and fibronectin aediates binding to collagen by its collagen binding domain. The major portion of the IgA antibodies did not bind to the heparin sep- harose and no collagen binding IgA antibodies were found in this unbound fraction. Contrasting results were obtained with a serum sample from a healthy blood donor. No immune complexes were detected neither in the fraction bound to the heparin-Sepharose nor in the unbound fraction ( fig 1 ) . EXAMPLE 9 liD^iD9-_.Si_i5!DyQ§_£9!!Bl§SSS-i2-3Di9D_.f>l£h*nge_cglumn i Since heparin binds not only fibronectin, but other plasma proteins as well (9) an ion exchange column was selected for the next step in the purification pro¬ cedure. Material eluted from the heparin column was applied to a Mono Q, anion exchange, HPLC column. Immune complexes, detected by the ELISA with collagen I coat, were eluted between 0.22-0.24 M NaCl (fig 2). The presence of free IgA antibodies (separated from the immune complexes) demonstrates that a small proportion of the IgA binds to the heparin column and/or IgA is liberated due to dissociation of immune complexes during the purification procedure. EXAMPLE 10 6^iStfi i n_ f_the_I.gA_cgmggnent_in_imfflune_cgmg^exes_tg_i^ £o_Li_. Fractions containing immune complexes (fig 2) were pooled and chromatographed on a jacalin-Sepharose column. Jacalin is a lectin that can be used for binding of IgA antibodies (18). This provides a possibi¬ lity to separate the complexes froa free fibronectin since this structu¬ re is not present on the fibronectin aolecule. As expected fibronictin is found both in the aaterial not bound to the jacalin coluan and in the bound fraction that was eluted with aelibiose, while IgA antibodies are present only in the bound material (fig 3). The fibronectin coapontnt of the immune complexes was bound to collagen I in an ELISA and fibronectin and IgA antibodies were identified (fig 3). The presence of components with mobilities corresponding to fibronectin and IgA in the material bound to the jacalin column was further demonstrated with SDS-PA6E both with and without reduction (fig 4). Further proof for the identity of the components was obtained by iamunoblotting using anti-IgA and anti- fibronektin respectively (fig 4). The major components visualized by SDS-PA6E was thus shown to represent IgA and fibronectin , respectively. The above examples show that patients with IgA nephrop thy have circulating immune complexes containing fibronectin IgA antibodies. This is in agreement with a proposed immune plex mediated nature of primary IgA neprhopathy (11). fo References 1. Cederholm, B., Wieslander, J. and Heinegard, D. (1986) Proc. Natl. Acad. Sci. USA 83, 6151-6155. 2. Mustonen, J., Pasternack, A., Helin, H. and Nikkila, M. (1985) Am, 3. Nephrol. _>, 150-157. 3. Baart De La Faille- uyper, E., Kater, L., Kuijten, R. f Kooiker, C, Wagenaar, 5., Van Der Zouwen, P. and Mees, E. (1976) Kidney Int. 9, 424-429. 4. Nomoto, Y., Sakai, H. and Arimori, S. (1979) Am. 3. Clin. Pathol. 71, 158-160. 5. Sakai, H., Nomoto, Y. and Arimori, S. (1979) Clin. Exp. Immu¬ nol. 38, 243-298. 6. Sakai, H., Endoh, M. and Tomino, Y. (1982) Clin. Exp. Immunol. 50, 77-82. 7. Hakomori, S., Fukuda, M., Sekiguchi, K. and Carter, W. (1984) In: Extracellular Matrix Biocemistry. Elsevier Science Publis¬ hing Co, New York, USA, 229-275. 8. Engvall, E., Rouslahti, E. and Miller, E. (1978) 3. Exp. Med. 147:2, 1584-1595. 9. Yamada, K (1982) Immunochemistry of the Extracellular Matrix 1, 111-123. 10. Pettersson, E. and Colvin, R. (1978) Clin. Immunol. Immunopat- hol. 11, 425-436. 11- Coppo, R., Basolo, B., Piccoli, G., Mazzucco, G., Bulzomi, M., Roccatello, D., De Marchi, M., Carbonara, A. and Di Belgiojoso, B. (1984) Clin. Exp. Immunol. 57, 583-590. 12. Vogel, K., Paulsson, M. and Heinegard, D. (1984) Biochem. 3. 223, 587-597. 13. Laemmli, U. (1970) Nature (London) .227, 680-685. if 14. Mornsey, 3. (1981) Anal. Biochem. 117, 307-310. 15. De Bias, A. and Cherwinski, M. (1983) Anal. Biochem. 133, 214-219. 16. Yamada, K. , (1983) Ann. Rev. Biochem. 52, 761-799. 17. Engvall, E. and Rouslahti, E. (1977) Int. J. Cancer 20, 1-5. 18. Roque-Berreira, M. and Campos-Neto, A. (1985) J. Immunol. 134, 1740-1743.

Claims: CLAIMS i. A method for the diagnosis of IgA nephropathy using an antigene-antibody interaction, characterized by the steps: a) preparing a substrate capable of binding fibronectin or IgA b) contacting the substrate resulting from step a) with a sample of body fluid drawn from a patient subject to diagnosis to bind any fibronectin-IgA-complex present in said sample to the substrate, and c) determining the presence of complex bound to the substrate using the reaction between the exposed part of such bound complex and a corresponding antibody thereto. 2. A method according to claim 1, characterized by preparing a substrate capable of binding via collagen, fibrin or heparin to the fibronectin part of the complex. 3. A method according to claim 1, characterized by preparing a substrate capable of binding via anti-fibronectin to the fibronectin part of the complex. 4. A method according to claim 1, characterized by preparing a substrate capable of binding via anti-IgA or lectin to the IgA-part of the complex. 5. A method according to any preceding claim, wherein the body fluid is constituted by human blood, serum, plasma or saliva. 6. A method according to any preceding claim, charactertized in that in step c> the presence of bound complex is measured quantitatively using a detection system based on enzymatic activity, radiation emitted by a radioactive isotope or fluorescence. 7. A method according to claim 2 or 3, characterized by using under step c) the reaction between the IgA-part of the complex and an anti-IgA- or lectin-enzyme complex. 8. A method according to claim 4, characterized by using under step c> the reaction between the fibronectin part of the complex and an anti- ibronectin-, heparin- , fibrin- or collagen-enzyme complex. 9. A method according to any preceding claim, wherein the substrate is constituted by a microtitβr plate. 10. A diagnostic kit for use in the diagnosis of IgA nephropathy, comprising: a) an object capable of binding fibronectin or IgA, and b) a reagent comprising a component capable of binding IgA or fibronectin and a detection component enabling determination of fibronectin-IgA complex bound to the ob ect. 11. A diagnostic kit according to claim 10, wherein the object carries attached to the surface thereof heparin, collagen, fibrin or anti-fibronectin, and wherein the reagent comprises lectin or antibody directed against IgA. 12. A diagnostic kit according to claim 10, wherein the object carries attached to the surface thereof anti-IgA or lectin, and wherein the reagent comprises heparin, collagen, fibrin or anti-fibronectin. 13. A diagnostic kit according to claim 10 or 11, wherein the function of the detection component is based on enzymatic activity, radiation emitted by a radioactive isotope or fluorescence.

Inventors: BYGREN PER, CEDERHOLM BO, HEINEGAARD DICK, WIESLANDER JOERGEN, BYGREN, PER, CEDERHOLM, BO, HEINEGAARD, DICK, WIESLANDER, JOERGEN
Applicants: BIOCARB AB
Application year: 1988
Main classification: G01N33
IPC codes: G01N33